Conjugates, compositions and methods for hydroxyapatite targeted imaging and therapy

Abstract

Hydroxyapatite-targeting conjugates, compositions comprising same, and methods of use for radioimaging or radiotherapy.

Description

[Technical field]

[0001] Priority This patent application is related to and claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 313,395, filed February 24, 2022. The contents of the aforementioned application are hereby incorporated by reference in their entirety into this disclosure.

[0002] The present disclosure relates to hydroxyapatite-targeting conjugates, compositions comprising same, and methods of using such conjugates and compositions for imaging and therapy. [Background technology]

[0003] It is difficult to deliver drugs to bone because its composition is different from other organs. Bone is mostly composed of calcium phosphate crystals known as hydroxyapatite. Therefore, current systemic treatments can be very ineffective for various bone diseases, because most of the drug is captured by internal organs instead of concentrating at diseased and damaged sites, and is eventually excreted through urine. Increasing the dosage can improve site localization, but can also increase systemic off-target side effects. Therefore, an effective bone targeting system is needed to improve the benefit of drugs in bone diseases.

[0004] Among various bone diseases, bone cancer (such as bone metastasis caused by breast cancer or prostate cancer) has a particularly high mortality rate, because current systemic chemotherapy and radiation therapy are ineffective in eradicating or reducing tumor growth.At the most, these treatments provide relief from the pain caused by tumor-induced bone lesions, and as a result, many bone cancers are widely accepted as incurable.Therefore, there is a great urgency in developing bone-targeted therapy to improve the quality of life and survival rate of patients. Summary of the Invention

[0005] Provided are conjugates of formula I: HB-L A -A (Formula I) or a pharma- ceutically acceptable salt thereof, HB is the radical of a (e.g., linear or branched) polyanionic, polyacidic and / or polyelectrolyte ligand (e.g., a biological targeting moiety) that binds to hydroxyapatite; A is a radiocontrast agent, a radiosensitizer, a radioprotector, or a radiotherapeutic agent; L A is a linker connecting HB and A or is absent. It is.

[0006] The HB can include a radical of a linear polyanionic, polyacidic and / or polyelectrolyte ligand that binds to hydroxyapatite. The HB can include an amino acid or a derivative thereof. The HB can include L-aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic acid, or a mixture of two or more of the foregoing, or a derivative thereof.

[0007] In some embodiments, HB is a combination of (i) a serum albumin binder (AB) directly conjugated to HB or (ii) AB and a linker L AB (AB-L AB ), wherein AB-L AB L AB In some embodiments, one or more peptide bonds of HB and / or A are arranged in a relative cis orientation. In some embodiments, one or more peptide bonds of HB and / or A are arranged in a relative trans orientation.

[0008] In some embodiments, L A , L AB , or L A and L AB In some embodiments, both of L A , L AB, or L A and L AB Both are in the D-configuration. AB may be attached to the amine or carboxyl terminus of a polyacidic peptide or an active group, such as a carboxyl, amine or thiol group, of the repeating anionic, acidic or electrolytic moieties of HB.

[0009] HB is the formula XA 1 , X.A. 2 , X.A. 3 or XA 4 :

[0010] [ka] [In the formula, R is COOH for L- or D-aspartic acid, and CH for L- or D-glutamic acid. 2 COOH, or for unnatural acidic amino acids, CH 2 CH 2 COOH, n=1 to 50,

[0011] [ka] L A represents the point of attachment to HB is AB or AB-L AB If further comprising:

[0012] [ka] AB or L AB represents the point of attachment to The linear polyanion may include a radical of

[0013] In some embodiments, HB is of the formula XA 3 and XA 4 HB may be free of (e.g., free of) an albumin binder as shown in the formula XA 5, X.A. 6 , X.A. 7 or XA 8 :

[0014] [ka] [In the formula, R is COO- in the case of L- or D-aspartic acid, and CH in the case of L- or D-glutamic acid. 2 COO- or, for unnatural acidic amino acids, CH 2 CH 2 COO- n=1 to 50,

[0015] [ka] L A represents the point of attachment to HB is AB or AB-L AB If further comprising:

[0016] [ka] AB or L AB represents the point of attachment to

[0017] [ka] represents a hydrogen or a point of attachment to HB. In some embodiments, HB can include a radical of a branched polyanion of the formula XA 7 and XA 8 These may be free of albumin binders, such as those shown in

[0018] L A can contain one or more amino acids. A can include a brush border membrane (BBM) linker. A can include a BBM linker comprising Met-Val-Lys.

[0019] L A and L AB Either or both of L may include a sustained release linker. A may include a sustained release linker. A may include a fast release linker. A is the following L 1 or L 2 :

[0020] [ka] wherein

[0021] [ka] L A and A or L A and HB.

[0022] L A and L AB Either or both of L may contain a spacer. A and L AB may be conjugated to the N- or C-terminus of HB, of any monomer of the polymer, or of any substituent of any monomer of the polymer.

[0023] A can include a chelating agent. A can include DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or a derivative thereof. A is DOTA or a derivative thereof; TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid) or a derivative thereof; HEHA (1,2,7,10,13-hexaazacyclooctadecane-1,4,7,10,13,16-hexaacetic acid) or a derivative thereof; PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N',N",N"',N""-pentaacetic acid); SarAr (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine) or a derivative thereof; NOTA (1,4,7-triazacyl) nonan-1,4,7-triacetic acid) or a derivative thereof; NETA (4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl)acetic acid or a derivative thereof; TRAP (1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid) or a derivative thereof; HBED (N,N0-bis(2-hydroxybenzyl)-ethylenediamine-N,N0-diacetic acid) or a derivative thereof; 2,3-HOPO (3-hydroxypyridin-2-one) or a derivative thereof; PCTA (3,6,9,15-tetraazabicyclo[9.3.1]-Pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid) or derivatives thereof;PDTA(1,3-propylenediaminetetraacetate) or derivatives thereof;NTA(nitrilotriacetic acid) or derivatives thereof;EDDS(ethylenediaminedisuccinate) or derivatives thereof;EDTA(ethylenediaminetetraacetic acid) or derivatives thereof;MGDA(N-(1-carboxylatoethyl)iminodiacetate) or derivatives thereof;DFO(desferrioxamine) or derivatives thereof;DTPA(diethylene triaminepentaacetic acid) or its derivatives;CDTA ((1,2-cyclohexylene dinitrilo)tetraacetic acid) or its derivatives;CPTA (1,4,8,11-tetraazacyclotetradecane derivatives) or its derivatives;OCTAPA (N,N0-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N0-diacetic acid) or its derivatives;H2-MACROPA (N,N'-bis[(6-carboxy-2-pyridylmethyl]-4,13-diaza-18-crown-6) or its derivatives;H. 2 The EC20 head group may be selected from the group consisting of dedpa (1,2-[[carboxy)-pyridin-2-yl]-methylamino]ethane or a derivative thereof; HEDP (hydroxyethylidenediphosphonic acid or etidronic acid) or a derivative thereof; HEBD (N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid) or a derivative thereof; HYNIC (6-hydrazinopyridine-3-carboxylic acid) or a derivative thereof; DMSA (meso-2,3-dimercaptosuccinic acid) or a derivative thereof; and β-l-diaminopropionic acid, an EC20 head group including Asp and Cys.

[0024] The chelating agent is 225 It is possible to chelate alpha-emitting radioisotopes such as Ac. The chelating agent is 177 Lu or 90 It is possible to chelate β-emitting radioisotopes such as Y. The chelating agent is 111 In or 67 It is possible to chelate gamma-emitting radioisotopes such as Ga. The chelating agent is 18 F, 44 Sc, 47 Sc,52 Mn, 55 Co, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, 90 Y, 99m Tc, 111 In, 114m In, 117m Sn, 124 I, 125 I, 131 I, 149 Tb, 153 Sm, 152 Tb, 155 Tb, 161 Tb, 177 Lu, 186 Re, 188 Re, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 224 Ra, 225 Ab, 225 Ac or 227 It can chelate Th.

[0025] A can include a chelating agent that chelates or binds to a radioisotope. The radioisotope can be a therapeutic radioisotope. 68 Positron Emission Tomography (PET) imaging radioisotopes such as Ga, or 11 C. 13 N, 15 O and 18 The radioisotope may be a PET imaging radioisotope selected from the group consisting of F. The radioisotope may be a therapeutic radioisotope, including an alpha-emitting or beta-emitting radioisotope. In certain embodiments, the beta-emitting radioisotope is 177 This is Lu.

[0026] The chelator can be an imaging radioisotope or a PET imaging radioisotope. In certain embodiments, the imaging radioisotope is a gamma-emitting radioisotope, 111In.

[0027] A is, 14 C. 3 H, 34 S, 32 P, 125 I and 131 The radioactive agent A can be linked to the N- or C-terminus of HB or to any active group on the repeating portion of HB via a linker L A It may be conjugated via

[0028] The conjugate has the following structure:

[0029] [ka] may have the following structure:

[0030] The conjugate has the following structure:

[0031] [ka] may have the following structure:

[0032] The conjugate has the following structure:

[0033] [ka] may have the following structure:

[0034] The conjugate has the following structure:

[0035] [ka] may have the following structure:

[0036] The conjugate is 177 It is chelated to Lu and has the structure:

[0037] [ka] The conjugate may include a conjugate having the formula:

[0038] The conjugate is 111 Chelated to In, structure:

[0039] [ka] The conjugate may include a conjugate having the formula:

[0040] Pharmaceutical compositions are also provided. The pharmaceutical compositions include a conjugate of formula I or formula IA or a pharma- ceutically acceptable salt of formula I or formula IA and a pharma- ceutically acceptable carrier or excipient. The pharmaceutical compositions can further include a radiosensitizer, a radioprotector, an immunotherapeutic agent, a chemotherapeutic agent, an anticancer agent, and / or a hormonal therapy agent.

[0041] Further provided is a method of imaging and / or treating bone in a subject.In certain embodiments, the method comprises administering to the subject an effective amount (e.g., a therapeutically effective amount): (i) a conjugate or a pharma- ceutically acceptable salt thereof described herein, a first pharmaceutical composition (e.g., comprising a conjugate or a pharma- ceutically acceptable salt thereof) described herein, or (ii) a first pharmaceutical composition comprising a conjugate of formula I or IA and a first pharma- ceutically acceptable carrier or excipient, and simultaneously or sequentially, in any order, (b) (i') an active agent (e.g., free drug) or (ii') a second pharmaceutical composition comprising an active agent and a second pharma- ceutically acceptable carrier or excipient.The active agent in (b) can be / include a radiosensitizer, a radioprotector, an immunotherapeutic agent, a chemotherapeutic agent, an anticancer agent, or a hormonal therapy agent.The active agent in (b) can include lysine (Lys).

[0042] The subject may have cancer, for example, a primary bone cancer, such as osteosarcoma, chondrosarcoma, Ewing's sarcoma, or chordoma, or a secondary bone cancer, such as metastatic breast cancer, prostate cancer, multiple myeloma, thyroid cancer, lung cancer, renal cancer, ovarian cancer, colon cancer, or melanoma.

[0043] The method may further include imaging the bone of the subject. If the bone is imaged in the subject, the method may further include diagnosing whether the subject has cancer. If the subject is undergoing treatment for cancer and the bone is imaged in the subject, the method may further include evaluating or monitoring the efficacy of the treatment.

[0044] Also provided is a method of binding the conjugate or a pharma- ceutically acceptable salt thereof to hydroxyapatite in a subject. Such a method can include administering to the subject an effective amount of (i) the conjugate or a pharma- ceutically acceptable salt thereof, or (ii) a pharmaceutical composition (e.g., comprising the conjugate or a pharma- ceutically acceptable salt thereof). In some embodiments, the subject has cancer (e.g., bone cancer). In some embodiments, the subject has osteoblastic bone cancer. In some embodiments, the subject has osteolytic bone cancer.

