Patent Application
20250222143
Throughout this application, certain publications are referenced in parentheses. Full citations for these publications may be found immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention relates.
Picaga is a picolinic acid-functionalized triaza-cyclononane heptadentate chelator (
44Scandium is an ideal short-lived radioisotope with a half-life well matched to the typical pharmacokinetics of small molecules, peptides and small biologics and with ideal emission properties (t1/2=3.97 h, Emean β+=632 keV) for PET imaging. The isotope 47Sc, a low-energy β− emitter (t1/2=80.4 h, Emean β−=162 keV) is an isotope with identical chemical properties to 44Sc and highly suited for radiotherapeutic applications (Schmitt, M. et al. 2014). 177Lu is also ideally suited for a therapeutic radiometal. Its half-life (6.65 days) and β-emission energy (Avg Eβ−=134 keV, tissue penetration 1.5 mm) make it an attractive candidate for use in targeted radiotherapy (Price, E. W. & Orvig, C. 2014; Price, E. W. et al. 2013).
Although picaga provides a suitable approach to room temperature radiolabeling with Sc isotopes for preclinical studies, improvement of apparent molar activities (AMA) for clinical scale production of radiopharmaceuticals is desirable. Additionally, attempts to radiolabel picaga with 177Lu at room temperature have been unsuccessful. Towards this end, described herein is the preparation of novel Picaga analogs with the purpose of enabling improved chelation methods. These analogs along with the chelation methods can be used to prepare conjugates with improved properties with broad application for use in imaging and therapy.
Fluorine-18 remains the most widely clinically utilized radionuclide globally and plays a pivotal role in diagnostic cancer imaging with positron emission tomography (PET). The emergence of therapeutic isotopes for the management of disease has produced a pronounced interest in matched, theranostic isotope pairs that can be employed in tandem for the diagnosis and stratification of patients for subsequent radiotherapy. F-18 however does not have a suitable therapeutic isotopologue, thus F-18 PET probes represent suboptimal diagnostic partners to chemically dissimilar, frequently radiometal-based endoradiotherapies. Here, the formation of Sc-18F ternary complexes was demonstrated to be feasible under mild, aqueous conditions, producing chemically robust radiopharmaceuticals in high radiochemical yield and specific activity. A corresponding in vivo imaging and biodistribution study with a cancer-targeting Sc-18F tracer indicates excellent in vivo stability and produces exquisite PET image quality, rendering the 18F/47Sc isotope pair an unusual, yet chemically matched theranostic pair with excellent potential for clinical translation.
The present invention provides a compound having the structure:
or a pharmaceutically acceptable salt of the compound.
The present invention provides a process for producing a metal complex having the structure:
with a preformed M complex in a first suitable solvent to produce a metal complex having the structure:
The present invention provides a method of detecting cancer cells in a subject comprising administering an effective amount of a metal complex or a composition, and imaging the subject with a molecular imaging device to detect the metal complex or composition in the subject, wherein the cancer cells are prostate cancer cells, wherein the cancer cells have elevated levels of prostate-specific membrane antigen (PSMA), wherein the metal complex or composition comprising a compound having the structure:
or a pharmaceutically acceptable salt of the compound;
wherein the metal or metal-ion in the metal complex is Copper-62 (62Cu), Copper-64 (64Cu), Copper-67 (67Cu), Scandium-44 (44Sc), Scandium-47 (47Sc), Scandium-43 (43Sc), Lanthanum-132 (132La), Lanthanum-135 (135La), Yttrium-86 (86Y), Yttrium-90 (90Y), Lutetium-177 (177Lu), Terbium-149 (149Tb), Terbium-152 (152Tb), Terbium-155 (155Tb) or Terbium-161 (161Tb), or Scandium-Fluorine-18 (natSc-18F), Lanthanum-Fluorine-18 (natLa-18F), or Lutetium-Fluorine-18 (natLu-18F).
The present invention provides a method of imaging prostate cancer cells in a subject comprising:
or a pharmaceutically acceptable salt of the compound;
wherein the metal or metal-ion in the metal complex is Copper-62 (62Cu), Copper-64 (64Cu), Copper-67 (67Cu), Scandium-44 (44Sc), Scandium-47 (47Sc), Scandium-43 (43Sc), Lanthanum-132 (132La), Lanthanum-135 (135La), Yttrium-86 (86Y), Yttrium-90 (90Y), Lutetium-177 (177Lu), Terbium-149 (149Tb), Terbium-152 (152Tb), Terbium-155 (155Tb) or Terbium-161 (161Tb), or Scandium-Fluorine-18 (natSc-18F), Lanthanum-Fluorine-18 (natLa-18F), or Lutetium-Fluorine-18 (natLu-18F).
The present invention provides a method of detecting the presence of prostate cancer cells in a subject which comprises determining if an amount of a metal complex or a pharmaceutically acceptable salt thereof, or a composition or a pharmaceutically acceptable salt thereof, is present in the subject at a period of time after administration of the metal complex or composition to the subject, thereby detecting the presence of the prostate cancer cells based on the amount of the metal complex or composition determined to be present in the subject,
wherein the metal complex or composition comprising a compound having the structure:
or a pharmaceutically acceptable salt of the compound;
wherein the metal or metal-ion in the metal complex is Copper-62 (62Cu), Copper-64 (64Cu), Copper-67 (67Cu), Scandium-44 (44Sc), Scandium-47 (47Sc), Scandium-43 (43Sc), Lanthanum-132 (132La), Lanthanum-135 (135La), Yttrium-86 (86Y), Yttrium-90 (90Y), Lutetium-177 (177Lu), Terbium-149 (149Tb), Terbium-152 (152Tb), Terbium-155 (155Tb) or Terbium-161 (161Tb), or Scandium-Fluorine-18 (natSc-18F), Lanthanum-Fluorine-18 (natLa-18F), or Lutetium-Fluorine-18 (natLu-18F).
The present invention provides a method of reducing the size of a prostate tumor or of inhibiting proliferation of prostate cancer cells comprising contacting the tumor or cancer cells with the metal complex or a pharmaceutically acceptable salt thereof, or a composition or a pharmaceutically acceptable salt thereof, so as to thereby reducing the size of the tumor or inhibit proliferation of the cancer cells, wherein the metal complex or the composition comprising a compound having the structure:
or a pharmaceutically acceptable salt of the compound;
wherein the metal or metal-ion in the metal complex is Copper-62 (62Cu), Copper-64 (64Cu), Copper-67 (67Cu), Scandium-44 (44Sc), Scandium-47 (47Sc), Scandium-43 (43Sc), Lanthanum-132 (132La), Lanthanum-135 (135La), Yttrium-86 (86Y), Yttrium-90 (90Y), Lutetium-177 (177Lu), Terbium-149 (149Tb), Terbium-152 (152Tb), Terbium-155 (155Tb) or Terbium-161 (161Tb), or Scandium-Fluorine-18 (natSc-18F), Lanthanum-Fluorine-18 (natLa-18F), or Lutetium-Fluorine-18 (natLu-18F).
The present invention provides a compound having the structure:
or a pharmaceutically acceptable salt of the compound.
In some embodiments, Y3 is Z1-L(A), Z1-L(A)(B), L(A) or L(A)(B) and Y4 is —H.
In some embodiments, Y1 and Y2 are each, independently, —H, alkyl-CO2H, alkylaryl-CO2H, alkylheteroaryl-CO2H, alkylheteroaryl-(NO2)CO2H, alkyl-P(O)(OH)2, alkylaryl-P(O)(OH)2, alkylheteroaryl-P(O)(OH)2 or alkylheteroaryl-(NO2) P(O)(OH)2.
In some embodiments, Y1 and Y2 are each, independently, alkyl-CO2H, alkylaryl-CO2H, alkylheteroaryl-CO2H, alkylheteroaryl-(NO2)CO2H, alkyl-P(O)(OH)2, alkylaryl-P(O)(OH)2, alkylheteroaryl-P(O)(OH)2 or alkylheteroaryl-(NO2) P(O)(OH)2, Y3 is Z1-L(A) or Z1-L(A)(B) and Y4 is —H.
In some embodiments, the heteroaryl is pyridyl.
In some embodiments, the present invention provides a compound having the structure:
In some embodiments, the present invention provides a compound having the structure:
In some embodiments,
In some embodiments, the targeting moiety A is a moiety with specificity for a target protein on the surface of a cell.
In some embodiments, the targeting moiety A is a moiety with specificity for a target antigen on the surface of a cell.
In some embodiments, the targeting moiety A is a small molecule, a peptide, a protein or an antibody or a derivative or fragment thereof.
In some embodiments, the targeting moiety A is ((5-(2-(4-(aminomethyl)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propanamido)-1-carboxypentyl)carbamoyl)glutamic acid or a derivative or fragment thereof.
In some embodiments, the targeting moiety A is 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) or a derivative or fragment thereof.
In some embodiments, the targeting moiety A is trastuzumab, bombesin or somatostatin or a derivative or fragment thereof.
In some embodiments, the targeting moiety A is covalently attached to the chemical linker L.
In some embodiments, the bond between the targeting moiety A and the chemical linker L is formed by reacting a first terminal reactive group on the targeting moiety A with a second terminal reactive group on the chemical linker L.
In some embodiments, the bond between the targeting moiety A and the chemical linker L is formed by reacting an amine moiety on the targeting moiety A with a carboxylic acid moiety on the chemical linker L.
In some embodiments, the bond between the targeting moiety A and the chemical linker L is formed by reacting a carboxylic acid moiety on the targeting moiety A with an amine moiety on the chemical linker L.
In some embodiments, the chemical linker L is an alkyl, alkenyl, alkynyl, alkylether, alkylthioether, alkylamino, alkylamido, alkylester, alkylaryl, alklyheteroaryl, aryl, heteroaryl, a natural amino acid, an unnatural amino acid, a disulfide or thioether containing linker or combinations thereof.
In some embodiments, the present invention provides a compound having the structure:
In some embodiments, the albumin-binding moiety B is a small molecule, a peptide, a protein or an antibody or a derivative or fragment thereof.
In some embodiments, the albumin-binding moiety B is 4-(4-iodophenyl)butanoic acid or a derivative or fragment thereof.
In some embodiments, the albumin-binding moiety B is 4-(4-methyl)butanoic acid or a derivative or fragment thereof.
In some embodiments, the albumin-binding moiety B is covalently attached to the chemical linker L.
In some embodiments, both the targeting moiety A and the albumin-binding moiety B are both covalently attached to the chemical linker L.
In some embodiments, the bond between the albumin-binding moiety B and the chemical linker L is formed by reacting a first terminal reactive group on the albumin-binding moiety B with a second terminal reactive group on the chemical linker L.
In some embodiments, the bond between the albumin-binding moiety B and the chemical linker L is formed by reacting a carboxylic acid moiety on the albumin-binding moiety B with an amine moiety on the chemical linker L.
In some embodiments, the bond between the albumin-binding moiety B and the chemical linker L is formed by reacting an amine moiety on the albumin-binding moiety B with a carboxylic acid moiety on the chemical linker L.
In some embodiments, L has the structure:
In some embodiments, the chemical linker L is a releasable linker.
In some embodiments, the chemical linker L is a non-releasable linker.
In some embodiments, the present invention provides a compound having the structure:
In some embodiments, the present invention provides a compound having the structure:
In some embodiments, the present invention provides a compound having the structure:
In some embodiments, Y1 and Y2 are each, independently, —H, alkyl-CO2H, alkylaryl-CO2H, alkylheteroaryl-CO2H, alkylheteroaryl-(NO2)(CO2H), alkyl-P(O)(OH)2, alkylaryl-P(O)(OH)2, alkylheteroaryl-P(O)(OH)2 or alkylheteroaryl-(NO2)(P(O)(OH)2).
In some embodiments, one of Y1 or Y2 is —H, or each of Y1 and Y2 is —H.
In some embodiments, one of Y1 or Y2 is alkyl-CO2H, or each of Y1 and Y2 is alkyl-CO2H.
In some embodiments, one of Y1 or Y2 is alkylaryl-CO2H or alkylheteroaryl-CO2H, or each of Y1 and Y2 is alkylaryl-CO2H or alkylheteroaryl-CO2H.
In some embodiments, one of Y1 or Y2 is alkyl-P(O)(OH)2, or each of Y1 and Y2 is alkyl-P(O)(OH)2.
In some embodiments, one of Y1 or Y2 is alkylaryl-P(O)(OH)2 or alkylheteroaryl-P(O)(OH)2, or each of Y1 and Y2 is alkylaryl-P(O)(OH)2 or alkylheteroaryl-P(O)(OH)2.
In some embodiments, Y1 is alkyl-CO2H, and Y2 is alkylaryl-CO2H or alkylheteroaryl-CO2H.
In some embodiments, Y1 is alkyl-CO2H, and Y2 is alkylaryl-P(O)(OH)2 or alkylheteroaryl-P(O)(OH)2.
In some embodiments, Y1 is alkyl-P(O)(OH)2, and Y2 is alkylaryl-CO2H or alkylheteroaryl-CO2H.
In some embodiments, the heteroaryl is pyridyl.
In some embodiments, the present invention provides a compound having the structure:
In some embodiments, the present invention provides a compound having the structure:
In some embodiments, the present invention provides a compound having the structure:
The present invention also provides a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable carrier.
In some embodiments, a metal complex comprising the compound of the present invention, wherein the compound coordinates or chelates or complexes to a metal or metal-ion (M).
In some embodiments, the metal is Copper-62 (62Cu), Copper-64 (64Cu), Copper-67 (67Cu), Scandium-44 (44Sc), Scandium-47 (47Sc), Scandium-43 (43Sc), Lanthanum-132 (132La), Lanthanum-135 (135La), Yttrium-86 (86Y), Yttrium-90 (90Y), Lutetium-177 (177Lu), Terbium-149 (149Tb), Terbium-152 (152Tb), Terbium-155 (155Tb) or Terbium-161 (161Tb).
In some embodiments, the metal-ion is Scandium-Fluorine-18 (natSc-18F), Lanthanum-Fluorine-18 (natLa-18F), or Lutetium-Fluorine-18 (natLu-18F).
In some embodiments, the metal-ion is Scandium-44-Fluorine (44Sc—F), Scandium-47-Fluorine (47Sc—F), Lanthanum-132-Fluorine (132La—F), Lanthanum-135-Fluorine (135La—F) or Lutetium-177-Fluorine (177Lu—F).
In some embodiments, the Fluorine is Fluorine-18 (18F).
In some embodiments, the present invention provides a metal complex having the structure:
or a pharmaceutically acceptable salt thereof.
In some embodiments, the present invention provides a metal complex having the structure:
or a pharmaceutically acceptable salt thereof.
In some embodiments, the present invention provides a metal complex having the structure:
or a pharmaceutically acceptable salt thereof.
In some embodiments, the present invention provides a metal complex having the structure:
In some embodiments, the present invention provides a metal complex having the structure:
In some embodiments, a pharmaceutical composition comprising the metal complex of the present invention and a pharmaceutically acceptable carrier.
The present invention provides a method of detecting target cells in a subject comprising administering an effective amount of the metal complex of the present invention or the composition of the present invention to the subject, and imaging the subject with a molecular imaging device to detect the metal complex or composition in the subject.
In some embodiments, the target cells are cancer cells.
The present invention provides a method of imaging target cells in a subject comprising:
The present invention provides a method of detecting the presence of target cells in a subject which comprises determining if an amount of the metal complex of the present invention or a pharmaceutically acceptable salt thereof, or the composition of the present invention is present in the subject at a period of time after administration of the metal complex or composition to the subject, thereby detecting the presence of the target cells based on the amount of the metal complex or composition determined to be present in the subject.
In some embodiments, the detecting is performed by a Positron Emission Tomography (PET) device.
In some embodiments, the detecting is performed by a Single-Photon Emission Computed Tomography (SPECT) device.
