PSMA TARGETED CONJUGATE COMPOUNDS AND USES THEREOF
20250332265 ยท 2025-10-30
Inventors
Cpc classification
A61K49/0002
HUMAN NECESSITIES
A61K51/0485
HUMAN NECESSITIES
A61K47/55
HUMAN NECESSITIES
A61K47/64
HUMAN NECESSITIES
A61K47/542
HUMAN NECESSITIES
A61K47/65
HUMAN NECESSITIES
A61K41/0071
HUMAN NECESSITIES
C07B2200/05
CHEMISTRY; METALLURGY
A61K51/0497
HUMAN NECESSITIES
A61K41/0038
HUMAN NECESSITIES
International classification
Abstract
PSMA targeted conjugate compounds, pharmaceutical compositions comprising these compounds, methods for treating and detecting cancers in a subject, methods for identifying cancer cells in a sample are described herein.
Claims
1: A compound comprising the formula (I): ##STR00034## wherein: n.sup.1 is 1, 2, 3, or 4; Y.sup.1 and Y.sup.2 each independently include at least one detectable moiety, therapeutic agent, or a theranostic agent.
2: The compound of claim 1, wherein Y.sup.1 and Y.sup.2 include at least one radiolabel or radioisotope, radiosensitizer, chelating agent, photosensitizer, fluorescent labeling agent, magnetic resonance imaging (MRI) agent, or anti-cancer agent.
3-11. (canceled)
12: The compound of claim 2, wherein the anticancer agent is linked to the thio group or amine group via a protease cleavable or acid-labile linker.
13: The compound of claim 12, wherein the linker includes a maleimido-caproyl linker-valine-citrulline cleavable peptide-p-aminobenzyl carbamate spacer (MC-VC-PABC) protease cleavable linker.
14: The compound of any of claim 1, wherein at least one of Y.sup.1 or Y.sup.2 includes an anti-cancer agent and the other Y.sup.1 or Y.sup.2 includes a photosensitizer, radiosensitizer, or fluorescent imaging agent.
15: The compound of claim 1 selected from the following: ##STR00035## ##STR00036## or a pharmaceutically acceptable salt thereof.
16: A compound comprising the following formula: ##STR00037## optionally complexed with a metal, or a pharmaceutically acceptable salt thereof.
17: The compound of claim 16, wherein the metal ion includes at least one of .sup.186Re, .sup.188Re, .sup.99mTc, .sup.153Gd, .sup.111In, .sup.67Ga, .sup.68Ga, .sup.201TI, .sup.82Rb, .sup.64Cu, .sup.89Zr, .sup.90Y, .sup.177Lu, T(tritium), .sup.149Tb, .sup.161Tb, .sup.153Sm, .sup.89Sr, .sup.211At, .sup.225Ac, .sup.227Ac, .sup.123-125I, .sup.131I, .sup.67Cu, .sup.203Pb, .sup.212Pb, .sup.211Bi, .sup.213Bi, or .sup.227-233Th.
18: The compound of claim 1, not binding to aquaporin.
19: The compound of claim 1 having a selectivity for PSMA expressing cancer tissue versus non-PSMA expressing non-cancer tissue 5 times.
20: The compound of claim 1, upon administration to a subject in need thereof showing minimal accumulation or uptake in salivary glands of the subject.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0072] The following is a brief description of the drawings which are presented for the purpose of illustrating the invention and not for the purpose of limiting them.
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DETAILED DESCRIPTION
[0103] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the application.
[0104] The articles a and an are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, an element means one element or more than one element.
[0105] The terms comprise, comprising, include, including, have, and having are used in the inclusive, open sense, meaning that additional elements may be included. The terms such as, e.g., as used herein are non-limiting and are for illustrative purposes only. Including and including but not limited to are used interchangeably.
[0106] The term or as used herein should be understood to mean and/or unless the context clearly indicates otherwise.
[0107] The term agent is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.
[0108] The term sample can refer to a specimen or culture obtained from any source, as well as clinical, research, biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass cells, fluids, solids, tissues, and organs, and whole organisms.
[0109] The terms patient, subject, mammalian host, and the like are used interchangeably herein, and refer to humans and non-human animals (e.g., rodents, arthropods, insects, fish (e.g., zebrafish)), non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, or canines felines, aves, etc.).
[0110] The terms cancer or tumor refer to any neoplastic growth in a subject, including an initial tumor and any metastases. The cancer can be of the liquid or solid tumor type. Liquid tumors include tumors of hematological origin, including, e.g., myelomas (e.g., multiple myeloma), leukemias (e.g., Waldenstrom's syndrome, chronic lymphocytic leukemia, other leukemias), and lymphomas (e.g., B-cell lymphomas, non-Hodgkin's lymphoma). Solid tumors can originate in organs and include cancers of the lungs, brain, breasts, prostate, ovaries, colon, kidneys, and liver.
[0111] Terms cancer cell or tumor cell can refer to cells that divide at an abnormal (i.e., increased) rate. Cancer cells include, but are not limited to, carcinomas, such as squamous cell carcinoma, non-small cell carcinoma (e.g., non-small cell lung carcinoma), small cell carcinoma (e.g., small cell lung carcinoma), basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma, choriocarcinoma, semonoma, embryonal carcinoma, mammary carcinomas, gastrointestinal carcinoma, colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamous cell carcinoma of the neck and head region; sarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, synoviosarcoma and mesotheliosarcoma; hematologic cancers, such as myelomas, leukemias (e.g., acute myelogenous leukemia, chronic lymphocytic leukemia, granulocytic leukemia, monocytic leukemia, lymphocytic leukemia), lymphomas (e.g., follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, malignant lymphoma, plasmocytoma, reticulum cell sarcoma, or Hodgkin's disease), and tumors of the nervous system including glioma, glioblastoma multiform, meningoma, medulloblastoma, schwannoma and epidymoma.
[0112] The term polypeptide refers to a polymer composed of amino acid residues related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds or modified peptide bonds (i.e., peptide isosteres), related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof, glycosylated polypeptides, and all mimetic and peptidomimetic polypeptide forms. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The term can refer to an oligopeptide, peptide, polypeptide, or protein sequence, or to a fragment, portion, or subunit of any of these. The term protein typically refers to large polypeptides. The term peptide typically refers to short polypeptides.
[0113] A portion of a polypeptide or protein means at least about three sequential amino acid residues of the polypeptide. It is understood that a portion of a polypeptide may include every amino acid residue of the polypeptide.
[0114] Mutants, derivatives, and variants of a polypeptide (or of the DNA encoding the same) are polypeptides which may be modified or altered in one or more amino acids (or in one or more nucleotides) such that the peptide (or the nucleic acid) is not identical to the wild-type sequence, but has homology to the wild type polypeptide (or the nucleic acid).
[0115] A mutation of a polypeptide (or of the DNA encoding the same) is a modification or alteration of one or more amino acids (or in one or more nucleotides) such that the peptide (or nucleic acid) is not identical to the sequences recited herein, but has homology to the wild type polypeptide (or the nucleic acid).
[0116] Recombinant, as used herein, means that a protein is derived from a prokaryotic or eukaryotic expression system.
[0117] The term derivative refers to an amino acid residue chemically derivatized by reaction of a functional side group. Such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-benzylhistidine. Also included as derivatives are those amino acid residues, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids, such as non-standard amino acids.
[0118] PSMA refers to Prostate Specific Membrane Antigen, a potential carcinoma marker that has been hypothesized to serve as a target for imaging and cytotoxic treatment modalities for cancer.
[0119] The term small molecule can refer to lipids, carbohydrates, polynucleotides, polypeptides, or any other organic or inorganic molecules.
[0120] The term imaging probe can refer to a biological or chemical moiety that may be used to detect, image, and/or monitor the presence and/or progression of a cell cycle, cell function/physiology, condition, pathological disorder and/or disease.
[0121] The terms treating or treatment of a disease can refer to executing a treatment protocol to eradicate at least one diseased cell. Thus, treating or treatment does not require complete eradication of diseased cells.
[0122] The term nanoparticle refers to any particle having a diameter of less than 1000 nanometers (nm). In some embodiments, nanoparticles can be optically or magnetically detectable. In some embodiments, intrinsically fluorescent or luminescent nanoparticles, nanoparticles that comprise fluorescent or luminescent moieties, plasmon resonant nanoparticles, and magnetic nanoparticles are among the detectable nanoparticles that are used in various embodiments. In general, the nanoparticles should have dimensions small enough to allow their uptake by eukaryotic cells. Typically, the nanoparticles have a longest straight dimension (e.g., diameter) of 200 nm or less. In some embodiments, the nanoparticles have a diameter of 100 nm or less. Smaller nanoparticles, e.g., having diameters of 50 nm or less, e.g., about 1 nm to about 30 nm or about 1 nm to about 5 nm, are used in some embodiments.
[0123] The term radiosensitizer refers to compounds or agents that increase the cytotoxicity of ionizing radiation. For example, heavy-metal nanomaterials with high atomic number (Z) values, such as gold nanomaterials.
[0124] An effective amount can refer to that amount of a therapeutic agent that results in amelioration of symptoms or a prolongation of survival in the subject and relieves, to some extent, one or more symptoms of the disease or returns to normal (either partially or completely) one or more physiological or biochemical parameters associated with or causative of the disease.
[0125] Therapeutic agents can include any agent (e.g., molecule, drug, pharmaceutical composition, etc.) capable of preventing, inhibiting, or arresting the symptoms and/or progression of a disease.
[0126] Embodiments described herein relate to PSMA targeted conjugate compounds, pharmaceutical compositions comprising these compounds, methods for treating and detecting cancers (e.g., prostate cancer) in a subject using these PSMA targeted conjugate compounds, and methods for identifying cancer cells (e.g., prostate cancer cells) in a sample using these compounds. It has been shown that PSMA targeted compounds conjugated to anti-cancer agents can increase uptake of the conjugate compound in PSMA expressing cells while also improving cell killing compared to agents administered alone. In addition, PSMA targeted conjugate compounds described herein can decrease non-PSMA target toxicity of the therapeutic agent, or a theranostic agent administered (e.g., systemically) to a subject. Moreover, surprisingly it was found that the PSMA targeted conjugate compound upon systemic administration to a subject show minimal accumulation and/or uptake in non-PSMA targets in non-cancer tissue, such as salivary glands, lacrimal glands, and kidney of the subject.
[0127] Pathological studies indicate that PSMA is expressed by virtually all prostate cancers, and its expression is further increased in poorly differentiated, metastatic, and hormone-refractory carcinomas. Higher PSMA expression is also found in cancer cells from castration-resistant prostate cancer patients. Increased PSMA expression is reported to correlate with the risk of early prostate cancer recurrence after radical prostatectomy. In addition to being overexpressed in prostate cancer (PCa), PSMA is also expressed in the neovasculature of neoplasms including but not limited to conventional (clear cell) renal carcinoma, transitional cell carcinoma of the urinary bladder, testicular embryonal carcinoma, colonic adenocarcinoma, neuroendocrine carcinoma, gliobastoma multiforme, malignant melanoma, pancreatic ductal carcinoma, non-small cell lung carcinoma, soft tissue carcinoma, breast carcinoma, and prostatic adenocarcinoma.
[0128] In some embodiments, the PSMA targeted conjugate compounds described herein, can selectively recognize PSMA-expressing tumors, cancer cells, and/or cancer neovasculature in vivo and be used to deliver a therapeutic agent, detectable moiety, and/or theranostic agent to the PSMA-expressing tumors, cancer cells, and/or cancer neovasculature to treat and/or detect the PSMA-expressing tumors, cancer cells, and/or cancer neovasculature in a subject.
[0129] In some embodiments, the PSMA expressing cancer that is treated and/or detected is prostate cancer. In other embodiments, the cancer that is treated and/or detected can include malignant neoplasms, such a conventional (clear cell) renal carcinoma, transitional cell carcinoma of the urinary bladder, testicular embryonal carcinoma, colonic adenocarcinoma, neuroendocrine carcinoma, gliobastoma multiforme, malignant melanoma, pancreatic ductal carcinoma, non-small cell lung carcinoma, soft tissue carcinoma, breast carcinoma, and prostatic adenocarcinoma.
[0130] In some embodiments, the compound can include the general formula (I):
##STR00013## [0131] wherein: [0132] n.sup.1 and n.sup.2 are each independently 1, 2, 3, or 4; [0133] A, B, and C each independently include at least one of a negatively charged amino acid, an acidic amino acid, a non-standard negatively charged or acidic amino acid thereof, a derivative thereof, or an analogue thereof; [0134] L is an optionally substituted aliphatic or heteroaliphatic linking group; and [0135] Y.sup.1 and Y.sup.2 are each independently a H of B or can include at least one detectable moiety, therapeutic agent, or a theranostic agent that is directly or indirectly linked to B, wherein at least one of Y.sup.1 or Y.sup.2 is not H.
[0136] In some embodiments, Y.sup.1 and Y.sup.2 are not H.
[0137] In some embodiments, the compound of formula (I) does not bind to aquaporin (e.g., AQP3), such as aquaporin expressed in the salivary glands, upon administration of the compound to a subject with cancer.
[0138] In other embodiments, the compound has a selectivity for PSMA expressing cancer tissue versus non-PSMA expressing non-cancer tissue 5 times, 10 times, 20 times, 30 times, 40 times, 50 times or more times.
[0139] In other embodiments, the compound upon administration to a subject in need thereof shows minimal accumulation or uptake in salivary glands of the subject.
[0140] In other embodiments, Y.sup.1 and Y.sup.2 can include at least one radiolabel or radioisotope, radiosensitizer, chelating agent, photosensitizer, fluorescent labeling agent, magnetic resonance imaging (MRI) agent, or anti-cancer agent.
[0141] In some embodiments, the anticancer agent is linked to B via a protease cleavable or acid-labile linker. The linker can include a maleimido-caproyl linker-valine-citrulline cleavable peptide-p-aminobenzyl carbamate spacer (MC-VC-PABC) protease cleavable linker.
[0142] In some embodiments, at least one of Y.sup.1 or Y.sup.2 includes a chelating agent optionally complexed with a metal or metal ion. In some embodiments, the chelating agent includes at least one of diethylenetriaminepentaacetate (DTPA), 1,4,7,10-tetraazadodecanetetraacetate (DOTA), 1,4,7,10-tetraazadodecane-1,4,7-triacetate (DO3A), ethylenediaminetetraacetate (EDTA), 1,4,7,10-tetraazacyclotridecanetetraacetic acid (TRITA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazadodecanetetramethylacetate (DOTMA), 1,4,7,10-tetraazadodecane-1,4,7-trimethylacetate (DO3MA), N,N,N,N-tetraphosphonatomethyl-1,4,7,10-tetraazacyclododecane (DOTP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene methylphosphonic acid) (DOTMP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phenylphosphonic acid) (DOTPP), N,N-ethylenedi-L-cysteine, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane (TACN), N,N-Bis(2-hydroxy-5-(ethylene-beta-carboxy)benzyl)ethylenediamine N,N-diacetic acid (HBED-CC), S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacylododecane tetracetic acid (p-SCN-Bn-DOTA), MeO-DOTA-NCS, [(R)-2-Amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid (CHX-A-DTPA-NCS), 2-[4-nitrobenzyl]-1,4,7,10,13-pentaazacyclopentadecane-N,N,N,N,N-pentaacetic acid (PEPA), 1,4,7,10,13,16-hexaazacyclooctadecane-N,N,N,N,N-hexaacetic acid (HEHA), desferrioxamine B (DFO), or derivatives thereof.
[0143] In some embodiments, the metal ion includes at least one of a radioactive isotope of Ga, I, In, Y, Lu, Bi, Ac, Re, In, Th, Tc, Tl, Tb, Zr, Cu, Rb, At, Pb, Gd, Sm, or Sr. In other embodiments, the metal ion includes at least one of .sup.186Re, .sup.188Re, .sup.99mTc .sup.153Gd, .sup.111In .sup.67Ga, .sup.68Ga, .sup.201Tl, .sup.82Rb, .sup.64Cu, .sup.89Zr, .sup.90Y, .sup.177Lu, T(tritium), .sup.149P, .sup.161T, .sup.153Sm, .sup.89Sr, .sup.211At, .sup.225Ac, .sup.227Ac, .sup.123-125I, .sup.131I, .sup.67Cu, .sup.203Pb, .sup.212Pb, .sup.211Bi, .sup.213Bi, or .sup.227-233Th.
[0144] In other embodiments, L can be an optionally substituted aliphatic or heteroaliphatic group that includes at least one ring selected from the group consisting of an optionally substituted 4 to 7 membered nonaromatic heterocyclic ring and an optionally substituted C4-C7 cycloalkyl ring.
[0145] An aliphatic group is a straight chained, branched or cyclic non-aromatic hydrocarbon, which is completely saturated or which contains one or more units of unsaturation. An alkyl group is a saturated aliphatic group. Typically, a straight chained or branched aliphatic group has from 1 to about 10 carbon atoms, preferably from 1 to about 4, and a cyclic aliphatic group has from 3 to about 10 carbon atoms, preferably from 3 to about 8. An aliphatic group is preferably a straight chained or branched alkyl group, e.g., methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl or octyl, or a cycloalkyl group with 3 to about 8 carbon atoms. C1-C4 straight chained or branched alkyl or alkoxy groups or a C3-C8 cyclic alkyl or alkoxy group (preferably C1-C4 straight chained or branched alkyl or alkoxy group) are also referred to as a lower alkyl or lower alkoxy groups; such groups substituted with F, Cl, Br, or I are lower haloalkyl or lower haloalkoxy groups; a lower hydroxyalkyl is a lower alkyl substituted with OH; and the like.
[0146] Optional substituents for a substitutable atom in alkyl, cycloalkyl, aliphatic, cycloaliphatic, heterocyclic, benzylic, aryl, or heteroaryl groups described herein are those substituents that do not substantially interfere with the activity of the disclosed compounds. A substitutable atom is an atom that has one or more valences or charges available to form one or more corresponding covalent or ionic bonds with a substituent. For example, a carbon atom with one valence available (e.g., C(H)) can form a single bond to an alkyl group (e.g., C(-alkyl)=), a carbon atom with two valences available (e.g., C(H.sub.2)) can form one or two single bonds to one or two substituents (e.g., C(alkyl)(Br)), C(alkyl)(H)) or a double bond to one substituent (e.g., CO)), and the like. Substitutions contemplated herein include only those substitutions that form stable compounds.