[0045] The disclosed embodiments and other features, advantages and aspects contained herein, as well as problems related to achieving the same, will become apparent in light of the following detailed description of various exemplary embodiments of the present disclosure, which will be better understood when read in conjunction with the accompanying drawings. [Brief description of the drawings]

[0046] [Figure 1] FIG. 1 shows luciferin bioluminescence in the right femur of a mouse two weeks after initial tumor challenge, demonstrating tumor growth in the bone (bioluminescence circled). [Diagram 2]FIG. 13 shows micro-computed tomography (micro-CT) images of the right tibia of a mouse two weeks after initial tumor challenge demonstrating severe bone resorption due to tumor growth. [Diagram 3] FIG. 2 shows the fluorescence of DD10-S0456 localized to tumor-induced bone lesions in the mice shown in FIG. 1 (fluorescence circled). [Figure 4] FIG. 1 shows the reaction scheme for fluorescent imaging conjugate DD10-S0456 with the following additions / protocols at each identified step as follows: (1) 1 equivalent of S0456 and 5 equivalents of CsCO3 in dimethylsulfoxide (DMSO) at room temperature (RT) for 4 hours; (2) a) Boc-deprotection 40% trifluoroacetic acid (TFA) in dichloromethane (DCM) at room temperature for 40 minutes; and b) 1 equivalent of 3-maleimidopropionic acid, 1 equivalent of benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and 5 equivalents of N,N-diisopropylethylamine (DIPEA) in anhydrous DMSO at room temperature for 4 hours; and (3) 1 equivalent of DD10-Cys-Fmoc in anhydrous DMSO at room temperature for 4 hours. [Figure 5-1] 1 shows a reaction scheme for radioimaging and radiotherapy conjugates K(DOTA)-VMF-DD10 and K(DOTA)-F-DD10. DOTA is the chelator shown in this scheme, but it will be appreciated that other chelators with similar functional groups can be attached in a similar manner. [Figure 5-2] Same as above. [Figure 6] FIG. 1 shows the structure of Lys(DOTA)-Phe-D-Asp10 (also called K(DOTA)-F-DD10 or KDFDD10). [Figure 7] FIG. 1 shows the structure of Lys(DOTA)-Val-Met-Phe-D-Asp10 (also called K(DOTA)-VMF-DD10 or KDVMFDD10). [Figure 8]Single photon emission computed tomography / computed tomography (SEPCT / CT) images of a tibial tumor-bearing mouse injected with 111In-chelated K(DOTA)-F-DD10 (220 μCi of 200 μCi / 10 nmol dosage was injected). Arrows indicate tumor growth sites and white boxes indicate kidneys. From left to right: images taken at 1, 4, 12, 24, 48, 72, 96 and 120 hours after radiocontrast injection. [Figure 9] Single photon emission computed tomography (SPECT) / computed tomography (CT) images of tibial tumor-bearing mice injected with 111In-chelated K(DOTA)-VMF-DD10 (375 μCi of 300 μCi / 10 nmol dosage was injected). Arrows indicate tumor growth sites and white boxes indicate kidneys. From left to right: images were taken at 1, 4, 12, 24, 48, 72, 96 and 120 hours after radiocontrast injection. [Figure 10] SPECT / CT images of healthy mice injected with 111In-chelated K(DOTA)-F-DD10 (160 μCi of 200 μCi / 10 nmol dose injected) in the top row and 111In-chelated K(DOTA)-VMF-DD10 (350 μCi of 300 μCi / 10 nmol dose injected) in the bottom row. Arrows point to kidneys. From left to right: Imaging at 1 h, 4 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h after radiocontrast injection. [Figure 11] SPECT / CT images of a tibial tumor-bearing mouse injected with 177Lu-chelated K(DOTA)-F-DD10 (1.5 mCi / 10 nmol dosage of 0.962 mCi was injected). Arrows indicate tumor growth sites and white boxes indicate kidneys. From left to right: Imaged at 3, 24, 48, 72, 96, 120 and 168 hours after radiotherapy injection. [Figure 12]SPECT / CT images of tibial tumor-bearing mice injected with 177Lu-chelated K(DOTA)-F-DD10 (3.0 mCi / 10 nmol dosage injected). Arrows indicate tumor growth sites and white boxes indicate kidneys. From left to right: imaged at 3, 24, 48, 72, 96, 120 and 168 hours after radiotherapy injection. [Figure 13] SPECT / CT images of a tibial tumor-bearing mouse injected with 177Lu-chelated K(DOTA)-F-DD10 (3.57 mCi of 3.0 mCi / 10 nmol dosage was injected). Arrows indicate tumor growth sites and white boxes indicate kidneys. Top row, left to right: images taken 2, 12, 24 and 48 hours after radiotherapy injection. Bottom row, left to right: images taken 72, 96, 144 and 196 hours after radiotherapy injection. [Figure 14] SPECT / CT images of a tibial tumor-bearing mouse injected with 177Lu-chelated K(DOTA)-F-DD10 (5.50 mCi of 6.0 mCi / 10 nmol dosage injected). Arrows indicate tumor growth site, white square indicates kidney. Top row, left to right: images taken 2, 12, 24, 48, and 72 hours after radiotherapy injection. Bottom row, left to right: images taken 96, 144, 196, and 240 hours after radiotherapy injection. [Figure 15] Figure 1 shows the reaction scheme for the radioimaging and radiotherapy conjugate K(DD10F2)VM-DOTA, constructed from the carboxyl terminus to the amine terminus. Steps 1-4, identified by the third arrow, include: 1) Alloc deprotection; 2) KVM-DOTA+DD10F2-Fmoc coupling; 3) Fmoc deprotection; and 4) deprotection and cleavage. [Figure 16] FIG. 1 shows the structure of Lys(D-Asp10-Phe2)-Val-Met-DOTA (also called K(DD10F2)VM-DOTA). [Figure 17]FIG. 7 shows the structure of a 177Lu-complexed DOTA conjugate, where the underlying conjugate comprises the conjugate of FIG. [Figure 18] FIG. 7 shows the structure of an 111In-conjugated DOTA conjugate, where the underlying conjugate comprises the conjugate of FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] While the disclosure is susceptible to various modifications and alternative forms, exemplary embodiments thereof have been shown by way of example in the drawings and will herein be described in detail.

[0048] The present disclosure is predicated, at least in part, on the use of polyanionic, polyacidic and / or polyelectrolyte polymers, such as polymers comprising or consisting of acidic amino acids (e.g., L and / or D, aspartic acid and / or glutamic acid), to target hydroxyapatite (e.g., the positively charged calcium heads of hydroxyapatite). Provided are conjugates comprising radicals of hydroxyapatite-binding polyanionic, polyacidic and / or polyelectrolyte ligands (e.g., the carboxyl terminus, amino terminus and / or carboxylic acid side chains of acidic peptides) coupled to active agents, such as imaging, radiotherapy, radiosensitizing and / or radioprotective agents. In certain embodiments, the conjugation of the polyanionic, polyacidic and / or polyelectrolyte ligands to the active agents may be via a linker (optionally further comprising a spacer). Such conjugates may be used to target the delivery of the aforementioned active agents to damaged or diseased bones, such as bones containing cancer-induced lesions, for diagnostic and / or therapeutic (e.g., "theranostics") purposes.

[0049] When cancer cells invade bone, they excrete various compounds (such as cytokines or growth factors) that induce osteoclasts (i.e., bone-degrading cells) to hyper-proliferate and become active. Osteoclasts can then act to support tumor growth by degrading bone and releasing various growth factors and compounds previously trapped within the bone matrix. During this process of bone resorption, hydroxyapatite can be exposed to blood vessels and become targetable. Although hydroxyapatite can be exposed in normal healthy bones, as bones undergo continuous turnover to maintain bone homeostasis (balance between formation and degradation), the exposure is not significant compared to exposure in diseased or damaged bones. In addition, unlike other receptors, exposed hydroxyapatite is highly abundant and therefore not easily saturated by clinical therapeutic dosages. These characteristics make hydroxyapatite a potential receptor for selective delivery of active agents.

[0050] Conjugates The conjugates can bind to hydroxyapatite and can deliver imaging, radiotherapy, radiosensitizing and / or radioprotective agents to damaged or diseased bone, such as bone containing a cancer-induced lesion, for theranostic purposes.

[0051] In certain embodiments, the conjugate has formula I: HB-L A -A (Formula I) or a pharma- ceutically acceptable salt thereof, wherein HB is a radical of an anionic, acidic and / or electrolytic ligand (e.g., one that binds hydroxyapatite); A is an active agent, including a radiocontrast agent, a radiosensitizer, a radioprotector, or a radiotherapeutic agent; and optionally, L A is a linker connecting HB and A, or is absent.

[0052] In certain embodiments, L Ais optional, and if not included, HB is directly conjugated to A. For example, in certain embodiments, the conjugate has the formula IA: HB-A (Formula IA) or a pharma- ceutically acceptable salt thereof, where HB is a radical of an anionic, acidic and / or electrolytic ligand (e.g., one that binds to hydroxyapatite); and A is an active agent, including a radiocontrast agent, a radiosensitizer, a radioprotector, or a radiotherapeutic agent.

[0053] In certain embodiments, the anionic, acidic and / or electrolytic ligand is a linear (e.g., unbranched) anionic, acidic and / or electrolytic ligand. In certain embodiments, the anionic, acidic and / or electrolytic ligand is a branched anionic, acidic and / or electrolytic ligand. The ligand (e.g., HB) can be a biological targeting moiety that has one or more negative charges or can otherwise interact with the positively charged calcium of hydroxyapatite. In certain embodiments, the ligand comprises a peptide or peptide analog (which can be a linear peptide, a cyclic peptide, a branched peptide or a combination thereof) and / or one or more polymers. The ligand can be of synthetic or natural origin.

[0054] The ligand (e.g., HB) can include from 1 to about 50 (e.g., 1-50) anionic, acidic, and / or electrolytic moieties. In certain embodiments, the ligand includes a polyacid ligand. The polyacid ligand includes repeating units that carry acidic moieties. Examples of acidic moieties include, but are not limited to, carboxylic acid, sulfonic acid, boronic acid, and phosphonic acid moieties. The acidic moieties can be acidic macromolecules, such as, for example, nucleic acids, actin, or proteoglycans. In certain embodiments, the ligand (e.g., HB) includes polyacidic peptides and / or polymers, such as, for example, polyglutamic acid peptides.

[0055] In certain embodiments, the ligand comprises a polyelectrolyte ligand. The polyelectrolyte ligand can include repeating units that carry electrolyte moieties. Examples of electrolyte moieties include, but are not limited to, sulfonates, acrylates, and phosphates.

[0056] In certain embodiments, the ligand comprises from 1 to about 50 (e.g., 1-50) anionic moieties. In certain embodiments, the ligand comprises a polyanion. Polyanions are a subgroup of polyelectrolytes composed of repeating anionic moieties (e.g., polymer-based moieties). Examples of anionic moieties include, but are not limited to, phosphates and polyphosphates.

[0057] The ligand (e.g., HB) can include an amino acid or a derivative thereof. As used herein, "amino acid" refers to an L- or D-amino acid, an amino acid analog, or an amino acid mimetic (i.e., a single enantiomer and therefore a chiral, or mixture of enantiomers) that can be naturally occurring or of pure synthetic origin, and can optionally be pure. As used herein, "amino acid mimetic" refers to a synthetic analog of a naturally occurring amino acid that is isosteric (i.e., designed to mimic the stereochemical and electronic structure of the natural compound). The amino acid can be natural or non-natural, essential or non-essential. The amino acid can have any suitable relative configuration (such as the D- or L-configuration described above). The HB can include L-aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic acid, or a mixture of two or more of the foregoing, or a derivative thereof.

[0058] The ligand (e.g., HB) can further comprise one or more hydrophobic moieties coupled to the radical of the anionic, acidic and / or electrolytic ligand, e.g., from 1 to about 30 (e.g., 1-30) hydrophobic moieties, e.g., alkyl or alkenyl, ultraviolet (UV) active moieties, e.g., phenylalanine (Phe), tyrosine (Tyr) and / or tryptophan (Trp), or active group protecting moieties, e.g., 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc) and / or t-butyl. "Alkyl" generally refers to those having 1 to 15 carbon atoms (e.g., C 1 ~C 15 "Alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, such as alkyl, aryl ... 1 ~C 13 Alkyl can contain from 1 to 8 carbon atoms (e.g., C 1 ~C 8 Alkyl can contain 1 to 5 carbon atoms (e.g., C 1 ~C 5 Alkyl can contain 1 to 4 carbon atoms (e.g., C 1 ~C 4 Alkyl can contain 1 to 3 carbon atoms (e.g., C 1 ~C 3 Alkyl can contain 1 to 2 carbon atoms (e.g., C 1 ~C 2 Alkyl can contain one carbon atom (e.g., C 1 Alkyl can contain 5 to 15 carbon atoms (e.g., C 5 ~C 15 Alkyl can contain 5 to 8 carbon atoms (e.g., C 5 ~C 8Alkyl can contain from 2 to 5 carbon atoms (e.g., C 2 ~C 5 Alkyl can contain 3 to 5 carbon atoms (e.g., C 3 ~C 5 In other embodiments, the alkyl group is selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (iso-propyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), and 1-pentyl (n-pentyl). The alkyl is attached to the remainder of the molecule by a single bond.

[0059] The ligand (e.g., HB) can be (i) a serum albumin binder (AB) directly conjugated to HB or (ii) AB and a linker L AB (AB-L AB ), wherein AB-L AB L AB In certain embodiments, AB is conjugated to HB via a linker L. In certain embodiments, AB is attached to the amine or carboxyl terminus of a polyacidic peptide or active or functional groups (such as, for non-limiting examples, carboxyl, amine or thiol groups) of the repeating anionic, acidic or electrolytic moieties of HB. AB may be directly attached to HB or may be attached via a linker L. AB In certain embodiments, AB is linked to the thiol of HB by using a maleimide linker (see, for example, the maleimide-thiol linker in FIG. 4 used in this case to conjugate the dye to the targeting moiety), a disulfide linker, or any other linker that can react with a sulfur group. In some embodiments, HB does not contain AB.