In some embodiments, A has the structure:
In some embodiments, the present invention provides a compound having the structure:
In some embodiments, the present invention provides a compound having the structure:
In some embodiments, the chemical linker L is an alkyl, alkenyl, alkynyl, alkylether, alkylthioether, alkylamino, alkylamido, alkylester, alkylaryl, alklyheteroaryl, aryl, heteroaryl, a natural amino acid, an unnatural amino acid, a disulfide or thioether containing linker or combinations thereof.
In some embodiments, the chemical linker L is alkyl, alkenyl, alkynyl, alkyl-O-alkyl, alkyl-O-alkyl-O-alkyl, alkyl-NH, alkyl-NH-alkyl, alkyl-C(O)O-alkyl, alkyl-OC(O)-alkyl alkyl-CO-alkyl, alkyl-C(O)NH-alkyl, alkyl-NHC(O)-alkyl or alkyl-C(O)NH-alkyl-NH or combinations thereof.
In some embodiments, Z1 is
In some embodiments, Y1 and Y2 are each, independently, —H, alkyl-CO2H, alkylaryl-CO2H, alkylheteroaryl-CO2H, alkylheteroaryl-(NO2)(CO2H), alkyl-P(O)(OH)2, alkylaryl-P(O)(OH)2, alkylheteroaryl-P(O)(OH)2 or alkylheteroaryl-(NO2)(P(O)(OH)2).
In some embodiments, Y1 and Y2 are each, independently, —H,
In some embodiments, the present invention provides a compound having the structure:
In some embodiments, the present invention provides a compound having the structure:
In some embodiments, the present invention provides a compound having the structure:
In some embodiments, the chemical linker L is an alkyl, alkenyl, alkynyl, alkylether, alkylthioether, alkylamino, alkylamido, alkylester, alkylaryl, alklyheteroaryl, aryl, heteroaryl, a natural amino acid, an unnatural amino acid, a disulfide or thioether containing linker or combinations thereof.
In some embodiments, the chemical linker L is alkyl, alkenyl, alkynyl, alkyl-O-alkyl, alkyl-O-alkyl-O-alkyl, alkyl-NH, alkyl-NH-alkyl, alkyl-C(O)O-alkyl, alkyl-OC(O)-alkyl alkyl-CO-alkyl, alkyl-C(O)NH-alkyl, alkyl-NHC(O)-alkyl, alkyl-C(O)NH-alkyl-NH, alkyl-C(O)NH-(alkyl-C(O)) (alkyl-NH) or combinations thereof.
In some embodiments, L has the structure:
In some embodiments, the present invention provides a compound having the structure:
In some embodiments, Z1 is
In some embodiments, Y1 and Y2 are each, independently, —H, alkyl-CO2H, alkylaryl-CO2H, alkylheteroaryl-CO2H, alkylheteroaryl-(NO2)(CO2H), alkyl-P(O)(OH)2, alkylaryl-P(O)(OH)2, alkylheteroaryl-P(O)(OH)2 or alkylheteroaryl-(NO2)(P(O)(OH)2).
In some embodiments, Y1 and Y2 are each, independently, —H,
In some embodiments, B has the structure:
In some embodiments, the halogne is F, Br, I.
In some embodiments, the halogne is I.
In some embodiments, the present invention provides a compound having the structure:
and
In some embodiments, the present invention provides a compound having the structure:
In some embodiments, Y1 and Y2 are each, independently
or a pharmaceutically acceptable salt of the compound.
In some embodiments, the present invention provides a compound having the structure:
or a pharmaceutically acceptable salt of the compound.
In some embodiments, the present invention provides a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable carrier.
In some embodiments, the present invention provides a metal complex comprising the compound of the present invention, wherein the compound coordinates or chelates or complexes to a metal or metal-ion (M).
In some embodiments, the metal is Copper-62 (62Cu), Copper-64 (64Cu), Copper-67 (67Cu), Scandium-44 (44Sc), Scandium-47 (47Sc), Scandium-43 (43Sc), Lanthanum-132 (132La), Lanthanum-135 (135La), Yttrium-86 (86Y), Yttrium-90 (90Y), Lutetium-177 (177Lu), Terbium-149 (149Tb), Terbium-152 (152Tb), Terbium-155 (155Tb) or Terbium-161 (161Tb).
In some embodiments, the metal-ion is Scandium-Fluorine-18 (natSc-18F), Lanthanum-Fluorine-18 (natLa-18F), or Lutetium-Fluorine-18 (natLu-18F).
In some embodiments, the metal-ion is Scandium-44-Fluorine (44Sc—F), Scandium-47-Fluorine (47Sc—F), Lanthanum-132-Fluorine (132La—F), Lanthanum-135-Fluorine (135La—F) or Lutetium-177-Fluorine (177Lu—F).
In some embodiments, wherein the Fluorine is Fluorine-18 (18F).
In some embodiments, the present invention provides a metal complex having the structure:
In some embodiments, the present invention provides a metal complex having the structure:
or a pharmaceutically acceptable salt of the compound.
In some embodiments, the present invention provides a pharmaceutical composition comprising the metal complex of the present invention and a pharmaceutically acceptable carrier.
The present invention also provides a method of detecting cancer cells in a subject comprising administering an effective amount of the metal complex of the present invention or the composition of the present invention to the subject, and imaging the subject with a molecular imaging device to detect the metal complex or composition in the subject, wherein the cancer cells are prostate cancer cells, wherein the cancer cells have elevated levels of prostate-specific membrane antigen (PSMA).
The present invention provides a method of imaging prostate cancer cells in a subject comprising:
The present invention provides a method of detecting the presence of prostate cancer cells in a subject which comprises determining if an amount of the metal complex of the present invention or a pharmaceutically acceptable salt thereof, or the composition of the present invention is present in the subject at a period of time after administration of the metal complex or composition to the subject, thereby detecting the presence of the prostate cancer cells based on the amount of the metal complex or composition determined to be present in the subject.
The present invention provides a method of reducing the size of a prostate tumor or of inhibiting proliferation of prostate cancer cells comprising contacting the tumor or cancer cells with the metal complex of the present invention or a pharmaceutically acceptable salt thereof, or the composition of the present invention, thereby reducing the size of the tumor or inhibit proliferation of the cancer cells.
The present invention also provides a process for producing a metal complex having the structure:
with a preformed M complex in a first suitable solvent to produce a metal complex having the structure:
In some embodiments, the present invention provides a peptide consists of between 1-500 residues, wherein the residues can be natural and unnatural amino acids, and wherein the amino acids may be linear, cyclic and bicyclic.
The present invention provides a method of detecting target cells in a subject comprising administering an effective amount of the metal complex of the present invention or the composition of the present invention to the subject, and imaging the subject with a molecular imaging device to detect the metal complex or composition in the subject.
In some embodiments, the compound or composition specifically accumulates at the target cells.
In some embodiments, the target cells are cancer cells.
In some embodiments, the target cells are prostate cancer cells.
In some embodiments, a detection of the compound or composition in the target cells of the subject is an indication that cancers cells are present in subject.
In some embodiments, the compound or composition is detected using a PET imaging device.
The present invention provides a method of imaging target cells in a subject comprising:
In some embodiments, the compound or composition is detected using a PET imaging device.
In some embodiments, the image obtained is a three-dimensional image.
The present invention provides a method of detecting the presence of target cells in a subject which comprises determining if an amount of the metal complex of the present invention or a pharmaceutically acceptable salt thereof, or the composition of the present invention is present in the subject at a period of time after administration of the metal complex or composition to the subject, thereby detecting the presence of the target cells based on the amount of the metal complex or composition determined to be present in the subject.
In some embodiments, the detecting is performed by a Positron Emission Tomography (PET) device.
In some embodiments, the detecting is performed by a Single-Photon Emission Computed Tomography (SPECT) device.
In some embodiments, the method further comprising quantifying the amount of the compound in the subject and comparing the quantity to a predetermined control.
In some embodiments, the method further comprising determining whether the subject is afflicted with cancer based on the amount of the compound in the subject.
In some embodiments, the method further comprising determining the stage of the cancer.
The present invention provides a method of reducing the size of a tumor or of inhibiting proliferation of cancer cells comprising contacting the tumor or cancer cells with the metal complex of the present invention or a pharmaceutically acceptable salt thereof, or the composition of the present invention, so as to thereby reducing the size of the tumor or inhibit proliferation of the cancer cells.
In some embodiments, chemical linker L is C2-C12 alkyl, C2-C12 alkyl-NH, C2-C12 alkyl-NHC(O)—C2-C12 alkyl, C2-C12 alkyl-C(O)NH—C2-C12 alkyl or C2-C12 alkyl-C(O)NH—C2-C12 alkyl-NH.
In some embodiments, chemical linker L is C4-alkyl-NH.
In some embodiments, chemical linker L is C5-alkyl-NH.
In some embodiments, chemical linker L is C2-alkyl-C(O)NH—C4 alkyl-NH or C2-alkyl-C(O)NH—C5 alkyl-NH.
In some embodiments, chemical linker L is C4-alkyl-NH or C5-alkyl-NH.
In some embodiments, chemical linker L is C2-alkyl-C(O)NH—C4 alkyl-NH or C2-alkyl-C(O)NH—C5 alkyl-NH.
In some embodiments, chemical linker L is C2-C12 alkyl, C2-C12 alkyl-NH, C2-C12 alkyl-NHC(O)—C2-C12 alkyl, C2-C12 alkyl-C(O)NH—C2-C12 alkyl or C2-C12 alkyl-C(O)NH—C2-C12 alkyl-NH.
In some embodiments, chemical linker L is
In some embodiments, chemical linker L is
In some embodiments, each of Y1 and Y2 is
In some embodiments, the present invention provides a metal complex comprising the compound of the present invention, wherein the compound coordinates to a metal.
In some embodiments, the metal is Copper-62 (62Cu), Copper-64 (64Cu), Copper-67 (67Cu), Scandium-44 (44Sc), Scandium-47 (47Sc), Scandium-43 (43Sc), Lanthanum-132 (132La), Lanthanum-135 (135La), Yttrium-86 (86Y), Yttrium-90 (90Y), Lutetium-177 (177Lu), Terbium-149 (149Tb), Terbium-152 (152Tb), Terbium-155 (155Tb) or Terbium-161 (161Tb).
In some embodiments, the metal is Scandium-47 (47Sc) or Copper-67 (67Cu). Furthermore, X-Fluoride-18, where X corresponds to the metal ion bound to the chelator and may be any of the elements mentioned above in its stable (natLa, natSc, natLu) or radioactive form, with Fluorine-18 or Fluorine-19 bound directly to the metal center.
In some embodiments, the present invention provides a pharmaceutical composition comprising the metal complex of the present invention and a pharmaceutically acceptable carrier.
The present invention provides a method of detecting cancer cells in a subject comprising administering an effective amount of the metal complex of the present invention or the composition of the present invention to the subject, and imaging the subject with a molecular imaging device to detect the metal complex or composition in the subject.
In some embodiments, the cancer cells are prostate cancer cells.
In some embodiments, the compound or composition specifically accumulates at prostate cancer cells.
In some embodiments, a detection of the compound or composition in the prostate gland of the subject is an indication that cancers cells are present in the prostate gland.
In some embodiments, the cancer cells have elevated levels of prostate-specific membrane antigen (PSMA).
In some embodiments, the compound or composition is detected using a PET imaging device.
In some embodiments, the compound or composition is detected using a SPECT imaging device.
The present invention provides a method of imaging prostate cancer cells in a subject comprising:
In some embodiments, the image obtained is a three-dimensional image.
The present invention provides a method of detecting the presence of prostate cancer cells in a subject which comprises determining if an amount of the metal complex of the present invention or a pharmaceutically acceptable salt thereof, or the composition of the present invention is present in the subject at a period of time after administration of the metal complex or composition to the subject, thereby detecting the presence of the prostate cancer cells based on the amount of the metal complex or composition determined to be present in the subject.
In some embodiments, the detecting is performed by a Positron Emission Tomography (PET) device.
In some embodiments, the compound or composition is detected using a SPECT imaging device.
In some embodiments, the method further comprising quantifying the amount of the compound in the subject and comparing the quantity to a predetermined control.
In some embodiments, the method further comprising determining whether the subject is afflicted prostate cancer based on the amount of the compound in the subject.
In some embodiments, the method further comprising determining the stage of the prostate cancer.
The present invention provides a method of reducing the size of a prostate tumor or of inhibiting proliferation of prostate cancer cells comprising contacting the tumor or cancer cells with the metal complex of the present invention or a pharmaceutically acceptable salt thereof, or the composition of the present invention, so as to thereby reducing the size of the tumor or inhibit proliferation of the cancer cells.
In some embodiments, Y1 is —H and Y2 is other than H.
In some embodiments, Y1 and Y2 are each —H.
In some embodiments, Y1 and Y2 are each other than —H.
In some embodiments, the present invention provides a method for reducing one or more symptoms of disease in a subject, comprising administering an effective amount of the compound of the present invention or the composition of the present invention to the subject so as to treat the disease in the subject.
In some embodiments, the disease is cancer.
In some embodiments, the cancer cells have elevated levels of proteins or antigens or both.
In some embodiments, the metal (M) is a radioisotope.
The present invention provides a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable carrier.
The present invention provides a method for detecting cancer cells in a subject comprising administering an effective amount of a compound of the present invention or a composition of the present invention to the subject, and imaging the subject with a molecular imaging device to detect the compound or composition in the subject.
In some embodiments of the method, the compound or composition specifically accumulates in cancer cells relative to non-cancer cells.
In some embodiments of the method, a detection of the compound or composition in an organ of the subject is an indication that cancers cells are present in the organ.
In some embodiments of the method, the cancer cells are lung cancer, breast cancer, prostate cancer, cervical cancer, pancreatic cancer, colon cancer, ovarian cancer, stomach cancer, esophagus cancer, skin cancer, heart cancer, liver cancer, bronchial cancer, testicular cancer, kidney cancer, bladder cancer, spleen cancer, thymus cancer, thyroid cancer, brain cancer, or gall bladder cancer cells.
In some embodiments, the present invention provides a method of reducing one or more symptoms of cancer or of imaging cancer cells. Cancers or cells thereof include, but are not limited to, lung cancer, breast cancer, prostate cancer, cervical cancer, pancreatic cancer, colon cancer, ovarian cancer; stomach cancer, esophagus cancer, mouth cancer, tongue cancer, gum cancer, skin cancer (e.g., melanoma, basal cell carcinoma, Kaposi's sarcoma, etc.), muscle cancer, heart cancer, liver cancer, bronchial cancer, cartilage cancer, bone cancer, testis cancer, kidney cancer, endometrium cancer, uterus cancer, bladder cancer, bone marrow cancer, lymphoma cancer, spleen cancer, thymus cancer, thyroid cancer, brain cancer, neuron cancer, mesothelioma, gall bladder cancer, ocular cancer (e.g., cancer of the cornea, cancer of uvea, cancer of the choroids, cancer of the macula, vitreous humor cancer, etc.), joint cancer (such as synovium cancer), glioblastoma, lymphoma, and leukemia. Malignant neoplasms are further exemplified by sarcomas (such as osteosarcoma and Kaposi's sarcoma).
In some embodiments of the method, the compound or composition is detected using a PET imaging device.
In some embodiments, the compound or composition is detected using a SPECT imaging device.
In some embodiments of the above method, the image obtained is a two-dimensional image.
In some embodiments of the above method, the image obtained is a three-dimensional image.
The present invention provides methods relate to the administration of a compound containing an imaging moiety linked to a targeting moiety, i.e. an antibody, peptide or small molecule, that recognizes target proteins or antigens in or on target cells in a subject, and to an albumin-binding moiety, i.e. an antibody, peptide or small molecule, that recognizes albumin.
In some embodiments, the imaging moiety is linked to both a targeting moiety and to an albumin-binding moiety.