[0147] For example, suitable optional substituents for substitutable carbon atoms include F, Cl, Br, I, CN, NO.sub.2, OR.sup.a, C(O)R.sup.a, OC(O)R.sup.a, C(O)OR.sup.a, SR.sup.a, C(S)R.sup.a, OC(S)Ra, C(S)OR.sup.a, C(O)SR.sup.a, C(S)SR.sup.a, S(O)R.sup.a, SO.sub.2R.sup.a, SO.sub.3R.sup.a, POR.sup.aR.sup.b, PO.sub.2R.sup.aR.sup.b, PO.sub.3R.sup.aR.sup.b, PO.sub.4R.sup.aR.sup.b, P(S)R.sup.aR.sup.b, P(S)OR.sup.aR.sup.b, P(S)O.sub.2R.sup.aR.sup.b, P(S)O.sub.3R.sup.aR.sup.b, N(R.sup.aR.sup.b), C(O)N(R.sup.aR.sup.b), C(O)NR.sup.aNR.sup.bSO.sub.2R.sup.c, C(O)NR.sup.aSO.sub.2R.sup.c, C(O)NR.sup.aCN, SO.sub.2N(R.sup.aR.sup.b) SO.sub.2N(R.sup.aR.sup.b), NR.sup.cC(O)R.sup.a, NR.sup.cC(O)OR.sup.a, NR.sup.cC(O)N(R.sup.aR.sup.b), C(NR.sup.c)N(R.sup.aR.sup.b), NR.sup.dC(NR.sup.c)N(R.sup.aR.sup.b), NR.sup.aN(R.sup.aR.sup.b), CR.sup.cCR.sup.aR.sup.b, CCR.sup.a, O, S, CR.sup.aR.sup.b, NR.sup.a, NOR.sup.a, NNR.sup.a, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aliphatic, optionally substituted cycloaliphatic, optionally substituted heterocyclic, optionally substituted benzyl, optionally substituted aryl, and optionally substituted heteroaryl, wherein R.sup.a-R.sup.d are each independently H or an optionally substituted aliphatic, optionally substituted cycloaliphatic, optionally substituted heterocyclic, optionally substituted benzyl, optionally substituted aryl, or optionally substituted heteroaryl, or, N(R.sup.aR.sup.b), taken together, is an optionally substituted heterocyclic group. Also contemplated are isomers of these groups.
[0148] Suitable substituents for nitrogen atoms having two covalent bonds to other atoms include, for example, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aliphatic, optionally substituted cycloaliphatic, optionally substituted heterocyclic, optionally substituted benzyl, optionally substituted aryl, optionally substituted heteroaryl, CN, NO.sub.2, OR.sup.a, C(O)R.sup.a, OC(O)R.sup.a, C(O)OR.sup.a, SR.sup.a, S(O)R.sup.a, SO.sub.2R.sup.a, SO.sub.3R.sup.a, N(R.sup.aR.sup.b), C(O)N(R.sup.aR.sup.b), C(O)NR.sup.aNR.sup.bSO.sub.2R.sup.c, C(O)NR.sup.aSO.sub.2R.sup.c, C(O)NR.sup.aCN, SO.sub.2N(R.sup.aR.sup.b), SO.sub.2N(R.sup.aR.sup.b), NR.sup.cC(O)R.sup.a, NR.sup.cC(O)OR.sup.a, NR.sup.cC(O)N(R.sup.aR.sup.b), and the like.
[0149] Suitable substituents for nitrogen atoms having three covalent bonds to other atoms include OH, alkyl, and alkoxy (preferably C1-C4 alkyl and alkoxy). Substituted ring nitrogen atoms that have three covalent bonds to other ring atoms are positively charged, which is balanced by counteranions such as chloride, bromide, fluoride, iodide, formate, acetate and the like. Examples of other suitable counter anions are provided in the section below directed to suitable pharmacologically acceptable salts.
[0150] In some embodiments B includes at least one, two, three, four, five, or more negatively charged or acidic amino acids.
[0151] In some embodiments, the negatively charged or acidic amino acids can be selected from aspartic acid or aspartate or glutamic acid or glutamate.
[0152] In other embodiments, B further includes at least one of lysine, tyrosine, or cysteine.
[0153] In other embodiments, the compound can include the general formula (II):
##STR00014## [0154] wherein: [0155] n.sup.1 is 1, 2, 3, or 4; [0156] B includes a negatively charged amino acid, an acidic amino acid, a non-standard negatively charged or acidic amino acid thereof, a derivative thereof, or an analogue thereof; [0157] L is an optionally substituted aliphatic or heteroaliphatic linking group; and [0158] Y.sup.1 and Y.sup.2 are each independently a H of B or can include at least one detectable moiety, therapeutic agent, or a theranostic agent that is directly or indirectly linked to B, wherein at least one of Y.sup.1 or Y.sup.2 is not H.
[0159] In some embodiments, Y.sup.1 and Y.sup.2 are not H.
[0160] In some embodiments, Y.sup.1 and Y.sup.2 each independently include at least one radiolabel or radioisotope, radiosensitizer, chelating agent, photosensitizer, fluorescent labeling agent, magnetic resonance imaging (MRI) agent, or anti-cancer agent.
[0161] In some embodiments, B has the following formula:
##STR00015## [0162] wherein m is 1, 2, 3, or 4, X.sup.1 includes at least one amino acid, and at least one of Y.sup.1 or Y.sup.2 includes at least one detectable moiety, therapeutic agent, or theranostic agent that is directly or indirectly linked to X.sup.1.
[0163] In some embodiments, X.sup.1 further includes at least one of lysine, tyrosine, or cysteine.
[0164] In some embodiments, two of Y.sup.1 and Y.sup.2 include at least one radiolabel or radioisotope, radiosensitizer, chelating agent, photosensitizer, fluorescent labeling agent, magnetic resonance imaging (MRI) agent, or anti-cancer agent
[0165] In other embodiments, B has the following formula:
##STR00016## [0166] wherein m is 1, 2, 3, or 4; [0167] X.sup.2 is H or includes at least one amino acid; [0168] Y.sup.1 includes at least one detectable moiety; therapeutic agent, or theranostic agent that is directly or indirectly linked to X.sup.2 or is absent if X.sup.2 is H; and [0169] Y.sup.2 includes at least one detectable moiety, therapeutic agent, or theranostic agent.
[0170] In other embodiments, one of Y.sup.1 or Y.sup.2 includes at least one radiolabel or radioisotope, radiosensitizer, chelating agent, photosensitizer, fluorescent labeling agent, magnetic resonance imaging (MRI) agent, or anti-cancer agent.
[0171] In some embodiments, the compound can include the general formula:
##STR00017##
complexed with at least one metal contrast agent or metal ion. In some embodiments, the metal ion can include at least one of .sup.186Re, .sup.188Re, .sup.99mTc, .sup.153Gd, .sup.111In, .sup.67Ga, .sup.68Ga, .sup.201Tl, .sup.82Rb, .sup.64Cu, .sup.89Zr, .sup.90Y, .sup.177Lu, T(tritium), .sup.149T, .sup.161Tb, .sup.153Sm, .sup.89Sr, .sup.211At, .sup.225Ac, .sup.227Ac, .sup.123-125I, .sup.131I, .sup.67Cu, .sup.203Pb, .sup.212Pb, .sup.211Bi, .sup.213Bi, or .sup.227-233Th or a pharmaceutically acceptable salt thereof.
[0172] In some embodiments, the compound includes the following formula:
##STR00018##
or a pharmaceutically acceptable salt thereof, wherein the I is .sup.123-125I or .sup.131I.
[0173] In some embodiments, B has the following formula:
##STR00019## [0174] wherein m is 1, 2, 3, or 4; [0175] X.sup.3 is H or includes at least one of an amino acid or linker; [0176] Y.sup.1 includes at least one of H, detectable moiety, therapeutic agent, or theranostic agent that is directly or indirectly linked to X.sup.3, or is absent if X.sup.3 is a H; and [0177] Y.sup.2 includes at least one detectable moiety, therapeutic agent, or theranostic agent.
[0178] In other embodiments, Y.sup.1 and Y.sup.2 include at least one radiolabel or radioisotope, radiosensitizer, chelating agent, photosensitizer, fluorescent labeling agent, magnetic resonance imaging (MRI) agent, or anti-cancer agent.
[0179] In some embodiments, the radiosensitizer is a Au ion that is conjugated to a free thiol.
[0180] In some embodiments, the compound includes the following formula:
##STR00020##
[0181] In some embodiments, a plurality of such compounds can aggregate form a PSMA targeted gold nanocluster (AuNC) and MMAE conjugates (PSMA-AuNC-MMAE), for radiotherapy and chemotherapy. In an exemplary embodiment, PSMA-AuNC-MMAE synthesis can occur by CY-PSMA-1-MMAE ligand reduction and stabilization where the cysteine residue captures Au.sup.3+ ions in solution with the thiol group and a pH increase from the addition of NaOH in the solution turns the phenolic group of the tyrosine residue into a phenoxide ion, which can reduce Au.sup.3+ ions. The resultant Au atoms can then aggregate to form gold nanoclusters where the gold nanoclusters are stabilized via thiol groups of the CY-PSMA-1-MMAE cysteine residues.
[0182] The PSMA-AuNC-MMAE can exhibit discrete optical and electronic states characterized by, for example, strong quantum effects and fluorescence, which allows the gold nanoclusters to behave as molecular, rather than metallic substances. This molecular optical and electronic behavior sharply distinguishes the PSMA-AuNC-MMAE from gold nanoparticles whose optical characteristics can be driven by plasmon resonance.
[0183] In some embodiments, an PSMA-AuNC-MMAE formed from a plurality of compounds that include formula (I) as described herein can each include about 10 to about 150 gold atoms. In some embodiments, the gold nanoclusters include about 10 to about 40 gold atoms or about 10 to about 12 gold atoms. In particular embodiments, a gold nanocluster can include 15, 18, 22, 25, 39 or 144 gold atoms. In an exemplary embodiment, a PSMA-AuNC-MMAE formed from a plurality of compounds as described herein can include 25 gold atoms.
[0184] In some embodiments, PSMA-AuNC-MMAE for use in a composition described herein can have an average diameter less than about 10 nm. In order to reduce nanoparticle-induced toxicity compared to larger particles, PSMA-AuNC-MMAE formed as described herein can be small enough (e.g., less than about 5.5 nm) to be excreted from urine. In some embodiments, PSMA-AuNC-MMAE for use in a composition describe herein can have an average diameter less than about 5 nm. In particular embodiments, a PSMA-AuNC-MMAE can have an average diameter of about 1.5 nm. An exemplary PSMA-AuNC-MMAE for use in a composition describe herein can have an average diameter of 1.50.5 nm and a hydrodynamic diameter of 3.0 nm.
[0185] In other embodiments, the compound can include the general formula:
##STR00021## [0186] wherein n.sup.1 is independently 1, 2, 3, or 4; [0187] L is an optionally substituted aliphatic or heteroaliphatic linking group; [0188] wherein B has the following formula:
##STR00022## [0189] wherein m is 1, 2, 3, or 4; and [0190] Y.sup.1 is a first imaging agent, therapeutic agent, or theranostic agent that is directly or indirectly linked to the thiol group and Y.sup.2 is a second imaging agent, therapeutic agent, or theranostic agent that is directly or indirectly linked to the amine group.
[0191] In some embodiments, each of Y.sup.1 or Y.sup.2 independently includes at least one radiolabel or radioisotope, radiosensitizer, chelating agent, photosensitizer, fluorescent labeling agent, magnetic resonance imaging (MRI) agent, or anti-cancer agent.
[0192] In some embodiments, L can include at least one ring selected from the group consisting of an optionally substituted 4 to 7 membered nonaromatic heterocyclic ring and an optionally substituted C4-C7 cycloalkyl ring.
[0193] In some embodiments, at least one of Y.sup.1 or Y.sup.2 includes a chelating agent optionally complexed with a metal contrast agent or metal ion.
[0194] In some embodiments, the chelating agent includes at least one of diethylenetriaminepentaacetate (DTPA), 1,4,7,10-tetraazadodecanetetraacetate (DOTA), 1,4,7,10-tetraazadodecane-1,4,7-triacetate (DO3A), ethylenediaminetetraacetate (EDTA), 1,4,7,10-tetraazacyclotridecanetetraacetic acid (TRITA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazadodecanetetramethylacetate (DOTMA), 1,4,7,10-tetraazadodecane-1,4,7-trimethylacetate (DO3MA), N,N,N,N-tetraphosphonatomethyl-1,4,7,10-tetraazacyclododecane (DOTP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene methylphosphonic acid) (DOTMP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phenylphosphonic acid) (DOTPP), N,N-ethylenedi-L-cysteine, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane (TACN), N,N-Bis(2-hydroxy-5-(ethylene-beta-carboxy)benzyl)ethylenediamine N,N-diacetic acid (HBED-CC), S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacylododecane tetracetic acid (p-SCN-Bn-DOTA), MeO-DOTA-NCS, [(R)-2-Amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid (CHX-A-DTPA-NCS), 2-[4-nitrobenzyl]-1,4,7,10,13-pentaazacyclopentadecane-N,N,N,N,N-pentaacetic acid (PEPA), 1,4,7,10,13,16-hexaazacyclooctadecane-N,N,N,N,N-hexaacetic acid (HEHA), desferrioxamine B (DFO), or derivatives thereof.
[0195] In some embodiments, the metal ion includes at least one of a radioactive isotope of Ga, I, In, Y, Lu, Bi, Ac, Re, In, Th, Tc, Tl, Tb, Zr, Cu, R.sup.b, At, Pb, Gd, Sm, or Sr. In other embodiments, the metal ion includes at least one of .sup.186Re, .sup.188Re, .sup.99mTc, .sup.153Gd, .sup.111In, .sup.67Ga, .sup.68Ga, .sup.201Tl, .sup.82Rb, .sup.111Cu, .sup.89Zr, .sup.90Y, .sup.177Lu, T(tritium), .sup.149T, .sup.161Tb, .sup.153Sm, .sup.89Sr, .sup.211At, .sup.225Ac, .sup.227Ac, .sup.123-125I, .sup.131I, .sup.67Cu, .sup.203Pb, .sup.212Pb, .sup.211Bi, .sup.213Bi, or .sup.227-233Th.
[0196] In some embodiments, the anticancer agent is linked to B via a protease cleavable or acid-labile linker. The linker can include a maleimido-caproyl linker-valine-citrulline cleavable peptide-p-aminobenzyl carbamate spacer (MC-VC-PABC) protease cleavable linker.
[0197] In some embodiments, the compound has the general formula:
##STR00023## [0198] wherein m and n.sup.1 are each independently 1, 2, 3, or 4; and wherein Y.sup.1 includes a radiolabel or radioisotope, radiosensitizer, chelating agent, photosensitizer, fluorescent labeling agent, magnetic resonance imaging (MRI) agent, or anti-cancer agent that is directly or indirectly linked to the thiol group and Y.sup.2 includes a radiolabel or radioisotope, radiosensitizer, chelating agent, photosensitizer, fluorescent labeling agent, magnetic resonance imaging (MRI) agent, or anti-cancer agent that is directly or indirectly linked to the amine group.
[0199] In some embodiments, a therapeutic, imaging and/or a theranostic agent can be coupled directly or indirectly to the PSMA targeted compound via a linker. The linker can include a heterobifunctional crosslinker capable of conjugating to the cysteine residue of the PSMA targeted compound via a maleimide group that is sulfhydryl (thiol; SH) reactive.
[0200] In some embodiments, the heterobifunctional crosslinker can include a protease cleavable or acid-labile linker. In particular embodiments, the detectable moiety, the therapeutic agent, or the theranostic agent can be coupled directly or indirectly to the PSMA targeted compound via an acid-labile linker, such as a 4-(4-maleimidomethyl)cyclohexane-1-carboxyl hydrazide (MMCCH) linker. The MMCCH linker includes a hydrazine bond that can be cleaved to release the detectable moiety, therapeutic agent, or the theranostic agent coupled to the PSMA targeted compound. In alternative embodiments, the detectable moiety, the therapeutic agent, or the theranostic agent can be coupled directly or indirectly to the PSMA targeted portion of the compound (PSMA-1(Cys)) via a non-cleavable linker, such as Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).
[0201] In other embodiments, the detectable moiety, therapeutic agent, or the theranostic agent can be coupled directly or indirectly to the PSMA targeted portion of the compound (PSMA-1(Cys)) via a protease cleavable linker. The protease cleavable linker can include a MC-Val-Cit-PABC protease cleavable linker where the Valine-Citrulline-PABC portion is protease cleaveable.
[0202] In some embodiments, the PSMA targeted conjugate compounds described herein are prepared by coupling a vc-MMAE linker-drug combination construct to the thiol group of the cysteine residue of the PSMA targeted compound. The vc-MMAE construct is also referred to as the linker-drug mc-vc-PABC-MMAE. The construct utilizes a maleimidocaproyl (mc) spacer, a protease-sensitive dipeptide, valine-citrulline (vc), a self-immolative spacer, para-amino benzyloxycarbonyl (PABC), and the antimitotic agent, monomethyl auristatin E (MMAE). The me spacer provides enough room for the vc group to be recognized by the lysosomal cysteine protease cathepsin B in a PSMA expressing cell, which cleaves the citrulline-PABC amide bond. The resultant PABC-substituted MMAE is not a stable intermediate and spontaneously undergoes a 1,6-elimination with a loss of p-iminoquinone methide and carbon dioxide (self-immolation) leaving MMAE as the product to exert anti-mitotic effects.
[0203] In some embodiments, the PSMA targeted conjugate compound can include a detectable moiety directly or indirectly coupled to B. Examples of detectable moieties include, but are not limited to: various ligands, imaging agents, contrast agents, radionuclides, fluorescent dyes, near-infrared dyes, chemiluminescent agents, microparticles (such as, for example, quantum dots, nanocrystals, phosphors and the like), enzymes (such as, for example, those used in an ELISA, i.e., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric labels, magnetic labels, chelating groups, and biotin, dioxigenin or other haptens and proteins for which antisera or monoclonal antibodies are available.