[0060] Examples of AB include, but are not limited to, Evans Blue, Fmoc, diphenylcyclohexanol phosphate, naphthalene acylsulfonamide, 4-(pX-phenyl)butyric acid, where X is -H, -C1 ~C 6 Alkyl, -F, -Cl, -Br, -I, -OC 1~6 Alkyl, -CN, -CHO, -B(OH) 2 , -C=CC(O)aryl, -C=CS(O) 2 Aryl, -CO 2 H, -SO 3 H, -SO 2 NH 2 , -PO 3 H 2 , -SO 2 F, C.F. 3 or a derivative of any of the foregoing. Additional examples of AB include

[0061] [ka]

[0062] [ka] Including but not limited to:

[0063] The AB can comprise a hapten. Examples of haptens include, but are not limited to, 2,4-dinitrophenol (DNP), 2,4,6-trinitrophenol (TNP), rhamnose, galactose-α-1,3-galactose (α-Gal) or an antibody binding agent. Examples of antibody binding agents include Fab, scFv, V H , V L , V H These include, but are not limited to, H, V-NARs, monobodies, anticalins, affibodies and DARPins.

[0064] HB is the formula XA 1 , X.A. 2 , X.A. 3 or XA 4 :

[0065] [ka] [In the formula, R is COOH for L- or D-aspartic acid, and CH for L- or D-glutamic acid. 2 COOH, or for unnatural acidic amino acids, CH 2 CH 2 COOH, n=1 to 50,

[0066] [ka] AB or L AB represents the point of attachment to The linear polyanion may include a radical of

[0067] In some embodiments, HB is of the formula XA 3 and XA 4 HB does not contain an albumin binder as shown in formula XA 5 , X.A. 6 , X.A. 7 or XA 8 :

[0068] [ka] [In the formula, R is COO- in the case of L- or D-aspartic acid, and CH in the case of L- or D-glutamic acid. 2 COO- or, for unnatural acidic amino acids, CH 2 CH 2 COO- n=1 to 50,

[0069] [ka] L A represents the point of attachment to HB is AB or AB-L AB If further comprising:

[0070] [ka] AB or L AB represents the point of attachment to

[0071] [ka] represents a hydrogen or a point of attachment to HB. In certain embodiments, HB can include a radical of a branched polyanion of the formula XA 7 and XA 8 The composition does not contain any albumin binders, such as those shown in

[0072] L A and L AB The linker represented by can be any suitable linker. The linker can include atoms selected from C, N, O, S, Si and P; C, N, O, S and P; or C, N, O and S. The linker can have a backbone that ranges in length, from as few as two atoms in the backbone of the linker to as many as 100 or more consecutive atoms in the backbone of the linker. The "backbone" of the linker is the shortest chain of consecutive atoms that forms a covalent connection between HB and A. In some embodiments, the polyvalent linker has a branched backbone, with each branch serving as part of the backbone linker until a terminal end is reached.

[0073] For example, the linker can have a chain length of at least about 7 atoms. In some embodiments, the linker is at least about 10 atoms in length. In some embodiments, the linker is at least about 14 atoms in length. In some embodiments, the linker is between about 7 and about 31 atoms (such as about 7 to 31, 7 to about 31, or 7 to 31), between about 7 and about 24 atoms (such as about 7 to 24, 7 to about 24, or 7 to 24), or between about 7 and about 20 atoms (such as about 7 to 20, 7 to about 20, or 7 to 20). In some embodiments, the linker is between about 14 and about 31 atoms (such as about 14 to 31, 14 to about 31, or 14 to 31), between about 14 and about 24 atoms (such as about 14 to 24, 14 to about 24, or 14 to 24), or between about 14 and about 20 atoms (such as about 14 to 20, 14 to about 20, or 14 to 20). In some embodiments, the linker has a chain length of at least 7 atoms, at least 14 atoms, at least 20 atoms, at least 25 atoms, at least 30 atoms, or at least 40 atoms; or from 1 to 15 atoms, 1 to 5 atoms, 5 to 10 atoms, 5 to 20 atoms, 10 to 40 atoms, or 25 to 100 atoms.

[0074] L A and L AB may contain at least one carbon-carbon bond and / or at least one amide bond. A and L AB can comprise one or more L- or D-configured, natural or unnatural amino acids, polyethylene glycol (PEG) monomers, PEG oligomers, PEG polymers, or combinations of any of the foregoing. In linkers that include one or more PEG units, all carbon and oxygen atoms of the PEG units are part of the backbone, unless otherwise specified.

[0075] L A and L AB L may comprise an oligomer of a peptidoglycan, a glycan, an anion, or a combination of any of the foregoing. A and L ABcan contain at least one 2,3-diaminopropionic acid group, at least one glutamic acid group, at least one cysteine ​​group, or a combination of two or more of the foregoing.

[0076] In certain embodiments, L A and / or L AB In certain embodiments, L A and / or L AB comprises an amino acid linker (eg, a lysine (Lys) linker as described in Example 4 below).

[0077] L A and / or L AB Each of L can independently be a fast release linker. A and / or L ABcan each independently be a sustained release linker. As used herein, the term "fast-release" in the context of a linker refers to a linker that includes at least one bond that is releasable as known in the art and / or can be cleaved to various degrees under certain conditions, and in particular can be fragmented or cleaved in less than about one week when under or otherwise exposed to certain metabolic, physiological or cellular conditions that can initiate a cascade of fragmentation or bond cleavage (e.g., can result in the release of one or more of the moieties connected via one or more moieties (e.g., HB and A) of the linker). Bond cleavage can occur by standard chemical hydrolysis reactions that occur, for example, at physiological pH or as a result of compartmentalization into organelles such as endosomes that have a pH lower than the cytoplasmic pH. Bond cleavage can also occur by acid-catalyzed elimination. Alternatively, fragmentation can be initiated by nucleophilic attack on the disulfide group of the fast-release linker, causing cleavage to form, for example, a thiolate. In any of these cases, the rapid release nature of such linkers may be achieved by any mechanism that may relate to the chemical, metabolic, physiological or biological conditions present, hi certain embodiments, the rapid release linker comprises one or more sulfide bridges.

[0078] In contrast, "sustained release" in the context of a linker refers to a linker that includes at least one bond that is not easily or rapidly broken (i.e., the bond is not cleaved), but is potentially cleavable or fragmentable to various degrees under certain conditions, and does not cleave, fragment, or otherwise release one or more of the moieties connected via one or more moieties (e.g., HB and A) of the linker when subjected to certain metabolic, physiological, or cellular conditions that may initiate a cascade of fragmentation (e.g., after administration to a subject) for more than about one week (e.g., one week), more than about one month (e.g., one month), more than about four months (e.g., four months), more than about six months (e.g., six months), or more than about one year (e.g., one year). In certain embodiments, a sustained release linker includes one or more amide bonds.

[0079] The conjugate herein can be formulated with a fast-release linker or a slow-release linker, as desired and / or suitable for a particular application.In general, in imaging and radiotherapy applications, it may be generally desirable that the active agent and / or imaging agent is not easily released after administration to a subject.Therefore, the conjugate for use in imaging or radiotherapy applications may preferentially include a slow-release linker, as opposed to a fast-release linker.

[0080] On the other hand, high and / or sustained renal uptake can be a common feature of peptide-based pharmaceuticals, which can lead to reduced sensitivity to lesions adjacent to the kidney and nephrotoxicity, since the kidney is the main organ responsible for excreting these drugs from the body. There are various approaches to reduce renal uptake and retention, such as co-administering positively charged amino acids (e.g., lysine) with the drug. Another conventional method is to utilize unnatural amino acids (UAA), such as D-amino acids, because they are not easily recognized by renal proteins compared to those using natural amino acids. Additionally or alternatively, fast-release linkers can be incorporated into the conjugate, which can be fragmented or cleaved in the kidney to prevent uptake.

[0081] Brush border is the name for the microvilli-covered surface of pseudostratified epithelium found in several places in the body, the two main places being the small intestine and the kidney. In the kidney, the brush border is useful for distinguishing the proximal tubules (which have a brush border) from the distal tubules (which do not). In the proximal tubules of the kidney, various brush border membrane (BBM) peptidases can bind to peptides and cleave them so that they can be recycled or excreted. Several BBM peptidases (which are fast-release linkers) are known in the art, including the tripeptide Met-Val-Lys, the dipeptide Gly-Lys, and the tripeptide Gly-Phe-Lys. The term "peptidase" refers to an enzyme that hydrolyzes peptide bonds between amino acids. Previous studies have tested these sequences as linkers between targeting moieties and bioactive agents and confirmed that inclusion of these linkers in conjugates can enhance renal clearance.

[0082] In certain embodiments, L A comprises a BBM peptidase linker. The BBM linker can be a fast-release linker. Insertion of the BBM linker between the ligand / targeting moiety (HB) and the imaging or therapeutic agent (A) of the conjugate can result in recognition and cleavage by renal brush border enzymes.

[0083] In certain embodiments, the BBM linker comprises a cleavable tripeptide Met-Val-Lys. When a conjugate comprising the BBM linker is administered to a subject (e.g., systemically), the BBM linker can be cleaved and release therefrom the ligand / targeting moiety (HB) and the imaging or therapeutic agent (A) (e.g., under physiological conditions).

[0084] The BBM linker, in certain embodiments, can include one or more UAAs, including, but not limited to, one or more of the D-amino acids, citrulline, hydroxyproline, norleucine, 3-nitrotyrosine, nitroarginine, naphthylalanine, aminobutyric acid (Abu), 2,4-diaminobutyric acid (DAB), methionine sulfoxide, methionine sulfone, and the like.

[0085] It should be appreciated that physiological conditions that result in BBM linker breakdown include standard chemical hydrolysis reactions that occur, for example, at physiological pH or as a result of compartmentalization into organelles such as endosomes that have a pH lower than the cytoplasmic pH. By way of illustration, the BBM linkers described herein can undergo cleavage (e.g., rapid release) under certain metabolic, physiological, or cellular conditions that can initiate a cascade of fragmentation or bond cleavage (e.g., resulting in the release of one or more of the moieties connected via one or more portions of the linker (e.g., HB and A)).

[0086] L A and L AB The atoms used in forming L may be combined in any chemically relevant manner, such as a chain of carbon atoms forming an alkylene group, a chain of carbon and oxygen atoms forming a polyoxyalkylene group, a chain of carbon and nitrogen atoms forming a polyamine, etc. In addition, the bonds connecting the atoms in the chain may be either saturated or unsaturated, whereby, for example, alkanes, alkenes, alkynes, cycloalkanes, arylenes, imides, etc., can be formed from L. A and L AB In addition, the atoms forming the linker can be cyclized together to form a saturated or unsaturated divalent cyclic radical within the linker.

[0087] In some embodiments, L A and L AB In some embodiments, L can include a moiety that is neutral under physiological conditions. A and LAB can include a moiety that can be protonated or deprotonated to carry one or more positive or one or more negative charges, respectively. A and L AB can include neutral moieties and moieties that can be protonated to carry one or more positive charges.

[0088] Examples of neutral moieties include sugars, carbohydrates, saccharides, polyhydroxyl groups such as inositol, and / or polyether groups such as polyoxyalkylene groups including polyoxyethylene, polyoxypropylene, and the like.

[0089] Examples of moieties that can be protonated to carry one or more positive charges include amino groups such as polyamino alkylenes, including ethylenediamine, propylenediamine, butylenediamine, and the like, and / or heterocycles, including pyrrolidine, piperidine, piperazine, and other amino groups, each of which may be optionally substituted. In certain embodiments, L A and L AB can include a positive portion that includes one or more lysine residues.

[0090] Examples of moieties that can be deprotonated to carry one or more negative charges include carboxylic acids, such as aspartic acid, glutamic acid, and long chain carboxylic acid groups, and sulfate esters, such as alkyl esters of sulfuric acid.

[0091] Alternatively or in addition to the chain length, in some embodiments, L A and L AB has suitable substituents that can affect its hydrophobicity or hydrophilicity. Thus, for example, L A and L AB L can have a hydrophobic side group such as an alkyl, cycloalkyl, aryl, arylalkyl or similar group, each of which is optionally substituted. A and L ABIf L contains one or more amino acids, A and L AB can contain hydrophobic amino acid side chains, such as one or more amino acid side chains derived from Phe and Tyr, including substitution variants thereof, as well as analogs and derivatives of such side chains.

[0092] L A and L AB Either or both of may comprise a sustained release linker. In some embodiments, the sustained release linker comprises a backbone that is stable under physiological conditions (e.g., the backbone is not susceptible to hydrolysis (e.g., aqueous or enzymatic hydrolysis)). In some embodiments, the sustained release linker does not release any component to which it is conjugated. In some embodiments, the sustained release linker lacks a disulfide bond (e.g., SS) or an ester in the backbone. In some embodiments, HB and A are connected by a backbone that is substantially stable throughout the circulation (e.g., in vivo) of the composition. The sustained release linker may comprise an alkyl(ene), an anhydride, an amide, an ester, an ether, an amine, and / or a thioether (e.g., thio-maleimide). Any sustained release linker may be used, provided that at least one bond is formed that is not easily or rapidly broken under physiological conditions.