In some embodiments, the claimed conjugates are capable of high affinity binding to receptors on cancer cells or other cells to be visualized. The high affinity binding can be inherent to the targeting moiety or the binding affinity can be enhanced by the use of a derivative or fragment of the targeting moiety or by the use of particular chemical linkage between the imaging agent and targeting moiety that is present in the conjugate.
In some embodiments, the claimed conjugates are capable of high affinity binding to receptors on cancer cells or other cells to be visualized and to albumin. The high affinity binding can be inherent to the targeting moiety and to the albumin-binding moiety or the binding affinity can be enhanced by the use of a derivative or fragment of the targeting moiety or by the use of particular chemical linkage between the imaging agent, targeting moiety and/or the albumin-binding moiety that is present in the conjugate.
In some embodiments, the present invention provides a compound having the structure:
or a pharmaceutically acceptable salt of the compound.
In some embodiments, the present invention provides a compound having the structure:
wherein L is a chemical linker, A is a targeting moiety, and B is an albumin-binding moiety.
In some embodiments, Y1 and Y2 are each, independently, —H, alkylheteroaryl, carboxylic acid, alkyl-carboxylic acid, alkyl-CO2H, alkylaryl-CO2H, alkylheteroaryl-CO2H, alkylheteroaryl-(NO2)(CO2H), alkyl-CO2R1, alkylaryl-CO2R1, alkylheteroaryl-CO2R1, alkyl-OH, alkylaryl-OH, alkylheteroaryl-OH, or alkyl-N(alkylaryl)2.
In some embodiments, Y1 and Y2 are each, independently, —H, alkylheteroaryl, carboxylic acid, alkyl-carboxylic acid, alkyl-CO2H, alkylaryl-CO2H, alkylheteroaryl-CO2H, or alkylheteroaryl-(NO2)(CO2H),
In some embodiments, Y1 and Y2 are independently H or carboxylic acid.
In some embodiments, the carboxylic acid is methanoic acid, ethanoic acid, propanoic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, or decanoic acid.
In some embodiments, the carboxylic acid is pentanoic acid.
In some embodiments, Y1 and Y2 are each, independently, —H, alkylheteroaryl, carboxylic acid, alkyl-carboxylic acid, alkyl-CO2H, alkylaryl-CO2H, alkylheteroaryl-CO2H, alkylheteroaryl-(NO2)(CO2H), alkylheteroaryl-P(O)(OH)2, —P(O)(OH)2, alkyl-CO2R1, alkylaryl-CO2R1, alkylheteroaryl-CO2R1, alkyl-OH, alkylaryl-OH, alkylheteroaryl-OH, or alkyl-N(alkylaryl)2.
In some embodiments, Y1 and Y2 are each, independently, —H, alkylheteroaryl, carboxylic acid, alkyl-carboxylic acid, alkyl-CO2H, alkylaryl-CO2H, alkylheteroaryl-CO2H, alkylheteroaryl-P(O)(OH)2, —P(O)(OH)2, or alkylheteroaryl-(NO2)(CO2H).
In some embodiments, Y1 and Y2 are independently alkyl-CO2H, alkylheteroaryl-P(O)(OH)2, alkylheteroaryl-CO2H, —P(O)(OH)2 or carboxylic acid.
In some embodiments, Y1 or Y2 is
wherein R is H, alkyl, alkenyl, alkynyl, alkyl-aryl, alkyl-heteroaryl, aryl, heteroaryl, alkyl-CF3 or —Si(alkyl)3.
In some embodiments, the present invention provides a compound having the structure:
wherein R is H, alkyl, alkenyl, alkynyl, alkyl-aryl, alkyl-heteroaryl, aryl, heteroaryl, alkyl-CF3 or —Si(alkyl)3.
In some embodiments, the present invention provides a compound having the structure:
wherein R is H, alkyl, alkenyl, alkynyl, alkyl-aryl, alkyl-heteroaryl, aryl, heteroaryl, alkyl-CF3 or —Si(alkyl)3.
In some embodiments, the present invention provides a compound having the structure:
wherein R is H, alkyl, alkenyl, alkynyl, alkyl-aryl, alkyl-heteroaryl, aryl, heteroaryl, alkyl-CF3 or —Si(alkyl)3.
In some embodiments, A is ((5-(2-(4-(aminomethyl)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propanamido)-1-carboxypentyl)carbamoyl)glutamic acid or a derivative or fragment thereof; 2-[3-(1,3-dicarboxypropyl)ureido]pentanedioic acid (DUPA) or a derivative or fragment thereof; trastuzumab, bombesin or somatostatin or a derivative or fragment thereof.
In some embodiments, the albumin-binding moiety B is a small molecule, a peptide, a protein or an antibody or a derivative or fragment thereof, more preferably, the albumin-binding moiety B is 4-(4-iodophenyl)butanoic acid or a derivative or fragment thereof, or the albumin-binding moiety B is 4-(4-methyl)butanoic acid or a derivative or fragment thereof.
In some embodiments, the present invention provides a metal complex comprising the compound disclosed in this application.
In some embodiments, the metal in the metal complex is Copper-62 (62Cu), Copper-64 (64Cu), Copper-67 (67Cu), Scandium-44 (44Sc), Scandium-47 (47Sc), Scandium-43 (43Sc), Lanthanum-132 (132La), Lanthanum-135 (135La), Yttrium-86 (86Y), Yttrium-90 (90Y), Lutetium-177 (177Lu), Terbium-149 (149Tb), Terbium-152 (152Tb), Terbium-155 (155Tb) or Terbium-161 (161Tb).
In some embodiments, the metal-ion in the metal complex is Scandium-Fluorine-18 (natSc-18F), Lanthanum-Fluorine-18 (natLa-18F), or Lutetium-Fluorine-18 (natLu-18F).
In some embodiments, the present invention provides a compound having the structure:
In some embodiments, the present invention provides a pharmaceutical composition comprising the compound of or the metal complex disclosed in this application, and a pharmaceutically acceptable carrier.
In some embodiments, the present invention provides a method of detecting target cells in a subject comprising administering an effective amount of the metal complex or the composition disclosed in this application to the subject, and imaging the subject with a molecular imaging device to detect the metal complex or composition in the subject.
In some embodiments, the present invention provides a method of imaging target cells in a subject comprising:
In some embodiments, the present invention provides a method of detecting the presence of target cells in a subject which comprises determining if an amount of the metal complex or a pharmaceutically acceptable salt thereof, or the composition disclosed in this application is present in the subject at a period of time after administration of the metal complex or composition to the subject, thereby detecting the presence of the target cells based on the amount of the metal complex or composition determined to be present in the subject.
In some embodiments, the detecting and imaging is performed by a Positron Emission Tomography (PET) device or a Single-Photon Emission Computed Tomography (SPECT) device.
In some embodiments, the target cells are cancer cells; preferably, the cancer cells are prostate cancer cells, more preferably, the cancer cells have elevated levels of prostate-specific membrane antigen (PSMA).
In some embodiments, the present invention provides a method of reducing the size of a prostate tumor or of inhibiting proliferation of prostate cancer cells comprising contacting the tumor or cancer cells with the metal complex or a pharmaceutically acceptable salt thereof, or the composition disclosed in this application, so as to thereby reducing the size of the tumor or inhibit proliferation of the cancer cells.
As used herein, the term “imaging agent” refers to any agent or portion (i.e. imaging moiety) of an agent that is used in medical imaging to visualize or enhance the visualization of the body including, but not limited to, internal organs, cells, cancer cells, cellular processes, tumors, and/or normal tissue. Imaging agents or imaging moieties include, but are not limited to, PET imaging agents, SPECT imaging agents. Imaging agents or moieties include, but are not limited to, any compositions useful for imaging cancer cells.
The imaging moiety of the compound of the present invention has the structure:
The targeting moiety may comprise, consist of, or consist essentially of an antibody, peptide, protein or small molecule.
The targeting moiety may comprise, consist of, or consist essentially of Brentuximab (targets cell-membrane protein CD30), Inotuzumab targets CD22), Gemtuzumab (targets CD33), Milatuzumab (targets CD74), Trastuzumab (targets HER2 receptor), Glembatumomab (targets transmembrane glycoprotein NMB-GPNMB), Lorvotuzumab (targets CD56), or Labestuzumab (targets carcinoembryonic cell adhesion molecule 5) or derivatives or fragments thereof.
The targeting moiety may comprise, consist of, or consist essentially of ((5-(2-(4-(aminomethyl)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propanamido)-1-carboxypentyl)carbamoyl)glutamic acid (targets prostate-specific membrane antigen (PSMA)), or derivatives or fragments thereof.
The targeting moiety may comprise, consist of, or consist essentially of (((S)-5-((R)-2-((1r,4R)-4-(aminomethyl)cyclohexane-1-carboxamido)-3-(naphthalen-2-yl)propanamido)-1-carboxypentyl)carbamoyl)-L-glutamic (targets prostate-specific membrane antigen (PSMA)), or derivatives or fragments thereof.
The targeting moiety may comprise, consist of, or consist essentially of DUPA [(2-[3-(1, 3-dicarboxy propyl)ureido]pentanedioic acid)] (targets prostate-specific membrane antigen (PSMA)), or derivatives or fragments thereof.
The targeting moiety may comprise, consist of, or consist essentially of bombesin (targets G-protein-coupled receptors BBR1, -2, and -3) or somatostatin (targets Somatostatin receptor subtypes 1-5), or derivatives or fragments thereof.
The targeting moiety is capable of selectively binding to the population of cells to be visualized due to preferential expression on the targeted cells of a receptor for the targeting moiety. The binding site for the targeting moiety can include receptors or other proteins that are uniquely expressed, overexpressed, or preferentially expressed by the population of cells to be visualized. A surface-presented protein uniquely expressed, overexpressed, or preferentially expressed by the cells to be visualized is a receptor not present or present at lower amounts on other cells providing a means for selective, rapid, and sensitive visualization of the cells targeted for diagnostic imaging using the conjugates of the present invention.
Exemplary targeting moieties are described in U.S. Pat. Nos. 10,005,820 B2, 9,801,951 B2 or U.S. Patent Application Publication No. 2015/0105540 A1, the contents of which are hereby incorporated by reference.
The albumin-binding moiety may comprise, consist of, or consist essentially of an antibody, peptide, protein or small molecule.
The albumin-binding moiety may comprise, consist of, or consist essentially of 4-(4-iodophenyl)butanoic or 4-(4-methyl)butanoic acid or derivatives or fragments thereof.
The albumin-binding moiety is capable of selectively binding to plasma proteins such as human serum albumin (HSA).
The term “chemical linker” or “linker” refers to a chemical moiety or bond that covalently attaches two or more molecules, such as an imaging moiety, a targeting moiety and an albumin-binding moiety. The linker may be a cleavable linker, e.g. pH-sensitive (acid-labile) linker, disulfide linker, a peptide linker, a β-glucuronide linkers or a hydrazine linker. The linker may be a non-cleavable linker, e.g. thioether, maleimidocaproyl, maleimidomethyl cyclohexane-carboxylate, alkyl, alkylamido or amide linker.
Covalent bonding of the imaging agent and chemical linker to both the targeting moiety and albumin-binding moiety can occur through the formation of amide, ester or imino bonds between acid, aldehyde, hydroxy, amino, or hydrazo groups. For example, a carboxylic acid on the targeting moiety can be activated using carbonyldiimidazole or standard carbodiimide coupling reagents such as 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and thereafter reacted with the other component of the conjugate, or with a linker, having at least one nucleophilic group, i.e. hydroxy, amino, hydrazo, or thiol, to form the vitamin-chelator conjugate coupled, with or without a linker, through ester, amide, or thioester bonds.
Linkage of a targeting moiety and albumin-binding moiety to the imaging moiety may be achieved by any means known to those in the art, such as genetic fusion, covalent chemical attachment, noncovalent attachment (e.g., adsorption) or a combination of such means. Selection of a method for linking a targeting moiety and an albumin-binding moiety to an imaging moiety will vary depending, in part, on the chemical nature of the targeting moiety and the albumin-binding moiety.
Linkage may be achieved by covalent attachment, using any of a variety of appropriate methods. For example, the targeting moiety, albumin-binding moiety and imaging moiety may be linked using trifunctional reagents (linkers) that are capable of reacting with each of the targeting moiety, albumin-binding moiety and imaging moiety and forming a bridge between the three.
The term “non-covalent linker” is used in accordance with its ordinary meaning and refers to a divalent or trivalent moiety which includes at least two molecules that are not covalently linked to each other but do interact with each other via a non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond) or van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion).
The terms “cleavable linker” or “cleavable moiety” as used herein refers to a divalent or monovalent, respectively, moiety which is capable of being separated (e.g., detached, split, disconnected, hydrolyzed, a stable bond within the moiety is broken) into distinct entities. A cleavable linker is cleavable (e.g., specifically cleavable) in response to external stimuli (e.g., enzymes, nucleophilic/basic reagents, reducing agents, photo-irradiation, electrophilic/acidic reagents, organometallic and metal reagents, or oxidizing reagents). A chemically cleavable linker refers to a linker which is capable of being split in response to the presence of a chemical (e.g., acid, base, oxidizing agent, reducing agent, Pd(0), tris-(2-carboxyethyl)phosphine, dilute nitrous acid, fluoride, tris(3-hydroxypropyl)phosphine), sodium dithionite (Na2S2O4), hydrazine (N2H4)). A chemically cleavable linker is non-enzymatically cleavable. In embodiments, the cleavable linker is cleaved by contacting the cleavable linker with a cleaving agent. In embodiments, the cleaving agent is sodium dithionite (Na2S2O4), weak acid, hydrazine (N2H4), Pd(0), or light-irradiation (e.g., ultraviolet radiation).
A photocleavable linker (e.g., including or consisting of a o-nitrobenzyl group) refers to a linker which is capable of being split in response to photo-irradiation (e.g., ultraviolet radiation). An acid-cleavable linker refers to a linker which is capable of being split in response to a change in the pH (e.g., increased acidity). A base-cleavable linker refers to a linker which is capable of being split in response to a change in the pH (e.g., decreased acidity). An oxidant-cleavable linker refers to a linker which is capable of being split in response to the presence of an oxidizing agent. A reductant-cleavable linker refers to a linker which is capable of being split in response to the presence of an reducing agent (e.g., Tris(3-hydroxypropyl)phosphine). In embodiments, the cleavable linker is a dialkylketal linker, an azo linker, an allyl linker, a cyanoethyl linker, a 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or a nitrobenzyl linker.
The term “orthogonally cleavable linker” or “orthogonal cleavable linker” as used herein refer to a cleavable linker that is cleaved by a first cleaving agent (e.g., enzyme, nucleophilic/basic reagent, reducing agent, photo-irradiation, electrophilic/acidic reagent, organometallic and metal reagent, oxidizing reagent) in a mixture of two or more different cleaving agents and is not cleaved by any other different cleaving agent in the mixture of two or more cleaving agents. For example, two different cleavable linkers are both orthogonal cleavable linkers when a mixture of the two different cleavable linkers are reacted with two different cleaving agents and each cleavable linker is cleaved by only one of the cleaving agents and not the other cleaving agent. In embodiments, an orthogonally is a cleavable linker that following cleavage the two separated entities (e.g., fluorescent dye, bioconjugate reactive group) do not further react and form a new orthogonally cleavable linker.
Exemplary linkers are described in U.S. Patent Application No. 2012/0322741 A1, U.S. Patent Application No. 2018/0289828 A1 and U.S. Pat. No. 8,461,117 B2, the contents of which are hereby incorporated by reference.
An “antibody” as used herein is defined broadly as a protein that characteristically immunoreacts with an epitope (antigenic determinant) of an antigen. As is known in the art, the basic structural unit of an antibody is composed of two identical heavy chains and two identical light chains, in which each heavy and light chain consists of amino terminal variable regions and carboxy terminal constant regions. The antibodies of the present invention include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, CDR-grafted antibodies, humanized antibodies, human antibodies, catalytic antibodies, multispecific antibodies, as well as fragments, regions or derivatives thereof provided by known techniques, including, for example, enzymatic cleavage, peptide synthesis or recombinant techniques.