[0204] In one example, the detectable moiety can include a radiolabel, that is directly or indirectly linked (e.g., attached or complexed) with B using general organic chemistry techniques. The detectable peptide can also include radiolabels, such as .sup.68Ga, .sup.123, .sup.131I, .sup.125, .sup.18F, .sup.11C, .sup.75Br, .sup.76Br, .sup.124, .sup.13N, .sup.64Cu, .sup.32P, .sup.3S, for PET by techniques well known in the art and are described by Fowler, J. and Wolf, A. in POSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M., Mazziota, J., and Schelbert, H. eds.) 391-450 (Raven Press, NY 1986) the contents of which are hereby incorporated by reference. The detectable moiety can also include .sup.123I for SPECT. The .sup.123I can be coupled to B can by any of several techniques known to the art. See, e.g., Kulkarni, Int. J. Rad. Appl. & Inst. (Part B) 18: 647 (1991), the contents of which are hereby incorporated by reference. In addition, detectable moiety can include any radioactive iodine isotope, such as, but not limited to .sup.131I, .sup.125, or .sup.123I. The radioactive iodine isotopes can be coupled to B by iodination of a diazotized amino derivative directly via a diazonium iodide, see Greenbaum, F. Am. J. Pharm. 108: 17 (1936), or by conversion of the unstable diazotized amine to the stable triazene, or by conversion of a non-radioactive halogenated precursor to a stable tri-alkyl tin derivative which then can be converted to the iodo compound by several methods well known to the art.
[0205] The detectable moiety can further include known metal radiolabels, such as Technetium-99m (.sup.99mTc), .sup.153Gd, .sup.111In, .sup.67Ga, .sup.201Tl, .sup.82Rb, .sup.64Cu, .sup.90Y, .sup.177Lu.sup.188Rh, T(tritium), .sup.153Sm, .sup.89Sr, and .sup.211At, .sup.227Ac. The metal radiolabeled compounds can then be used to detect cancers, such as prostate cancer in the subject. Preparing radiolabeled derivatives of Tc99m is well known in the art. See, for example, Zhuang et al., Neutral and stereospecific Tc-99m complexes: [99mTc]N-benzyl-3,4-di-(N-2-mercaptoethyl)-amino-pyrrolidines (P-BAT) Nuclear Medicine & Biology 26(2):217-24, (1999); Oya et al., Small and neutral Tc(v)O BAT, bisaminoethanethiol (N2S2) complexes for developing new brain imaging agents Nuclear Medicine & Biology 25(2):135-40, (1998); and Hom et al., Technetium-99m-labeled receptor-specific small-molecule radiopharmaceuticals: recent developments and encouraging results Nuclear Medicine & Biology 24(6):485-98, (1997).
[0206] In still other embodiments, a radiolabel or radioisotope can be converted from an imaging agent to a radiotherapy. The radiolabel of the PSMA targeting conjugate compound can be injected into the subject's body and targeted to the cancer in the subject by the PSMA targeting conjugate compound. Alpha or gamma irradiation treatment can be used to switch the radiolabel from an imaging isotope of lower energy to a therapeutic agent of higher energy. Switching the isotope converts the agent from an imaging agent to an alpha or gamma emitting therapeutic agent.
[0207] In some embodiments, the detectable moiety can include a chelating agent (with or without a chelated metal group). Exemplary chelating agents can include those disclosed in U.S. Pat. No. 7,351,401, which is herein incorporated by reference in its entirety. In some embodiments, the chelating agent includes at least one of diethylenetriaminepentaacetate (DTPA), 1,4,7,10-tetraazadodecanetetraacetate (DOTA), 1,4,7,10-tetraazadodecane-1,4,7-triacetate (DO3A), ethylenediaminetetraacetate (EDTA), 1,4,7,10-tetraazacyclotridecanetetraacetic acid (TRITA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), 1,4,7,10-tetraazadodecanetetramethylacetate (DOTMA), 1,4,7,10-tetraazadodecane-1,4,7-trimethylacetate (DO3MA), N,N,N,N-tetraphosphonatomethyl-1,4,7,10-tetraazacyclododecane (DOTP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene methylphosphonic acid) (DOTMP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phenylphosphonic acid) (DOTPP), N,N-ethylenedi-L-cysteine, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane (TACN), N,N-Bis(2-hydroxy-5-(ethylene-beta-carboxy)benzyl)ethylenediamine N,N-diacetic acid (HBED-CC), S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacylododecane tetracetic acid (p-SCN-Bn-DOTA), MeO-DOTA-NCS, [(R)-2-Amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid (CHX-A-DTPA-NCS), 2-[4-nitrobenzyl]-1,4,7,10,13-pentaazacyclopentadecane-N,N,N,N,N-pentaacetic acid (PEPA), 1,4,7,10,13,16-hexaazacyclooctadecane-N,N,N,N,N-hexaacetic acid (HEHA), desferrioxamine B (DFO), or derivatives thereof.
[0208] The chelating agent can be complexed with a radionuclide or metal ion. In some embodiments, the metal ion includes at least one of a radioactive isotope of Ga, I, In, Y, Lu, Bi, Ac, Re, In, Th, Tc, Tl, Tb, Zr, Cu, Rb, At, Pb, Gd, Sm, or Sr. In other embodiments, the metal ion includes at least one of .sup.186Re, .sup.188Re, .sup.99mTc, .sup.153Gd, .sup.111In, .sup.67Ga, .sup.68Ga, .sup.201Tl, .sup.82Rb, .sup.64Cu, .sup.89Zr, .sup.90Y, .sup.177Lu, T(tritium), .sup.149Tb, .sup.161Tb, .sup.153Sm, .sup.89Sr, .sup.211At, .sup.225Ac, .sup.227Ac, .sup.123-125I, .sup.131I, .sup.67Cu, .sup.203Pb, .sup.212Pb, .sup.211Bi, .sup.213Bi, or .sup.227-233Th.
[0209] Fluorescent labeling agents or infrared agents include those known to the art, many of which are commonly commercially available, for example, fluorophores, such as ALEXA 350, PACIFIC BLUE, MARINA BLUE, ACRIDINE, EDANS, COUMARIN, BODIPY 493/503, CY2, BODIPY FL-X, DANSYL, ALEXA 488, FAM, OREGON GREEN, RHODAMINE GREEN-X, TET, ALEXA 430, CAL GOLD, BODIPY R6G-X, JOE, ALEXA 532, VIC, HEX, CAL ORANGE, ALEXA 555, BODIPY 564/570, BODIPY TMR-X, QUASAR 570, ALEXA 546, TAMRA, RHODAMINE RED-X, BODIPY 581/591, CY3.5, ROX, ALEXA 568, CAL RED, BODIPY TR-X, ALEXA 594, BODIPY 630/650-X, PULSAR 650, BODIPY 630/665-X, TEXAS RED, ALEXA 647, TideFluor, ICG, IR700, IR800, and QUASAR 670. Fluorescent labeling agents can include other known fluorophores, or proteins known to the art, for example, green fluorescent protein.
[0210] In other embodiments, the detectable moiety includes a fluorescent dye. Exemplary Fluorescent dyes include fluorescein isothiocyanate, cyanines such as Cy5, Cy5.5, Cy7.5, and analogs thereof (e.g., sulfo-Cyanine 5 NHS ester and Cy5.5 maleimide). See also Handbook of Fluorescent Probes and Research Chemicals, 6.sup.th Ed., Molecular Probes, Inc., Eugene Oreg, which is incorporated herein by reference.
[0211] In some embodiments, the fluorescence labeling agent can include the cyanine monosuccinimidyl ester, CY5.5. In certain embodiments, the near-infrared (NIR) labeling agent can include IRDye700DX (IR700). In some embodiments, a CY5.5 fluorescence labeling agent or IR700 NIR labeling agent can be coupled to the amine group of lysine linked to the PSMA ligand via a carbon spacer arm linker. A carbon spacer arm can include a standard (C6) or a longer spacer arm, such as a (C12) spacer arm. For example, the carbon spacer arm can be a C6, C7, C8, C9, C10, C11, or C12 spacer arm. In certain embodiments, the C6 carbon spacer arm is a 6-Aminohexanoic acid (Ahx) spacer arm.
[0212] The detectable moiety can further include a near infrared imaging group. Near infrared imaging groups are disclosed in, for example, Tetrahedron Letters 49(2008) 3395-3399; Angew. Chem. Int. Ed. 2007, 46, 8998-9001; Anal. Chem. 2000, 72, 5907; Nature Biotechnology vol 23, 577-583; Eur Radiol(2003) 13: 195-208; and Cancer 67: 1991 2529-2537, which are herein incorporated by reference in their entirety. Applications may include the use of a NIRF (near infra-red) imaging scanner. In one example, the NIRF scanner may be handheld. In another example, the NIRF scanner may be miniaturized and embedded in an apparatus (e.g., micro-machines, scalpel, neurosurgical cell removal device).
[0213] Quantum dots, e.g., semiconductor nanoparticles, can be employed in a molecular probe as described in Gao, et al In vivo cancer targeting and imaging with semiconductor quantum dots, Nature Biotechnology, 22, (8), 2004, 969-976, the entire teachings of which are incorporated herein by reference. The disclosed PSMA ligangs can be coupled to the quantum dots, administered to a subject or a sample, and the subject/sample examined by fluorescence spectroscopy or imaging to detect the labeled compound.
[0214] In certain embodiments, a detectable moiety includes a MRI contrast agent. MRI relies upon changes in magnetic dipoles to perform detailed anatomic imaging and functional studies. MRI can employ dynamic quantitative T.sub.1 mapping as an imaging method to measure the longitudinal relaxation time, the Ti relaxation time, of protons in a magnetic field after excitation by a radiofrequency pulse. Ti relaxation times can in turn be used to calculate the concentration of a molecular probe in a region of interest, thereby allowing the retention or clearance of an agent to be quantified. In this context, retention is a measure of molecular contrast agent binding.
[0215] Numerous magnetic resonance imaging (MRI) contrast agents are known to the art, for example, positive contrast agents and negative contrast agents. The disclosed targeting peptides can be coupled to the MRI agents, administered to a subject or a sample, and the subject/sample examined by MRI or imaging to detect the labeled compound. Positive contrast agents (typically appearing predominantly bright on MRI) can include typically small molecular weight organic compounds that chelate or contain an active element having unpaired outer shell electron spins, e.g., gadolinium, manganese, iron oxide, or the like. Typical contrast agents include macrocycle-structured gadolinium(III)chelates, such as gadoterate meglumine (gadoteric acid), gadopentetate dimeglumine, gadoteridol, mangafodipir trisodium, gadodiamide, and others known to the art. In certain embodiments, the detectable moiety includes gadoterate meglumine. Negative contrast agents (typically appearing predominantly dark on MRI) can include small particulate aggregates comprised of superparamagnetic materials, for example, particles of superparamagnetic iron oxide (SPIO). Negative contrast agents can also include compounds that lack the hydrogen atoms associated with the signal in MRI imaging, for example, perfluorocarbons (perfluorochemicals).
[0216] In some embodiments, a PSMA targeted conjugate compound that is coupled to a fluorescence labeling agent, near-infrared or infrared agent can have the following general formula:
##STR00024## [0217] wherein Y is selected from at least one of a detectable moiety, therapeutic agent, or theranostic agent that is directly or indirectly linked to thiol group. This PSMA targeted conjugate compound can be prepared by linking the cysteine residue of the PSMA ligand to a lysine residue via a C6 linker, which then allows for coupling a detectable moiety, therapeutic agent, or theranostic agent, for example, the fluorescence labeling agent, CY5.5 to the amine on the lysine amino acid.
[0218] In some embodiments, a PSMA targeted conjugate compound that is coupled to a fluorescence labeling agent and a therapeutic agent can have the formula:
##STR00025##
or a pharmaceutically acceptable salt thereof.
[0219] In some embodiments, a PSMA targeted conjugate compound that is coupled to a fluorescence labeling agent, near-infrared agent or infrared agent can have the following general formula:
##STR00026## [0220] wherein Y is a therapeutic agent, such as an anti-cancer agent or chemotherapeutic, that is directly or indirectly linked to the thiol group. This PSMA targeted conjugate compound can be prepared by linking the cysteine residue of the PSMA ligand to a lysine residue via a C6 linker, which then allows for coupling the near-infrared (NIR) labeling agent, IR700 to the amine on the lysine amino acid.
[0221] In some embodiments, a PSMA targeted conjugate compound that is coupled to a near-infrared labeling agent IR700 and a therapeutic agent can have the formula:
##STR00027##
or a pharmaceutically acceptable salt thereof.
[0222] In some embodiments, a PSMA targeted conjugate compound that is coupled to an anti-cancer agent, such as MMAE, and a chelating agent optionally complexed with a metal ion can have the following formula:
##STR00028## [0223] or a pharmaceutically acceptable salt thereof. The metal ion complexed with the chelating agent can include at least one of a radioactive isotope of Ga, I, In, Y, Lu, Bi, Ac, Re, In, Th, Tc, Tl, Zr, Cu, R.sup.b, At, Pb, Gd, Sm, or Sr. In other embodiments, the metal ion includes at least one of .sup.186Re, .sup.188Re, .sup.99mTc .sup.153Gd, .sup.111In, .sup.67Ga, .sup.68Ga, .sup.201Tl, .sup.82Rb, .sup.64Cu, .sup.89Zr, .sup.90Y, .sup.177Lu, T(tritium), .sup.149Tb, .sup.161Tb, .sup.153Sm, .sup.89Sr, .sup.211At, .sup.225Ac, .sup.227Ac, .sup.123-125I, .sup.131I, .sup.67Cu, .sup.203Pb, .sup.212Pb, .sup.211Bi, .sup.213Bi, or .sup.227-23Th.
[0224] In some aspects, the PSMA ligands coupled to a detectable moiety described herein may be used in conjunction with non-invasive imaging techniques for in vivo imaging, such as magnetic resonance spectroscopy (MRS) or imaging (MRI), or gamma imaging, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT), to determine the location or distribution of cancer cells. The term in vivo imaging refers to any method, which permits the detection of a labeled PSMA targeted conjugate compound, as described above. For gamma imaging, the radiation emitted from the organ or area being examined is measured and expressed either as total binding or as a ratio in which total binding in one tissue is normalized to (for example, divided by) the total binding in another tissue of the same subject during the same in vivo imaging procedure. Total binding in vivo is defined as the entire signal detected in a tissue by an in vivo imaging technique without the need for correction by a second injection of an identical quantity of molecular probe along with a large excess of unlabeled, but otherwise chemically identical compound.
[0225] For purposes of in vivo imaging, the type of detection instrument available is a major factor in selecting a given detectable moiety. For instance, the type of instrument used will guide the selection of the stable isotope. The half-life should be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that the host does not sustain deleterious effects.
[0226] The PSMA targeted conjugate compounds coupled to a detectable moiety described herein can be administered to the subject by, for example, systemic, topical, and/or parenteral methods of administration. These methods include, e.g., injection, infusion, deposition, implantation, or topical administration, or any other method of administration where access to the tissue desired. In one example, administration can be by intravenous injection in the subject. Single or multiple administrations of the probe can be given. Administered, as used herein, means provision or delivery is in an amount(s) and for a period of time(s) effective to label cancer cells in the subject.
[0227] The PSMA targeted conjugate compounds coupled to a detectable moiety described herein can be administered to a subject in a detectable quantity of a pharmaceutical composition containing the PSMA targeted conjugate compounds coupled to a detectable moiety described herein or a pharmaceutically acceptable water-soluble salt thereof, to a patient. A detectable quantity means that the amount of the detectable compound that is administered is sufficient to enable detection of binding of the compound to the cancer cells. An imaging effective quantity means that the amount of the detectable compound that is administered is sufficient to enable imaging of binding of the compound to the cancer cells.
[0228] The PSMA targeted conjugate compounds coupled to a detectable moiety described herein administered to a subject can be used to determine the presence, location, and/or distribution of cancer cells, i.e., PSMA expressing cancer cells or PSMA expressing neovaculature of the cancer cells, in an organ or body area of a patient. The presence, location, and/or distribution of the PSMA ligands coupled to a detectable moiety in the animal's tissue, e.g., brain tissue, can be visualized (e.g., with an in vivo imaging modality described above). Distribution as used herein is the spatial property of being scattered about over an area or volume. In this case, the distribution of cancer cells is the spatial property of cancer cells being scattered about over an area or volume included in the animal's tissue, e.g., prostate tissue. The distribution of the PSMA targeted conjugate compounds coupled to a detectable moiety may then be correlated with the presence or absence of cancer cells in the tissue. A distribution may be dispositive for the presence or absence of a cancer cells or may be combined with other factors and symptoms by one skilled in the art to positively detect the presence or absence of migrating or dispersing cancer cells, cancer metastases or define a tumor margin in the subject.
[0229] In some embodiments, the PSMA targeted conjugate compounds coupled to a detectable moiety may be administered to a subject to assess the distribution prostate cells in a subject and correlate the distribution to a specific location. Surgeons routinely use stereotactic techniques and intra-operative MRI (iMRI) in surgical resections. This allows them to specifically identify and sample tissue from distinct regions of the tumor such as the tumor edge or tumor center. Frequently, they also sample regions of prostate on the tumor margin that are outside the tumor edge that appear to be grossly normal but are infiltrated by dispersing tumor cells upon histological examination.
[0230] The PSMA targeted conjugate compounds coupled to a detectable moiety can be used in intra-operative imaging techniques to guide surgical resection and eliminate the educated guess of the location of the tumor by the surgeon. Previous studies have determined that more extensive surgical resection improves patient survival. Thus, PSMA targeted conjugate compounds coupled to a detectable moiety that function as diagnostic molecular imaging agents have the potential to increase patient survival rates. PSMA-targeted conjugates are easily detectable at real-time imaging exposures (i.e., at <67 ms) and thus capable of being used for real-time image-guided surgery (IGS) during urological surgery. Thus, the compositions described herein for use in IGS can increase patient survival rates especially when including a therapeutic agent and/or when combined with subsequent photodynamic therapy (PDT) in accordance with a method described herein.
[0231] In some embodiments, IGS can be performed real-time using an in vivo imaging system, such as an intraoperative near-infrared fluorescence imaging system. In certain embodiments, image guided surgery can be performed using a FLARE (Fluorescence-Assisted Resection and Exploration) intraoperative NIR fluorescence imaging system where the targeted cancer is imaged at a wavelength of about 671 nm to about 705 nm. In an exemplary embodiment, the targeted PMSA expressing cancer is imaged using a PSMA conjugate compound coupled to a NIR-imaging agent during image guided surgical resection at about 690 to about 700 nm.