[0093] In some embodiments, non-releasable linkers include linkers that will hydrolyze within a period of time (e.g., 24 hours) in an aqueous (e.g., buffered (e.g., phosphate buffer)) solution, for example, less than ten percent (10%) (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.1%, less than 0.01%, or less than 0.001%) at neutral pH. In some embodiments, when a sustained release linker is used, less than about ten percent (10%) and preferably less than five percent (5%) of the administered conjugate releases A or not at all (e.g., in the systemic circulation prior to uptake by the target cell / tissue). In some embodiments, less than five percent (5%) of A is released from the conjugate within one (1) hour after administration while the compound is in the systemic circulation.

[0094] L A L can include a fast-release linker. A fast-release linker can release A from the conjugate relatively quickly, for example, when exposed to physiological conditions (e.g., when circulating in serum). Examples of fast-release linkers include, but are not limited to, esters, disulfides, and thiols. A can include a fast release linker. The cleavable bond for the fast release linker can be part of the backbone.

[0095] Linker L Acan include biodegradable, pH-sensitive, self-immolative, peptidase-sensitive or hydrolyzable linkers. Examples of these linkers include, but are not limited to, β-glucuronide-based linkers, maleimide-based thiol linkers, cathepsin K-sensitive linkers, cathepsin B-sensitive linkers, matrix metalloproteinase-sensitive linkers and BBM-cleavable linkers. Fast-release groups also include photochemically cleavable groups. Examples of photochemically cleavable groups include linkers containing 2-(2-nitrophenyl)-ethan-2-ol groups and o-nitrobenzyl, decyl, trans-o-cinnamoyl, m-nitrophenyl or benzylsulfonyl groups (see, e.g., Dorman and Prestwich, Trends Biotech. 18: 64-77 (2000); Greene & Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons, New York (1991)). Linker L A and L AB may be conjugated to the N- or C-terminus of HB, of any monomer of the polymer, or of any substituent of any monomer of the polymer.

[0096] Both fast and slow release linkers can be engineered to optimize biodistribution, bioavailability and PK / PD (e.g., of A) and / or to increase uptake (e.g., of A) into target tissues according to methodologies generally known in the art or hereafter developed, such as via PEGylation, etc. In some embodiments, the linker is configured to avoid significant release of pharma- ceutically active amounts of A in the circulation prior to cellular uptake.

[0097] It is understood that the instability of the fast-release bond can be adjusted by including functional groups or fragments in the linker that can assist or facilitate such bond cleavage, also referred to as proximity assistance.The instability of the fast-release bond can also be adjusted by, for example, substitution modifications at or near the fast-release bond, such as including an alpha branch adjacent to the fast-release disulfide bond, increasing the hydrophobicity of the substituent on the silicon in the moiety with the hydrolyzable silicon-oxygen bond, homologating the alkoxy group that forms part of the hydrolyzable ketal or acetal, etc.In addition, it is understood that, if present, additional functional groups or fragments can be included in the linker that can assist or facilitate further fragmentation of the compound after the bond breakage of the fast-release linker.

[0098] L A L 1 or L 2 :

[0099] [ka] wherein

[0100] [ka] L A and A or L A and HB.

[0101] L A and L AB Either or both of L may include (e.g., be conjugated to and / or include) a spacer. The spacer may be any suitable spacer. A and L ABThe spacers may include hydrophilic, hydrophobic, amphipathic, non-peptidic, peptidic and / or aromatic monomers. The length of the spacer may range from 1 to 30 (e.g., PEG with 1 to 30 carbon atoms, 1-30 units, etc.). Examples of hydrophilic spacers include, but are not limited to, polyethylene glycol polymers and their derivatives. Examples of hydrophobic spacers include, but are not limited to, pure or mixed branched hydrocarbon, fluorocarbon, alkane, alkene and / or alkyne polymers. Examples of amphipathic spacers include, but are not limited to, pure or mixed phospholipids and / or their derivatives. Examples of peptidic spacers include, but are not limited to, pure and mixed single, branched, L- or D-configuration, essential, non-essential, natural and non-natural amino acids and their derivatives. Examples of aromatic spacers include, but are not limited to, pure and mixed repeating quinoids.

[0102] In some embodiments, the linker is formed via click chemistry / click chemistry derived synthetic methods. Those skilled in the art will understand that the terms "click chemistry" and "click chemistry derived" generally refer to a class of small molecule reactions commonly used in conjugation, which allow the conjugation of a substrate of choice with a specific molecule. Click chemistry describes a methodology that produces products that are not a single specific reaction, but follow examples from nature, which also produces substances by conjugating small modular units. In many applications, click reactions conjugate biomolecules and reporter molecules. Click chemistry is not limited to biological conditions, and the concept of "click" reactions has been used in pharmacological and various biomimetic applications. However, they have become significantly useful in the detection, localization and qualification of biomolecules.

[0103] The click reaction can occur in one pot, is typically not hindered by water, can produce minimal by-products, is "spring loaded", and is characterized by a high thermodynamic driving force that drives it rapidly and irreversibly to a high yield of a single reaction product with high reaction specificity (in some cases both positional and stereospecificity). These qualities make the click reaction well suited to the problem of isolating and targeting molecules in complex biological environments. Thus, in such environments, the product must be physiologically stable and any by-products must be non-toxic (e.g., to in vivo systems).

[0104] A can be an active agent, including a radiocontrast agent, a radiosensitizer, a radioprotector, or a radiotherapeutic agent. A can include a chelator (e.g., a chelator). In certain embodiments, the chelator (A) is coupled to a radical of a ligand / biological targeting moiety (HB), either directly or via a linker (L A The chelator is covalently linked to the radical of the ligand / biological targeting moiety (HB) either directly or via a linker (L A The term "functional group suitable for conjugation" can include a functional group suitable for conjugation via HB or L, as desired. A By "CH3" is meant a functional group that will react with a corresponding functional group (such as, but not limited to, an amine, carboxyl, or thiol group) of to chemically link a chelator thereto.

[0105] In certain embodiments, A comprises DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or a derivative thereof (see, for example, the conjugates shown in Figures 6, 7 and 16). In certain embodiments, A is selected from the group consisting of DOTA, TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid) and derivatives thereof; HEHA (1,2,7,10,13-hexaazacyclooctadecane-1,4,7,10,13,16-hexaacetic acid) and derivatives thereof; PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N',N",N"',N""-pentaacetic acid) and derivatives thereof; SarAr (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine) and derivatives thereof; NOTA (1, 4,7-Triazacyclononane-1,4,7-triacetic acid) or its derivatives;NETA(4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl)acetic acid or its derivatives;TRAP(1,4,7-Triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid) or its derivatives;HBED(N,N0-bis(2-hydroxybenzyl)-ethylenediamine-N,N0-diacetic acid) or its derivatives;2,3-HOPO(3-hydroxypyridin-2-one) or its derivatives;PCTA(3,6,9,15-tetraazabicyclo[9.3.1]-Pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid) or derivatives thereof;PDTA(1,3-propylenediaminetetraacetate) or derivatives thereof;NTA(nitrilotriacetic acid) or derivatives thereof;EDDS(ethylenediaminedisuccinate) or derivatives thereof;EDTA(ethylenediaminetetraacetic acid) or derivatives thereof;MGDA(N-(1-carboxylatoethyl)iminodiacetate) or derivatives thereof;DFO(desferrioxamine) or derivatives thereof;DTPA(diethylene triaminepentaacetic acid) or its derivatives;CDTA ((1,2-cyclohexylene dinitrilo)tetraacetic acid) or its derivatives;CPTA (1,4,8,11-tetraazacyclotetradecane derivatives) or its derivatives;OCTAPA (N,N0-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N0-diacetic acid) or its derivatives;H2-MACROPA (N,N'-bis[(6-carboxy-2-pyridylmethyl]-4,13-diaza-18-crown-6) or its derivatives;H. 2 The chelators are selected from the group consisting of dedpa (1,2-[[carboxy)-pyridin-2-yl]-methylamino]ethane or a derivative thereof; HEDP (hydroxyethylidenediphosphonic acid or etidronic acid) or a derivative thereof; HEBD (N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid) or a derivative thereof; HYNIC (6-hydrazinopyridine-3-carboxylic acid) or a derivative thereof; DMSA (meso-2,3-dimercaptosuccinic acid) or a derivative thereof; and β-l-diaminopropionic acid, an EC20 head group including Asp and Cys.

[0106] The chelator is capable of chelating a radioisotope. In certain embodiments, the radioisotope is, for example, an alpha-emitting radioisotope (e.g., 225 Ac) or β-emitting radioisotopes (e.g. 177 Lu or 90 In certain embodiments, the conjugate is a therapeutic radioisotope such as 177 Lu-DOTA conjugates.

[0107] A of formula I or formula IA can include a chelator that includes an imaging radioisotope. In certain embodiments, the imaging radioisotope is 111 In or 67 The isotope can be a gamma-emitting radioisotope, such as Ga. In certain embodiments, the conjugate is shown in FIG. 111 The chelating agent includes an In-conjugated DOTA conjugate. 18 F, 44 Sc, 47 Sc, 52 Mn, 55 Co, 64 Cu, 6 7Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, 90 Y, 99m Tc, 111 In, 114m In, 117m Sn, 124 I, 125 I, 131 I, 149 Tb, 153 Sm, 152 Tb, 155 Tb, 161 Tb, 177 Lu, 186 Re, 188 Re, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 224 Ra, 225 Ab, 225 Ac or 227 In certain embodiments, the chelator is 68 Ga, 11 C. 13 N, 15 O and 18 A is a positron emission tomography (PET) imaging radioisotope such as F. 14 C. 3 H, 34 S, 32 P, 125 I and 131The radioactive agent A can be linked to the N- or C-terminus of HB or to any active group on the repeating portion of HB via a linker L A The conjugation may be via

[0108] The above conjugates can be synthesized using methods known in the art and exemplified herein, see, e.g., Example 4.

[0109] The conjugate can have the structure shown in Figure 4. The conjugate can have the structure shown in Figure 6. The conjugate can have the structure shown in Figure 7. The conjugate can have the structure shown in Figure 16.

[0110] Conjugates can be presented as pharmaceutically acceptable salts. Examples of acceptable salts include, but are not limited to, alkali metal (e.g., sodium, potassium or lithium) or alkaline earth metal (e.g., calcium) salts, but any salt that is generally non-toxic and effective when administered to the subject being treated is acceptable. Similarly, "pharmaceutically acceptable salts" refers to salts with counterions that can be used in pharmaceutical products. Such salts can include, but are not limited to, (1) acid addition salts, which may be obtained by reaction of the free base of the parent compound with inorganic acids, such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, perchloric acid, and the like, or with organic acids, such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or (2) salts formed when any of the acidic protons present in the parent compound are replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion, or coordinated with an organic base, e.g., ethanolamine, diethanolamine, triethanolamine, trimethamine, N-methylglucamine, and the like. Pharmaceutically acceptable salts are well known to those of skill in the art, and all such pharma-ceutically acceptable salts are contemplated.

[0111] Acceptable salts can be obtained using standard procedures known in the art, including (but not limited to) reacting sufficiently acidic compounds with a suitable base to give physiologically acceptable anions. Suitable acid addition salts are formed from acids which form non-toxic salts. Illustrative, but non-limiting examples include acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, saccharate, stearate, succinate, tartrate, tosylate, and trifluoroacetate. Suitable base salts of the compounds may be formed from bases which form non-toxic salts. Illustrative, but non-limiting, examples include arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemi-salts of acids and bases can also be formed, such as hemisulfate and hemicalcium salts.

[0112] The conjugates herein may be "deuterated", meaning that one or more hydrogen atoms may be replaced with deuterium. Since deuterium and hydrogen have nearly the same physical properties, deuterium substitution is the smallest structural change that can be made. Deuteration is well known to those skilled in the art.

[0113] The conjugates, in some embodiments, contain one or more asymmetric centers, and thus can give rise to enantiomers, diastereomers and other stereoisomeric forms defined in terms of absolute stereochemistry as (R)- or (S)-. In certain embodiments, the conjugates are of the R-configuration. In certain embodiments, the conjugates are of the S-configuration. Unless otherwise stated, all stereoisomeric forms of the conjugates are intended to be contemplated. When the conjugates contain an alkene double bond, both E and Z geometric isomers (e.g., cis or trans) are intended to be included unless otherwise specified. In certain embodiments, HB and A are arranged in a relative cis orientation. In certain embodiments, HB and A are arranged in a relative trans orientation. Similarly, all possible isomers and their racemic and optically pure forms, as well as all tautomeric forms, are intended to be included. The term "geometric isomer" refers to the E or Z geometric isomers (e.g., cis or trans) of the alkene double bond. The term "positional isomers" refers to structural isomers around a central ring, e.g., ortho, meta and para isomers around a benzene ring.

[0114] Pharmaceutical Compositions In view of the above, further provided is a pharmaceutical composition comprising any of the conjugates. In certain embodiments, provided herein is a pharmaceutical composition comprising a conjugate (e.g., a conjugate of formula (I) or formula (IA)) and one or more pharma- ceutically acceptable carriers or excipients. The term "composition" generally refers to any product that comprises more than one ingredient, including a conjugate. The composition can be prepared from an isolated conjugate, or from a salt, solution, hydrate, solvate and other forms of the conjugate.