As used herein, “monoclonal antibody” means an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256:495-97 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The monoclonal antibodies may also be isolated from phage display libraries using the techniques described, for example, in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222 (3): 581-97 (1991).
The term “hybridoma” or “hybridoma cell line” refers to a cell line derived by cell fusion, or somatic cell hybridization, between a normal lymphocyte and an immortalized lymphocyte tumor line. In particular, B cell hybridomas are created by fusion of normal B cells of defined antigen specificity with a myeloma cell line, to yield immortal cell lines that produce monoclonal antibodies. In general, techniques for producing human B cell hybridomas, are well known in the art [Kozbor et al., Immunol. Today 4:72 (1983); Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. 77-96 (1985)].
The term “epitope” refers to a portion of a molecule (the antigen) that is capable of being bound by a binding agent, e.g., an antibody, at one or more of the binding agent's antigen binding regions. Epitopes usually consist of specific three-dimensional structural characteristics, as well as specific charge characteristics.
“Humanized antibodies” means antibodies that contain minimal sequence derived from non-human immunoglobulin sequences. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hyper variable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. See, for example, U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205, each herein incorporated by reference. In some instances, framework residues of the human immunoglobulin are replaced by corresponding non-human residues (see, for example, U.S. Pat. Nos. 5,585,089; 5,693,761; 5,693,762, each herein incorporated by reference). Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance (e.g., to obtain desired affinity). In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details see Jones et al., Nature 331:522-25 (1986); Riechmann et al., Nature 332:323-27 (1988); and Presta, Curro Opin. Struct. Biol. 2:593-96 (1992), each of which is incorporated herein by reference.
Also encompassed by the term “antibody” are xenogeneic or modified antibodies produced in a non-human mammalian host, more particularly a transgenic mouse, characterized by inactivated endogenous immunoglobulin (Ig) loci. In such transgenic animals, competent endogenous genes for the expression of light and heavy subunits of host immunoglobulins are rendered non-functional and substituted with the analogous human immunoglobulin loci. These transgenic animals produce human antibodies in the substantial absence of light or heavy host immunoglobulin subunits. See, for example, U.S. Pat. No. 5,939,598, the entire contents of which are incorporated herein by reference.
Those skilled in the art will be aware of how to produce antibody molecules of the present invention. For example, polyclonal antisera or monoclonal antibodies can be made using standard methods. To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art. Hybridoma cells can be screened immunochemically for production of antibodies which are specifically reactive with the oligopeptide, and monoclonal antibodies isolated.
The term “target cells” refers to the cells that are involved in a pathology and so are preferred targets for imaging or therapeutic activity. Target cells can be, for example and without limitation, one or more of the cells of the following groups: primary or secondary tumor cells (the metastases), stromal cells of primary or secondary tumors, neoangiogenic endothelial cells of tumors or tumor metastases, macrophages, monocytes, polymorphonuclear leukocytes and lymphocytes, and polynuclear agents infiltrating the tumors and the tumor metastases. The term “targeting moiety” and “targeting agent” refer to an antibody, aptamer, peptide, small molecule or other substance that binds specifically to a target. A targeting moiety may be an antibody targeting moiety (e.g. antibodies or fragments thereof) or a non-antibody targeting moiety (e.g. aptamers, peptides, small molecules or other substances that bind specifically to a target).
The term “target tissue” refers to target cells (e.g., tumor cells) and cells in the environment of the target cells.
The term “cancer” refers to any of a number of diseases characterized by uncontrolled, abnormal proliferation of cells, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body (e.g., metastasize), as well as any of a number of characteristic structural and/or molecular features. A “cancerous cell” or “cancer cell” is understood as a cell having specific structural properties, which can lack differentiation and be capable of invasion and metastasis. Examples of cancers are, breast, lung, brain, bone, liver, kidney, colon, and prostate cancer.
Exemplary targets are described in Avicenna J Med Biotechnol. 2019 January-March; 11 (1): 3-23, Nature Reviews Drug Discovery Volume 16, pages 315-337 (2017), the contents of which are hereby incorporated by reference.
As used herein, the term “amino acid” refers to any natural or unnatural amino acid including its salt form, ester derivative, protected amine derivative and/or its isomeric forms. Amino Acids comprise, by way of non-limiting example: Agmatine, Alanine Beta-Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamine, Glutamic Acid, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Phenyl Beta-Alanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, and Valine. The amino acids may be L or D amino acids.
The terms “peptide”, “polypeptide”, peptidomimetic and “protein” are used to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. These terms also encompass the term “antibody”. “Peptide” is often used to refer to polymers of fewer amino acid residues than “polypeptides” or “proteins”. A protein can contain two or more polypeptides, which may be the same or different from one another.
As used herein, the term “oligopeptide” refers to a peptide comprising of between 2 and 20 amino acids and includes dipeptides, tripeptides, tetrapeptides, pentapeptides, etc.
An amino acid or oligopeptide may be covalently bonded to an amine of another molecule through an amide linkage, resulting in the loss of an “OH” from the amino acid or oligopeptide.
As used herein, the term “activity” refers to the activation, production, expression, synthesis, intercellular effect, and/or pathological or aberrant effect of the referenced molecule, either inside and/or outside of a cell. Such molecules include, but are not limited to, cytokines, enzymes, growth factors, pro-growth factors, active growth factors, and pro-enzymes. Molecules such as cytokines, enzymes, growth factors, pro-growth factors, active growth factors, and pro-enzymes may be produced, expressed, or synthesized within a cell where they may exert an effect. Such molecules may also be transported outside of the cell to the extracellular matrix where they may induce an effect on the extracellular matrix or on a neighboring cell. It is understood that activation of inactive cytokines, enzymes and pro-enzymes may occur inside and/or outside of a cell and that both inactive and active forms may be present at any point inside and/or outside of a cell. It is also understood that cells may possess basal levels of such molecules for normal function and that abnormally high or low levels of such active molecules may lead to pathological or aberrant effects that may be corrected by pharmacological intervention.
This invention also provides isotopic variants of the compounds disclosed herein, including wherein the isotopic atom is 2H and/or wherein the isotopic atom 13C. Accordingly, in the compounds provided herein hydrogen can be enriched in the deuterium isotope. It is to be understood that the invention encompasses all such isotopic forms.
It is understood that the structures described in the embodiments of the methods hereinabove can be the same as the structures of the compounds described hereinabove.
It is understood that where a numerical range is recited herein, the present invention contemplates each integer between, and including, the upper and lower limits, unless otherwise stated.
Except where otherwise specified, if the structure of a compound of this invention includes an asymmetric carbon atom, it is understood that the compound occurs as a racemate, racemic mixture, and isolated single enantiomer. All such isomeric forms of these compounds are expressly included in this invention. Except where otherwise specified, each stereogenic carbon may be of the R or S configuration. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis, such as those described in “Enantiomers, Racemates and Resolutions” by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, NY, 1981. For example, the resolution may be carried out by preparative chromatography on a chiral column.
The subject invention is also intended to include all isotopes of atoms occurring on the compounds disclosed herein. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.
It will be noted that any notation of a carbon in structures throughout this application, when used without further notation, are intended to represent all isotopes of carbon, such as 12C, 13C, or 14C. Furthermore, any compounds containing 13C or 14C may specifically have the structure of any of the compounds disclosed herein.
It will also be noted that any notation of a hydrogen in structures throughout this application, when used without further notation, are intended to represent all isotopes of hydrogen, such as 1H, 2H, or 3H. Furthermore, any compounds containing 2H or 3H may specifically have the structure of any of the compounds disclosed herein.
Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically-labeled reagents in place of the non-labeled reagents employed.
In the compounds used in the method of the present invention, the substituents may be substituted or unsubstituted, unless specifically defined otherwise.
In the compounds used in the method of the present invention, alkyl, heteroalkyl, monocycle, bicycle, aryl, heteroaryl and heterocycle groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.
It is understood that substituents and substitution patterns on the compounds used in the method of the present invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure result.
In choosing the compounds used in the method of the present invention, one of ordinary skill in the art will recognize that the various substituents, i.e. R1, R2, etc. are to be chosen in conformity with well-known principles of chemical structure connectivity.
As used herein, “alkyl” includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and may be unsubstituted or substituted. Thus, C1-Cn as in “C1-Cn alkyl” is defined to include groups having 1, 2, . . . , n−1 or n carbons in a linear or branched arrangement. For example, C1-C6, as in “C1-C6 alkyl” is defined to include groups having 1, 2, 3, 4, 5, or 6 carbons in a linear or branched arrangement, and specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, pentyl, hexyl, and octyl.
As used herein, “alkenyl” refers to a non-aromatic hydrocarbon radical, straight or branched, containing at least 1 carbon to carbon double bond, and up to the maximum possible number of non-aromatic carbon-carbon double bonds may be present, and may be unsubstituted or substituted. For example, “C2-C6 alkenyl” means an alkenyl radical having 2, 3, 4, 5, or 6 carbon atoms, and up to 1, 2, 3, 4, or 5 carbon-carbon double bonds respectively. Alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl.
The term “alkynyl” refers to a hydrocarbon radical straight or branched, containing at least 1 carbon to carbon triple bond, and up to the maximum possible number of non-aromatic carbon-carbon triple bonds may be present, and may be unsubstituted or substituted. Thus, “C2-C6 alkynyl” means an alkynyl radical having 2 or 3 carbon atoms and 1 carbon-carbon triple bond, or having 4 or 5 carbon atoms and up to 2 carbon-carbon triple bonds, or having 6 carbon atoms and up to 3 carbon-carbon triple bonds. Alkynyl groups include ethynyl, propynyl and butynyl.
“Alkylene”, “alkenylene” and “alkynylene” shall mean, respectively, a divalent alkane, alkene and alkyne radical, respectively. It is understood that an alkylene, alkenylene, and alkynylene may be straight or branched. An alkylene, alkenylene, and alkynylene may be unsubstituted or substituted.
As used herein, “aryl” is intended to mean any stable monocyclic, bicyclic or polycyclic carbon ring of up to 10 atoms in each ring, wherein at least one ring is aromatic, and may be unsubstituted or substituted. Examples of such aryl elements include phenyl, p-toluenyl (4-methylphenyl), naphthyl, tetrahydro-naphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl. In cases where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring.
As used herein, the term “polycyclic” refers to unsaturated or partially unsaturated multiple fused ring structures, which may be unsubstituted or substituted.
The term “alkylaryl” refers to alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an aryl group as described above. It is understood that an “alkylaryl” group is connected to a core molecule through a bond from the alkyl group and that the aryl group acts as a substituent on the alkyl group. Examples of arylalkyl moieties include, but are not limited to, benzyl (phenylmethyl), p-trifluoromethylbenzyl (4-trifluoromethylphenylmethyl), 1-phenylethyl, 2-phenylethyl, 3-phenylpropyl, 2-phenylpropyl and the like.
The term “heteroaryl”, as used herein, represents a stable monocyclic, bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Bicyclic aromatic heteroaryl groups include phenyl, pyridine, pyrimidine or pyridizine rings that are (a) fused to a 6-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom; (b) fused to a 5- or 6-membered aromatic (unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom together with either one oxygen or one sulfur atom; or (d) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one heteroatom selected from O, N or S. Heteroaryl groups within the scope of this definition include but are not limited to: benzoimidazolyl, benzofuranyl, benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl, oxazolyl, isoxazoline, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl, tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, acridinyl, carbazolyl, quinolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, isoxazolyl, isothiazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl. In cases where the heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively. If the heteroaryl contains nitrogen atoms, it is understood that the corresponding N-oxides thereof are also encompassed by this definition.
The term “alkylheteroaryl” refers to alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an heteroaryl group as described above. It is understood that an “alkylheteroaryl” group is connected to a core molecule through a bond from the alkyl group and that the heteroaryl group acts as a substituent on the alkyl group. Examples of alkylheteroaryl moieties include, but are not limited to, —CH2—(C5H4N), —CH2—CH2—(CH4N) and the like.
The term “heterocycle”, “heterocyclyl” or “heterocyclic” refers to a mono- or poly-cyclic ring system which can be saturated or contains one or more degrees of unsaturation and contains one or more heteroatoms. Preferred heteroatoms include N, O, and/or S, including N-oxides, sulfur oxides, and dioxides. Preferably the ring is three to ten-membered and is either saturated or has one or more degrees of unsaturation. The heterocycle may be unsubstituted or substituted, with multiple degrees of substitution being allowed. Such rings may be optionally fused to one or more of another “heterocyclic” ring(s), heteroaryl ring(s), aryl ring(s), or cycloalkyl ring(s). Examples of heterocycles include, but are not limited to, tetrahydrofuran, pyran, 1,4-dioxane, 1,3-dioxane, piperidine, piperazine, pyrrolidine, morpholine, thiomorpholine, tetrahydrothiopyran, tetrahydrothiophene, 1,3-oxathiolane, and the like.
The alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl substituents may be substituted or unsubstituted, unless specifically defined otherwise.
In the compounds of the present invention, alkyl, alkenyl, alkynyl, aryl, heterocyclyl and heteroaryl groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups. These include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.
As used herein, the term “halogen” refers to F, Cl, Br, and I.
As used herein, “heteroalkyl” includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and at least 1 heteroatom within the chain or branch.
As used herein, “heterocycle” or “heterocyclyl” as used herein is intended to mean a 5- to 10-membered nonaromatic ring containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S, and includes bicyclic groups. “Heterocyclyl” therefore includes, but is not limited to the following: imidazolyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, dihydropiperidinyl, tetrahydrothiophenyl and the like. If the heterocycle contains a nitrogen, it is understood that the corresponding N-oxides thereof are also encompassed by this definition.
As sued herein, “cycloalkyl” shall mean cyclic rings of alkanes of three to eight total carbon atoms, or any number within this range (i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl).
As used herein, “monocycle” includes any stable polyatomic carbon ring of up to 10 atoms and may be unsubstituted or substituted. Examples of such non-aromatic monocycle elements include but are not limited to: cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Examples of such aromatic monocycle elements include but are not limited to: phenyl.
As used herein, “bicycle” includes any stable polyatomic carbon ring of up to 10 atoms that is fused to a polyatomic carbon ring of up to 10 atoms with each ring being independently unsubstituted or substituted. Examples of such non-aromatic bicycle elements include but are not limited to: decahydronaphthalene. Examples of such aromatic bicycle elements include but are not limited to: naphthalene.
The term “ester” is intended to a mean an organic compound containing the R—O—CO—R′ group.
The term “amide” is intended to a mean an organic compound containing the R—CO—NH—R′ or R—CO—N—R′R″ group.
The term “phenyl” is intended to mean an aromatic six membered ring containing six carbons and five hydrogens.
The term “benzyl” is intended to mean a —CH2R1 group wherein the R1 is a phenyl group.
The term “substitution”, “substituted” and “substituent” refers to a functional group as described above in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms, provided that normal valencies are maintained and that the substitution results in a stable compound. Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Examples of substituent groups include the functional groups described above, and halogens (i.e., F, Cl, Br, and I); alkyl groups, such as methyl, ethyl, n-propyl, isopropryl, n-butyl, tert-butyl, and trifluoromethyl; hydroxyl; alkoxy groups, such as methoxy, ethoxy, n-propoxy, and isopropoxy; aryloxy groups, such as phenoxy; arylalkyloxy, such as benzyloxy (phenylmethoxy) and p-trifluoromethylbenzyloxy (4-trifluoromethylphenylmethoxy); heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl, methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto; sulfanyl groups, such as methylsulfanyl, ethylsulfanyl and propylsulfanyl; cyano; amino groups, such as amino, methylamino, dimethylamino, ethylamino, and diethylamino; and carboxyl. Where multiple substituent moieties are disclosed or claimed, the substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or plurally. By independently substituted, it is meant that the (two or more) substituents can be the same or different.
It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure result.