[0232] Following surgical resection, residual PSMA expressing cancer cells that have penetrated beyond the resection site may revert to a proliferative state to produce a more aggressive recurrent tumor that continues to disperse into nonneoplastic tissue adjacent the resection site and beyond. A therapeutic method described herein can be used to minimize cancer metastasis after a surgical resection procedure targeting PSMA positive cancer cells and/or tumor tissue using PDT.
[0233] Therefore, a therapeutic method described herein further includes the step of irradiating the PSMA-targeted conjugate compounds coupled to a photosensitizing agent that are bound to and/or complexed with the PSMA expressing cancer cells remaining in, and/or adjacent to, the surgical site to induce the cytotoxic effects of a coupled photosensitizing agent, such as the NIR-imaging agent IR700, on residual cancer cells following surgical resection of the cancer.
[0234] Methods and photosensitizing agents for conducting PDT are known in the art. See for example Thierry Patrice. Photodynamic Therapy; Royal Society of Chemistry, 2004. PDT is a site specific treatment modality that requires the presence of a photosensitizer, light, and adequate amounts of molecular oxygen to destroy targeted tumors (Grossweiner, Li, The science of phototherapy. Springer: The Netherlands, 2005). Upon illumination, a photoactivated sensitizer transfers energy to molecular oxygen that leads to the generation of singlet oxygen (O.sup.2) and other reactive oxygen species (ROS), which initiate apoptosis and oxidative damage to cancer cells. Only the cells that are exposed simultaneously to the targeted PDT compound (which is non-toxic in the dark) and light are destroyed while surrounding healthy, non-targeted and nonirradiated cells are spared from photodamage. Furthermore, the fluorescence of the photosensitizing compound, such as IR700, coupled to the PSMA targeting moiety enable simultaneous diagnostic optical imaging that can be used to guide the PDT step of the method of treating cancer described herein.
[0235] PDT agent photosensitizer compounds for use in an agent described herein can include compounds that are excited by an appropriate light source to produce radicals and/or reactive oxygen species. Typically, when a sufficient amount of photosensitizer appears in diseased tissue (e.g., tumor tissue), the photosensitizer can be activated by exposure to light for a specified period. The light dose supplies sufficient energy to stimulate the photosensitizer, but not enough to damage neighboring healthy tissue. The radicals or reactive oxygen produced following photosensitizer excitation kill the target cells (e.g., cancer cells). In some embodiments, the targeted tissue can be locally illuminated. For example, light can be delivered to a photosensitizer via an argon or copper pumped dye laser coupled to an optical fiber, a double laser consisting of KTP (potassium titanyl phosphate)/YAG (yttrium aluminum garnet) medium, LED (light emitting diode), or a solid state laser.
[0236] PDT sensitizers for use as a theranostic or therapeutic agent can include a first generation photosensitizer (e.g., hematoporphyrin derivatives (HpDs) such as Photofrin (porfimer sodium), Photogem, Photosan-3 and the like). In some embodiments, PDT sensitizers can include second and third generation photosensitizers such as porphyrinoid derivatives and precursors. Porphyrinoid derivatives and precursors can include porphyrins and mettaloporphrins (e.g., meta-tetra(hydroxyphenyl)porphyrin (m-THPP), 5,10,15,20-tetrakis(4-sulfanatophenyl)-21H,23H-porphyrin (TPPS.sub.4), and precursors to endogenous protoporphyrin IX (PpIX): 5-aminolevulinic acid (5-ALA, which has been used for photodynamic therapy (PDT) of gliomas with some success (Stummer, W. et al. J Neurooncol. 2008, 87(1):103-9.).), IR700 dye, methyl aminolevulinate (MAL), hexaminolevulinate (HAL)), chlorins (e.g., benzoporphyrin derivative monoacid ring A (BPD-MA), meta-tetra(hydroxyphenyl)chlorin (m-THPC), N-aspartyl chlorine6 (NPe6), and tin ethyl etiopurpurin (SnET2)), pheophorbides (e.g., 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide (HPPH)), bacteriopheophorbides (e.g., bacteriochlorphyll a, WST09 and WST11), Texaphyrins (e.g., motexafin lutetium (Lu-Tex)), and phthalocyanines (PCs) (e.g., aluminum phthalocyanine tetrasulfonate (AlPcS4) and silicon phthalocyanine (Pc4)). In some embodiments, the PDT sensitizer can include cationic zinc ethynylphenyl porphyrin. Although porphyrinoid structures comprise a majority of photosensitizers, several non-porphyrin chromogens exhibit photodynamic activity. These compounds include anthraquinones, phenothiazines, xanthenes, cyanines, and curcuminoids.
[0237] In some embodiments, a theranostic agent or therapeutic agent described herein can include a phthalocyanine compound. Phthalocyanines, hereinafter also abbreviated as Pcs, are a group of photosensitizer compounds having the phthalocyanine ring system. Phthalocyanines are azaporphyrins consisting of four benzoindole groups connected by nitrogen bridges in a 16-membered ring of alternating carbon and nitrogen atoms (i.e., C.sub.32H.sub.16N.sub.8) which form stable chelates with metal and metalloid cations. In these compounds, the ring center is occupied by a metal ion (either a diamagnetic or a paramagnetic ion) that may, depending on the ion, carry one or two ligands. In addition, the ring periphery may be either unsubstituted or substituted. Phthalocyanines strongly absorb clinically useful red or near IR radiation with absorption peaks falling between about 600 and 810 nm, which potentially allows deep tissue penetration by the light. The synthesis and use of a wide variety of phthalocyanines in photodynamic therapy is described in International Publication WO 2005/099689.
[0238] In some embodiments, the phthalocyanine compound is Pc4. Pc4 is relatively photostable and virtually non-toxic. In some embodiments, the phthalocyanine compound is an analog of the PDT photosensitizing drug Pc4 found to be effective in targeted bioimaging and targeted PDT of cancer in a subject, see for example, U.S. Pat. No. 9,889,199, the contents of which are hereby incorporated by reference. In some embodiments, the Pc4 analog can include Pc413.
[0239] In certain embodiments, anear-infrared (NIR) labeling agent, such as IRDye700DX (IR700), can be used as the photosensitizing agent.
[0240] Following administration and detection/localization of PSMA-targeted conjugate compounds, the targeted cancer cells can be exposed to therapeutic amount of light that causes cancer cell damage and/or suppression of cancer cell growth. The light, which is capable of activating the PDT therapeutic agent can delivered to the targeted cancer cells, using for example, semiconductor laser, dye laser, optical parametric oscillator or the like. It will be appreciated that any source light can be used as long as the light excites the phthalocyanine compound bound or complexed with a PSMA expressing cancer cell.
[0241] In some embodiments, the surgical resection site can be irradiated using visible laser diodes to photoactivate the photosensitizing compound coupled to the PSMA targeting moiety. For example, in some embodiments, the surgical resection site can be irradiated using visible laser diodes emitting at 672 nm. In certain embodiments, the surgical resection site can be irradiated with an amount of radiation effective to inhibit tumor recurrence in the subject. In an exemplary embodiment, the PDT step of the method of treating cancer described herein can include irradiating the resection site bed with a 672 nm laser for 12.5 minutes with total radiant exposure of 75 J/cm.sup.2.
[0242] In some embodiments, where the sensitizing agent coupled to the PSMA targeting moiety is IR700, the surgical resection site can be irradiated using visible laser diodes emitting at 690 nm. It is contemplated that the absorption peak of IR700 at the near infra-red permits increased light penetration across tissues. Photoactivation of IR700 specifically kills PSMA-expressing tumor cells while sparing the tumor microenvironment as well as the surrounding healthy tissues.
[0243] Following the PDT step of a method of treating cancer described herein, the resection site of a subject can be further imaged and irradiated after a period of time(s) to detect and ablate residual PSMA expressing cancer cells that may have survived previous irradiation. This optional step may or may not include additional administration of a pharmaceutical composition including a PSMA targeted conjugate compound described herein.
[0244] The PSMA targeted conjugate compounds can be used to deliver a therapeutic anti-cancer agent to a PSMA expressing cancer cell of a subject or mammal. Therefore, some embodiments relate to the administration of a PSMA targeted conjugate compound coupled to an anticancer agent to a subject having or suspected of having cancer, such as a PSMA expressing cancer for the treatment of cancer. In particular embodiments, a PSMA targeted conjugate compounds coupled to an anticancer agent can be administered to a subject having prostate cancer.
[0245] The PSMA targeted conjugate compounds can target and transiently interact with, bind to, and/or couple with a cancer cell, such as a prostate cancer cell, and once interacting with, bound to, or coupled to the targeted cell or tissue advantageously facilitate delivery of a therapeutic agent within cell by, for example, receptor mediated endocytosis. Without being bound by theory, where the PSMA targeted conjugate compound includes a therapeutic agent, it is believed that once delivered to a PSMA expressing cell, endosomal conditions can release the therapeutic agent, e.g., doxorubicin, where it travels to the nucleus, binds to DNA and exerts anti-proliferative effects.
[0246] Examples of anticancer agents that can be directly or indirectly coupled to a PSMA targeting conjugate compound as described herein can include, but are not limited to chemotherapeutic agents and other agents including Taxol, Adriamycin, Dactinomycin, Bleomycin, Vinblastine, Cisplatin, acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflomithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon -2a; interferon -2b; interferon -n1; interferon -n3; interferon -I a; interferon -I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride.
[0247] Other examples of anti-cancer agents include, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; 9-dioxamycin; diphenyl spiromustine; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen-binding protein; silicon phthalocyanine (PC4) sizofuran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans (GAGs); tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
[0248] Still other examples of anti-cancer agents can include the following marketed drugs and drugs in development: Erbulozole (also known as R-55104), Dolastatin 10 (also known as DLS-10 and NSC-376128), Mivobulin isethionate (also known as CI-980), Vincristine, NSC-639829, Discodermolide (also known as NVP-XX-A-296), ABT-751 (Abbott, also known as E-7010), Altorhyrtins (such as Altorhyrtin A and Altorhyrtin C), Spongistatins (such as Spongistatin 1, Spongistatin 2, Spongistatin 3, Spongistatin 4, Spongistatin 5, Spongistatin 6, Spongistatin 7, Spongistatin 8, and Spongistatin 9), Cemadotin hydrochloride (also known as LU-103793 and NSC-D-669356), Epothilones (such as Epothilone A, Epothilone B, Epothilone C (also known as desoxyepothilone A or dEpoA), Epothilone D (also referred to as KOS-862, dEpoB, and desoxyepothilone B), Epothilone E, Epothilone F, Epothilone B N-oxide, Epothilone A N-oxide, 16-aza-epothilone B, 21-aminoepothilone B (also known as BMS-310705), 21-hydroxyepothilone D (also known as Desoxyepothilone F and dEpoF), 26-fluoroepothilone), Auristatin PE (also known as NSC-654663), Soblidotin (also known as TZT-1027), LS-4559-P (Pharmacia, also known as LS-4577), LS-4578 (Pharmacia, also known as LS-477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR-112378 (Aventis), Vincristine sulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, also known as WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2 (Hungarian Academy of Sciences), BSF-223651 (BASF, also known as ILX-651 and LU-223651), SAH-49960 (Lilly/Novartis), SDZ-268970 (Lilly/Novartis), AM-97 (Armad/Kyowa Hakko), AM-132 (Arnad), AM-138 (Armad/Kyowa Hakko), IDN-5005 (Indena), Cryptophycin 52 (also known as LY-355703), AC-7739 (Ajinomoto, also known as AVE-8063A and CS-39.HCl), AC-7700 (Ajinomoto, also known as AVE-8062, AVE-8062A, CS-39-L-Ser.HCl, and RPR-258062A), Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (also known as NSC-106969), T-138067 (Tularik, also known as T-67, TL-138067 and TI-138067), COBRA-1 (Parker Hughes Institute, also known as DDE-261 and WHI-261), H10 (Kansas State University), H16 (Kansas State University), Oncocidin A1 (also known as BTO-956 and DIME), DDE-313 (Parker Hughes Institute), Fijianolide B, Laulimalide, SPA-2 (Parker Hughes Institute), SPA-1 (Parker Hughes Institute, also known as SPIKET-P), 3-IAABU (Cytoskeleton/Mt. Sinai School of Medicine, also known as MF-569), Narcosine (also known as NSC-5366), Nascapine, D-24851 (Asta Medica), A-105972 (Abbott), Hemiasterlin, 3-BAABU (Cytoskeleton/Mt. Sinai School of Medicine, also known as MF-191), TMPN (Arizona State University), Vanadocene acetylacetonate, T-138026 (Tularik), Monsatrol, Inanocine (also known as NSC-698666), 3-IAABE (Cytoskeleton/Mt. Sinai School of Medicine), A-204197 (Abbott), T-607 (Tularik, also known as T-900607), RPR-115781 (Aventis), Eleutherobins (such as Desmethyleleutherobin, Desaetyleleutherobin, Isoeleutherobin A, and Z-Eleutherobin), Caribaeoside, Caribaeolin, Halichondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica), Diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), Taccalonolide A, TUB-245 (Aventis), A-259754 (Abbott), Diozostatin, ()-Phenylahistin (also known as NSCL-96F037), D-68838 (Asta Medica), D-68836 (Asta Medica), Myoseverin B, D-43411 (Zentaris, also known as D-81862), A-289099 (Abbott), A-318315 (Abbott), HTI-286 (also known as SPA-110, trifluoroacetate salt) (Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC-12983 (NCl), Resverastatin phosphate sodium, BPR-OY-007 (National Health Research Institutes), and SSR-250411 (Sanofi).
[0249] Other examples of anti-cancer agents include alkylating agents, such as nitrogen mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, melphalan, etc.), ethylenimine and methylmelamines (e.g., hexamethlymelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne, semustine, streptozocin, etc.), or triazenes (decarbazine, etc.), antimetabolites, such as folic acid analog (e.g., methotrexate), or pyrimidine analogs (e.g., fluorouracil, floxouridine, Cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin, vinca alkaloids (e.g., vinblastin, vincristine), epipodophyllotoxins (e.g., etoposide, teniposide), platinum coordination complexes (e.g., cisplatin, carboblatin), anthracenedione (e.g., mitoxantrone), substituted urea (e.g., hydroxyurea), methyl hydrazine derivative (e.g., procarbazine), adrenocortical suppressant (e.g., mitotane, amino glutethimide).
[0250] In particular embodiments, the anti-cancer agent is coupled to the PSMA targeted conjugate compound. In certain embodiments, the anti-cancer agent coupled to a PSMA targeted conjugate compound is selected from doxorubicin or an ant-mitotic agent or monomethyl auristatin E (MMAE).
[0251] In an embodiment, a PSMA targeted conjugate compound that is coupled to a therapeutic anti-cancer agent can have the formula:
##STR00029## ##STR00030## ##STR00031##
or pharmaceutical salts thereof.
[0252] In some embodiments, PSMA targeted conjugate compounds can be coupled to both a therapeutic agent and a NIR imaging agent to provide targeted delivery of the therapeutic agent, such as an anti-cancer agent, while also allowing for tumor imaging and targeted photodynamic therapy.
[0253] In an embodiment, a PSMA targeted conjugate compound can be coupled to the anti-cancer agent MMAE via a MC-VC-PABC protease cleavable linker and coupled to the near-infrared labeling agent or photosensitizer IR700 (i.e., a PSMA-1-VcMMAE-IR700 conjugate compound) and have the formula:
##STR00032##
or a pharmaceutical salt thereof.
[0254] In an embodiment, PSMA-1-VcMMAE-IR700 conjugate compounds administered to subject can provide for targeted delivery of the MMAE chemotherapeutic agent to PSMA+prostate cancer cells while the NIR imaging agent IR700 coupled to PSMA-1 allows for image guidance during prostate tumor resection and subsequent targeted PDT to eliminate unresectable or remaining cancer cells.
[0255] In another embodiment, a PSMA targeted conjugate compound can be coupled to the anti-cancer agent MMAE via a MC-VC-PABC protease cleavable linker and coupled to chelating agent optionally complexed metal ion. For example, the PSMA targeted conjugate compound including MMAE and a chelating agent optionally complexed with a metal ion can have the following formula:
##STR00033## [0256] or a pharmaceutically acceptable salt thereof. The metal ion complexed with the chelating agent can include at least one of a radioactive isotope of Ga, I, In, Y, Lu, Bi, Ac, Re, In, Th, Tc, Tl, Tb, Zr, Cu, R.sup.b, At, Pb, Gd, Sm, or Sr. In other embodiments, the metal ion includes at least one of .sup.186Re, .sup.188Re, .sup.99mTc, .sup.153Gd, .sup.111In, .sup.67Ga, .sup.68Ga, .sup.201Tl, .sup.82Rb, .sup.64Cu, .sup.89Zr, .sup.90Y, .sup.177Lu, T(tritium), .sup.149T, .sup.161Tb, .sup.153Sm, .sup.89Sr, .sup.211At, .sup.225Ac, .sup.227Ac, .sup.123-125I, .sup.131I, .sup.67Cu, .sup.203Pb, .sup.212Pb, .sup.211Bi, .sup.213Bi, or .sup.227-233Th.
[0257] The PSMA targeting conjugate compounds can be administered alone as a monotherapy, or in conjunction with or in combination with one or more additional therapeutic agents. In some embodiments, a PSMA targeting conjugate compound described herein can be administered to the subject in combination with an additional anti-cancer agent. In a particular embodiment, a PSMA targeting conjugate compound coupled to doxorubicin as described herein can be administered to the subject in combination with the anti-cancer agent docetaxel.
[0258] It will be appreciated that additional detectable moieties, therapeutic agents, and/or theranostic agents administered to a subject need not be conjugated directly or indirectly to the PSMA ligand of the PSMA targeting compound and can optionally be provided in a pharmaceutical composition or preparation with the PSMA targeting conjugate compounds described herein or in a separate pharmaceutical composition.
[0259] The term in conjunction with or in combination with indicates that the PSMA targeting conjugate compound is administered at about the same time as the additional agent. The PSMA targeting conjugate compound can be administered to the subject in need thereof as part of a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier or excipient and, optionally, one or more additional therapeutic agents. The compound and additional therapeutic agent can be components of separate pharmaceutical compositions, which can be mixed together prior to administration or administered separately. The PSMA targeting conjugate compound can, for example, be administered in a composition containing the additional therapeutic agent, and thereby, administered contemporaneously with the agent. Alternatively, the PSMA targeting conjugate compound can be administered contemporaneously, without mixing (e.g., by delivery of the compound on the intravenous line by which the compound is also administered, or vice versa). In another embodiment, the PSMA targeting conjugate compound can be administered separately (e.g., not admixed), but within a short timeframe (e.g., within 24 hours) of administration of the compound.