[0115] In certain embodiments, the conjugate of the composition is a conjugate of formula I (or a pharma- ceutically acceptable salt thereof): HB-L A -A (Formula I) where HB is a radical of an anionic (e.g., linear or branched) anionic, acidic and / or electrolytic ligand (e.g., one that binds to hydroxyapatite); A is an active agent, including a radiocontrast agent, radiosensitizer, radioprotector, or radiotherapeutic agent; L A is a linker (as described herein) connecting HB and A. In certain embodiments, L A is optional and if not included, HB is directly conjugated to A.

[0116] In certain embodiments, L A is optional, and if not included, HB is directly conjugated to A. For example, in certain embodiments, the conjugate has the formula IA: HB-A (Formula IA) or a pharma- ceutically acceptable salt thereof, where HB is a radical of an anionic, acidic and / or electrolytic ligand (e.g., one that binds hydroxyapatite); A is an active agent, including a radiocontrast agent, a radiosensitizer, a radioprotector, or a radiotherapeutic agent. In certain embodiments, HB serves as a targeting moiety for the conjugate.

[0117] In certain embodiments, the ligand (e.g., HB) can comprise from 1 to about 50 (e.g., 1-50) anionic, acidic and / or electrolytic moieties. In certain embodiments, the ligand (e.g., HB) can comprise an amino acid or a derivative thereof. In certain embodiments, the ligand (e.g., HB) can comprise an L-configuration. In certain embodiments, the ligand (e.g., HB) can comprise a D-configuration. In certain embodiments, the ligand (e.g., HB) can further comprise one or more hydrophobic moieties. The ligand (e.g., HB) can be directly conjugated to the HB or via a linker L AB (AB-L AB ) conjugated to HB (e.g., AB-L ABL AB (when conjugated to HB via). In certain embodiments, one or more peptide bonds of HB and / or A are arranged in a relative cis orientation. In certain embodiments, one or more peptide bonds of HB and / or A are arranged in a relative trans orientation. In certain embodiments, the conjugate is in the R-configuration. In certain embodiments, the conjugate is in the S-configuration.

[0118] In certain embodiments, the ligand (e.g., HB) has the formula XA 1 , X.A. 2 , X.A. 3 or XA 4 :

[0119] [ka] [In the formula, R is COOH for L- or D-aspartic acid, and CH for L- or D-glutamic acid. 2 COOH, or for unnatural acidic amino acids, CH 2 CH 2 COOH, n=1 to 50,

[0120] [ka] AB or L AB represents the point of attachment to The linear polyanion radicals include:

[0121] HB is the formula XA 5 , X.A. 6 , X.A. 7 or XA 8 :

[0122] [ka] [In the formula, R is COO- in the case of L- or D-aspartic acid, and CH in the case of L- or D-glutamic acid. 2 COO- or, for unnatural acidic amino acids, CH 2 CH 2 COO- n=1 to 50,

[0123] [ka] L A represents the point of attachment to HB is AB or AB-L AB If further comprising:

[0124] [ka] AB or L AB represents the point of attachment to

[0125] [ka] represents a hydrogen or a point of attachment to HB. The branched polyanion may include a radical of

[0126] In certain embodiments, HB is an albumin binder (e.g., a compound of formula XA 3 , X.A. 4 , X.A. 7 and XA 8 ) is not included (may not be included). A and L AB The linker represented by can be any suitable linker (e.g., a fast-release or slow-release linker).

[0127] A can be an active agent, including a radio-imaging agent, a radiosensitizer, a radioprotector, or a radiotherapeutic agent. For example, A can include a chelator (e.g., any suitable chelator, including, but not limited to, those specified herein in connection with the conjugate). A can include a radioisotope. A can include a PET imaging radioisotope. The active agent A can be linked to the N- or C-terminus of HB or to any active group on the repeating portion of HB via a linker (e.g., L A ) may be conjugated.

[0128] Certain functional groups such as hydroxy, amino and similar groups can form complexes with water and / or various solvents resulting in different physical forms of the conjugate.

[0129] Compositions may be prepared from various amorphous, non-amorphous, partially crystalline, crystalline and / or other morphological forms of the conjugates, and compositions may be prepared from various hydrates and / or solvates of the conjugates. Thus, such pharmaceutical compositions may contain each of the conjugates, or any combination thereof, or its individual forms, various morphological forms and / or solvates or hydrate forms.

[0130] The pharmaceutical composition may comprise one or more pharma- ceutically acceptable carriers, adjuvants, diluents, excipients and / or vehicles (e.g., conventional non-toxic pharma- ceutically acceptable carriers, adjuvants and vehicles) and combinations thereof. Any pharma- ceutically acceptable carriers and excipients as known in the art may be used. Examples include, but are not limited to, excipients, color additives, preservatives and stabilizers. More specific examples include crystalline cellulose, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, ethyl cellulose and magnesium stearate.

[0131] Solutions of active conjugates or pharmaceutical compositions can be aqueous, optionally mixed with non-toxic surfactants, and / or can contain carriers or excipients, such as salts, carbohydrates, and buffers (preferably at pH 3 to 9), although for some applications they may be more suitably formulated as sterile non-aqueous solutions or as dry forms used in conjunction with a suitable vehicle, such as sterile pyrogen-free water or phosphate-buffered saline. For example, dispersions can be prepared in glycerol, liquid PEG, triacetin, and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations may further contain a preservative to prevent the growth of microorganisms.

[0132] The pharmaceutical composition may further comprise a radiosensitizer, a radioprotector, an immunotherapeutic agent, a chemotherapeutic agent, an anti-cancer agent, and / or a hormonal therapy agent. Alternatively, the conjugate (or a pharmaceutical composition comprising it) may be administered simultaneously or sequentially with a radiosensitizer, a radioprotector, an immunotherapeutic agent, an anti-cancer agent, a chemotherapeutic agent, and / or a hormonal therapy agent (or a pharmaceutical composition comprising any of the foregoing), in any order.

[0133] The radiosensitizer can be any suitable radiosensitizer. Examples of suitable radiosensitizers include peptides or proteins such as HER3-ADC, SYM004, cetuximab, nimotuzumab, AMG102, C-reactive peptide, HSP, paraoxonase-2, and ECI301; gold (Au), GSH-modified Au, PEG-modified Au, silver (Ag), PEG-modified Ag, PVP-modified Ag, gadolinium (Gd), PEG-modified Gd, DTPA-modified Gd, hafnium (Hf), tantalum (Ta), tungsten (W), and the like. Nanomaterials such as bismuth (Bi) and platinum (Pt); including, but not limited to, apaziquone, curcumin, misonidazole, tirapazamine, AQ4N, paclitaxel, fluorouracil, cisplatin, dihydroartemisinin, resveratrol, etanidazole, porfiromycin, TH-302, mitomycin C, nelfinavir, docetaxel, hydrogen peroxide, RRx-001, gemcitabine or papaverine hydrochloride. See, e.g., Gong et al., Int J Nanomedicine 16: 1083-1102 (2021).

[0134] The radioprotector can be any suitable radioprotector. A non-limiting example of a suitable radioprotector is amifostine. See, for example, Gong et al. (2021) above.

[0135] The immunotherapeutic agent can be any suitable immunotherapeutic agent. Examples of suitable immunotherapeutic agents include, but are not limited to, transforming growth factor beta (TGF-β) inhibitors such as R268712, or programmed cell death ligand 1 (PD-L1) inhibitors such as Keytruda.

[0136] The anti-cancer drug can be any suitable anti-cancer drug. Examples of suitable anti-cancer drugs include, but are not limited to, kinase inhibitors such as dasatinib. In some embodiments, A is not dasatinib, such as when HB comprises at least 4 amino acids (e.g., 4-20 amino acids).

[0137] The chemotherapy agent can be any suitable chemotherapy agent. Examples of suitable chemotherapy agents include, but are not limited to, anthracyclines such as doxorubicin, taxanes such as docetaxel, cyclophosphamides such as cytoxan, or 5-fluoro-uracil.

[0138] The hormone or hormone-related therapeutic agent can be any suitable hormone or hormone-related therapeutic agent. Examples include, but are not limited to, hormone production inhibitors such as Zoladex or Letrozole.

[0139] The conjugates may be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms adapted to a selected route of administration. Pharmaceutical compositions may, for example, be formulated for a given route of administration, and may be prepared, for example, as described in the art and in, for example, Remington, The Science and Practice of Pharmacy, 22 nd The composition may be prepared according to the methods described in the New York edition (2012). The composition may be an injectable or injectable composition, such as a composition that may be injected subcutaneously or intravenously.

[0140] Pharmaceutical compositions may be administered to a mammalian host, such as a human patient, in a variety of forms adapted to the chosen route of administration. In certain embodiments, pharmaceutical compositions are formulated to be administered subcutaneously. In certain embodiments, pharmaceutical compositions are formulated to be administered orally. In certain embodiments, pharmaceutical compositions are formulated to be administered intramuscularly, intravenously, intraarterially, intraperitoneally, or as any other art-recognized route of parenteral administration.

[0141] In certain embodiments, pharmaceutical compositions are administered systemically in combination with pharmaceutically acceptable vehicles.The percentage of components of compositions and preparations may vary and may be between about 1 and about 99% by weight of active ingredient (e.g., conjugate), as well as binders, excipients, disintegrants, lubricants and / or sweeteners (as known in the art).The amount of active conjugate in such therapeutically useful compositions is such that an effective dosage level can be obtained (e.g., in serum or target tissue).

[0142] Illustrative parenteral administration means include needle (including microneedle) injectors, needle-free injectors and infusion techniques, and any other parenteral administration means recognized in the art. Parenteral formulations are typically aqueous solutions, which can contain excipients, such as salts, carbohydrates, and buffers (preferably at a pH of about 3 to about 9), although in some applications they may be more suitably formulated as sterile non-aqueous solutions or as dry forms used in combination with a suitable vehicle, such as sterile pyrogen-free water. Preparation of parenteral formulations under sterile conditions, for example by lyophilization, can be easily accomplished using standard pharmaceutical techniques well known to those skilled in the art.

[0143] The pharmaceutical dosage form suitable for administration can include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, which are suitable for extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes, nanocrystals or polymeric nanoparticles. In any case, the final dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be, for example, a solvent or liquid dispersion medium, including, but not limited to, water, electrolytes, sugars, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters and / or suitable mixtures thereof. In at least one embodiment, the desired fluidity can be maintained by the formation of liposomes, by maintaining the required particle size in the case of dispersions, or by the use of surfactants.

[0144] Sterile injectable solution can be prepared by incorporating pharmaceutical composition in the required amount of suitable solvent with one or more of the other ingredients described above, as required, followed by filtration sterilization.In the case of sterile powders for preparing sterile injectable solution, vacuum drying and freeze-drying technology can be used, which can produce a powder of active ingredient plus any additional desired ingredient that exists in the solution previously sterile-filtered.

[0145] method Further provided is a method of imaging and / or treating bone in a subject.The method comprises administering to the subject an effective amount of (i) a conjugate of formula I, formula IA, or a pharmaceutically acceptable salt of formula I or formula IA, (ii) a first pharmaceutical composition comprising a conjugate of formula I, a conjugate of formula IA, or a pharmaceutically acceptable salt of formula I or IA, optionally wherein the first pharmaceutical composition further comprises a radiosensitizer, a radioprotector, an immunotherapeutic agent, a chemotherapeutic agent, an anticancer drug, or a hormonal therapy agent and a first pharmaceutically acceptable carrier or excipient.In addition, as desired, the method can further comprise administering to the subject (i) or (ii) alone or in combination with the administration of a second pharmaceutical composition comprising an active agent or an active agent and a second pharmaceutically acceptable carrier or excipient. The active agent can be, for example, a radiosensitizer, a radioprotector (eg, Lys), an immunotherapeutic agent, a chemotherapeutic agent, an anti-cancer agent, or a hormonal therapy agent.

[0146] As used herein, the term "administering" and variations thereof include all means of introducing the compounds and compositions described herein into a subject, including, but not limited to, orally (po), intravenously (iv), intramuscularly (im), subcutaneously (sc), transdermally, via inhalation (e.g., intranasally (in)), bucally, ocularly, sublingually, intravaginally, rectally, and the like.

[0147] As used herein, "effective amount" refers to an amount that is sufficient to achieve a desired result or have an effect on an undesired condition. For example, an effective amount can refer to an amount that is sufficient to achieve a desired therapeutic result or have an effect on a symptom. The specific effective dose level (e.g., amount) for any subject will depend on a variety of factors, including the disorder / condition and the severity of the disorder / condition (e.g., type, location and severity of fracture, bone injury or cancer) being treated; the specific composition and / or conjugate used (i.e., its potency); the subject's age, weight, general health, sex and dietary habits; the subject's response; administration time; administration route; excretion rate of the specific conjugate used; duration of treatment; drugs / active agents used in combination or concomitantly with the specific conjugate used, and similar factors well known in the medical field. For example, it is well within the skill of the art to start with a lower level of dose of the conjugate / composition than is required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. If desired, the effective amount can be divided into multiple doses for purposes of administration. As a result, a single dose conjugate / composition can contain such amounts or submultiples thereof that constitute a daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. In addition, pharmacogenomic (effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of an antigen or composition) information about a particular patient can affect the dosage used to achieve an effective amount.