In choosing the compounds of the present invention, one of ordinary skill in the art will recognize that the various substituents, i.e. R1, R2, etc. are to be chosen in conformity with well-known principles of chemical structure connectivity.
The various R groups attached to the aromatic rings of the compounds disclosed herein may be added to the rings by standard procedures, for example those set forth in Advanced Organic Chemistry: Part B: Reaction and Synthesis, Francis Carey and Richard Sundberg, (Springer) 5th ed. Edition. (2007), the content of which is hereby incorporated by reference.
The compounds used in the method of the present invention may be prepared by techniques well known in organic synthesis and familiar to a practitioner ordinarily skilled in the art. However, these may not be the only means by which to synthesize or obtain the desired compounds.
The compounds used in the method of the present invention may be prepared by techniques described in Vogel's Textbook of Practical Organic Chemistry, A. I. Vogel, A. R. Tatchell, B. S. Furnis, A. J. Hannaford, P. W. G. Smith, (Prentice Hall) 5th Edition (1996), March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Michael B. Smith, Jerry March, (Wiley-Interscience) 5th Edition (2007), and references therein, which are incorporated by reference herein. However, these may not be the only means by which to synthesize or obtain the desired compounds.
Another aspect of the invention comprises a compound used in the method of the present invention as a pharmaceutical composition.
In some embodiments, the present invention provides a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable carrier.
As used herein, the term “pharmaceutically active agent” means any substance or compound suitable for administration to a subject and furnishes biological activity or other direct effect in the treatment, cure, mitigation, diagnosis, or prevention of disease, or affects the structure or any function of the subject. Pharmaceutically active agents include, but are not limited to, substances and compounds described in the Physicians' Desk Reference (PDR Network, LLC; 64th edition; Nov. 15, 2009) and “Approved Drug Products with Therapeutic Equivalence Evaluations” (U.S. Department Of Health And Human Services, 30th edition, 2010), which are hereby incorporated by reference. Pharmaceutically active agents which have pendant carboxylic acid groups may be modified in accordance with the present invention using standard esterification reactions and methods readily available and known to those having ordinary skill in the art of chemical synthesis. Where a pharmaceutically active agent does not possess a carboxylic acid group, the ordinarily skilled artisan will be able to design and incorporate a carboxylic acid group into the pharmaceutically active agent where esterification may subsequently be carried out so long as the modification does not interfere with the pharmaceutically active agent's biological activity or effect.
The compounds used in the method of the present invention may be in a salt form. As used herein, a “salt” is a salt of the instant compounds which has been modified by making acid or base salts of the compounds. In the case of compounds used to treat an infection or disease caused by a pathogen, the salt is pharmaceutically acceptable. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols. The salts can be made using an organic or inorganic acid. Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like. Phenolate salts are the alkaline earth metal salts, sodium, potassium or lithium. The term “pharmaceutically acceptable salt” in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).
As used herein, “treating” means preventing, slowing, halting, or reversing the progression of a disease or infection. Treating may also mean improving one or more symptoms of a disease or infection.
The compounds used in the method of the present invention may be administered in various forms, including those detailed herein. The treatment with the compound may be a component of a combination therapy or an adjunct therapy, i.e. the subject or patient in need of the drug is treated or given another drug for the disease in conjunction with one or more of the instant compounds. This combination therapy can be sequential therapy where the patient is treated first with one drug and then the other or the two drugs are given simultaneously. These can be administered independently by the same route or by two or more different routes of administration depending on the dosage forms employed.
As used herein, a “pharmaceutically acceptable carrier” is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the animal or human. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmaceutically acceptable carrier.
The dosage of the compounds administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of a specific chemotherapeutic agent and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.
A dosage unit of the compounds used in the method of the present invention may comprise a single compound or mixtures thereof with additional antibacterial agents. The compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection, topical application, or other methods, into or onto a site of infection, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.
The compounds used in the method of the present invention can be administered in admixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration. The compounds can be administered alone or mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. The active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
Techniques and compositions for making dosage forms useful in the present invention are described in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol. 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.
Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
The compounds used in the method of the present invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamallar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. The compounds may be administered as components of tissue-targeted emulsions.
The compounds used in the method of the present invention may also be coupled to soluble polymers as targetable drug carriers or as a prodrug. Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.
Gelatin capsules may contain the active ingredient compounds and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
For oral administration in liquid dosage form, the oral drug components are combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.
Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance. In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.
The compounds used in the method of the present invention may also be administered in intranasal form via use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.
Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
The compounds and compositions of the present invention can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by topical administration, injection or other methods, to the afflicted area, such as a wound, including ulcers of the skin, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.
Specific examples of pharmaceutical acceptable carriers and excipients that may be used to formulate oral dosage forms of the present invention are described in U.S. Pat. No. 3,903,297 to Robert, issued Sep. 2, 1975. Techniques and compositions for making dosage forms useful in the present invention are described-in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.
The term “prodrug” as used herein refers to any compound that when administered to a biological system generates the compound of the invention, as a result of spontaneous chemical reaction(s), enzyme catalyzed chemical reaction(s), photolysis, and/or metabolic chemical reaction(s). A prodrug is thus a covalently modified analog or latent form of a compound of the invention.
The active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, powders, and chewing gum; or in liquid dosage forms, such as elixirs, syrups, and suspensions, including, but not limited to, mouthwash and toothpaste. It can also be administered parentally, in sterile liquid dosage forms.
Solid dosage forms, such as capsules and tablets, may be enteric coated to prevent release of the active ingredient compounds before they reach the small intestine. Materials that may be used as enteric coatings include, but are not limited to, sugars, fatty acids, waxes, shellac, cellulose acetate phthalate (CAP), methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), and methyl methacrylate-methacrylic acid copolymers.
The compounds and compositions of the invention can be coated onto stents for temporary or permanent implantation into the cardiovascular system of a subject.
The compounds of the present invention can be synthesized according to general Schemes. Variations on the following general synthetic methods will be readily apparent to those of ordinary skill in the art and are deemed to be within the scope of the present invention.
Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention.
This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.
While the invention has been shown and described with reference to certain embodiments of the present invention thereof, it will be understood by those skilled in the art that various changes in from and details may be made therein without departing from the spirit and scope of the present invention and equivalents thereof.
All starting materials were purchased from Acros Organics, Alfa Aesar, Macrocyclics, Sigma Aldrich, or TCI America and used without further purification. Mass spectrometry: low-resolution electrospray ionization (ESI) mass spectrometry was carried out on an Agilent 1260 Infinity II HPLC with a G6125B single quadrupole mass detector. High-resolution (ESI) mass spectrometry was carried out at the Stony Brook University Center for Advanced Study of Drug Action (CASDA) mass spectrometry facility with an Agilent LC-UV-TOF spectrometers. Inductively coupled plasma spectroscopy (ICP) was performed on an Agilent Technologies ICP-OES (Model 5110). UV-VIS spectra were collected with the NanoDrop 1C instrument (AZY1706045). Spectra were recorded from 190 to 850 nm in a quartz cuvette with 1 cm path length. HPLC: Preparative HPLC was carried out using a Shimadzu HPLC-20AR equipped with a Binary Gradient, pump, UV-Vis detector, manual injector on a Phenomenex Luna C18 column (250 mm×21.2 mm, 100 A, AXIA packed). Method A (preparative purification method): A=0.1% TFA in water, B=0.1% TFA in MeCN. Gradient: 0-5 min: 5% B. 5-24 min: 5-95% B. RadioHPLC analysis was carried out using a Shimadzu HPLC-20AR equipped with a binary gradient, pump, UV-Vis detector, autoinjector and Laura radiodetector on a Phenomenex Luna C18 column (150 mm×3 mm, 100 A). Method B: A=0.1% TFA in water, B=0.1% TFA in MeCN with a flow rate of 0.8 mL/min, UV detection at 220 and 254 nm. Gradient 0-1 min: 5% B. 1-16 min 5-95% B. Compounds 1, PSMA-617, picaga tris t-butyl ester, and Lu-mpaten were synthesized according to previously published procedures. 177Lu was obtained from the DOE isotope program, produced at the University of Missouri reactor.
Picaga derivatives described herein are improved Sc chelators suitable for kit formulations. To be compatible with clinical radiopharmacies these formulations must exhibit accelerated room temperature complexation kinetics and enhanced apparent molar activity of ideally >0.1 Ci/μmol. The azamacrocycle-based chelator L2/m-phospaten is a phosphonate-containing chelator characterized by enhanced inner sphere crowding and a first deprotonation event occurring at lower pH which accelerates kon for lanthanides and Cu(II) (Anderson, C. J. & Ferdani, R. 2009). A drastic improvement of the achievable apparent molar activity (AMA) with 44Sc: at room temperature, AMA was improved to 0.4 Ci/μmol, which corresponds to an improvement of two orders of magnitude when compared to picaga (0.004 Ci/μmol) (Vaughn, B. A. et al. 2020) and a greater than 2-fold improvement compared to the AMA of DOTA at 80° C. (0.14 Ci/μmol) in comparative experiments (
Corresponding radiolabeling experiments with Lu-177 have revealed that L2 is indeed capable of chelating Lu-177 at room temperature. The concentration versus radiochemical yield plot in the dependence of reaction time is provided in
The diagnostic, positron-emitting isotope fluorine-18 is widely produced on a clinical scale. Sc-18F/177Lu represents a potential clinically viable theranostic pair. The development of Sc-18F radiochelation chemistry has the potential to mitigate long synthesis and purification protocols associated with covalent 18F radiolabeling.
Initial studies identified mpaten as a lead ligand for complexation with Sc-18F (
Complexation of picaga-DUPA conjugate with preformed Sc-18F complex was also achieved to form [Sc-18F(picaga-DUPA)]− (
The stability of [Sc-18F(picaga-DUPA)]− in both DPBS and saline was evaluated for up to 4 h. It was determined that [Sc-18F(picaga-DUPA)]− was stable in both DPBS and saline for at least 4 h (
To assess the in vivo behavior of [Sc-18F(picaga-DUPA)], the radiolabeled compound was administered to mice bearing PSMA+ and PSMA-tumor xenografts on the right and the left flank respectively. The results show that [Sc-18F(picaga-DUPA)]− exhibits high PSMA+tumor uptake, high in vivo stability and favorable biodistribution in normal tissues (
Preparative HPLC was carried out on a Phenomenex Luna C18 column (250 mm×21.2 mm, 100 Å, AXIA packed) at a flow rate of 15 mL/min using a Shimadzu HPLC-20AR equipped with a binary gradient pump, UV-vis detector, and manual injector. UV absorption was recorded at 254 nm.
Method A: Gradient: 0-1 min: 5% B; 1-14 min: 5-50% B; 14-23 min: 50-95% B; 23-26 min: 95% B; 26-27 min: 95-5% B; 27-30 min: 5% B.
Diethyl (6-(chloromethyl)pyridine-2-yl)phosphonate (2) was synthesized in two steps according to a previously published procedure) (Salaam et al. 2018).
A suspension of 1,4,7-triazacyclononane (1) (140.7 mg, 1.091 mmol), compound 2 (286.3 mg, 1.089 mmol) and K2CO3 (224.7 mg, 1.628 mmol) in acetonitrile (3 mL) was stirred at room temperature for 12 hours. The reaction mixture was filtered and the filtrated was concentrated in vacuo. The crude mixture was purified with reverse phase preparative HPLC (Method A) with the product eluting at 11.6 min. The fractions containing product were combined and the solvent was removed in vacuo to afford 3 as an amber oil (102.3 mg, 26% yield).
A suspension of 3 (102.3 mg, 0.2872 mmol), tert-butyl bromoacetate (128 μL, 0.817 mmol) and K2CO3 (200.7 mg, 1.454 mmol) in acetonitrile (7.5 mL) was stirred at room temperature for 12 hours. The reaction mixture was filtered and the filtrated was concentrated in vacuo. The resulting mixture was purified with reverse phase preparative HPLC (Method A) with the product eluting at 19.2 min. The fractions containing product were combined and the solvent was removed in vacuo to afford 4 as a yellow oil (49.3 mg, 29% yield).
6-{[4,7-Bis(carboxymethyl)-1,4,7-triazonan-1-yl]methyl}-2-pyridinephosphonate (L2), mphospatcn Compound 4 (25.5 mg, 0.0436 mmol) was dissolved in 6 M HCl (2 mL) and refluxed for 14 hours. The solvent was removed under vacuum to afford L2 as an off-white solid in HCl salt (14.7 mg, 81%) without further purification.
All HPLC purification and analytical methods were conducted using a binary solvent system in which solvent A was water+0.1% TFA and solvent B was MeCN+0.1% TFA. Preparative HPLC was carried out using a Shimadzu HPLC-20AR equipped with a binary gradient pump, UV-vis detector, and manual injector. UV absorption was recorded at 254 nm.
Method A: Phenomenex Luna C18 column (250 mm×21.2 mm, 100 Å, AXIA packed) at a flow rate of 15 mL/min. Gradient: 0-1 min: 5% B; 1-14 min: 5-50% B; 14-23 min: 50-95% B; 23-26 min: 95% B; 26-27 min: 95-5% B; 27-30 min: 5% B.
Method B: Phenomenex Luna C18 column (250 mm×10 mm, 100 Å, AXIA packed) at a flow rate of 5 mL/min. Gradient: 0-1 min: 5% B; 1-2 min: 5-20% B; 2-25 min: 20-30% B; 25-26 min: 30-95% B; 26-28 min: 95% B; 28-29 min: 95-5% B; 29-30 min: 5% B. Analytical HPLC was carried out on a Phenomenex Luna 5 μm C18 column (150 mm×3 mm, 100 Å, AXIA packed) at a flow rate of 0.8 mL/min using either a Shimadzu HPLC-20AR equipped with a binary gradient pump, UV-vis detector, autoinjector, and Laura radiodetector or Agilent 1260 Infinity II HPLC. UV absorption was recorded at 254 nm.
Method C: Gradient: 0-2 min: 5% B; 2-14 min: 5-95% B; 14-16 min: 95% B; 16-16.5 min: 95-5% B; 16.5-20 min 5% B.
Diethyl (6-(chloromethyl)pyridine-2-yl)phosphonate (1) was synthesized in two steps according to a previously published procedure (Salaam et al. 2018).
To a solution of(S)-2-(4-nitrobenzyl)-1,4,7-triazonane (30.0 mg, 0.1135 mmol) in dry acetonitrile was added K2CO3 (78.3 mg, 0.5675 mmol) to afford a suspension. The mixture was cooled to 0° C. in an ice-water bath and a solution of 2 (29.8 mg, 0.1135 mmol) in dry acetonitrile was added dropwise. Following stirring at room temperature for 12 hours, the reaction mixture was filtered, and the filtrate was concentrated in vacuo. This procedure afforded two of three possible structural isomers, where X=2, 3 or 5; X=5 is shown. Reverse phase preparative HPLC (Method B) enabled purification and separation of structural isomers 2a (6.9 mg, 0.0140 mmol) and 2b (7.4 mg, 0.0151 mmol) in a combined yield of 26%.
Di-tert-butyl 2,2′-(7-((6-(diethoxyphosphoryl)pyridin-2-yl)methyl)-X-(4-nitrobenzyl)-1,4,7-triazonane-1,4-diyl) (S)-diacetate (3a-b). X=2, 5, or 6. X=2 is shown.
A suspension of 2a (6.9 mg, 0.0140 mmol) or 2b (7.4 mg, 0.0151 mmol), tert-butyl bromoacetate (3.5 μL, 0.028 mmol or 4.0 μL, 0.030 mmol) and K2CO3 (12.7 mg, 0.092 mmol or 10.4 mg, 0.075 mmol) in dry acetonitrile was stirred at room temperature for 12 hours. Upon completion, the reaction mixture was filtered, and the filtrate was concentrated in vacuo. The crude product was purified with reverse phase preparative HPLC (Method B) to afford 3a (4.4 mg, 0.00612 mmol) in 44% yield and 3b (4.0 mg, 0.00556 mmol) in 37% yield.