[0260] The disclosed PSMA targeting conjugate compounds and additional therapeutic agents, detectable moieties, and/or theranostic agents described herein can be administered to a subject by any conventional method of drug administration, for example, orally in capsules, suspensions or tablets or by parenteral administration. Parenteral administration can include, for example, intramuscular, intravenous, intraventricular, intraarterial, intrathecal, subcutaneous, or intraperitoneal administration. The disclosed compounds can also be administered orally (e.g., in capsules, suspensions, tablets or dietary), nasally (e.g., solution, suspension), transdermally, intradermally, topically (e.g., cream, ointment), inhalation (e.g., intrabronchial, intranasal, oral inhalation or intranasal drops) transmucosally or rectally. Delivery can also be by injection into the brain or body cavity of a patient or by use of a timed release or sustained release matrix delivery systems, or by onsite delivery using micelles, gels and liposomes. Nebulizing devices, powder inhalers, and aerosolized solutions may also be used to administer such preparations to the respiratory tract. Delivery can be in vivo, or ex vivo. Administration can be local or systemic as indicated. More than one route can be used concurrently, if desired. The preferred mode of administration can vary depending upon the particular disclosed compound chosen. In specific embodiments, oral, parenteral, or systemic administration (e.g., intravenous) are preferred modes of administration for treatment.
[0261] The methods described herein contemplate either single or multiple administrations, given either simultaneously or over an extended period of time. The PSMA targeting conjugate compound (or composition containing the compound) can be administered at regular intervals, depending on the nature and extent of the inflammatory disorder's effects, and on an ongoing basis. Administration at a regular interval, as used herein, indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time dose). In one embodiment, the PSMA targeting conjugate compound is administered periodically, e.g., at a regular interval (e.g., bimonthly, monthly, biweekly, weekly, twice weekly, daily, twice a day or three times or more often a day).
[0262] The administration interval for a single individual can be fixed, or can be varied over time, depending on the needs of the individual. For example, in times of physical illness or stress, or if disease symptoms worsen, the interval between doses can be decreased. Depending upon the half-life of the agent in the subject, the agent can be administered between, for example, once a day or once a week.
[0263] For example, the administration of the PSMA targeting conjugate compound described herein and/or the additional therapeutic agent can take place at least once on day 1, 2,3, 4,5, 6, 7, 8, 9,10,11,12,13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least once on week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or any combination thereof, using single or divided doses of every 60, 48, 36, 24, 12, 8, 6, 4, or 2 hours, or any combination thereof. Administration can take place at any time of day, for example, in the morning, the afternoon or evening. For instance, the administration can take place in the morning, e.g., between 6:00 a.m. and 12:00 noon; in the afternoon, e.g., after noon and before 6:00 p.m.; or in the evening, e.g., between 6:01 p.m. and midnight.
[0264] A disclosed PSMA targeting conjugate compound and/or additional therapeutic agent can be administered in a dosage of, for example, 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day. Dosage forms (composition) suitable for internal administration generally contain from about 0.1 milligram to about 500 milligrams of active ingredient per unit. In these pharmaceutical compositions, the active ingredient will ordinarily be present in an amount of about 0.5-95% by weight based on the total weight of the composition.
[0265] The amount of disclosed PSMA targeting conjugate compound and/or additional therapeutic agent administered to the subject can depend on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs as well as the degree, severity and type of rejection. The skilled artisan will be able to determine appropriate dosages depending on these and other factors using standard clinical techniques.
[0266] In addition, in vitro or in vivo assays can be employed to identify desired dosage ranges. The dose to be employed can also depend on the route of administration, the seriousness of the disease, and the subject's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. The amount of the compound can also depend on the disease state or condition being treated along with the clinical factors and the route of administration of the compound.
[0267] For treating humans or animals, the amount of disclosed PSMA targeting conjugate compound and/or additional therapeutic agent administered (in milligrams of compound per kilograms of subject body weight) is generally from about 0.1 mg/kg to about 100 mg/kg, typically from about 1 mg/kg to about 50 mg/kg, or more typically from about 1 mg/kg to about 25 mg/kg. In a preferred embodiment, the effective amount of agent or PSMA targeting conjugate compound is about 1-10 mg/kg. In another preferred embodiment, the effective amount of agent or PSMA targeting conjugate compound is about 1-5 mg/kg. The effective amount for a subject can be varied (e.g., increased or decreased) over time, depending on the needs of the subject.
[0268] The term unit dose refers to a physically discrete unit suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material that can produce the desired therapeutic effect in association with the required diluent; e.g., carrier or vehicle. In addition to the ingredients particularly mentioned above, the formulations of the present invention may include other agents conventional in the art having regard to the type of formulation in question.
[0269] The disclosed PSMA targeting conjugate compound and/or additional therapeutic agent described herein can be administered to the subject in conjunction with an acceptable pharmaceutical carrier or diluent as part of a pharmaceutical composition for therapy. Formulation of the PSMA targeting conjugate compound to be administered will vary according to the route of administration selected (e.g., solution, emulsion, capsule, and the like). Suitable pharmaceutically acceptable carriers may contain inert ingredients that do not unduly inhibit the biological activity of the PSMA targeting conjugate compounds. The pharmaceutically acceptable carriers should be biocompatible, e.g., non-toxic, non-inflammatory, non-immunogenic and devoid of other undesired reactions upon the administration to a subject. Standard pharmaceutical formulation techniques can be employed, such as those described in Remington's Pharmaceutical Sciences, ibid. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer's-lactate and the like. Methods for encapsulating compositions (such as in a coating of hard gelatin or cyclodextran) are known in the art (Baker, et al., Controlled Release of Biological Active Agents, John Wiley and Sons, 1986).
[0270] The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art. Typically, such compositions are prepared as injectables as either liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. Formulation will vary according to the route of administration selected (e.g., solution, emulsion, capsule).
[0271] A pharmaceutically acceptable carrier for a pharmaceutical composition can also include delivery systems known to the art for entraining or encapsulating drugs, such as anticancer drugs. In some embodiments, the disclosed compounds can be employed with such delivery systems including, for example, liposomes, nanoparticles, nanospheres, nanodiscs, dendrimers, and the like. See, for example Farokhzad, 0. C., Jon, S., Khademhosseini, A., Tran, T. N., Lavan, D. A., and Langer, R. (2004). Nanoparticle-aptamer bioconjugates: a new approach for targeting prostate cancer cells. Cancer Res., 64, 7668-72; Dass, C. R. (2002). Vehicles for oligonucleotide delivery to tumours. J. Pharm. Pharmacol., 54, 3-27; Lysik, M. A., and Wu-Pong, S. (2003). Innovations in oligonucleotide drug delivery. J. Pharm. Sci., 92, 1559-73; Shoji, Y., and Nakashima, H. (2004). Current status of delivery systems to improve target efficacy of oligonucleotides. Curr. Pharm. Des., 10, 785-96; Allen, T. M., and Cullis, P. R. (2004). Drug delivery systems: entering the mainstream. Science, 303, 1818-22. The entire teachings of each reference cited in this paragraph are incorporated herein by reference.
[0272] The following examples are for the purpose of illustration only and are not intended to limit the scope of the claims, which are appended hereto.
Example 1
[0273] In this Example, we used our PSMA ligand to selectively deliver the very potent microtubule disruption drug, monomethyl auristatin E (MMAE), to prostate cancer cells. Advantageously, using the PSMA ligand to selectively target a chemotherapeutic drug to PSMA was found to improve pharmacokinetics and enable more precise chemotherapy with lower side-effects. Using a prodrug strategy we developed a therapeutic small molecule, PSMA-1-VcMMAE, and a theranostic molecule, PSMA-1-VcMMAE-Cy5.5, in which the near-infrared dye Cy5.5 was incorporated in the structure to allow modification of pharmacokinetics and to follow tumor response to the treatment in mouse models. In heterotopic, orthotopic, and metastatic prostate cancer models in mice we found that both conjugates selectively and effectively inhibited PSMA-expressing tumor growth and prolonged animal survival with no obvious toxicity. PSMA-1-VcMMAE also had a much more favorable therapeutic index than either PSMA-1-VcMMAE-Cy5.5 or PSMA-ADC.
Materials and Methods
General
[0274] PSMA targeting peptide Glu-CO-Glu-Amc-Ahx-Glu-Glu-Glu-Cys-C6-Lys (PSMA-1-Cys-C6-Lys) was synthesized by Fmoc chemistry. (S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido)pentanedioic acid (Cys-CO-Glu) was custom made by Bachem Bioscience Inc. All the other chemicals were purchased from Sigma-Aldrich Inc. HPLC was performed on a Shimadzu HPLC system equipped with a SPD-20A prominence UV/visible detector and monitored at 220 nm and 254 nm. Preparative HPLC was achieved using Luna 5m C18(2) 100A column (250 mm10 mm5 mm; Phenomenex) at a flow rate of 2.5 m/min. Analytical HPLC was performed using an analytical Luna 5m C18(2) 100A column (250 mm4.6 mm 5 mm; Phenomenex) at a flow rate of 0.8 mL/min. The gradient used was 10% to 90% acetonitrile against 0.1% trifluoroacetic acid over 30 minutes.
Synthesis of PSMA-1-VcMMAE
[0275] PSMA-1-VcMMAE was synthesized as a prodrug by conjugating MMAE to the Cys residue in PSMA-1-Cys-C6-Lys via a maleimido caproyl valine-citruline (Vc) cathepsin-cleavable linker with a self immolative p-aminobenzyl carbamate (PABC) spacer. PSMA-1-Cys-C6-Lys (2.6 mg, 2 mol) was dissolved in 500 L of phosphate buffer; then 2.2 mol of Vc-MMAE (3.0 mg) (BOC Sci.) in 500 L of DMF was added. The pH of the reaction mixture was adjusted to 8 by triethylamine. The mixture was stirred at room temperature for 1 hour, then went through HPLC to get purified PSMA-1-VcMMAE. Yield: 4.4 mg (85%). Retention time: 16.8 min. MS (C.sub.123H.sub.195N.sub.23O.sub.37S). calculated: 2618.3. found: 1310.7 ([M+2H]/2), 874.1 ([M+3H]/3).
Synthesis of PSMA-1-VcMMAE-Cy5.5
[0276] PSMA-1-Cys-C6-Lys (2.6 mg, 2 mol) was dissolved in 500 L of phosphate buffer; then 2.2 mol of Vc-MMAE (3.0 mg) (BOC Sci.) in 500 L of DMF was added. The pH of the reaction mixture was adjusted to 8 by trimethylamine. After stirring at room temperature for 1 hour, Cy5.5 NHS ester 2.5 mol in 200 L of DMF was added. The reaction mixture was stirred overnight, then the product was purified by semi-preparative HPLC. Yield: 3.1 mg (50%). Retention time: 18.8 min. MS (C.sub.163H.sub.236N.sub.25O.sub.38S). calculated: 3185.8. found: 1062.2 ([M+3H]/3), 796.9 ([M+4H]/4).
Synthesis of PSMA-1-McMMAE-Cy5.5
[0277] PSMA-1-McMMAE-Cy5.5 was synthesized using a non-cleavable maleimido caproyl (Mc) linker. It was synthesized in the same way as PMA-1-MMAE-Cy5.5. Yield: 45%. Retention time: 20.9 min. MS (C.sub.144H.sub.209N.sub.20O.sub.33S). calculated: 2780.4. found: 1041.2 ([M+H+Na]/2), 942.2 ([M+H+2Na]/3).
Cell Culture
[0278] Retrovirally transfected PSMA positive PC3pip cells and transfection control PC3flu cells were obtained from Dr. Michel Sadelain in 2000 (Laboratory of Gene Transfer and Gene Expression, Gene Transfer and Somatic Cell Engineering Facility, Memorial-Sloan Kettering Cancer Center, New York, NY). C4-2 cells were from ATCC. The cells were last sorted and checked by western blot in 2019; no genetic authentication was performed. Cells were maintained in RPMI1640 medium (Invitrogen) with 2 mM L-glutamine and 10% Fetal Bovine Serum at 37 C. and 5% CO.sub.2 under a humidified atmosphere.
Competitive Binding Assay
[0279] The assay was carried as previously reported by incubation PC3pip cells with different concentrations of drug conjugates in the presence of 10 nM N[N[(S)-1,3-dicarboxypropyl]carbamoyl]-S-[.sup.3H]-methyl-L-cysteine (.sup.3H-ZJ24) (GE Healthcare Life Sciences, Pittsburgh, PA). Radioactivity of cell pellet was counted by scintillation counter. The concentration required to inhibit 50% of binding was determined (IC.sub.50) by GraphPad Prism 3.0.
Enzymatic Cleavage of PSMA-1-MMAE-Cy5.5 by Cathepsin B
[0280] PSMA-1-VcMMAE-Cy5.5 or PSMA-1-McMMAE-Cy5.5 was added to 500 L of activated human liver cathepsin B (Anthens Research and Technology) solution to a final concentration of 2 M and incubated at 37 C. At different time intervals, 40 L of the solution was taken out into tubes loaded with 1 L of 1 mM E64 protease inhibitor. The mixture was vortexed and then stored at 80 C. for future HPLC analysis to assess degradation of PSMA-1-VcMMAE-Cy5.5 or PSMA-1-McMMAE-Cy5.5. Studies were performed in triplicate.
In Vitro Cytotoxicity Assay
[0281] Cells (1,000/well) were seeded in 96-well culture plates the day before treatment. Cells were incubated with various concentrations of drugs for 72 hours and cell viability evaluated by CCK-8 (Dojindo). The concentration required to reach 50% of cell proliferation was determined (IC.sub.50) by GraphPad Prism 3.0.
In Vitro Cellular Uptake Studies
[0282] PC3pip and PC3flu cells were seeded in p-Slide 8-Well Chamber Slide (ibidi GmbH) at 2,000 cells/well. When cells grew to 70% confluency, PSMA-1-VcMMAE-Cy5.5 or PSMA-1-McMMAE-Cy5.5 conjugations were added at 50 nM and incubated at 37 C. for 4 hours. Cells were washed with PBS and stained with DAPI and LysoOrange (Abcam) for 30 minutes at 37 and 5% CO.sub.2, after which they were washed again with PBS and filled with fresh medium. Selectivity was determined by including 20-fold excess of the PSMA-1 ligand. The uptake and localization of the drug conjugates were visualized under a Leica HyVolution SP8 confocal microscope at 40.
Immunofluorescence Analysis of Alpha-Tubulin
[0283] Cells on coverslips at about 70% confluency were incubated with 5 nM of MMAE, PSMA-1-VcMMAE-Cy5.5 or PSMA-1-McMMAE-Cy5.5 for 24 hours. Cells were washed and fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. Alpha-tubulin (B5-1-2) Alexa Fluor 488 Mouse Monoclonal Antibody (Invitrogen) was then added at 2 g/mL in 0.1% BSA and incubated for 3 hours at room temperature. Cells were counterstained with DAPI, mounted with Fluor-Mount aqueous mounting solution, and observed under Leica DM4000B fluorescence microscopy at 40.
In Vivo NIR Imaging Studies
[0284] Under guidelines of the animal care and use committee at Case Western Reserve University (IACUC #150033) 6 to 8-week old male Balb/cathymic nude mice were implanted subcutaneously with 110.sup.6 of PC3flu and PC3pip on the left and right dorsum respectively. Mice received 40 nmol/kg of PSMA-1-VcMMAE-Cy5.5 or PSMA-1-McMMAE-Cy5.5 in PBS via tail vein injection when tumors reached 10 mm in diameter. Fluorescence imaging was performed using the Maestro In Vivo Imaging system (Perkin-Elmer). Multispectral images were unmixed into their component spectra (PSMA probes, autofluorescence, and background) to quantitate the average fluorescence intensity associated with the tumors using regions of interest (ROIs) around the tumors.
Maximum Tolerated Dose (MTD)
[0285] Groups of three male mice received single injections of MMAE, PSMA-1-Vc-MMAE, PSMA-1-VcMMAE-Cy5.5 or PSMA-ADC via the tail vein to determine single-dose MTD. Mice were monitored daily for 14 days. The MTD was defined as the highest dose that did not cause serious overt toxicities or 20% weight loss in any of the animals.
Heterotopic Survival Study
[0286] Male athymic nude mice were implanted subcutaneously with 110.sup.6 of PC3pip cells under the right dorsum of the mice. When tumor size reached approximately 100 mm.sup.3 (tumor volume=Lengthwidth.sup.2/2), mice received drugs through tail vein injection. Mice were treated every four days with a total of five doses. Animals were weighed and tumor size measured every other day for 90 days. Cures were defined as no tumor present at end of the 90-day study. When tumors became too large or animals were moribund they were euthanized. Five mice were used in each group.
Orthotopic Survival Study
[0287] Surgical orthotopic tumor implantation was performed as previously reported. Tumor growth/size was monitored using Siemens Acuson S2000 ultrasound scanner. When the tumors were at the appropriate size (5 mm as measured by ultrasound, approximately 2 weeks) animals were given PSMA-1-VcMMAE, PSMA-1-VcMMAE-Cy5.5, or PBS every four days with a total of 5 doses. Tumors were monitored every other day by ultrasound. Each group had five mice.
Metastatic Survival Study
[0288] Male athymic nude mice were injected into the left ventricle of the heart with 110.sup.5 GFP labeled PC3pip cells to generate bone metastasis. One week later mice received 160 nmol/kg PSMA-1-VcMMAE-Cy5.5 or PBS every 4 days with a total of 5 doses and the progression of disease was monitored by GFP imaging. Five mice were used in each group.
Statistics
[0289] Student t-test was used to compare inter-group differences. Kaplan-Meier survival data were analyzed by SAS9.4 using log-rank tests. A p value <0.05 was considered statistically significant for all comparisons.