[0148] Depending on the route of administration, a wide range of acceptable dosages is contemplated.For example, the effective amount of the conjugate and / or pharmaceutical composition can range from about 0.1 μg / kg / day, for example, 0.5 μg / kg / day, 0.7 μg / kg / day, or 0.01 mg / kg / day to about 1,000 mg / kg / day.Intravenous doses can be several orders of magnitude lower.

[0149] When the conjugate and / or the first pharmaceutical composition is administered together with an active agent or a second pharmaceutical composition comprising an active agent, the conjugate / first pharmaceutical composition and the active agent / second pharmaceutical composition may be administered to a subject simultaneously or sequentially, in any order and using any delivery modality. For example, but not limited to, the conjugate / first pharmaceutical composition may be administered intravenously (e.g., as an iv solution) and the active agent / second pharmaceutical composition may be administered subcutaneously. In other embodiments, both the conjugate / first pharmaceutical composition and the active agent / second pharmaceutical composition are administered intravenously. The use of pharmaceutical compositions comprising more than one drug / active agent is within the scope of this disclosure.

[0150] The conjugate and any other active agent administered therewith (or sequentially therewith) may be administered in a therapeutically effective dosage to obtain a clinically acceptable result, such as reduction or elimination of symptoms or of tumors. Thus, the conjugate and any active agent may be administered concurrently or sequentially in a treatment protocol. The administration of any active agent may be according to treatment protocols known in the art.

[0151] Those skilled in the art will understand that treatment protocol can vary according to the needs of the subject.Thus, the combination of conjugate and other compounds (drugs) used in the method herein can be administered with the variation of the protocol described herein.For example, conjugate and / or active agent can be administered intermittently rather than continuously during the treatment cycle.

[0152] The conjugate of formula I (or a pharma- ceutically acceptable salt thereof) can be any of the conjugates described herein. In certain embodiments, the conjugate or a pharma- ceutically acceptable salt thereof has the formula: HB-L A -A (Formula I) where HB is a radical of an anionic (e.g., linear or branched) anionic, acidic and / or electrolytic ligand (e.g., one that binds to hydroxyapatite); A is an active agent, including a radiocontrast agent, radiosensitizer, radioprotector, or radiotherapeutic agent; L A is a linker connecting HB and A, or is absent.

[0153] In certain embodiments, L A is optional, and if not included, HB is directly conjugated to A. For example, in certain embodiments, the conjugate has the formula IA: HB-A (Formula IA) or a pharma- ceutically acceptable salt thereof, where HB is a radical of an anionic, acidic and / or electrolytic ligand (e.g., one that binds to hydroxyapatite); and A is an active agent, including a radiocontrast agent, a radiosensitizer, a radioprotector, or a radiotherapeutic agent.

[0154] In certain embodiments, the ligand (e.g., HB) comprises from 1 to about 50 (e.g., 1-50) anionic, acidic and / or electrolytic moieties. In certain embodiments, the ligand (e.g., HB) comprises an amino acid or a derivative thereof (e.g., in the L- or D-configuration). In certain embodiments, the ligand (e.g., HB) further comprises one or more hydrophobic moieties. The ligand (e.g., HB) can be conjugated directly to the HB or via a linker L. AB (AB-L AB ) conjugated to HB (e.g., AB-L AB L AB (when conjugated to HB via

[0155] In certain embodiments, the ligand (e.g., HB) has the formula XA 1 , X.A. 2 , X.A. 3 or XA 4 :

[0156] [ka] [In the formula, R is COOH for L- or D-aspartic acid, and CH for L- or D-glutamic acid. 2 COOH, or for unnatural acidic amino acids, CH 2 CH 2 COOH, n=1 to 50,

[0157] [ka] AB or L AB represents the point of attachment to The linear polyanion radicals include:

[0158] HB is the formula XA 5 , X.A. 6 , X.A. 7 or XA 8 :

[0159] [ka] [In the formula, R is COO- in the case of L- or D-aspartic acid, and CH in the case of L- or D-glutamic acid. 2 COO- or, for unnatural acidic amino acids, CH 2 CH 2 COO- n=1 to 50,

[0160] [ka] L A represents the point of attachment to HB is AB or AB-L AB If further comprising:

[0161] [ka] AB or L AB represents the point of attachment to

[0162] [ka] represents a hydrogen or a point of attachment to HB. The branched polyanion may include a radical of

[0163] In certain embodiments, HB is an albumin binder (e.g., a compound of formula XA 3 , X.A. 4 , X.A. 7 and XA 8 ) is not included (may not be included). A and L AB The linker represented by can be any suitable linker (e.g., a fast-release or slow-release linker).

[0164] A of formula I or formula IA can be an active agent, including a radio-imaging agent, a radiosensitizer, a radioprotector, or a radiotherapeutic agent. For example, A includes a chelator (e.g., any suitable chelator, including but not limited to those specified herein in connection with the conjugate). A can include a radioisotope. A can include a PET imaging radioisotope. The active agent of A can be linked to the N- or C-terminus of HB or to any active group on the repeating portion of HB via a linker (e.g., L A ) may be conjugated.

[0165] In certain embodiments, the HB and A of the conjugate are arranged in a relative cis orientation. In certain embodiments, the HB and A of the conjugate are arranged in a relative trans orientation. In certain embodiments, the conjugate is in the R-configuration. In certain embodiments, the conjugate is in the S-configuration.

[0166] The method can further include simultaneous or sequential administration, in any order, of an effective amount of an active agent that is a free radiosensitizer, radioprotector, immunotherapeutic agent, chemotherapeutic agent, anticancer agent, or hormonal therapy agent, or a pharmaceutical composition (e.g., a second pharmaceutical composition) comprising the same and a pharma- ceutically acceptable carrier or excipient. In certain embodiments, the active agent comprises a radioprotector. In certain embodiments, the radioprotector active agent comprises Lys.

[0167] The subject may have cancer, for example, a primary bone cancer, such as osteosarcoma, chondrosarcoma, Ewing's sarcoma, or chordoma, or a secondary bone cancer, such as metastatic breast cancer, prostate cancer, multiple myeloma, thyroid cancer, lung cancer, renal cancer, ovarian cancer, colon cancer, or melanoma.

[0168] In certain embodiments, the method may further include imaging the bone of the subject and / or the cancer cell population in the subject (e.g., after administration of the conjugate, the first pharmaceutical composition comprising the conjugate, or the first pharmaceutical composition comprising the conjugate and a radiosensitizer, radioprotector, immunotherapeutic agent, chemotherapeutic agent, anticancer agent, and / or hormonal therapy agent). Imaging may be performed through any currently known or later developed imaging technique related to the medical field. In certain embodiments, imaging may be performed through the use of functional imaging modalities such as hybrid scanning, single photon emission computed tomography (SPECT) or PET in combination with computed tomography (CT) and / or magnetic resonance imaging (MRI) techniques, and combinations thereof. Ultrasound imaging may also be used. The conjugates herein may be used to label bone cancer cells (e.g., by binding to hydroxyapatite), and thus the methods herein may be used to visualize, characterize, monitor, and facilitate the treatment of bone cancer or other diseases.

[0169] If bone is imaged in the subject (either in conjunction with the method or separately therefrom), the method may further include the step of diagnosing whether the subject has cancer.

[0170] When the subject is undergoing cancer treatment and bone is imaged in the subject, the method can further include evaluating or monitoring the efficacy of the treatment. For example, the conjugate and / or pharmaceutical composition can be used to quantitatively monitor the growth and proliferation of tumors or lesions in vivo. In certain embodiments, a method is provided for monitoring the progression of cancer (e.g., bone cancer) in a subject, comprising administering to the subject a conjugate, a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition comprising the conjugate or a pharma- ceutically acceptable salt thereof. Such a method can further include imaging the cancer (and / or bone) of the subject.

[0171] The subject's bone may be imaged periodically over the course of therapeutic treatment, and the practitioner can then compare the images and / or otherwise quantify the lesion or cancer growth in the bone to determine therapeutic efficacy (e.g., if there is a differential effect on cancer cell killing or a relative increase in lesion size or cancer growth in the bone over the course of therapeutic treatment). Thus, methods are provided for determining the likelihood of success of a therapeutic treatment in a subject.

[0172] Also provided is a method of binding a conjugate or a pharma- ceutically acceptable salt thereof to hydroxyapatite (e.g., in damaged or diseased bone) in a subject (e.g., in a subject in need thereof), comprising administering to the subject an effective amount of: (i) any of the conjugates or pharma- ceutically acceptable salts thereof described herein, or (ii) a pharmaceutical composition described herein. In certain embodiments, the conjugate or pharma- ceutically acceptable salt thereof has the formula: HB-L A -A (Formula I) where HB is a radical of an anionic (e.g., linear or branched) anionic, acidic and / or electrolytic ligand (e.g., one that binds to hydroxyapatite); A is an active agent, including a radiocontrast agent, radiosensitizer, radioprotector, or radiotherapeutic agent; L A is a linker connecting HB and A, or is absent. In certain embodiments, the conjugate or a pharma- ceutically acceptable salt thereof has the formula: HB-A (Formula IA) or a pharma- ceutically acceptable salt thereof, wherein HB is a radical of an anionic, acidic and / or electrolytic ligand (e.g., one that binds to hydroxyapatite); A is an active agent, including a radiocontrast agent, a radiosensitizer, a radioprotector, or a radiotherapeutic agent. In some embodiments, HB binds to hydroxyapatite in damaged or diseased bone. In some embodiments, the subject has a bone disease, bone injury, or bone disorder (e.g., a disease, disorder, or injury described herein). In certain embodiments, the subject has cancer (e.g., bone cancer). In some embodiments, the subject has osteoblastic bone cancer. In some embodiments, the subject has osteolytic bone cancer.

[0173] The conjugates and pharmaceutical compositions may be administered in unit dosage forms and / or compositions.

[0174] In the methods described herein, the conjugates and compositions may be administered in a single dose or via a combination of multiple doses, which may be administered contemporaneously, simultaneously, sequentially or separately by any suitable means. When the doses are administered in separate dosage forms, the number of doses administered per day for each compound or composition may be the same or different. The conjugate and / or composition dosages may be administered via the same or different administration routes. The conjugates or compositions may be administered in parallel, in divided or single forms, at the same or different times during the course of therapy, according to simultaneous or alternating regimens.

[0175] Dosage can vary and may be administered daily, for one or several days, in one or multiple doses.Guidelines can be found in the literature for the appropriate dosage of a given class of pharmaceutical product.In further various embodiments, the preparation may be administered in a "protectively effective amount", i.e., an amount effective for the prevention of a disease or condition.

[0176] The conjugates / compositions may be administered more than once, for example daily (1-3 or more times per day; qd (once a day), bid (twice a day), tid (three times a day)), weekly (including 1-3 or more times on a given day), biweekly (including 1-3 or more times on a given day), monthly (including 1-3 or more times on a given day), or bimonthly (including 1-3 or more times on a given day). In each case, it is understood that the effective amounts described herein correspond to the dosage examples, or alternatively, to the total daily, weekly, monthly or quarterly dose as determined by the dosing protocol.

[0177] Specific Definitions As used herein, the following terms and phrases shall have the meanings set forth below: Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0178] The term "about" can allow for a degree of variation within a value or range, e.g., within 10%, within 5%, or within 1% of a stated value or a stated limit of a range. The term "substantially" can allow for a degree of variation within a value or range, e.g., within 90%, 95%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more of a stated value or a stated limit of a range.

[0179] The terms "a," "an," or "the" are used to include one or more than one unless context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated.

[0180] The term "pharmaceutically acceptable carrier" is recognized in the art and refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting any subject composition or its components. Each carrier must be "acceptable" in the sense of being compatible with the subject composition and its components and not harmful to the patient. Some examples of materials that can serve as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) cellulose, such as corn oil, and soybean oil; (11) cellulose, such as corn oil, and soybean oil; (12) cellulose, such as corn oil, and soybean oil; (13) cellulose, such as corn oil, and soybean oil; (14) cellulose, such as corn oil, and soybean oil; (15) cellulose, such as corn oil, and soybean oil; (16) cellulose, such as corn oil, and soybean oil; (17) cellulose, such as corn oil, and soybean oil; (18) cellulose, such as corn oil, and soybean oil; (19) cellulose, such as corn oil, and soybean oil; (20) cellulose, such as corn oil, and soybean oil; (21) cellulose, such as corn (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical preparations.

[0181] The terms "patient" and "subject" are used interchangeably and include human patients, laboratory animals such as rodents (e.g., mice, rats, or hamsters), rabbits, monkeys, chimpanzees, domestic animals such as dogs, cats, or rabbits, agricultural animals such as cows, horses, pigs, sheep, or goats, or captive wild animals such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins, or whales. The patient to be treated is preferably a mammal, in particular a human.