(S)-2,2′-(X-(4-nitrobenzyl)-7-((6-phosphonopyridin-2-yl)methyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (4a-b). X=2, 5, or 6. X=2 is shown.
Compound 3a (4.4 mg, 0.00612 mmol) and 3b (4.0 mg, 0.00556 mmol) were independently dissolved in 6 M HCl and refluxed for 14 hours. Solvent was removed under vacuum to afford 4a and 4b as off-white solids in HCl salt, respectively, and used without further purification.
All HPLC purification and analytical methods were conducted using a binary solvent system in which solvent A was water+0.1% TFA and solvent B was MeCN+0.1% TFA. Preparative HPLC was carried out using a Shimadzu HPLC-20AR equipped with a binary gradient pump, UV-vis detector, and manual injector. UV absorption was recorded at 254 nm.
Method A: Phenomenex Luna C18 column (250 mm×21.2 mm, 100 Å, AXIA packed) at a flow rate of 15 mL/min. Gradient: 0-1 min: 5% B; 1-14 min: 5-50% B; 14-23 min: 50-95% B; 23-26 min: 95% B; 26-27 min: 95-5% B; 27-30 min: 5% B. Analytical HPLC was carried out on a Phenomenex Luna 5 μm C18 column (150 mm×3 mm, 100 Å, AXIA packed) at a flow rate of 0.8 mL/min using either a Shimadzu HPLC-20AR equipped with a binary gradient pump, UV-vis detector, autoinjector, and Laura radiodetector or Agilent 1260 Infinity II HPLC. UV absorption was recorded at 254 nm.
Method B: Gradient: 0-2 min: 5% B; 2-14 min: 5-95% B; 14-16 min: 95% B; 16-16.5 min: 95-5% B; 16.5-20 min 5% B.
6-methylpicolinic acid (298.1 mg, 2.18 mmol) was dissolved in dichloromethane (15 mL), followed by addition of tert-butyl 2,2,2-trichloroacetimidate (783 μL, 4.38 mmol) and boron trifluoride, BF3·OEt2 (43 μL) The reaction mixture was stirred overnight at room temperature and monitored via TLC (90:10 DCM: MeOH, UV visualized on silica plates). Upon completion, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting solid was resuspended in hexanes, filtered and the solid was washed with hexanes (3×10 mL). The crude product was purified via column chromatography (silica solid phase, 0-5% MeOH in DCM mobile phase) to afford the product as a yellow oil in a 32% yield.
Compound 1 (133.7 mg, 0.69 mmol) was dissolved in 2 mL carbon tetrachloride to afford a yellow solution. N-bromosuccinimide (98.5 mg, 0.8 eq, 0.55 mmol) and benzoyl peroxide (502.76 mg, 2.08 mmol based on 75% w/w in water) were added and the reaction mixture was heated to reflux for 4 hours. The reaction was monitored via TLC (mobile phase: 9:1 hexane:ethyl acetate, UV visualized). The crude mixture was filtered, and the filtrate concentrated to dryness. Needle-like crystals were resuspended in DCM, filtered, dried, and purified via column chromatography (silica solid phase, 0-25% ethyl acetate in hexane).
1,4,7-triazacyclononane (TACN, 129.4 mg, 1.00 mmol) was dissolved in chloroform (30 mL), followed by the addition of triethyl amine (279 μL, 2.00 mmol). A solution of di-tert-butyl dicarbonate (460 μL, 2.00 mmol) in chloroform (9 mL) was added dropwise to the reaction mixture to afford a final TACN concentration of 0.02 M. The solution was stirred at room temperature for 2 hours. Upon completion, the solvent was removed under reduced pressure and the resulting residue was partitioned between 10% NaOH (10 mL) and diethyl ether (30 mL). The organic layer was then sequentially washed with 10% NaOH (4×10 mL) and water (3×10 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to afford a transparent yellow oil in a 78% yield (Chong, et al. 2018).
Compound 3 (135 mg, 0.41 mmol) was dissolved in dry acetonitrile, followed by addition of oven dried K2CO3 (185 mg, 1.34 mmol) to afford a suspension. Ethyl bromoacetate (49.6 μL, 0.45 mmol) was added, and the reaction was allowed to stir overnight at room temperature. The reaction mixture was filtered and concentrated to afford a yellow oil (crude yield=84%).
Compound 4 (143.2 mg, 0.34 mmol) was dissolved in 20% trifluoroacetic acid in dichloromethane (4 mL) and allowed to stir at room temperature for 2 hours. Following completion, the reaction mixture was concentrated under reduced pressure.
Compound 5 (41.7 mg, 0.19 mmol) was dissolved in dry acetonitrile (5 mL), followed by addition of oven dried K2CO3 (130.8 mg, 0.95 mmol) to afford a suspension. The reaction mixture was cooled to 0° C. over an ice-water bath, followed by dropwise addition of compound 2 in acetonitrile (2 mL). The reaction mixture was stirred at room temperature overnight. Upon completion, the reaction was filtered, concentrated and HPLC purified (Method A). The product was afforded as red/brown oil in 45% yield following lyophilization.
To a solution of compound 6 (26 mg, 0.064 mmol) in 6 M trace metal grade HCl (1.65 mL, 0.04 M) was added phosphorous acid (64 μL, 1.28 mmol) and the reaction mixture heated to reflux. Paraformaldehyde (8.7 mg, 0.29 mmol) was added at the reaction stirred overnight. The reaction mixture was filtered, concentrated, and HPLC purified (Method A). The desired product was obtained in a 29% yield.
(2S)-2-({[(2S)-1,5-bis(tert-butoxy)-1,5-dioxopentan-2-yl]carbamoyl}amino)-6-[(2R)-3-(naphthalen-2-yl)-2-{[(1r,4r)-4-{[(2S)-6-amino-2-{[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl|amino}hexanamido]methyl}cyclohexyl]formamido}propanamido]hexan oic acid (2). Compound 1 on 2-chlorotrityl resin beads (0.212 mmol, 1.0 eq) and HBTU (0.241 g, 0.636, 3.0 eq) were dissolved in DMF (6 mL), DIPEA (0.137 g, 1.06 mmol, 5.0 eq) was added. Dde-Lys (Fmoc)-OH (0.169 g, 0.318 mmol, 1.5 eq) was added and reaction mixture was stirred overnight at room temperature. Resin beads were washed four times with dichloromethane (5.0 mL) and four times with DMF (5.0 mL). Fmoc protecting group was removed by shaking resin beads in 20% piperidine in DMF for 45 minutes (5.0 mL). Resin beads were washed four times with DMF (5.0 mL). 2 was cleaved from the resin in a 50% TFA: DCM mixture (1.0 mL). Supernatant was transferred to a vial to afford tert-butyl deprotected 2 in solution. Calculated monoisotopic mass for afford tert-butyl deprotected 2 (C49H69N7O12): 947.50; found m/z=948.6 [M+H]+.
(2S)-2-({[(2S)-1,5-bis(tert-butoxy)-1,5-dioxopentan-2-yl]carbamoyl}amino)-6-[(2R)-3-(naphthalen-2-yl)-2-{[(1r,4r)-4-{[(2S)-2-amino-6-[4-(4-iodophenyl)butanamido]hexanamido]methyl}cyclohexyl]formamido}propanamido]hexanoic acid (3). Compound 2 on 2-chlorotrityl resin beads (0.212 mmol, 1.0 eq) and HBTU (0.241 g, 0.636, 3.0 eq) were dissolved in DMF (6 mL), DIPEA (0.137 g, 1.06 mmol, 5.0 eq) was added. 4-(p-Iodophenyl) butyric acid (0.0923 g, 0.318 mmol, 1.5 eq) was added and reaction mixture was stirred overnight at room temperature. Resin beads were washed four times with dichloromethane (5.0 mL) and four times with dimethylformamide (5.0 mL). Dde-protecting group was removed by shaking resin beads in 2% hydrazine in DMF for 1 hour. Resin beads were washed four times with DMF (5.0 mL). 3 was cleaved from the resin in a 1% TFA: DCM mixture (1.0 mL). Supernatant was transferred to a vial to afford 3 in solution. Calculated monoisotopic mass for 3 (C57H82IN7O11): 1167.51; found m/z=1168.5 [M+H]+.
(2S)-2-({[(2S)-1,5-bis(tert-butoxy)-1,5-dioxopentan-2-yl]carbamoyl}amino)-6-[(2R)-3-(naphthalen-2-yl)-2-{[(1r,4r)-4-{[(2S)-2-[(4R)-5-(tert-butoxy)-4-{4-[2-(tert-butoxy)-2-oxoethyl]-7-({6-[(tertbutoxy)carbonyl]pyridin-2-yl}methyl)-1,4,7-triazonan-1-yl}-5-oxopentanamido]-6-[4-(4-iodophenyl)butanamido]hexanamido]methyl}cyclohexyl]formamido}propanamido]hexanoic acid (4). Compound 3 2-chlorotrityl resin beads (0.086 mmol, 1.0 eq) and HBTU (0.098 g, 0.258, 3.0 eq) were dissolved in DMF (1 mL), DIPEA (0.055 g, 0.430 mmol, 5.0 eq) was added. Ditert-butyl 2-{3-[(R)-4-(5-aminopentylamino)-4-oxo-1-tert-butoxycarbonylbutyl]ureido}glutarate (0.086 g, 0.130 mmol, 1.5 eq) was added and reaction mixture was stirred overnight at room temperature. Resin beads were washed four times with dichloromethane (5.0 mL) and four times with dimethylformamide (5.0 mL). 4 was cleaved from the resin in a 1% TFA: DCM mixture (1.0 mL). Supernatant was transferred to a vial and solvent was removed in vacuo. Product was purified by semi-preparative HPLC (Method A) to afford 4 (0.0085 g, 0.005 mmol, 6%) as an off-white solid. Calculated monoisotopic mass for 4 (C89H132IN11O18): 1769.88; found: m/z=1770.8858 [M+H]+, 886.4491 [M+2H]2+.
(2S)-2-({[(1S)-1-carboxy-5-[(2R)-3-(naphthalen-2-yl)-2-{[(1r,4r)-4-{[(2S)-2-[(4R)-4-carboxy-4-[4-(carboxymethyl)-7-[(6-carboxypyridin-2-yl)methyl]-1,4,7-triazonan-1-yl]butanamido]-6-[4-(4-iodophenyl)butanamido]hexanamido]methyl}cyclohexyl]formamido}propanamido]pentyl]carbamo yl}amino) pentanedioic acid, picaga-HSA (5). Compound 4 (0.007 g, 0.004 mmol, 1 eq) was dissolved into a solution of 2:1 TFA and DCM (0.3 mL). The reaction mixture was stirred overnight at room temperature. Solvent was removed in vacuo and 5 isolated as an off-white solid (0.0064 g, 0.004 mmol, 86%). Calculated monoisotopic mass for 5 (C69H92IN11O18): 1489.57; found: m/z=1490.6 [M+H]+, 745.9 [M+2H]2+.
To determine the concentration and molar absorptivity of picaga-HSA and PSMA-617, a spectrophotometric titration was carried out with Cu2+. The formation of [Cu(picaga-HSA)]− or [Cu(PSMA-617)]− was monitored at 280 nm or 290 nm using a 1 cm path length cuvette and a NanoDrop spectrophotometer. The pH was adjusted to 5.5 using 0.25 M ammonium acetate buffer. 100 μM ligand stock solutions were titrated with addition of 98 μM Cu2+ aliquots (as determined by ICP-OES) to determine the concentration of ligand by equivalents of Cu2+. The titration endpoint was determined by the inflection point of the change to the absorbance intensity at 280 nm or 290 nm, diagnostic of complex formation, was detected. A standard curve from A standard curve from 0.005 to 0.07 mM picaga-HSA or 0.00049 to 0.16 mM PSMA-617 was measured at 277 nm and the slope was determined using simple linear regression in Graph Pad Prism. The molar absorptivity (ε) was calculated from the slope of the standard curve as ε=1603 M−1 cm−1 for picaga-HSA. The molar absorptivity (ε) was calculated from the slope of the standard curve as ε=4268 M−1 cm−1 for PSMA-617. (See
natLu-complexes were formed in a 0.40 M solution of ammonium acetate at pH 5.5 at 80° C. for 30 minutes. Complex formation was monitored and characterized by HPLC-MS as described below.
Lu-PSMA-617. H3PSMA-617 in deionized water (70 μL) and LuCl3·6H2O in deionized water (5 μL) was added to a 0.40 M solution of ammonium acetate at pH 5.5 (500 μL). The resulting solution was heated at 80° C. for 30 minutes. Complex formation was confirmed by mass spectrometry. Calculated monoisotopic mass for (C49H68LuN9O16): 1213.42; found: m/z=1214.4 [M+H], m/z=607.9 [M+2H]. Rt of 177Lu radiolabeled product on HPLC (Method B: gradient: A: H2O, 0.1% TFA, B: CH3CN. 5-100% B gradient 20 min): 7.15±0.2 minutes (n≥9).
Lu-(picaga)-HSA. H3(picaga)-DUPA in dimethylsulfoxide (100 μL) and LuCl3·6H2O in deionized water (5 μL) in was added to a 0.40 M solution of ammonium acetate at pH 5.5 (500 μL). The resulting solution was heated at 80° C. for 30 minutes. Complex formation was confirmed by mass spectrometry. Calculated monoisotopic mass for (C69H89ILuN11O18): 1661.48; found: m/z=1662.6 [M+H], m/z=831.8 [M+2H]. Rt of 177Lu radiolabeled product on HPLC (Method B: gradient: A: H2O, 0.1% TFA, B: CH3CN. 5-100% B gradient 20 min): 9.01±0.2 minutes (n≥9).
To determine the concentration and molar absorptivity of picaga-HSA and PSMA-617, a spectrophotometric titration was carried out with Cu2+. The formation of [Cu(picaga-HSA)]− or [Cu(PSMA-617)] was monitored at 280 nm or 290 nm using a 1 cm path length cuvette and a NanoDrop spectrophotometer. The pH was adjusted to 5.5 using 0.25 M ammonium acetate buffer. 100 μM ligand stock solutions were titrated with addition of 98 μM Cu2+ aliquots (as determined by ICP-OES) to determine the concentration of ligand by equivalents of Cu2+. The titration endpoint was determined by the inflection point of the change to the absorbance intensity at 280 nm or 290 nm, diagnostic of complex formation, was detected. A standard curve from 0.005 to 0.07 mM picaga-HSA or 0.00049 to 0.16 mM PSMA-617 was measured at 277 nm and the slope was determined using simple linear regression in Graph Pad Prism. The molar absorptivity (ε) was calculated from the slope of the standard curve as ε=1603 M−1 cm−1 for picaga-HSA. The molar absorptivity (ε) was calculated from the slope of the standard curve as ε=4268 M−1 cm−1 for PSMA-617.
Stock solutions of Lu-(picaga)-HSA in (DMSO: H2O, 1:5) were prepared and concentrations were determined by ICP-OES in accordance with previously published method. In brief, 12 concentrations ranging 1 mM-10 pM Lu (picaga)-HSA, and 1 mM-10 pM (DCFPyL) N-{[(1S)-1-carboxy-5-{[(6-fluoro-3-pyridinyl) carbonyl]amino}-pentyl]carbamoyl}-L-glutamic acid were used as cold displacers to 99mTc(CO)3-MIP-1427, which was synthesized according to literature procedure (Hillier, S. M. et al. 2013).
The Ki values were calculated using the equation below (Szabo, Z. et al. 2015), where the Ki value of DCFPyL was given as 1.1 nM. The Ki was determined by nonlinear regression analysis using GraphPad Prism software.