Results
A Prodrug Strategy is Crucial for Antitumor Activity
[0290] We initially synthesized two PSMA-targeted MMAE-Cy5.5 conjugates, one with a cathepsin-cleavable linker, PSMA-1-VcMMAE-Cy5.5, and the other with a non-cleavable linker, PSMA-1-McMMAE-Cy5.5, (
[0291] Competition binding experiments demonstrated that the complexity of the drug conjugates did not impact their binding affinity to PSMA; PSMA-1-VcMMAE-Cy5.5 showed an IC.sub.50 of 3.65 nM and PSMA-1-McMMAE-Cy5.5 had an IC.sub.50 of 4.88 nM, both similar to unconjugated PSMA-1, IC.sub.50=2.30 nM and significantly lower than the parent ZJ24 ligand, IC.sub.50=11.73 nM (
[0292] To investigate if PSMA-targeted drug conjugates were cleavable by cathepsin B, the two conjugates were incubated with or without the enzyme and chromatographed by HPLC. When the conjugates were incubated with PBS, both PSMA-1-VcMMAE-Cy5.5 and PSMA-1-McMMAE-Cy5.5 were stable (
[0293] To test if PSMA-targeted drug conjugates could selectively kill PSMA-expressing cells, cytotoxicity studies were performed in both PSMA-positive PC3pip and PSMA-negative PC3flu cells. PSMA-1-VcMMAE-Cy5.5 was about 20-fold more potent at killing PSMA-positive PC3pip cells (IC.sub.50=0.84 nM) than PSMA-negative PC3flu cells (IC.sub.50=17.0 nM) (
[0294] To determine if PSMA-targeted drug conjugates would result in selective cellular binding and uptake, in vitro uptake studies were performed. Similar levels of fluorescence uptake into PC3pip cells was observed after treatment with either PSMA-1-VcMMAE-Cy5.5 or PSMA-1-McMMAE-Cy5.5 and was localized to the lysosomal compartment of the cells (
[0295] It is known that MMAE inhibits cell division by destabilization of -tubulin. To further validate the biological consequence of MMAE-conjugate treatment, in vitro immunofluorescence staining of -tubulin was performed (
[0296] To demonstrate selective tumor uptake in vivo, mice bearing both PC3flu and PC3pip tumors were injected with 40 nmol/kg of PSMA-1-VcMMAE-Cy5.5 or PSMA-1-McMMAE-Cy5.5 and uptake monitored via fluorescence imaging over time (
[0297] We next compared the antitumor activity of the two conjugates in mice bearing a flank PC3pip tumor. Each animal received 160 nmol/kg of PSMA-1-VcMMAE-Cy5.5 or PSMA-1-McMMAE-Cy5.5 through tail vein injection every four days with a total of 5 doses. (Dosing was based on previously published dose and schedules for antibody-drug conjugates). Mice were then imaged and tumors were measured by caliper. Untreated PC3pip tumor grew rapidly achieving a 50-fold increase in size by day 22 (
Efficacy of PSMA-Targeted Prodrug Conjugates In Vitro
[0298] Having confirmed that prodrug strategy is crucial for the antitumor activity, we performed studies to further explore the antitumor activity of PSMA-1-VcMMAE and PSMA-1-VcMMAE-Cy5.5 (
In Vivo Studies of Toxicity and Efficacy
[0299] The maximum tolerated dose (MTD) was determined in tumor-free athymic nude mice to determine the toxicity of the conjugates. Loss of 20% body weight or any overt signs of toxicity was used as an end point. We compared the MTD of free drug MMAE, PSMA-targeted drug conjugates and PSMA-ADC. MTDs of single dose i.v. injection of MMAE, PSMA-1-VcMMAE, PSMA-1-VcMMAE-Cy5.5 and PSMA-ADC were 700 nmol/kg, 7640 nmol/kg, 480 nmol/kg and 640 nmol/kg, respectively, highlighting the superior safety (10-fold or greater) of PSMA-1-VcMMAE compared to all the other drug derivatives.
[0300] For in vivo antitumor activity, we first studied in vivo potency of PSMA-1-VcMMAE in nude mice bearing heterotopic PC3pip tumors. Mice received drugs intravenously every 4 days with a total of 5 doses. In PBS control groups the tumor grew rapidly resulting in animal death within 30 days. In contrast treatment with PSMA-1-VcMMAE showed the ability to inhibit tumor growth and prolong animal survival in a dose dependent manner. At the highest dose tested (3820 nmol/kg, of its MDT), all 5 mice survived the 90-day experimental time and 3 out of 5 mice were tumor free resulting in 60% cure. No body weight loss was observed even at the highest dose tested. It was also found that PSMA-1-VcMMAE was significantly more effective at inhibiting PSMA-positive PC3pip tumor growth than PSMA-negative PC3flu tumor growth when used at a lower dose of 955 nmol/kg (P=0.0493). We then administered PSMA-1-VcMMAE in mice bearing orthotopic PC3pip tumors, which mimic human prostate cancer in a more realistic way. Inhibition of tumor growth and extension of animal survival time were observed in the orthotopic PC3pip tumor models, and significant differences were observed at the dose of 1910 (P=0.0449) and 3860 nmol/kg (P=0.0019) when compared to the PBS control, with 1 mouse that was tumor free at the dose of 3860 nmol/kg. Initial changes in body weight were similar among the tested groups.
[0301] We have reported that Cy5.5 can change the pharmacokinetics of the conjugate drastically. We therefore tested antitumor activity of PSMA-1-VcMMAE-Cy5.5 and also included equimolar doses of free MMAE and PSMA-ADC as controls in the study. Free MMAE was relatively ineffective at inhibiting tumor growth, even at the dose of 160 nmol/kg, all mice died within 36 days. In contrast, PSMA-1-VcMMAE-Cy5.5 effectively inhibited tumor growth, and expanded animal survival times. Significantly at 160 nmol/kg of PSMA-1-VcMMAE-Cy5.5 the tumors were completely eliminated in every case for a period of time and resulted in a cure rate (no tumors at end of study) of 60% for the 90-day experimental period. Changes in body weight were similar in each regimen and control group in the first 16 days, however, increased body weight was observed in PSMA-1-VcMMAE-Cy5.5 treated groups due to reduced disease burden. Compared to PSMA-1-VcMMAE-Cy5.5, all animals treated with 160 nmol/kg of PSMA-ADC survived the 90-day experimental time with 40% cure, the difference between the two groups was not significant (P=1.000).
[0302] We then tried PSMA-1-VcMMAE-Cy5.5 in orthotopic PC3pip tumor. No significant body weight loss was observed in mice treated with PSMA-1-VcMMAE-Cy5.5 while the PBS control mice lost body weight due to increased tumor burden. Tumor volume in PSMA-1-VcMMAE-Cy5.5 treated animals was significantly reduced and animal survival were dramatically extended (P=0.0026) with 40% of cure at day 90.
[0303] The mortality of prostate cancer is mainly due to metastasis and metastatic castration resistant prostate cancer is the most deadly and difficult form of the disease to treat. To test the effectiveness of PSMA-1-MMAE-Cy5.5 against metastatic disease, we developed a metastatic prostate cancer model using intracardiac injection of GFP-expressing PC3pip cells. We then used this model to assess the effectiveness of our drug conjugate. Treatment was initiated 1 week after cardiac injection of tumors cells, and mice received 160 nmol/kg of PSMA-1-VcMMAE-Cy5.5 every 4 days with a total of 5 doses. After treatment, no significant body weight loss was observed. The control mice died with 28 days. Maestro images of control mice showed GFP signal in tibia, spine, liver, spleen, kidney and testis, indicating tumors had metastasized to these sites. In contrast, all 5 treated mice survived the 90-day experimental period, and no GFP signal was observed in throughout the mice body, indicating a 100% response. PSMA-1-VcMMAE was also found to have the ability to prolong animal survival time with no body weight loss in the metastatic PC3pip model.
[0304] To show versatility of the antitumor activity of PSMA-1-VcMMAE-Cy5.5, we tested it in mice bearing heterotopic C4-2 tumors, which are androgen-independent prostate cancer cells endogenously expressing PSMA. PSMA-1-VcMMAE-Cy5.5 showed the ability to successfully inhibit C4-2 tumor growth with no weight loss and prolonged animal survival significantly (P=0.0018) with a 40% cure rate.
[0305] The targeted delivery of potent cytotoxic agents has emerged as a promising strategy for the treatment of cancer. In this study, we demonstrate that targeted delivery of MMAE can be more efficacious and less toxic than MMAE. More remarkably, PSMA-1-MMAE has an improved therapeutic index compared to PSMA-ADC.
Prodrug Strategy is Vital for Antitumor Activity
[0306] Targeted strategies require three components: the targeting ligand which can be an antibody, small molecular ligand or aptamer; the cytotoxic payload; and the linker. PSMA is a well-established biomarker for prostate cancer. We used our PSMA-1 ligand to target and deliver the potent microtubule polymerization inhibitor, MMAE, to PSMA-expressing prostate cancer cells. For linker selection, there are two types of linker-cleavable and non-cleavable linkers. To determine which linker would be most suitable, we tried both the cathepsin cleavable maleimido-caproyl-Val-Cit-PABC linker (Vc) (PSMA-1-VcMMAE-Cy5.5) (
Targeting Improves the Therapeutic Profile of MMAE
[0307] MMAE is a favored drug payload for ADCs, with many in clinical trials, and is usually conjugated to the antibody through a cathepsin B cleavable Vc linker, exploiting the ability to release drug intracellularly. Here, we replaced the antibody with a PSMA targeting ligand (PSMA-1) and developed a small-molecule-drug conjugate to reduce the cost and shorten the circulation time, potentially reducing the off-target toxicities resulting from longer blood half-lives. In vitro comparison of the cytotoxicity of free MMAE (
[0308] Comparing the maximum tolerated dose (MTD) of MMAE, PSMA-ADC and PSMA-1-drug conjugates, PSMA-1-VcMMAE was more than 10-fold better than MMAE. This is likely due to targeted drug delivery. Interestingly, the MTD for PSMA-ADC was much lower than our lower affinity small molecule conjugate PSMA-1-VcMMAE, but very similar to PSMA-1-VcMMAE-Cy5.5. Although the MTDs of PSMA-ADC and PSMA-1-VcMMAE-Cy5.5 were lower than MMAE itself, their in vivo effective doses were disproportionately better. At the dose of 160 nmol/kg, PSMA-1-VcMMAE-Cy5.5 and PSMA-ADC potently inhibited PC3pip tumor growth leading to significant prolonged survival time, while MMAE did not show any effectiveness at the same dose. No significant difference in animal survival time was observed between PSMA-1-VcMMAE-Cy5.5 and PSMA-ADC treatment groups (P=1.000), suggesting that PSMA-1-VcMMAE-Cy5.5 has similar efficacy to PSMA-ADC. Compared to PSMA-ADC and PSMA-1-VcMMAE-Cy5.5, PSMA-1-VcMMAE needed a higher dose to achieve the same treatment effect when given at the same dosing schedule, which is likely attributed to differences in pharmacokinetic characteristics. Our previous studies have shown that the hydrophobicity/hydrophilicity of conjugated dyes can dramatically impact the pharmacokinetics of the conjugates, with more hydrophilic conjugates resulting in a shorter time-to-peak accumulation in tumors and shorter circulation times. It may be that integration of hydrophobic Cy5.5 in PSMA-1-VcMMAE-Cy5.5 provided elongated circulation of the drug conjugate in vivo, maximizing antitumor activity. The difference in pharmacokinetics may contribute to the different MTDs of PSMA-1-VcMMAE, PSMA-1-VcMMAE-Cy5.5 and PSMA-ADC. Future pharmacokinetic studies are required to confirm our hypothesis and optimize these agents. It was noticed that animals treated by the drug conjugates showed no body weight loss due to the treatment, indicating low toxicity of the drug conjugates. Biodistribution studies using PSMA-1-VcMMAE-Cy5.5 showed that the fluorescence from the conjugate was associated with PC3pip tumors with minimal signals observed in the liver, spleen, lungs etc. This may indirectly indicate the low toxicity of the conjugate in other organs. However, MMAE will be detached from Cy5.5 in vivo, the information obtained from fluorescence imaging may not be directly correlated to the biodistribution of the drug. Detailed biodistribution and toxicity studies are still needed.
[0309] Notably, PSMA-1-VcMMAE and PSMA-1-VcMMAE-Cy5.5 showed the ability to significantly prolong the survival time of animals in metastatic PC3pip tumor model. In spite of the fact that the treatment initiated one week after tumor inoculation, before the metastasis was detectable using imaging, it is encouraging that it exhibited the ability to successfully prevent tumor metastasis. As a significant percentage of patients with prostate cancer die from the metastatic form of the disease due to lack of effective treatment options, it is critical to develop potent treatments that can effectively eradicate cancer cells and control metastatic tumors. We also found that both PSMA-1-VcMMAE and PSMA-1-VcMMAE-Cy5.5 greatly inhibited tumor growth when given every 7 days.
[0310] PSMA-1-VcMMAE utilizes the same targeting strategy as PSMA-ADC, but has shorter circulation time due to its smaller size and can be easily excreted from the body leading to reduced toxicity. PSMA-1-VcMMAE effectively inhibited tumor growth starting at the dose of 191 nmol/kg, and its' MTD was at 7640 nmol/kg, resulting in a therapeutic index of 40. The TI of PSMA-1-VcMMAE-Cy5.5 is 480/80=6. PSMA-ADC was reported to be effective starting at 2.0-3.0 mg/kg (53 nmol/kg-80 nmol/kg), its MTD was at 640 nmol/kg resulting in its TI at 8-12. By replacing the antibody with our PSMA-1 ligand, PSMA-1-VcMMAE dramatically improved the therapeutic index. These characteristics will help it avoid the problems of PSMA-ADC found in clinical trials. A theranostic such as PSMA-1-VcMMAE-Cy5.5 will allow modification of pharmacokinetics and will play a key role in initial drug development by enabling visualization of drug-target engagement, tumor size, and providing feedback information on the therapy status. Even though after prodrug activation by proteases Cy5.5 is released and physically separated from MMAE, this theranostic molecule provides significant data about drug targeting and allows greater ease and less time for small animal optimization studies. It is the first example of combining targeted small molecules with therapeutics and fluorophores for a theranostic approach to develop targeted drugs for prostate cancer.
[0311] In summary, we have developed small-molecule-based prodrugs for the treatment of prostate cancer. Their antitumor activities were demonstrated not only in androgen independent PC3pip heterotopic, orthotopic and metastatic models but also in androgen independent C4-2 tumor models that endogenously express PSMA, with no toxicity observed.
Example 2
[0312] In this Example, we developed a multifunctional theranostic approach that combines a cytotoxic drug (MMAE), a photosensitizer (IR700) and a low molecular weight PSMA targeting ligand (PSMA-1-Cys-C6-Lys) into a single molecule, PSMA-1-MMAE-IR700, that selectively and simultaneously delivers both chemotherapeutic drugs and photosensitizers to cancer cells (
Methods
Materials
[0313] PSMA-1-Cys-C6-Lys (Glu-CO-Glu-Amc-Ahx-Glu-Glu-Glu-Cys-C6-Lys) was synthesized manually by solid phase peptide synthesis method as reported previously. VcMMAE was purchased from Creative Biolabs (Shirley, NY). IRDye700 NHS ester was purchased from Li-Cor Biosciences (Lincoln, NE). PSMA-1-IR700 was synthesized as previously reported. (S)-2-(3-((S)-5-amino-1-carboxypentyl)-ureido)pentanedioic acid (ZJ24) was custom made by Bachem Bioscience Inc (Torrance, CA). Tritium labeled ZJ24 (N[N[(S)-1,3-dicarboxypropyl]-carbamoyl]-S-[3H]-methyl-L-cysteine, 3H-ZJ24) was custom synthesized by GE Healthcare Life Sciences (Chicago, IL). All the other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
High Performance Liquid Chromatography (HPLC)
[0314] HPLC was performed on a Shimadzu HPLC system equipped with an SPD-20A prominence UV/visible detector and monitored at 220 nm and 254 nm [57, 58, 60]. Semi-preparative HPLC was achieved using Luna 5m C18(2) 100 column (250 mm10 mm5 mm; Phenomenex) at a flow rate of 2.5 mL/min. Analytical HPLC was performed using an analytical Luna 5m C18(2) 100 column (250 mm4.6 mm5 mm; Phenomenex) at a flow rate of 0.8 m/min. The gradient used to purify PSMA-1-MMAE-IR700 was 10% to 90% acetonitrile against 25 mM triethyl-ammonium acetate (TEAA, pH 7.5) over 30 min.
Synthesis of PSMA-1-MMAE-IR700
[0315] PSMA-1-VcMMAE was synthesized as previously reported. Briefly, PSMA-1-Cys-C6-Lys (2.6 mg, 2 mol) was dissolved in phosphate buffered saline (PBS), the pH of the solution was adjusted to 7.5-8.0, then Vc-MMAE (3.0 mg, 2.2 mol) (BOC Sci.) in 500 L of DMF was added. The reaction mixture was allowed to react at room temperature for 1 h. PSMA-1-VcMMAE was then purified by semi-preparative HPLC and lyophilized. Retention time: 17.6 min. Mass spectrum (MS) (C.sub.123H.sub.195N.sub.23O.sub.37S). calculated: 2618.3. found: 2619 (M+1). Purified PSMA-1-VcMMAE (1.3 mg, 0.5 mol) was dissolved in 0.5 mL PBS. The pH of the solution was adjusted to 7.5, then IRDye700 NHS ester (1.9 mg, 1 mol, Li-Cor Inc.) in 0.5 mL PBS was added. The mixture was allowed to react at room temperature overnight. PSMA-1-MMAE-IR700 was then purified by HPLC. Yield: 1.6 mg, 76%. Retention time: 19.9 min. Mass spectrum (MS) (ammonium salt: C.sub.193H.sub.302N.sub.38O.sub.61S.sub.7Si.sub.3). calculated: 4439. found: 971 ([M4 NH.sub.4-C.sub.14H.sub.30NO.sub.10S.sub.3Si]/4), 1091 ([M4 NH]/4), 1295 ([M4 NH-.sub.4 C.sub.14H.sub.30NO.sub.10S.sub.3Si]/3), 1456 ([M4 NH.sub.4]/3), 1943 ([M4 NH.sub.4-C.sub.14H.sub.30NO.sub.10S.sub.3Si]/2).
Cell Culture
[0316] PSMA-positive PC3pip and PSMA-negative PC3flu cells were maintained in RPMI medium with 10% Fetal Bovine Serum at 37 C. and 5% CO.sub.2 under a humidified atmosphere. The cells were last sorted and checked by western blot in 2021.