[0182] While the concepts of the present disclosure have been illustrated and described in detail in the figures and description herein, it is understood that the results in the figures and their description are to be considered exemplary in character and not limiting, that only illustrative embodiments have been shown and described, and that all changes and modifications that fall within the spirit of the present disclosure are desired to be protected. Indeed, numerous specific details are provided and described in order to provide a thorough understanding of the present disclosure.

[0183] Any use of section headings is intended to aid in the reading of the document and should not be construed as limiting. Additionally, information related to a section heading may occur within or outside of that particular section.

[0184] All patents, patent application publications, academic papers, textbooks and other publications mentioned in this specification indicate the level of those skilled in the art to which this disclosure pertains.All such publications are incorporated herein by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.If there is a conflict between this document and the document so incorporated by reference, the usage in the incorporated reference should be considered as a supplement to that of this document, and in the case of irreconcilable conflict, the usage in this document shall prevail.

[0185] Various techniques and mechanisms of the present disclosure will sometimes describe a connection or coupling between two components. Words such as coupled, linked, coupled, connected, connected, and similar terms with their inflected morphemes are used interchangeably unless a distinction is noted or otherwise made clear from the context. These words and expressions do not necessarily convey a direct connection, but include a connection via an intermediate component. It should be noted that a connection between two components does not necessarily mean a direct and unobstructed connection, since various other components may be between the two components of interest. As a result, a connection does not necessarily mean a direct and unobstructed connection, unless otherwise noted. EXAMPLES

[0186] The following examples serve to illustrate the present disclosure and are not intended to limit the scope of the claimed invention in any way.

[0187] [Example 1] Animal models Female Balb / C mice (5-16 weeks old) were purchased from Charles River or The Jackson Laboratory. The femur or tibia of the subjects received 4T1 (murine breast cancer cells) transfected with luciferase (4T1 / L). First, the skin at the joint between the femur and tibia was incised. Next, a 26G needle was used to push aside the tendons connecting the femur and tibia, and the same needle was used to drill a hole at the top of the tibia or femur (location of the growth plate). The 26G needle was removed, and a 27G needle attached to a syringe was inserted into the hole created by the 26G needle to inject 1,000-100,000 4T1 / L cells into the bone. Finally, the incision site was sutured closed.

[0188] Three days after the first tumor challenge, the mice received an intraperitoneal injection of 100 μL of luciferin dissolved in PBS (15 mg / mL). 15 minutes after the injection, the mice were anesthetized and placed in an AMI small animal luminescence and fluorescence imaging instrument to measure and observe luciferin bioluminescence. Luciferin bioluminescence allowed visual confirmation of tumor development and progression. Figure 1 shows the luciferin bioluminescence of a tumor growing in the right femur of the first mouse two weeks after the first tumor challenge.

[0189] After confirming tumor development, the mice were imaged using micro-CT to track bone degradation. The mice were anesthetized and then placed on the micro-CT bed. Their left and right tibias and femurs were then scanned and the images were reconstructed to obtain 3D images of the bones. Figure 2 shows a micro-CT image of the right tibia of the mouse two weeks after initial tumor challenge, demonstrating severe bone resorption due to tumor growth.

[0190] [Example 2] Fluorescence imaging conjugate synthesis 2-Chlorotrityl resin was loaded with D-aspartic acid by reacting 1 equivalent of resin with 5 equivalents of Fmoc-D-Asp(t-butyl)-OH and 15 equivalents of N,N-diisopropylethylamine (DIPEA) in dichloromethane (DCM) for 4 hours. The resin was then capped by reacting it with 17:2:1 DCM:MeOH:DIPEA for 15 minutes three times. After loading, the resin was run on an AAPPTEC automated peptide synthesizer to couple 9 D-aspartic acids and 1 cysteine, resulting in the synthesis of 10 D-aspartic acid and 1 cysteine ​​peptide (DD10-C). Briefly, peptides were synthesized by deprotecting the Fmoc on the amine of the last amino acid bound to the resin, preactivating the resulting amino acid with 5 equivalents of PyBop plus 15 equivalents of NMM or HBTU, and reacting the resin with the preactivated amino acid for 40 minutes. Each amino acid coupling was repeated twice to ensure maximum yield of the final peptide. The Fmoc protecting group on the amine of the terminal cysteine ​​was not deprotected but served as an albumin binder and UV-active group to facilitate subsequent purification of the conjugate. After completion of the synthesis, DD10-C was deprotected and the DD10-C bound resin was washed with 95% TFA, 2.5% TIPS, 2.5% distilled H 2 The peptide was cleaved from the resin by reaction with 20 and 10 mM TCEP at room temperature (RT) for 2 h. The peptide was precipitated by saturating the collected cleavage solution with diethyl ether. The precipitate was purified using HPLC using a 5-50% ammonium acetate buffer (pH = 5, 20 mM) to acetonitrile method.

[0191] The near-infrared dye S0456 conjugate was prepared by first reacting 1 equivalent of N-Boc-tyramine with 1 equivalent of S0456 and 5 equivalents of CsCO. 3N-Boc-Tyramine-S0456 was prepared by reacting with 40% TFA in DMSO at room temperature for 4 hours. The conjugate was precipitated by saturating the reaction with ethyl acetate and the Boc protecting group was deprotected by reacting N-Boc-Tyramine-S0456 with 40% TFA in DCM for 40 minutes. The conjugate was purified using HPLC with a method from 5-60% ammonium acetate buffer (pH=7, 20 mM) to acetonitrile. Purified Tyramine-S0456 was conjugated to three maleimidopropionic acids (3MPA) by reacting 1 equivalent of Tyramine-S0456 with 1 equivalent of 3MPA that was preactivated with 1 equivalent of PyBOP and 5 equivalents of DIPEA. The reaction was accomplished at room temperature for 4 hours. The final 3MPA-Tyramine-S0456 was purified and collected using a HPLC method from 5-60% ammonium acetate buffer (pH=7, 20 mM) to acetonitrile.

[0192] Finally, 3MPA-tyramine-S0456 was coupled with DD10-Cys by reacting 1 equivalent of each reactant in anhydrous DMSO at room temperature for 4 h. The final fluorescent S0456-C-DD10 was purified and collected using an HPLC method from 5-50% ammonium acetate buffer (pH=7, 20 mM) to acetonitrile (Figure 4).

[0193] [Example 3] Fluorescence imaging After confirmation of the development of both tumors and bone lesions, the targeting potential of the hydroxyapatite binding moiety DD10 (or polyaspartic acid peptide) was tested. The subject of Example 1 received a subcutaneous injection of S0456-C-DD10 (4 nmol / 100 μL). 24 hours after injection, the mouse was anesthetized and placed in an AMI small animal luminescence and fluorescence imaging instrument to visualize the localization of the fluorescent conjugate. Figure 3 shows the fluorescence of the bone-targeting conjugate in the right femur of the first mouse two weeks after the first tumor challenge.

[0194] [Example 4] Radioimaging and radiotherapy conjugate synthesis Lysine was loaded onto the 2-chlorotrityl resin by reacting 1 equivalent of resin with 5 equivalents of Fmoc-Lys(Alloc)-OH and 15 equivalents of DIPEA in DCM for 4 hours. The resin was then capped by reacting it with 17:2:1 DCM:MeOH:DIPEA for 15 minutes three times. After loading, the resin was run on an AAPTEC automated peptide synthesizer to couple one phenylalanine and 10 D-aspartic acids with lysine. Briefly, peptides were synthesized by deprotecting the Fmoc on the amine of the last resin-bound amino acid, preactivating the resulting amino acid with 5 equivalents of PyBop plus 15 equivalents of NMM or HBTU, and reacting the resin with the preactivated amino acid for 40 minutes. Each amino acid coupling was repeated twice to ensure maximum yield of the final peptide. At the end of the synthesis, two peptides K(Alloc)-F-DD10 and K(Alloc)-VMF-DD10 were obtained.

[0195] After completion of the synthesis, alloc was removed from Lys by reacting the resin with 0.1-1 equivalents of tetrakis(triphenylphosphine)palladium(0) and 20 equivalents of phenylsilane in DCM. The deprotection lasted for 40 min and was repeated twice to obtain KF-DD10 and KVMF-DD10. The resin was then washed three times with dimethylformamide (DMF), three times with sodium diethyldithiocarbamate solution (0.03 M in DMF) and three times with DCM to ensure the removal of palladium. Then, 3 equivalents of DOTA-tris(t-butyl ester), preactivated with 3 equivalents of HATU and 10 equivalents of DIPEA, were reacted with the resin. The Fmoc protecting group on the amine of the terminal D-aspartic acid was not deprotected but served as an albumin binder and UV-active group to facilitate purification. Both peptides were deprotected and purified using 95% TFA, 2.5% TIPS, 2.5% distilled H 2The peptides were cleaved from the resin using O with 10 mM TCEP for 2 h at room temperature. The collected cleavage solution was saturated with diethyl ether to precipitate the peptides, and the products were purified using HPLC with 5-55% ammonium acetate buffer (pH = 5, 20 mM) to acetonitrile to yield K(DOTA)-F-DD10 and K(DOTA)-VMF-DD10 (Figures 5-7).

[0196] [Example 5] In-111 chelation to radiocontrast conjugates radioactive isotope 111 In (100-300 μCi) was chelated to DOTA in K(DOTA)-F-DD10 (Figure 6) and K(DOTA)-VM-DD10 (Figure 7). 111 The conjugate K(DOTA)-F-DD10 chelated to In is shown. First, stock solutions of both conjugates were prepared at a concentration of 10 mM in ultrapure sodium acetate buffer (pH 5, 1M). The conjugates were then reacted with 111-In to give a chelation concentration of 100-300 μCi / 10 nmol and a reaction concentration of at least 1 mM. The mixture was reacted at 90°C for 15 minutes, and the efficiency of chelation was examined by subjecting a diluted sample of the mixture to radio-HPLC using the method of ultrapure acetonitrile from 5-95% ultrapure ammonium acetate buffer (pH = 5, 20 mM). 5 mM DTPA was then added to a final concentration of 0.2 mM. DTPA is a chelating agent that will remove any free unchelated radioisotopes. Finally, the mixture was diluted with 1% DMSO in PBS with 100 mg / mL ascorbate and 5 mg / mL methionine to a final concentration of 10 nmol / 100 μL of conjugate, both of which are radiation stabilizers that will help prevent the conjugate from being degraded by the radioisotope.

[0197] [Example 6] radiographic contrast Female Balb / C mice (5 to 8 weeks old) bearing intratibial 4T1 / L murine mammary tumors were 111 Mice received a single tail vein injection of In-chelated K(DOTA)-F-DD10 (or K(DOTA)-VM-DD10) at a dosage of 100-300 μCi / 10 nmol. Single-photon emission computed tomography (SPECT) / computed tomography (CT) equipment was used to visualize the biodistribution of the radioactive chelated conjugate at 1, 4, 12, 24, 48, 72, 96 and 120 hours after injection. Their body weight was measured daily. One week after the first radiocontrast injection, mice were imaged for luciferin bioluminescence every other day to follow tumor growth and / or reduction. In addition, mice were scanned once or twice a week with micro-CT to monitor bone degradation and / or formation. After the study, mice were euthanized and their organs were collected and fixed for histology or any other further analysis.

[0198] Figure 8 shows a SPECT / CT image of a tibial tumor-bearing mouse injected with In-111 chelated K(DOTA)-F-DD10 (220 μCi of 200 μCi / 10 nmol dosage was injected). The arrow indicates the site of tumor growth and the white square indicates the kidney.

[0199] Figure 9 shows a SPECT / CT image of a tibial tumor-bearing mouse injected with In-111 chelated K(DOTA)-VMF-DD10 (375 μCi of 300 μCi / 10 nmol dosage was injected). The arrow indicates the site of tumor growth and the white square indicates the kidney.

[0200] Figure 10 shows 111 In-chelated K(DOTA)-F-DD10 (160 μCi of 200 μCi / 10) or 111 A SPECT / CT image of a healthy mouse injected with In-chelated K(DOTA)-VMF-DD10 (350 μCi of 300 μCi / 10 nmol dosage was injected). The white square indicates the kidney.

[0201] In all images, offsite localization to joints (particularly at the shoulder and between the femur and tibia) was visualized according to the age of the subject. Mice between 4 and 12 weeks of age are considered immature, at which age the mouse growth plates have not yet fully closed and bones and joints are rapidly and excessively building, exposing hydroxyapatite.

[0202] [Example 7] Lu-177 chelation to radiotherapy conjugates For chelation, 1–6 mCi of radioisotope 177 Lu was chelated to DOTA in K(DOTA)-F-DD10 (Figure 6). 177 The conjugate K(DOTA)-F-DD10 chelated to Lu is shown. First, a stock solution of the conjugate was prepared at a concentration of 10 mM in ultrapure sodium acetate buffer (pH 5, 1M). The conjugate was then reacted with 177-Lu to give a chelation concentration of 1-6 mCi / 10 nmol and a reaction concentration of at least 1 mM. The mixture was reacted at 90°C for 15 min, and the efficiency of chelation was examined by subjecting a diluted sample of the mixture to radio-HPLC using the method of ultrapure acetonitrile from 5-95% ultrapure ammonium acetate buffer (pH = 5, 20 mM). 5 mM DTPA was then added to a final concentration of 0.2 mM. DTPA is a chelating agent that will remove any free unchelated radioisotopes. Finally, the mixture was diluted with 1% DMSO in PBS with 100 mg / mL ascorbate and 5 mg / mL methionine to a final concentration of 10 nmol / 100 μL of conjugate. Both ascorbate and methionine are radiation stabilizers that will help prevent the conjugate from being degraded by the radioisotope.