To measure HSA binding of the complexes, a 0.1 mM solution (determined by ICP-OES) of the corresponding natLu complex in 4.5% w/v HSA was prepared and pipetted into an Amicon Ultra-0.5 Centrifugal Filter Unit (50 KDa cutoff, Millipore, UFC500396). The mixture was incubated at 37° C. for 15 min and subsequently centrifuged at 12000 rpm for 10 min. Binding is determined by measurement of Lu content in the filtrate by ICP-OES and compared to non-specific binding to the filter in absence of HSA.
The general radiolabeling protocol was used to radiolabel H3(picaga)-HSA and H3PSMA-617. Ligand was dissolved in dimethylsulfoxide to produce a stock solution. 10 nmol (PSMA-617) or 20 nmol (picaga-HSA) in 0.03 mL of the stock solution was added to an Eppendorf tube and 0.07 mL of 0.4 M ammonium acetate pH 5.5 was added to the solution. 177Lu was received in 0.05 M HCl (63 MBq) Activity was added to ligand solution and tube was placed in a heating block at 80° C. Tubes were intermittently vortexed and 5 μL was removed after 30 minutes to check complexation by radio-HPLC. The radiolabeled compounds were diluted in sterile PBS pH 7.4 at an activity concentration of 37 MBq/mL ready for injection. Radio-HPLC traces are shown in
The stability for 177Lu-radiolabeled ligands was evaluated by radio-HPLC for radiolytic degradation and decomplexation at relevant time points concentrations for dose preparation, storage, and administration. (picaga)-HSA was radiolabeled as previously described at a specific activity of 0.08 mCi/nmol (3.0 MBq/nmol) and diluted in PBS. A 0.1 mL aliquot was removed, stored at room temperature, and tested for stability by radio-HPLC. The activity concentration at t=0 was 0.12 mCi in 0.1 mL.
177Lu-(picaga)-HSA
177Lu-PSMA-617
The distribution coefficient was determined by liquid-liquid extraction and phase separation by centrifugation in 1-octanol and phosphate-buffered saline at pH 7.4 (PBS). 10 μL of either 177Lu-PSMA-617 or 177Lu-(picaga)-HSA in 0.4 M NH4OAc buffer was added to 490 μL PBS pH 7.4 (5 μCi, 0.3 nmol) in polypropylene tubes. 500 μL of 1-octanol was added and each tube was vortexed for two minutes. Tubes were centrifuged at 1600 ref for 3 minutes to accelerate phase separation. 50 μL was removed from each layer and radioactivity was measured on a gamma counter (Perkin Elmer Wallac Wizard 1470). The distribution coefficients were calculated as the logarithm of the ratio of the counts per minute (cpm) measured in the organic phase (1-octanol) over the cpm measured in the aqueous phase (PBS pH 7.4).
177Lu-PSMA-617
177Lu-(picaga)-HSA
The binding and internalization of 177Lu-(picaga)-HSA and 177Lu-PSMA-617 was evaluated in PSMA+PC3 PIP cells. In brief, 5×105 PC3 PiP cells are suspended, washed and aliquoted. The internalized fraction is determined using 177Lu-ligand in PC3 PiP cells, with ligands radiolabeled at a specific activity of 0.08 mCi/nmol (3.0 MBq/nmol) and diluted in PBS. Cells are incubated at 37° C. for 90 minutes with a 5 μCi aliquot of the 177Lu-ligand complex, followed by incubation with acidic stripping buffer (0.05 M glycine stripping buffer in 100 mM NaCl, pH 2.8) to remove surface-bound 177Lu-ligand. Subsequently, cell samples were lysed by addition of NaOH (1 M, 1 mL) to release cellular contents and internalized 177Lu-(picaga)-HSA. Radioactive counts are quantitated separately using a gamma well counter, with each incubation conducted in triplicate.
177Lu-(picaga)-
177Lu-PSMA-617
All animal experiments were conducted according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) at Stony Brook Medicine. Male NCr nude mice (6 weeks, Taconic Biosciences, Rensselaer, NY) were implanted subcutaneously on the right shoulder with 0.7-0.9×106 PC-3 PiP cells and on the left shoulder with 0.7-0.9×106 PC-3 flu cells suspended in Matrigel (1:2). When the tumors reached 50-100 mm3, the mice were anesthetized with isoflurane, and 1.7-2.2 MBq (45-60 μCi) of the tracer (0.4-0.9 nmol) was intravenously injected via tail vein catheter. At 4, 24 and 72 h, mice were sacrificed, and select organs were harvested. Radioactivity was counted by using a gamma counter, and the radioactivity associated with each organ was expressed as % ID/g. Biodistribution data were assessed by unpaired t-tests using GraphPad Prism to determine if differences between groups were statistically significant (p<0.05).
177Lu-(picaga)-HSA (n = 4)
177Lu-PSMA-617 (n = 4)
Whole body activity was measured by placing each mouse into a dose-calibrator. % Remaining activity was given by =[At/At=0] where At is the decay-corrected whole body activity at time t and At-0 is the total activity measured by dose calibrator p.i. The data were fit to a one-phase decay curve in GraphPad Prism and the biological decay constant K was calculated to be K=0.005638 h−1 for 177Lu-(picaga)-HSA and K=0.1415 h−1 for 177Lu-PSMA-617. The biological half-life is t1/2=122.9 h for 177Lu-(picaga)-HSA and 4.9 h for 177Lu-PSMA-617.
177Lu-(picaga)-HSA (%
177Lu-PSMA-617 (%
Twelve-week-old male mice were inoculated subcutaneously on the right shoulder with PSMA+PC-3 PIP cells (0.7×106/mouse in 1:2 DPBS pH 7.4: Matrigel). Tumors grew nine days before treatment with an approximate volume of 100 mm3 at day 0. Three groups of mice (n=6) (cohort A, B, and C) with statistically similar body weights and tumor volumes were injected at day 0 of the therapy study. Group A received saline only. Group B and C received 3.7 MBq of the radioligand via tail vein injection (177Lu-PSMA-617 or 177Lu-(picaga)-HSA respectively). Following administration of radioligand or vehicle, the mice were monitored by measuring the tumor size and body weight over 60 days. Mice were euthanized when the predefined end point criteria were reached, or when the study was terminated at day 60. The relative body weight (RBW) was defined as [BWx/BW0], where BWx is the body weight in grams at a given day x and BW0 is the body weight in grams on day 0. The tumor dimension was determined by measuring the longest tumor axis (L) and its perpendicular axis (W) with a digital caliper. The tumor volume (V) was calculated according to the equation [V=0.5×(LW2)]. The relative tumor volume (RTV) was defined as [TVx/TV0], where TVx is the tumor volume in mm3 at a given day x, and TV0 is the tumor volume in mm3 at day 0 (See
Twelve-week-old male mice were inoculated subcutaneously on the right shoulder with PSMA+PC-3 PIP cells (0.7×105/mouse in 1:1 DPBS pH 7.4: Matrigel). Tumors grew nine days before treatment with an approximate volume of 100 mm3 at day 0. One group of mice (n=4) (cohort D) with statistically similar body weights and tumor volumes were injected at day 0 of the therapy study. Group D received 3.3 MBq of the radioligand via tail vein injection (177Lu-picaga-DUPA). Following administration of radioligand, the mice were monitored by measuring the tumor size and body weight over 14 days. Mice were euthanized when the predefined end point criteria were reached, or when the study was terminated at day 14. The relative body weight (RBW) was defined as [BWx/BW0], where BWx is the body weight in grams at a given day x and BW0 is the body weight in grams on day 0. The tumor dimension was determined by measuring the longest tumor axis (L) and its perpendicular axis (W) with a digital caliper. The tumor volume (V) was calculated according to the equation [V=0.5×(LW2)]. The relative tumor volume (RTV) was defined as [TVx/TV0], where TVz is the tumor volume in mm3 at a given day x, and TV0 is the tumor volume in mm3 at day 0.
SPECT experiments were performed on select mice in the therapy cohorts. Scans were acquired at 4, 24, and 72 h post injection (p.i.) using a γ-Eye benchtop imaging system (BIOEMTECH, Athens, Greece). The reconstruction of SPECT data was performed using Visual-Eyes software (BIOEMTECH, Athens, Greece). Region of interest (ROI) analyses and post-processing on all images were performed using AMIDE.
Upon the conclusion of the therapy study all surviving mice and two naive mice were sacrificed and a kidney was harvested for histopathology. H&E stain was performed, and slides were evaluated by a certified pathologist blinded to the study (
All starting materials were purchased from commercial sources and used without further purification. All water used throughout radiochemistry and cold experiments was LCMS (trace metal) grade, including in the preparation of acids, bases, buffers, and stock solutions (1M HCl, 1M NaOH, 0.4M KHCO3, 0.25 M ammonium acetate (pH 4.1) buffer, ligand, and metal stock solutions).
NMR spectra (1H, 19F, 45Sc) were collected on a 400 MHz III Bruker instrument at 25° C. and processed using TopSpin 4.0.9. Chemical shifts are reported as parts per million (ppm). Liquid chromatography-mass spectrometry (LC-MS) was carried out on a Phenomenex Luna 5 μm C18 column (150 mm×3 mm, 100 Å, AXIA packed) at a flow rate of 0.8 mL/min using a single quadrupole Agilent 1200 Infinity II LC/MSD system equipped with a binary gradient pump, UV-vis detector, automatic injector, and an atmospheric pressure electrospray ionization (API-ES) source. UV absorption was recorded at 254 nm, and positive and negative mass spectra were collected from m/z 100-800. Gradient: 0-3 min: 5% B; 3-10 min: 5-95% B; 10-13 min: 95% B; 13-13.5 min: 95-5% B; 13.5-16 min: 5% B.
Method A: Binary solvent system (A: water+0.1% TFA; B: MeCN+0.1% TFA).
Method B: Binary solvent system (A: 10 mM ammonium acetate buffer, pH 5.6; B: MeCN). High resolution ESI mass spectrometry was carried out at the Stony Brook University Center for Advanced
Study of Drug Action (CASDA) with a Bruker Impact II UHR QTOF MS system. UV-VIS spectra were collected with the NanoDrop 1C instrument (AZY1706045). Spectra were recorded from 190 to 850 nm in a quartz cuvette with 1 cm path length. ICP-OES was carried out using an Agilent 5110 inductively coupled plasma optical emission spectrometer. A 6-point standard curve with respect to scandium or copper was used and fits were found to be at least R2 of 0.999. Concentrations were back calculated to determine the stock solution concentration.
All analytical HPLC methods were carried out using a Shimadzu HPLC-20AR equipped with a binary gradient pump, UV-vis detector, autoinjector, and Laura radiodetector. UV absorption was recorded at 254 nm. Gradient: 0-2 min: 5% B; 2-14 min: 5-95% B; 14-16 min: 95% B; 16-16.5 min: 95-5% B; 16.5-20 min 5% B.
Method C: Binary solvent system (A: water+0.1% TFA; B: MeCN+0.1% TFA). Phenomenex Luna 5 μm C18 column (150 mm×3 mm, 100 Å, AXIA packed) at a flow rate of 0.8 mL/min.
Method D: Binary solvent system (A: 10 mM sodium acetate buffer, pH 5.5; B: MeCN). Phenomenex Luna 10 μm C18 column (250 mm×4 mm, 100 Å, AXIA packed) at a flow rate of 0.5 mL/min.
The ligand 6-{[4,7-Bis(carboxymethyl)-1,4,7-triazonan-1-yl]methyl}-2-pyridinecarboxylic acid (4), H3mpaten, and the functionalized derivative 2-{3-[(R)-1-Carboxy-4-[5-(4-carboxy-4-{7-(carboxymethyl)-4-[(6-carboxy-2-pyridyl)methyl]-1,4,7-triazonan-1-yl}butyrylamino)pentyl amino]-4-oxobutyl]ureido}glutaric acid, picaga-DUPA, were synthesized according to previously reported procedures (Vaughn et al. 2020).
To a solution of H3mpaten (4.2 μmol) dissolved in water, 1 equivalent of ScCl3 salt was added. The pH was adjusted to 4.1 with 0.25 M ammonium acetate buffer. The reaction mixture was heated to 80° C. for 30 minutes, then checked via LC-MS to ensure complete complexation. Following addition of 5 equivalents NH4F, the reaction mixture was heated to 100° C. for 30 minutes. Fluorination of Sc(mpatcn) was monitored via LC-MS (Method A), and the reaction mixture was purified via analytical HPLC (Method D). The purified complex was concentrated under reduced pressure and dissolved in 450 μL 0.25 M ammonium acetate (pH 4.1) spiked with 50 μL D2O for 1H, 19F and 45Sc NMR studies. (See
1H chemical shift analysis of H3mpatcn (pH 4.5), Sc(mpatcn) (pH 4-6)
19F and 45Sc chemical shift analysis of internal chemical shift standards,
To determine the concentration of the picaga-DUPA and Hampaten samples used for radiolabeling experiments, spectrophotometric titrations were carried out with Cu2+. The formation of [Cu (mpaten)] or [Cu(picaga)-DUPA] was monitored at 300 nm using a 1 cm path length cuvette and a NanoDrop spectrophotometer. The pH was adjusted to 5.5 using 10 mM sodium acetate buffer. For H3mpaten, a 1.67 mM ligand stock solution (100.6 μL) was titrated with addition of 10 μL (9.9 nmol) Cu2+ aliquots (as determined by ICP-OES) to determine the concentration of ligand by equivalents of Cu2+. Due to limited sample availability, a 0.16 mM picaga-DUPA stock solution (97.3 μL) was titrated with addition of 10 μL (0.98 nmol) Cu2+ aliquots. The titration endpoint was determined when no further change to the absorbance intensity at 300 nm, diagnostic of complex formation, was detected. Different batches of H3mpaten were used for the various radiolabeling experiments. The concentration of each batch was determined using this method, and a representative titration is shown below. Analysis of the H3mpaten and picaga-DUPA samples reveals 49.7% and 79.8% w/w content of ligand in TFA salt following deprotection, respectively. (See
[18F]NaF was received from NCM USA (Bronx, NY) in 50 mCi batches. A Sep-Pak Waters QME cartridge was primed with 9 mL water, the received [18F]NaF solution was loaded onto the cartridge, washed with 9 mL water, then eluted with 500 μL 0.4 M potassium bicarbonate. The eluate was adjusted to pH 2.5 with 1M HCl to afford a [18F]KF stock solution (total volume 638-693 μL).
Inspired by previous success with pre-forming the [Al-18F]2+ complex prior to ligand addition, this approach was applied to [Sc-18F]2+ precursor (McBride et al. 2009). To do so, 0.615-1.023 [18F]KF stock was added to 200 μL 0.25 M ammonium acetate (pH 4.1) buffer, followed by addition of 0.2 equivalents of ScCl3 relative to the amount of ligand added in the next step. Following incubation at room temperature for 10 minutes, the appropriate amount of ligand was added (5-800 nmol) and the mixture was incubated at the specified temperature (40-100° C.) for the specified duration (30-45 minutes) prior to radioHPLC analysis (Method C). Total reaction volumes ranged from 270-480 μL.
Concentration-dependent Radiolabeling was shown in
Based on temperature- and concentration-dependent radiolabeling with mpaten, optimized radiolabeling conditions were determined. Incubate 10 nmol ScCl3 (0.2 eq.) with desired amount of [18F]KF stock at room temperature for 10 minutes in 0.25 M ammonium acetate (pH 4.1) buffer. Add 50 nmol ligand (1 eq.), then heat for 30 minutes at 60-100° C. Purify via radioHPLC (Method C), concentrate to dryness under reduced pressure, and formulate to desired volume in DPBS. Total time to formulation: 70 minutes. Rapid alternative purification methods are under investigation to further reduce this time.