Competition Binding Studies
[0317] PC3pip cells (5105) was suspended in 200 L of 50 mM Tris buffer, pH 7.5. The cells were incubated at 37 C. with different concentrations of PSMA-1-MMAE-IR700 or ZJ24 in the presence of 12 nM of 3H-ZJ24 for 1 h. The cells were then washed 3 times with cold PBS and cell-associated radioactivity was measured by scintillation counting. The concentration required to inhibit 50% of the binding (IC.sub.50) was determined by GraphPad Prism 3.0. Studies were performed in triplicate.
Cathespin Cleavage Studies
[0318] Enzymatic cleavage study of PSMA-1-MMAE-IR700 was performed as previously described. PSMA-1-MMAE-IR700 was incubated with activated human liver cathespin (Anthens Research and Technology, Anthens, GA) at 37 C. At different incubation time, 40 L of the solution was placed into tubes loaded with 1 L of 1 mM E64 protease inhibitor. The mixture was vortexed and then stored at 80 C. for future HPLC analysis. Studies were performed in triplicate. In vitro cellular uptake studies cells were plated on coverslips at about 60-70% confluency. Twenty-four hours later, cells were incubated with 50 nM of PSMA-1-MMAE-IR700. After incubation for various times (15 min, 30 min, 1 h and 4 h), cells were washed twice with cold RPMI 1640, fixed with 4% paraformaldehyde and counterstained with 4,6-diamidino-2-phenylindole (DAPI). Images were taken using a Leica DM4000B fluorescence microscope (Leica Biosystems, Buffalo Gove, IL) at 40. Blocking experiments were performed by co-incubation of cells with 50 nM of PSMA-1-MMAE-IR700 and 100 of PSMA-1 ligand. Studies were performed in triplicate.
In Vitro Cytotoxicity Studies
[0319] PC3pip and PC3flu cells were plated at 3,000 cells/well in 96-well plates. Twenty-four hours later, drugs (PSMA-1-MMAE-IR700 or PSMA-1-IR700) of different concentrations were added. After incubation at 37 C. in the dark for 24 h, cell viability was determined by CellTiter 96 aqueous one solution cell proliferation assay using absorbance at 490 nm (Promega Biotech, Madison, MI). The concentration required to inhibit 50% of cell growth (IC.sub.50) was determined by GraphPad Prism 3.0. To test the cytotoxicity of PSMA-1-MMAE-TR700 with light irradiation, drugs at a final concentration of 5 nM were added to cells and incubated in the dark for 24 h. Cells were then washed 3 times with RMPI 1640 and then irradiated with 690 nm light (L690-66-60, Marubeni America Co, New York, NY). Cells were incubated in the dark for another 24 h. Cell viability was then determined by CellTiter 96 aqueous one solution cell proliferation assay (Promega Biotech, Madison, MI). The coefficient drug interaction (CDI) was calculated as follows: CDI=AB/(AB), where AB is the ratio of the absorbance of combination treatment groups (PSMA-1-MMAE-IR700 with light irradiation) to the absorbance of control groups; A or B is the absorbance of single treatment group (PSMA-1-MMAE-IR700 without light or PSMA-1-IR700 with light irradiation) to the absorbance of control groups. CDI <1, =1 and >1 indicates synergistic, additive or antagonistic effect.
In Vivo Fluorescence Imaging Studies
[0320] Animal experiments were approved by the animal care and use committee at Case Western Reserve University (IACUC #150033). Six to eight-week-old male Balb/c athymic nude mice (Jackson Laboratory, Bar Harbor, ME) were implanted subcutaneously with 110.sup.6 of PC3pip (right flank) and PC3flu (left flank) cells in 100 l of matrigel. Animals were ready to use when tumor diameter reached 10 mm, about two weeks. Animals received 100 nmol/kg of PSMA-1-MMAE-IR700 through tail vein injection and were imaged at different time points by Maestro In vivo Image System (Perkin Elmer, Waltham, MI) using the yellow filter set (excitation 575-605 nm, emission 645 nm longpass). During imaging, mice were anesthetized by isoflurane. At 48 h post injection, mice were sacrificed and tissues such as heart, lung, liver, kidneys, stomach, tumors were harvested for ex vivo imaging. Multispectral images were unmixed into their component spectra and average fluorescence signals were quantified by creating regions of interest. Experiments were performed in 5 mice.
[0321] Orthotopic PC3pipGFP prostate cancer models were established as previously described using green fluorescence protein (GFP) transfected PC3pip cells. Mice bearing orthotopic PC3pipGFP tumors were injected with 100 nmol/kg of PSMA-1-MMAE-IR700. Mice were imaged at 1 h post injection by Maestro using the yellow filter set for PSMA-1-MMAE-IR700 and blue filter set for GFP (excitation 445-490 nm, emission 515 nm longpass). Mice were then euthanized and primary tumor was removed to expose lymph nodes. The mice were imaged again. Resected primary tumor and lymph nodes were snap-frozen in optimal cutting temperature (OCT) compound and sectioned. The slides were subjected to hematoxylin and eosin (H&E) staining, and adjacent set of slides were counter stained with DAPI and observed under Leica DM4000B fluorescence microscope at 10 to visualize DAPI, GFP and PSMA-1-MMAE-IR700. Experiments were repeated in three mice.
In Vivo Antitumor Efficacy Studies
[0322] The effect of PSMA-1-MMAE-IR700 were tested in mice bearing PC3pip tumors. Animals with tumor size at about 100 mm3 were used for the study (tumor volume=Lengthwidth2/2). Animals were divided into 7 groups: (1) mice receiving PBS; (2) mice receiving 100 nmol/kg PSMA-1-MMAE-IR700 with PDT; (3) mice receiving equal doses of PSMA-1-MMAE-IR700 to group 2, but not receiving PDT; (4) Mice receiving equal doses of PSMA-1-IR700 to group 2 with PDT treatment; (5) Mice receiving equal doses of PSMA-1-IR700 to group 2 without PDT; (6) mice receiving equal doses of PSMA-1-IR700 to group 2+free MMAE with PDT (MMAE normalized to that delivered by PSMA-1-MMAE-IR700); and (7) mice receiving equal doses of PSMA-1-IR700 to group 2+free MMAE without PDT. Each group had 5 mice. Animals received drugs through tail vein injection on day 0, 4, 8, 12 and 16 and treated with 50 J/cm2 of 690 nm light at 1 h post-injection on these injection days. The dose and schedule was based on previous PSMA-1-VcMMAE work and were not optimized. Animals were imaged before and after PDT. Mice were monitored every other day for 90 days. Animals were euthanized when tumors became too large (diameter >20 mm) or animals were moribund. Data were reported as body weight over time, tumor size over time and Kaplan-Meier survival plots.
Immunofluorescent Detection of Apoptosis
[0323] Animals bearing PC3pip tumors were divided into 7 groups: (1) mice receiving PBS; (2) mice receiving 100 nmol/kg PSMA-1-MMAE-IR700 with PDT; (3) mice receiving equal doses of PSMA-1-MMAE-IR700 to group 2, but not receiving PDT; (4) Mice receiving equal doses of PSMA-1-IR700 to group 2 with PDT treatment; (5) Mice receiving equal doses of PSMA-1-IR700 to group 2 without PDT; (6) mice receiving equal dose of PSMA-1-IR700 to group 2+free MMAE with PDT (MMAE normalized to that delivered by PSMA-1-MMAE-IR700); and (7) mice receiving equal dose of PSMA-1-IR700 to group 2+free MMAE without PDT. Animals were treated with one single dose and were sacrificed at 4-day post treatment. Tumors were snap-frozen in OCT, cut into 10 m thick sections and fixed on slides. Induction of apoptosis by the treatment was determined by rabbit polyclonal anti-Caspase-3 antibody (Abcam, Cambridge, UK). A goat anti-rabbit polyclonal antibody labeled by Alexa Fluor-594 was used as secondary antibody (Abcam, Cambridge, UK). The presence of apoptosis was determined by fluorescence images under Leica DM4000B fluorescence microscope at 10. H&E staining of tumor tissues was performed in adjacent sections to check the histology of the tumors. Experiments were repeated in 5 mice.
Statistics
[0324] Student t-test was used to compare inter-group differences. Kaplan-Meier survival data were analyzed by SAS 9.4 using log-rank tests. A p value <0.05 was considered statistically significant for all comparisons.
Results
Chemistry and In Vitro Competition Binding Studies
[0325] To synthesize PSMA-1-MMAE-IR700 (
In Vitro Uptake Studies
[0326] To determine the selectivity of PSMA-1-MMAE-IR700, uptake studies were performed using both PSMA-positive PC3pip and PSMA-negative PC3flu cells. Fluorescence signal in PC3pip cells was observed as early as 15 min after incubation with PSMA-1-MMAE-IR700 and the signal intensity increased with extended incubation time (
In Vitro Cytotoxicity Studies
[0327] Cytotoxicity of PSMA-1-MMAE-IR700 was performed in both PSMA-positive PC3pip and PSMA-negative PC3flu cells to test if PSMA-1-MMAE-IR700 would selectively kill PSMA-positive cells. We first tested the cytotoxicity of PSMA-1-MMAE-IR700 and PSMA-1-IR700 in PC3pip and PC3flu cells without light treatment. After 24 h of incubation in the dark, PSMA-1-IR700 did not show any activity indicating that PSMA-1-IR700 is not toxic without light activation (
[0328] Incubation of the cells with PSMA-1-MMAE-IR700 in the dark for 72 h showed that it was 50-fold more effective for killing PC3pip cells than for PC3flu cells. Our results suggest that PSMA-1-MMAE-IR700 selectively delivers MMAE to PC3pip cells leading to effective cell death. To test if combination of MMAE-based chemotherapy and PDT using PSMA-1-MMAE-IR700 would enhance the cytotoxicity, PC3pip and PC3flu cells were incubated with 5 nM of different targeted agents for 24 h, and then cells were washed and treated with or without light. PSMA-1-MMAE-IR700 with light treatment (1 J/cm2 and 3 J/cm2) significantly enhanced the cytotoxicity to PC3pip cells as compared to PSMA-1-IR700 with light treatment and compared to PSMA-1-MMAE-IR700 without light treatment (0 J/cm2) (
In Vivo Fluorescence Imaging Results
[0329] To evaluate the selectivity of PSMA-1-MMAE-IR700, in vivo fluorescence imaging was performed in mice bearing both PC3pip and PC3flu tumors. PSMA-1-MMAE-IR700 (100 nmol/kg) was administered through tail vein injection, and mice were imaged at various time points. As shown in
[0330] The orthotopic PC3pip prostate cancer mouse model can develop tumor metastases to lymph nodes. To test if PSMA-1-MMAE-IR700 can detect lymph node metastases, we performed fluorescence imaging in mice bearing PC3pipGFP tumors. Twenty-one days following orthotopic implantation of PC3pip cells into the prostate gland, mice received 100 nmol/kg PSMA-1-MMAE-IR700. At 1 hour post injection (peak tumor accumulation time determined above), bright fluorescence signal from PSMA-1-MMAE-IR700 was observed in primary orthotopic PC3pipGFP tumors and the signal correlated with the GFP fluorescence signal in the tumor (
In Vivo Antitumor Activity Studies
[0331] The effectiveness of PSMA-1-MMAE-IR700 to eliminate prostate tumors was performed in mice bearing PC3pip tumors. Mice received 100 nmol of PSMA-1-MMAE-IR700 through tail vein injection every 4 days with a total of 5 doses. Mice were irradiated by 50 J/cm.sup.2 of 690 nm light at the peak tumor accumulation of PSMA-1-MMAE-IR700, which was 1 h post injection. Controls included i.v. administration of PBS with no light treatment, 100 nmol/kg of PSMA-1-MMAE-IR700 with 50 J/cm.sup.2 of 690 nm light, 100 nmol/kg of PSMA-1-MMAE-IR700 with no light treatment, 100 nmol/kg of PSMA-1-IR700 with 50 J/cm.sup.2 of 690 nm light, 100 nmol of PSMA-1-IR700 with no light treatment, co-injection of 100 nmol/kg of PSMA-1-IR700 and 100 nmol/kg of free MMAE with 50 J/cm.sup.2 of 690 nm light, and co-injection of 100 nmol/kg of PSMA-1-IR700 and 100 nmol/kg of free MMAE with no light treatment. As shown in
Induction of Apoptosis by Treatment
[0332] To evaluate the apoptosis caused by the treatment, mice bearing PC3pip tumors were treated with drugs and tumors were collected four days after the treatment, sectioned, and examined by H&E and caspase 3 assay (
[0333] Prostate cancer is highly heterogeneous, which will affect treatment response, drug resistance and clinical outcome. The use of combination therapies with different mechanisms of action will offer potential advantages over a single therapy and it can be an effective way to deal with the heterogeneity of cancer cells. However, this is not a simple approach because different drugs may have different pharmacokinetics and do not necessarily get to the tumor at the same time and the drug can also go to other tissues in the body causing side effects. The use of anticancer drugs is therefore limited by unwanted side effects. To overcome these problems, we have developed a multifunctional molecule named PSMA-1-MMAE-IR700 that combines chemotherapy, PDT, and imaging in a single molecule that is targeted to PSMA. PSMA is over expressed almost exclusively on prostate cancer (
[0334] Our molecule is the first example that combines both chemotherapy and PDT in a single targeted small molecule for a dual-therapeutic approach to combat prostate cancer. The selective targeting and rapid clearance of the molecule should dramatically reduce off-target toxicity while simultaneously increasing anti-cancer efficacy. For localized prostate cancer, minimally invasive fiber optics have been developed to irradiate the prostate gland with light, e.g., TOOKAD, providing the needed infrastructure for implementation of the PDT approach. In addition to localized NIR light irradiation, the efficacy of PSMA-1-MMAE-IR700 can be extended to systemic cell killing by local release of active MMAE, which will overcome the problem that PDT cannot be used to treat large tumors due to limited light penetration. On the other hand, PDT will reduce the needed dose of the chemotherapeutic drug, therefore further reducing dose-related toxicity of MMAE. Compared to current clinical protocols for combination therapy of PDT and chemotherapy, our approach selectively delivers both drugs to cancer cells, reducing off-target toxicity related to untargeted drugs and achieving enhanced synergistic antitumor activity. Furthermore, the PDT agent, IR700, emits light at 700 nm when irradiated by 690 nm light. We have demonstrated that PSMA-1-MMAE-IR700 can identify cancer tissues, including metastases to lymph nodes, and delineate tumor margins (
[0335] In conclusion, we have synthesized a multi-functional theranostic molecule for simultaneous and targeted delivery of both PDT and chemotherapy to prostate cancer cells. The multifunctional molecule showed selective uptake in PSMA-positive tumors and significantly enhanced (synergistic) antitumor activity was observed as compared to individual treatment with PDT or chemotherapy alone. It can be used in the operating room to help surgeons detect tumors using real-time FIGS, and provide PDT and chemotherapy to kill any unresected cancer cells. It is also possible that the molecule can be used directly on prostate cancer patients that are not suitable for surgery, providing PDT and chemotherapy to cancer tissues.
Example 3
[0336] This example describes theranostic gold nanoclusters as prostate cancer (PCa) targeted radiosensitizers and chemotherapy delivery vehicles. We designed a peptide-prodrug conjugate, PSMA-MMAE, with protease-based MMAE release and used it to synthesize PCa-targeted PSMA-AuNC-MMAE having a renal-clearable size to reduce potential toxicity (
Methods
Synthesis of CY-PSMA-MMAE-SH Ligands
[0337] PSMA targeting peptide Glu-CO-Glu-Amc-Ahx-Glu-Glu-Glu-Lys-Cys (CY-PSMA-1) was synthesized by Fmoc chemistry as described previously. C(Trt)Y-PSMA-1 was also synthesized to keep the SH protected by Trt. All the amino acids were purchased from Peptides International Inc. The synthesized CY-PSMA-1 ligands were purified by a HPLC system (Shimadzu) with Luna 5m C18 semipreparative column and characterized by electrospray ionization mass spectrometry (ESI-MS, LCQ advantage, Thermo Finnigan).
[0338] The PSMA targeted prodrug, CY-PSMA-MMAE, was synthesized by conjugating monomethyl auristatin E (MMAE) to the Cys residue of CY-PSMA-1 ligands via a maleimido caproyl valine-citrulline (Vc) cathepsin-cleavable linker with a self-immolative p-aminobenzyl carbamate (PABC) spacer. Briefly, 1.5 equiv of MMAE (BOC Sci.) was dissolved in DMF and added to 1 equiv of CY-PSMA-1 peptides in DMF. The pH of reacting mixture was then adjusted to above 7.5 by using diisopropylethylamine (DIPEA) and stirred at room temperature for over 2 h. Purification was performed by using HPLC, and conjugates were confirmed by ESI-MS.
[0339] The synthesized CY-PSMA-MMAE or C(Trt)Y-PSMA-1 was reacted with N-succinimidyl S-acetylthioacetate (Pierce SATA), producing CY-PSMA-MMAE-SATA or C(Trt)Y-PSMA-SATA. Generally, 1.5 equiv of SATA was dissolved in DMF and added to 1 equiv of peptide conjugates. The pH of the reaction mixture was adjusted to around 8 by using trimethylamine (TEA) and incubated at room temperature for over 1 h. The final product was purified by HPLC, and the mass was confirmed by ESI-MS.
[0340] The purified CY-PSMA-MMAE-SATA or C(Trt)Y-PSMA-SATA was dissolved in DMF and added with 0.5 M hydroxylamine to deacetylate the SATA protection. The pH of mixture was adjusted to around 7.5 by using TEA and stirred at room temperature over 2 h. The final product, CY-PSMA-MMAE-SH or C(Trt)Y-PSMA-SH, was purified by HPLC and characterized by ESI-MS. The detailed reaction is shown schematically in the Supporting Information as well as the MS spectra of product from each step.
Synthesis of PSMA-AuNC-MMAE and PSMA-AuNCs Conjugates and Characterization
[0341] PSMA-AuNC-MMAE conjugates were synthesized by using a one-pot reaction according to a previously reported protocol with slight modifications in terms of pH, reacting temperature and time, and solvent. In a typical synthesis of the NCs, CY-PSMA-MMAE-SH or C(Trt)Y-PSMA-SH was dissolved in 2 mL of PBS with a final concentration of 1 mM. During vigorous stirring, 100 L of 25 mM HAuCl.sub.4 solution was added to the prodrug conjugates slowly. After about 2 min, 1 M NaOH solution was added to bring the reaction pH up to 12. The mixture was then kept at 37 C. to allow the reaction to proceed for 24 h. The formed NCs were purified, and excess peptides were removed by dialysis against 25 mM HEPES buffer for over 2 h and then against 12.5 mM HEPES buffer for 3 days. The final PSMA-AuNC-MMAE conjugates were lyophilized and stored at 20 C. until further use. PSMA-AuNCs without MMAE were also synthesized by using the same procedure with C(SH)Y-PSMA-1 ligands. PSMA-AuNC-MMAE conjugates were characterized by transmission electron microscope (TEM, FEI Tecnai F300 kV) and a dynamic light scattering system (DynoPro NanoStar). The optical properties (absorbance, excitation, emission) of NCs were measured by Tecan Infinite M200 plate reader and IVIS Spectrum (PerkinElmer).