[0203] [Example 8] Radiation therapy Female Balb / C mice (14 to 16 weeks old) bearing intratibial 4T1 / L murine mammary tumors received a single tail vein injection of Lu-111 chelated K(DOTA)-F-DD10 or K(DOTA)-VM-DD10 at a dosage of 1-6 mCi / 10 nmol. A SPECT / CT instrument was used to visualize the biodistribution of the radioactive chelated conjugates from 2 to 240 hours after injection. Their body weights were measured daily. One week after the first radiocontrast injection, the mice were imaged for luciferin bioluminescence every other day to follow tumor growth and / or reduction. In addition, the mice were scanned once or twice a week with microCT to monitor bone degradation and / or formation. After the study, the mice were euthanized and their organs were collected and fixed for histology or any other further analysis.

[0204] FIG. 11 shows SPECT / CT images of a tibial tumor-bearing mouse injected with Lu-177 chelated K(DOTA)-F-DD10 (injected at a dose of 0.962 mCi of 1.5 mCi / 10 nmol). The arrows point to the tumor growth site and the white squares point to the kidneys. From left to right: images taken at 3, 24, 48, 72, 96, 120 and 168 hours after radiotherapy injection. FIG. 12 shows SPECT / CT images of a tibial tumor-bearing mouse injected with Lu-177 chelated K(DOTA)-F-DD10 (injected at a dose of 3.0 mCi of 3.0 mCi / 10 nmol). The arrows point to the tumor growth site and the white squares point to the kidneys. From left to right: Imaging at 3, 24, 72, 96, 120 and 168 hours after radiotherapy injection. Figure 13 shows SPECT / CT images of a tibial tumor-bearing mouse injected with Lu-177 chelated K(DOTA)-F-DD10 (3.57mCi of 3.0mCi / 10nmol dosage was injected). Arrows indicate tumor growth sites and white boxes indicate kidneys. Top left to right: Imaging at 2, 12, 24 and 48 hours after radiotherapy injection. Bottom left to right: Imaging at 72, 96, 144 and 196 hours after radiotherapy injection. FIG. 14 shows SPECT / CT images of a tibial tumor-bearing mouse injected with Lu-177 chelated K(DOTA)-F-DD10 (5.50 mCi of 6.0 mCi / 10 nmol dosage was injected). Arrows indicate tumor growth sites and white boxes indicate kidneys. Top row, left to right: images taken 2, 12, 48, 48 and 72 hours after radiotherapy injection. Bottom row, left to right: images taken 96, 144, 196 and 240 hours after radiotherapy injection.

[0205] In all images, minimal extracellular localization was observed because the mice were at least 12 weeks of age, an age at which mice are considered skeletally mature and whose growth plates are mostly or completely closed.

[0206] All polyaspartic acid conjugates, S0456-C-DD10, K(DOTA)-F-DD10 and K(DOTA)-VMF-DD10, demonstrated specific localization to tumor-induced bone lesions. The conjugates attached to the bone lesions for at least 7 days after injection. Both conjugates were rapidly cleared, although extrasite localization was observed in the kidney. The majority of KDFDD10 was cleared from the kidney within 24-48 hours, whereas the majority of KDVMFDD10 was cleared within 48-72 hours. KDFDD10 showed faster renal clearance than KDVMFDD10. Extrasite localization was observed in the joints, which was expected since the mice were young (i.e., 5-6 weeks old) and their growth plates were still developing. This issue was resolved by using older mice (i.e., 12 weeks old or older).

[0207] [Example 9] Synthesis of novel radioimaging and radiotherapy conjugates To test whether a brush border membrane (BBM) peptidase linker (fast release) enhances renal clearance, bone-targeted radioimaging and radiotherapy conjugates with a BBM peptidase linker were incorporated to link a bone-targeted acidic peptide to a chelator.

[0208] 2-Chlorotrityl chloride resin was loaded with lysine by reacting 1 equivalent of resin with 5 equivalents of Fmoc-Lys(Alloc)-OH and 15 equivalents of DIPEA in DCM for 4 hours. The resin was then capped by reacting it with 17:2:1 DCM:MeOH:DIPEA for 15 minutes three times. After loading, the resin was run on an AAPTEC automated peptide synthesizer to couple chelators such as Val, Met, and DOTA.

[0209] Briefly, peptides were synthesized by deprotecting the Fmoc on the amine of the last resin-bound amino acid, preactivating the resulting amino acid with 5 equivalents of PyBop plus 15 equivalents of NMM or HBTU, and reacting the resin with the preactivated amino acid for 40 min. Each amino acid coupling was repeated twice to ensure maximum yield of the final peptide. At the end of the synthesis, K(Alloc)-Val-Met-DOTA was obtained.

[0210] 2-Chlorotrityl chloride resin was loaded with D-aspartic acid by reacting 1 equivalent of resin with 5 equivalents of Fmoc-D-Asp(OtBu)-OH and 15 equivalents of DIPEA in DCM for 4 hours. The resin was then capped by reacting it with 17:2:1 DCM:MeOH:DIPEA for 15 minutes three times. After loading, the resin was run on an AAPTEC automated peptide synthesizer to couple aromatic amino acids such as 9 D-Asp and one or more Phe. Briefly, peptides were synthesized by deprotecting the Fmoc on the amine of the last resin-bound amino acid, preactivating the resulting amino acid with 5 equivalents of PyBop plus 15 equivalents of NMM or HBTU, and reacting the resin with the preactivated amino acid for 40 minutes. Each amino acid coupling was repeated twice to ensure maximum yield of the final peptide. The peptide was cleaved in protected form by reacting the resin with 1:1:8 acetic acid:TFE:DCM for 30 min. At the end of the synthesis, D-Asp 10 (OtBu)-Phe 2 -Fmoc was obtained.

[0211] After completion of the synthesis, alloc was removed from Lys by reacting the resin with 0.1-1 equivalents of tetrakis(triphenylphosphine)palladium(0) and 20 equivalents of phenylsilane in DCM. The deprotection lasted for 40 min and was repeated twice to obtain KVM-DOTA. The resin was then washed three times with DMF, three times with sodium diethyldithiocarbamate solution (0.03 M in DMF) and three times with DCM to ensure the removal of palladium. Then, 1 equivalent of protected D-Asp, preactivated with 3 equivalents of HATU and 10 equivalents of DIPEA, was added. 10 (OtBu)-Phe 2 The -Fmoc peptide was reacted with the resin. The Fmoc protecting group on the amine of the terminal Phe was deprotected. The peptide was deprotected and purified with 95% TFA, 2.5% TIPS, 2.5% distilled H 2 The peptide was cleaved from the resin using 200 with 10 mM TCEP at room temperature for 2 h. The collected cleavage solution was saturated with diethyl ether to precipitate the peptide, and the product was purified using HPLC with 5-55% ammonium acetate buffer (pH = 5, 20 mM) to acetonitrile to obtain K(DD 10 F 2 ) to yield VM-DOTA (Figure 15). Figure 16 shows the structure of the conjugate.

Claims

1. Conjugates of Formula I: HB-L Ａ -A (Formula I) or a pharmaceutically acceptable salt thereof [In the formula, HB contains radicals of polyanionic, polyacidic and / or polyelectrolyte ligands that bind to hydroxyapatite; A is an active agent, including a radiocontrast agent, radiosensitizer, radioprotector, or radiotherapeutic agent; L Ａ is a linker connecting HB and A, or is absent].

2. 2. The conjugate of claim 1, wherein HB comprises a radical of a linear polyanionic, polyacidic and / or polyelectrolyte ligand that binds to hydroxyapatite.

3. The conjugate of claim 1 , wherein HB comprises an amino acid or a derivative thereof.

4. 4. The conjugate of claim 3, wherein HB comprises L-aspartic acid, D-aspartic acid, L-glutamic acid, D-glutamic acid, or a mixture of two or more of the foregoing, or a derivative thereof.

5. HB is a compound selected from the group consisting of (i) a serum albumin binder (AB) directly conjugated to HB or (ii) an AB and a linker L ＡＢ (AB-L ＡＢ ) further comprising AB-L ＡＢ Is, L ＡＢ 2. The conjugate of claim 1, which is conjugated to HB via

6. L Ａ , L ＡＢ , or L Ａ and L ＡＢ are both in the L-configuration; L A , L AB , or both L A and L AB are in the D-configuration; Either or both of L A and L AB comprise a sustained release linker; or 6. The conjugate of claim 1, wherein L A comprises a slow-release linker or a fast-release linker.

7. 6. The conjugate of claim 1, wherein one or more peptide bonds of HB and / or A are arranged in a relative cis orientation or a relative trans orientation.

8. HB is a compound of the formula X-A １ , X-A ２ , X-A ３ or X-A ４ : 【Chemistry 1】 [In the formula, R is COOH in the case of L- or D-aspartic acid, and CH in the case of L- or D-glutamic acid. ２ COOH, or in the case of unnatural acidic amino acids, CH ２ CH ２ COOH, n=1 to 50, 【Chemistry 2】 Is, L Ａ represents the point of attachment to HB is AB or AB-L ＡＢ If further comprising: 【Transformation 3】 is AB or L ＡＢ represents the point of attachment to 6. The conjugate of claim 1 , comprising a radical of a linear polyanion of the formula:

9. L Ａ The conjugate of claim 1 , wherein

10. L Ａ but, Brush border membrane (BBM) linker; a BBM linker comprising Met-Val-Lys; or L 1 or L 2 : 【Chemistry 4】 Including, During the ceremony, 【Transformation 5】 10. The conjugate of claim 1 or 9, wherein represents the point of attachment between LA and A or LA and HB.

11. L Ａ and L ＡＢ 10. The conjugate of claim 1, wherein either or both of the following groups comprise a spacer:

12. 10. The conjugate of any one of claims 1 to 5 or 9, wherein A comprises a chelator or DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid).

13. 13. The conjugate of claim 12, wherein the chelator chelates or binds to a radioisotope.

14. 14. The conjugate of claim 13, wherein the radioisotope is a therapeutic radioisotope comprising an alpha-emitting radioisotope or a beta-emitting radioisotope.

15. The beta-emitting radioisotope is １７７ 15. The conjugate of claim 14, wherein Lu is

16. 14. The conjugate of claim 13, wherein the chelator is an imaging radioisotope.

17. 17. The conjugate of claim 16, wherein the imaging radioisotope is a gamma-emitting radioisotope or a positron emission tomography (PET) imaging radioisotope.

18. the imaging radioisotope is a gamma-emitting radioisotope; １１１ 17. The conjugate of claim 16, wherein In is In.

19. The following structure: 【Transformation 6】 ； 【Transformation 7】 ； 【Transformation 8】 ； or 【Chemistry 9】 2. The conjugate of claim 1, having the formula:

20. １７７ chelated to Lu and has the structure: 【Chemistry 10】 or Chelated to 111 In and has the structure: 【Chemistry 11】 2. The conjugate of claim 1, having the formula:

21. 21. A pharmaceutical composition comprising the conjugate of any one of claims 1, 19, and 20, and a pharmaceutically acceptable carrier or excipient.

22. 22. The pharmaceutical composition of claim 21, further comprising a radiosensitizing agent, a radioprotector, an immunotherapeutic agent, a chemotherapeutic agent, an anti-cancer agent and / or a hormonal therapy agent.

23. for imaging and / or treating bone in a subject; 22. The pharmaceutical composition of claim 21.

24. 24. The pharmaceutical composition of claim 23, The pharmaceutical composition is used in combination, simultaneously or sequentially, in either order, with an active agent or a second pharmaceutical composition comprising the active agent and a second pharmaceutically acceptable carrier or excipient.

25. The pharmaceutical composition of claim 24, wherein the active agent comprises a radiosensitizer, a radioprotector, an immunotherapeutic agent, a chemotherapeutic agent, an anticancer agent or a hormonal therapy agent.

26. 26. The pharmaceutical composition of claim 25, wherein the active agent in (b) comprises lysine.

27. 24. The method of claim 23, wherein the subject has cancer.

28. 28. The method of claim 27, wherein the cancer is a primary or secondary bone cancer.

29. the primary bone cancer is osteosarcoma, chondrosarcoma, Ewing's sarcoma, or chordoma; or 29. The pharmaceutical composition of claim 28, wherein the secondary bone cancer is metastatic breast cancer, prostate cancer, multiple myeloma, thyroid cancer, lung cancer, kidney cancer, ovarian cancer, colon cancer or melanoma.

30. 24. The pharmaceutical composition of claim 23 for binding to hydroxyapatite.

31. 31. The pharmaceutical composition of claim 30, wherein the subject has cancer, bone cancer, osteoblastic bone cancer, osteolytic bone cancer.

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