The [18F]KF stock solution was prepared as previously described (39.4 mCi, 693 μL, pH 2.5). The Sc-18F precursor was formed via direct addition of 200 μL 0.25 M ammonium acetate (pH 4.1) buffer and 20 nmol ScCl3 stock (0.25 eq.) to the [18F]KF stock. Following incubation at room temperature for 10 minutes, 79.8 nmol picaga-DUPA (1 eq.) was added to 400 μL of the Sc-18F precursor stock solution (13.11 mCi), then incubated at 100° C. for 25 minutes. The major [18F]Sc—F(picaga)-DUPA species was separated the minor isomer and Sc-18F via analytical radioHPLC (Method C). (
All animal experiments and procedures were performed in accordance with the National Institute of Health's “Guide for the Care and Use of Laboratory Animals” and approved by Institutional Animal Care and Use Committee (IACUC) at Stony Brook Medicine.
Eight-week-old male mice were inoculated subcutaneously on the right shoulder with 1.0×106 PSMA (+) PC3 PIP cells in 1:1 DPBS pH 7.4: Matrigel or on the left shoulder with 1.0×106 PSMA (−) PC3 flu cells in 1:1 DPBS pH 7.4: Matrigel. At day 9 post-xenograft when tumors reached a suitable size, animals were administered [18F]Sc—F(picaga)-DUPA (206-273 μCi in 80 μL DPBS) via tail-vein injection. Mice were imaged at 90 min post injection (p.i.) using Siemens Inveon PET/CT Multimodality System, and image analysis was conducted using AMIDE. Upon completion of imaging at 120 min p.i., mice were sacrificed, select organs were harvested, and radioactivity was counted using a gamma counter. Counts per minute (CPM) values were decay corrected, and the radioactivity associated with each organ was expressed as % injected dose per gram (% ID/g). The processed data is plotted alongside previously published 47Sc(picaga)-DUPA biodistribution data collected at 2 hours post-injection (n=4) for ease of direct comparison.
47Sc(picaga)-DUPA data is reproduced from previous
47Sc(picaga)-DUPA
A solid phase synthesis approach was used for the construction of the trifunctional conjugate in lieu of the previously low yielding solution phase synthesis of picaga-DUPA (Scheme 1) (Umbricht, C. A. et al. 2018). This provided means to synthesize a more complex targeting scaffold than the first-generation molecule, while eliminating time-consuming, reverse-phase chromatography purification steps and significantly increasing over-all yields; an important aspect of relevance for scale-ups required for future clinical translation. The Glu-urea-Lys targeting moiety was synthesized according to a procedure previously described (Umbricht, C. A. et al. 2018). Similarly, PSMA-617 was also constructed on solid phase and synthesized as a reference compound of clinical relevance based on the approach previously reported (Eder, M. et al. 2012; Benesova, M. et al. 2015).
Picaga-HSA was synthesized from the resin-immobilized glu-urea-lys targeting moiety (Scheme 4). Dde-Lys (Fmoc) was activated with HBTU and coupled to the targeting moiety in the presence of diisopropylethyamine (DIPEA) and dimethylformamide (DMF). The Fmoc-protecting group was cleaved from the Dde-protected lysine in 20% piperidine in DMF. 4-(p-iodophenyl) butyric acid was coupled to the lysine via HBTU activation. Cleavage of the Dde-protecting group was accomplished in 2% hydrazine in DMF. The conjugation was performed with picaga by HBTU coupling in DMF. The tert-butyl protected chelator was cleaved from the resin in 1% TFA: DCM and then deprotected in 2:1 TFA: DCM overnight. Picaga-HSA was isolated following resin-cleavage in 0.6% overall non-optimized yield (0.0064 g) in 10 steps.
Prior to the assessment of the 177Lu-labeled compounds, binding affinity to the biological target PSMA was evaluated. The affinity (Ki) of natLu-(picaga)-HSA to the PSMA target was determined to be 1.4±0.6 nM by using a previously established displacement assay with the clinically investigated tracer 99mTc-MIP-1427 and non-radioactive, fluorinated small molecule DCFPyL as an internal reference (
Interaction with natLu-(picaga)-HSA and natLu-PSMA-617 to human serum albumin was evaluated to affirm that the iodophenyl-butyrate significantly enhanced the binding to serum albumin. To this end, each ligand was incubated at 37° C. for 15 minutes in PBS with or without 4.5% human serum albumin to simulate in vivo protein concentration. Each mixture was filtered through a 50 KDa MW cutoff filter and the natLu content of the filtrate was measured by ICP-OES. natLu-mpaten was used as a chelator-only control with no targeting functionality and thus no expected interaction with human serum albumin. Lu-(picaga)-HSA had 80±3.3% binding to HSA compared to PBS while PSMA-617 had 55±1.2% binding compared to the PBS, in good agreement with literature reported values (Benesova, M. et al. 2018). There was no significant difference in the compound in the filtrate with or without HSA for Lu-mpaten (
Following synthesis and validation of picaga-HSA, radiolabeling with 177Lu was carried out. Limited solubility in saline necessitated the addition of dimethylsulfoxide (DMSO) to solubilize the conjugate, a strategy commonly employed for other serum albumin targeting constructs (Umbricht, C. A. et al. 2018). As a consequence, the radiolabeling of picaga-HSA and PSMA-617 was performed in 30% DMSO. It was found that DMSO was necessary to solubilize the (picaga)-HSA ligand, but no precipitation or loss of labeled product was observed upon dilution of up to 0.5% DMSO in aqueous solution. At specific activities of (2.96 MBq/nmol), the construct labeled with a radiochemical yield (% RCY) of >99% as determined by radio-HPLC, requiring no further purification steps prior to dilution for injection (
Formulation stability was monitored, and no degradation was observed in the dose formulation solution at 4 h, 24 h, or 48 h in contrast with conjugates that exhibit radiolytic degradation (Table 1). When analyzed by radio-HPLC after 14 days, 177Lu-(picaga)-HSA was observed to be 74% intact. Under the same conditions, 177Lu-PSMA-617 was determined to be 80% intact. This indicates that at the relevant concentrations and time points, no significant degradation occurs.
When compared to the first-generation construct 177Lu-picaga-DUPA, the retention time of 177Lu-picaga-HSA and 177Lu-PSMA-617 indicated a significant difference in hydrophilicity as expected by the introduction of the HSA-binding moiety. Accordingly, the distribution coefficient in 1-octanol/PBS pH 7.4 (Log D7.4) revealed values of −2.11±0.03 for 177Lu-(picaga)-HSA and −2.71±0.08 for 177Lu-PSMA-617 (Table 2).
Cellular uptake and internalization of 177Lu-picaga-HSA and 177Lu-PSMA-617 was evaluated in PSMA±PC3 PIP cells (
To assess the in vivo behavior of the 177Lu-(picaga)-HSA and applicability to targeted therapy in comparison to 177Lu-PSMA-617, a 1.7-2.2 MBq (45-60 μCi) dose of the radiolabeled compound at 4.1 MBq/nmol for 177Lu-PSMA-617 and 2.0 MBq/nmol for 177Lu-(picaga)-HSA specific activity was administered to mice bearing PSMA+ (PC-3 PiP) and PSMA-(PC-3 Flu) tumor xenografts on the right and the left flank, respectively. Cohorts were sacrificed at 2, 24, and 72 hours post injection for biodistribution analysis (
To approximate clearance of the 177Lu-constructs, therapy cohort treated with 177Lu-(picaga)-HSA and 177Lu-PSMA-617 were monitored for residual whole-body activity remaining in the mouse. The whole-body activity remaining was plotted and the biological half-life in PSMA+tumor-bearing mice was determined using nonlinear regression in Graph Pad Prism (
A single dose radiotherapy study was conducted to evaluate therapeutic efficacy of 177Lu-(picaga)-HAS in a PSMA+ xenograft model in nude mice with a directly comparative study conducted with 177Lu-PSMA-617 (
Because the highest observed off-target uptake was observed in the kidney, H&E staining of kidneys of surviving mice in the therapy cohorts was performed and compared with non-treated animals of the same age. No abnormal histopathological changes were observed for any mice (
The results described herein show that 177Lu-(picaga)-HSA is a suitable candidate for further investigation toward translation for the management of prostate cancer using low dose radiotherapy and identifies picaga as a suitable chelator for theranostic applications.
Fluorine-18 is currently the most frequently and readily utilized PET isotope worldwide, for both diagnostic and research studies. The ease of production in large quantities using biomedical cyclotrons and automation of multi-step radiochemical syntheses with curie amounts of isotope has further streamlined and accelerated advancements in 18F-radiochemistry and de-novo tracer development, followed by subsequent preclinical and clinical translation (Fu et al. 2021 and Zheng et al. 2021). However, some shortcomings remain. Firstly, as conventional radiofluorination strategies involve C—F bond formation in anhydrous, aprotic solvents at high temperatures, the radiolabeling of thermally and chemically sensitive (bio) molecules remains inaccessible (Deng et al. 2019, Wright et al. 2021 and Sharninghausen et al. 2020). Previous attempts to address this issue involved the use of prosthetic group and the formation of B—F, Si—F and Al—F bonds. However, these strategies are hamstrung by production of low specific activity (B—F via isotope exchange reaction), incompatibility with aqueous solvent (Si—F) or high temperature reaction conditions and limited in vivo stability (Al—F) (Richter et al. 2015, Bernard-Gauthier et al. 2016, McBride et al. 2013 and Fersing et al. 2019). Secondly, as theranostic isotope pairs are becoming of imminent interest in nuclear medicine, the lack of a corresponding therapeutic partner to F-18 outlines a critical need (Hu et al. 2021). The Ga—F bond has been explored for direct radiofluorination, but while the Ga—F bond formation is feasible under forcing and low specific activity conditions, corresponding complexes are not sufficiently inert against defluorination in vitro and in vivo (Monzittu, et al. 2018, Koay et al. 2020, and Bhalla, et al. 2015). This is not surprising, considering the comparatively soft Lewis acid nature and kinetic lability of the Ga(III) ion. In contrast, Sc(III) exhibits greater chemical hardness and Lewis acidic character, thus it was considered as a viable alternative for the formation of ternary [18F][Sc(L)F] complexes sufficiently inert against radiodefluorination.
Previously, the chelation platform mpatcn/picaga (
Here, a rapid, aqueous in situ formation of Sc-18F coordination complex [18F][ScF(mpatcn)]− (
To evaluate the feasibility of the formation of and predict structural features of the [ScF (mpaten)] complex, a density functional theory calculation was employed (Frisch et al. 2009). Calculations were performed using the B3LYP(D3)-BJ functional with cc-PVDZ basis set and SMD solvation (Marenich et al., 2009). Complexes of mpaten were calculated starting from previously reported DFT structures (Vaughn et al. 2021). The [ScF(mpatcn)]− complex showed very similar overall bonding structure and geometry when compared with the parent aqua complex [Sc(mpatcn)(H2O)]. A significant lengthening of Sc—X bonds to all ligand donor atoms occurred by approximately 0.02-0.09 Å (Table 9). This is attributable to ionic repulsion caused by the negatively charged F and a significant reduction in the charge density at the metal center as compared to the weaker H2O ligand. Structures of the delta isomers of the Sc—F and Sc—H2O complexes of mpaten can be seen in
To assess the strength of the Sc—F bond relative to Al—F, the bond dissociation enthalpy (BDE) of relevant scandium complexes of mpaten were computed and compared to ternary aluminum complexes of N-benzyl-NODA. This chelator was chosen as a benchmark, as the corresponding [18F][AlF(N-benzyl-NODA)] complex has excellent PBS and serum stability, as well as low 18F decomplexation in-vivo (D'Souza et al. 2011). A summary of the BDE of relevant complexes can be seen in Tables 10 (a) and 10 (b). While for L=H2O, OH−, and F−, the Al-L BDE of the [Al(N-benzyl-NODA)]+ ternary complexes were higher than that of the corresponding Sc-L BDE of Sc(mpatcn) ternary complexes, it is important to note the competing species present in solution. The dominant species at physiological pH is the [Sc(mpatcn)(H2O)] complex, with the corresponding hydroxide species forming only above pH 9 (Vaughn, et al. 2021 and Vaughn, et al. 2020). This is further evidenced by comparing BDE values for [Sc(mpatcn)(H2O)]+ with [ScF(mpatcn)]−, with the Sc—F bond (229.22 kJ/mol) predicted to be nearly an order of magnitude stronger than the Sc—OH2 bond (27.15 kJ/mol). However, in the case of the [Al(N-benzyl-NODA)]+ complex, the hydroxide species [Al(OH)(N-benzyl-NODA)] is the dominant species observed above pH 5.0, thus it represents the main competing species to the [AlF(N-benzyl-NODA)]+ complex. With BDEs only exhibiting a 10 kJ/mol difference between these species, this likely contributes to the need for high-temperature radiolabeling conditions and use of organic solvents for the complex [18F][AlF(N-benzyl-NODA)]+, as well as reversible displacement with the OH bound species as the main competing defluorination process.
Supported by computational studies, [ScF(mpatcn)]− on macroscopic scale was produced using direct fluorination of the open coordination site of [Sc(mpatcn)(H2O)] with 5 equivalents of NH4F, followed by isolation of the desired species using reverse phase chromatographic separation. In addition to conventional 1H NMR, 19F and 45Sc-NMR spectroscopy was utilized to characterize the [ScF(mpatcn)]− ternary complex.
With solution chemical studies affirming the formation and aqueous stability of the desired species, the current application validates possible methods for the formation of the corresponding radiofluorinated species. The initial attempts to fluorinate the pre-formed [Sc(mpaten)(H2O)] complex in direct homology with the macroscopic fluorination strategy produced <5% product, possibly due to a lack of electrostatic interaction between the neutral aqua complex and the negatively charged F. In direct analogy with Al-18F chemistry, the in situ pre-formation of the [Sc-18F] species, followed immediately by the addition of the mpaten ligand produced the desired product at significantly increased radiochemical yields (>20%) at pH 4.5 and incubation at 60° C., without the need for any organic solvent additives. High radiofluorination yields are observed at 80° C. (62%) and 100° C. (89%) after incubation for 30 minutes.
Various aspects of these initial results indicated very significant differences to Al-18F radiochemistry; specifically, the formation of the desired radiochemical species at 60° C. using a macrocyclic chelator system, without the need for any organic solvent additive represent marked improvements; of note, the addition of ethanol to the radiolabeling reaction did not lead to significant changes in radiochemical yield. An apparent molar activity of 20 mCi/μmol was determined using high-temperature radiolabeling conditions, which is comparable to various targeted radiopharmaceuticals and motivated the validation of the Sc-18F approach in vivo as a logical next step.
A small-molecule peptide conjugate 44/47Sc(picaga)-DUPA as a preclinically validated theranostic pair was previously developed. The picaga-DUPA ligand would also efficiently produce the desired Sc-18F-complex species to form Sc-18F-(picaga)-DUPA. Indeed, using previously optimized radiolabeling conditions using the non-functionalized mpaten chelator, the desired complex forms readily. The target compound subsequently isolated using HPLC and formulated for injection in phosphate-buffered saline (PBS). Analysis of the formulated, purified Sc-18F-(picaga)-DUPA product demonstrated no detectable decomposition even after 4 hours (
In summary, it was shown for the first time that the formation of ternary natsc-18F complexes is not only feasible, but that these complexes are inert towards defluorination in vivo. Conclusively, Sc-18F complexes are ideally suited as an alternative to conventional C-18F bond formation or the use of large, lipoliphilic prosthetic groups to incorporate 18F using time-intensive and low-yielding radiochemical approaches. Furthermore, the demonstrated biologically homologous behavior of Sc-18F-ternary complex when directly compared with the corresponding 47Sc-complex renders the 18F/47Sc isotope pair an unusual, yet fully viable theranostic couple with prospective clinical utility.
Richter, S.; Wuest, M.; Bergman, C. N.; Way, J. D.; Krieger, S.; Rogers, B. E.; Wuest, F., Rerouting the metabolic pathway of 18F-labeled peptides: the influence of prosthetic groups. Bioconjugate Chem. 2015, 26 (2), 201-212.
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This application claims the benefit of U.S. Provisional Application No. 63/257,821, filed Oct. 20, 2021, the contents of which are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/078389 | 10/19/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63257821 | Oct 2021 | US |