MMAE Release Assay
[0342] Cathepsin B (human liver, Athens Research & Technology) was activated by using a protocol as described previously. PSMA-AuNC-MMAE conjugates were then added to 500 L of activated cathepsin B buffer, and the mixture was stored at 37 C. At the time points of 1, 7, 24, and 48 h, a 10 L solution was taken out for HPLC analysis. As a control, 1 mM E64 protease inhibitor was added to the PSMA-AuNC-MMAE conjugates, and the same procedure and analysis was carried out. The cleaved free MMAE from NCs was determined by the HPLC system equipped with a Luna 5 m C18 analytical column and SPD-20A prominence UV/vis detector at 220 and 254 nm. The amount of free MMAE was calculated according to an established calibration curve.
Selective Cell Uptake and Cytotoxicity
[0343] The cell uptake of PSMA-AuNC-MMAE conjugates was evaluated by using human prostate cancer cell lines: PC3pip cells (PSMA+) which were retrovirally transformed with PSMA and PC3flu cells (PSMA-) as a transformation control (Laboratory of Gene Transfer and Gene Expression, Gene Transfer and Somatic Cell Engineering Facility, Memorial-Sloan Kettering Cancer Center). Both cells were cultured in RPMI1640 medium with L-glutamine (2 mmol/L) and 10% FBS at 37 C. and 5% CO.sub.2. To image the uptake of PSMA-AuNC-MMAE conjugates and disruption of -tubulin, PC3pip and PC3flu cells were seeded in 8-well -slide (ibidi GmbH). When the cells growth reached 70% confluence, PSMA-AuNC-MMAE conjugates were added at calculated MMAE concentration of 5 nM and incubated for 1, 4, and 24 h at 37 C. Another group of cells were pretreated with 50 M E64 inhibitor before adding the PSMA-AuNCs-MMAE. Cells were then washed with PBS, fixed with 4% paraformaldehyde for 10 min, permeabilized by 0.1% Triton X-100 for 10 min, and blocked by 1% BSA for 1 h at 37 C. Then both PC3pip and PC3flu cells were incubated with 2 g/mL Alpha-tubulin (B5-1-2) Alexa Fluor 488 Mouse Monoclonal Antibody (Invitrogen) in 0.1% BSA at 37 C. for 3 h. Cells were then washed and counterstained with DAPI for 10 min before observation by using a Leica HyVolution SP8 confocal microscope (Leica Microsystem Inc.).
[0344] In vitro cytotoxicity was measured by MTT assay. Cells (10000 cells/well) were seeded in 96-well plates and incubated for 24 h before adding 5 nM free MMAE, PSMA-MMAE, or PSMA-AuNCs-MMAE. An equivalent amount of PSMA-AuNCs without MMAE was added as a control, and another group of cells pretreated with 50 M E64 inhibitor for 6 h was also incubated with PSMA-AuNCs-MMAE. After incubation for 1, 4, and 24 h, cells were rinsed with PBS and incubated with 1 mg/mL MTT solution in culture media for 3.5 h. After that media were removed, and 100 L of DMSO was added. The absorbance was measured 15 min later by a plate reader (Tecan Infinite M200) with absorbance at 570 nm and reference at 620 nm.
In Vitro Radiotherapy
[0345] Both PC3pip and PC3flu cells (110.sup.5 cells/well) were cultured in 6-well plates for 24 h, and then PSMA-AuNC-MMAE and PSMA-MMAE were added at an equivalent MMAE concentration of 1 nM. Additionally, PSMA-AuNCs without MMAE was added at 60 g Au/mL. Another group with the addition of a mixture of PSMA-AuNCs and PSMA-AuNC-MMAE at the equivalent dose of Au and MMAE was also evaluated. Mixing was performed so as to keep Au concentrations equivalent. This could not be achieved by adding more PSMA-AuNC-MMAE as this would result in toxic levels of MMAE. Cells without any treatment were used as blank control. Following incubation for 6 h, the media were removed, and cells were rinsed by PBS before exposing to X-ray radiation at doses of 0, 2, 4, and 6 Gy. Next, cells were trypsinized, counted, and reseeded in 6-well plate for colonies to grow. After 2 weeks, all the wells were rinsed with PBS, fixed with 4% paraformaldehyde, and stained by 0.4% crystal violet. The number of colonies from each well was counted to calculate the survive fraction.
In Vivo Tumor Targeting and Biodistribution
[0346] Under the guidelines of the animal care and use committee at Case Western Reserve University (IACUC #150033), 4- to 5-week-old male athymic nude mice were subcutaneously implanted with PC3pip or PC3flu cells (100 L matrigel containing 110.sup.6 cells) on the right flank. When the tumors reached a sufficient size, mice were intravenously injected with PSMA-AuNCs (15 g/g) and imaged by using the IVIS Spectrum system before and at 0.5, 1, 2, 4, 6, and 24 h postinjection (excitation filter at 500 nm and emission filter at 680-800 nm). Unmixing was performed by using the in vivo spectrum of PSMA-AuNCs to remove the autofluorescence background. All the images at different time points were demonstrated at the same optical threshold. The tumor region was determined in a free-drawn manner guided by the bright-field image, and average radiant efficiency (photos s.sup.1 cm.sup.1 sr.sup.1 (W cm.sup.2).sup.1) was calculated for each time point. At 24 h post injection, mice were euthanized, and tumor/organs were extracted for ex vivo imaging using the same setting, and the average radiant efficiency for all the tissues was calculated. The organs were also digested and analyzed by ICP-MS. Each group of treatment was done in triplicate.
In Vivo Chemoradiotherapy
[0347] Male athymic nude mice were subcutaneously implanted with the PC3pip tumor, and when the tumor size reached about 100 mm.sup.3 (tumor volume=lengthwidth.sup.2/2), the mice were divided into ten groups randomly. Each group was intravenously injected with PBS, PSMA-AuNCs, PSMA-AuNCs-MMAE, PSMA-MMAE, or a mixture of PSMA-AuNCs and PSMA-MMAE at MMAE dose of 400 nmol/kg and an equivalent dose of Au. At 4 h postinjection, five of the groups were irradiated with X-rays (6 Gy, Cs-137 with energy of 0.6616 MeV) focusing on the tumor region, and the other five groups received the same injections but no radiation and were used as controls. Each group contained five mice. All the treatments were given only once, and then the mice were monitored for tumor size and body weight every other day for one month. The coefficient of drug interaction (CDI) is calculated as follows: CDI=C/(AB),.sup.44 where C is the tumor volume ratio of PSMA-AuNCs-MMAE+RT group to control group, A is the tumor volume ratio of PSMA-AuNCs+RT group to control group, and B is the tumor volume ratio of PSMA-MMAE+RT group to control group; PBS+RT group is the control. A CDI value of <1, =1, or >1 indicates that the treatments are synergistic, additive, or antagonistic, respectively. When the tumors became too large or an animal was moribund, they were euthanized. At the end of monitoring, all mice were euthanized and tumors were isolated and weighed.
Histology, Detection of AuNPs, and Immunofluorescent Analysis
[0348] Groups of mice injected with PBS, PSMA-AuNCs, PSMA-AuNCs-MMAE, PSMA-MMAE, and a mixture of PSMA-AuNCs and PSMA-MMAE were euthanized at 24 h postinjection. Groups of mice that received radiation were euthanized 1 h after irradiation. All tumor samples were snap-frozen in optimum cutting temperature compound (OCT) for cryosectioning (Leica CM3050S). Sections, 12 m thick, were serially collected directly onto slides and stored at 80 C. for processing. Adjacent slides were stained for presence of NCs with a silver staining assay (Sigma Silver Enhancer Kit) followed by H&E standard procedures. -Tubulin of tumor samples was stained with a-tubulin (B5-1-2) Alexa Fluor 488 Mouse Monoclonal Antibody (Invitrogen) by using the sample protocol as described in the in vitro uptake section. Tissue nuclei were stained with DAPI and tumor tissues were imaged by Leica HyVolution SP8 confocal microscope (Leica Microsystem Inc.).
[0349] For immune-histochemical analysis (IHC), the slides were warmed to room temperature for 10 min, fixed with 10% buffered formalin, blocked in blocking buffer (5% normal goat serum/0.3% Triton X-100 in 1PBS) for 1 h at room temperature, and incubated in primary antibody for 1-3 h followed by three 5 min washes in 1PBS. The presence of apoptosis in the tumor was evaluated by rabbit anti-Cleaved Caspase-3 antibody (#700182, Cell Signal Tech) at a 1:400 dilution. The presence of the histones with double-strand breaks in DNA of the tumor nuclei was evaluated by rabbit anti-yH2AX (phospho-Ser139) antibody (SAB5600038, Sigma-Aldrich) at a 1:500 dilution. After washing, the slides were treated with the secondary ready-to-use antibody (goat anti-rabbit polyclonal antibody labeled by Alexa Fluor-594 (Invitrogen, Inc.)) for 20 min at room temperature followed by double washing with PBS for 5 min. Tissue nuclei were contrasted by using Fluoro-Gel-II with DAPI (Electron Microscopy Sciences, Hatfield, PA). Fluorescent images were viewed with a Leica-DM4000B microscope (bandpass=560/645, for Cleaved Caspase-3 or yH2AX) and analyzed with QCapturePro-7 software. An Olympus-VS120/S5 versatile microscope-based scanner was used to generate histological images larger than a single field of view.
Statistical Analysis
[0350] All numerical results are expressed as meanSD. Descriptive statistics and significant differences between groups were analyzed by using two-tailed Student's t tests, and the difference was considered significant if *p<0.05 and **p<0.01.
Results
[0351] PSMA-AuNC-MMAE conjugates were synthesized by using PSMA-MMAE as a template in a one-step reaction (
[0352] The synthesized PSMA-AuNC-MMAE conjugates had an average core size of 1.20.2 nm as revealed by transmission electron microscopy (TEM,
[0353] The PSMA-AuNCs show good stability in physiological solutions due to the zwitterionic peptide coating. A previous study of PSMA-AuNCs with no MMAE revealed a structure with a core consisting of 25 Au atoms and 18 PSMA ligands on the surface. We assumed that since MMAE was conjugated to the PMSA ligand, there would also be 18 MMAE drug molecules attached to each nanocluster. To confirm this, we incubated the PSMA-AuNC-MMAE conjugates with excess activated cathepsin B to release free MMAE. After 48 h incubation, 95% of MMAE (11.4 g) was released from the NCs according to the high performance liquid chromatography (HPLC) measurement, which was very close to the 12 g of MMAE that theoretically should be attached. When protease inhibitor E64 was added, release of intact MMAE was significantly reduced with only 17.5% MMAE released after 48 h. In addition, the MMAE release was dependent on the concentration of cathepsin B (
[0354] The selective uptake of PSMA-AuNC-MMAE conjugates was evaluated by incubating them with both PSMA-expressing PC3pip cells and PSMA-lacking PC3flu cells. Confocal fluorescence imaging of cells revealed that the PC3pip cells had avid internalization of NCs after 24 h incubation, while the PC3flu cells still showed no evidence of NCs fluorescence (
[0355] We then measured the cell viability after each treatment. MMAE has been shown to be highly efficatious for killing cancer cells in the form of a free drug or as antibody conjugates. Here, after incubation with PC3pip and PC3flu cells for 24 h (
[0356] We next investigated the radiation enhancement by both MMAE and AuNC conjugates. Au atoms have a high Z number and are well-known for RT sensitization. Au can absorb radiation energy and lead to DNA damage by the photoelectric and Auger effects. MMAE, on the other hand, can also increase radiosensitization by blocking the cells in G.sub.2-M, when cells have the highest sensitivity to radiation. By incubating PC3pip and PC3flu cells with PSMA-AuNCs alone, PSMA-AuNC-MMAE, PSMA-MMAE, and a mixture of PSMA-AuNCs and PSMA-AuNC-MMAE with equivalent Au and MMAE concentrations, we expected to measure a synergistic sensitizing effect. We added the mixture group to achieve optimal effective doses of gold for radiosensitization and of MMAE for chemotherapy, as the MMAE/Au ratio is fixed in the NCs. This approach normalized the amount of MMAE and Au that the cells received. As shown in
[0357] To investigate the selective tumor uptake and distribution in vivo, we injected the PSMA-AuNCs to the PC3pip or PC3flu tumor bearing mice at 0.25 g/kg and monitored the fluorescence of NCs with the Spectrum IVIS imager before injection and at 0.5, 1, 2, 4, 6, and 24 h postinjection. There was a strong fluorescence from the PC3pip tumor as early as 0.5 h postinjection that reached maximum at 1 h, after which the signal decayed over time (
[0358] We next evaluated the antitumor activity of PSMA-AuNC-MMAE conjugates in mice bearing flank PC3pip tumors. The PSMA-AuNC-MMAE were injected, and a radiation dose of 6 Gy was given 4 h postinjection, focused on the tumor area only. For the no radiation control groups (
[0359] To further compare the damage induced by MMAE, tumors for the five groups without radiation were retrieved and analyzed histologically (
[0360] The radiation-induced DNA double-strand breaks in tumors and sensitizing enhancement from different groups were evaluated by y-H2AX staining (
[0361] For successful clinical translation any possible side effect and toxicity related to RT and chemotherapy should be minimized. Advances in imaging technologies enable precise delivery of radiation doses to defined tumor areas, and radiation amplification can be achieved with radiosensitizers. For chemotherapy, the highly toxic therapeutic drugs can also be modified to improve specificity via tumor biomarker recognition. We developed a PSMA-AuNC-MMAE conjugate with MMAE conjugated via a self-immolative cathepsin cleavable linker. The PSMA-AuNC-MMAE conjugates showed excellent selectivity for PSMA-expressing tumor cells and antitumor activity upon cathepsin cleavage to release free MMAE. The prodrug approach, selective accumulation of PSMA-AuNC-MMAE in tumors, ensuing radiosensitization, and the conjugate's rapid urinary excretion may possibly reduce the toxicity to animals induced by either radiation or nonspecific release of MMAE. Biodistribution studies with PSMA-AuNC, which has an average size smaller than the threshold of renal clearance limit (around 5.5 nm), may result in a fast renal excretion and a reduced circulation time in vivo, which is beneficial for reducing the toxicity. According to the biodistribution, the PSMA-AuNCs peaked in tumor tissues around 1 h post injection and showed a low retention in the reticuloendothelial system (RES) after 24 h, which is in agreement with other AuNCs studies. In contrast, the majority AuNPs having a much larger size end up in RES organs.
[0362] On the other hand, the choice of a linker to conjugate chemotherapy drugs to targeting ligands also plays a critical role for their successful application. MMAE conjugated to antibody via uncleavable linkers is inactive until completely released via lysosomal proteolytic degradation of the antibody. A cathepsin self-imolative cleavable linker, MC-VC-PABC, was used here and showed controllable MMAE release upon cathepsin activity. This linker rendered the MMAE inactive until protease removal and liberation of free MMAE. This approach could take full advantage of upregulated cathepsin activities in tumor tissues and further reduce the toxicity of MMAE. Although antibodies conjugated with MMAE via the VC linker have been reported to exhibit instability in plasma, this may be related to the significantly extended half-life of ADCs in the blood. Our approach uses a much smaller carrier that is rapidly excreted from the body limiting nonselective release of MMAE. The PSMA-AuNC-MMAE showed good antitumor activity; however, they still caused some body weight loss the first 6 days after injection, which may also be related to the unavoidable nontumor selective cleavage of MMAE from PSMA-AuNC-MMAE during circulation. However, no obvious damage to the main organs was observed for the PSMA-AuNC-MMAE treated mice at the end of treatment (
[0363] A combined chemotherapy and RT has been prescribed to a wide range of patients with cancer. The PSMA-AuNC-MMAE conjugates integrated with both gold and MMAE enables precision chemotherapy-RT. Interestingly, without radiation, the PSMA-AuNC-MMAE showed better antitumor capability than the PSMA-MMAE counterpart, which was probably due to the enhanced binding to PSMA for nanoparticles and increased drug delivery by the NCs. With a single dose of 6 Gy radiation, we also observed a synergistic sensitizing effect by both gold and MMAE in PSMA-AuNC-MMAE (CDI=0.9). However, compared to PSMA-MMAE, PSMA-AuNCs had a much better sensitizing enhancement as measured by -H2AX staining for tumor tissues. The independent mechanism of the two therapies will also decrease the chance of tumor cells developing resistance. Moreover, X-ray irradiation may further enhance the accumulation of NCs in tumor tissues via disruption of the tumor vasculature. Though the retrovirally modified PSMA-expressing PC3 cells are a good model to evaluate the PSMA-targeted NCs, AR positive prostate cancer models that express PSMA, such as VCaP, C4-2b, and PDX models will be used in the future to evaluate the performance of NCs in different types of prostate cancers. We are hopeful that the future studies will move us closer to clinical translation of the PSMA-targeted nanoclusters.
[0364] In summary, we synthesized a PSMA-AuNC-MMAE conjugates for combined RT and chemotherapy of PCa. The NCs were functionalized with PSMA-1 targeting moieties and conjugated with MMAE via a cleavable linker, which demonstrated selective tumor cell uptake and active MMAE release upon cathepsin cleavage in vitro and in vivo. RT was enhanced by both gold and MMAE, and together with the chemotherapeutic effect of MMAE, the PSMA-AuNC-MMAE displayed excellent antitumor activity. Further studies for optimizing the dose and administration schedule for minimizing systemic toxicity will be performed to help translate the PSMA-AuNC-MMAE conjugates for clinical applications.
Example 4
[0365] Magnetic resonance (MR) imaging is a clinically relevant, noninvasive diagnostic tool for high-resolution anatomic and functional imaging. Molecular MR imaging enables the visualization of biological markers in vivo. Gd-based contrast agents are widely accepted by clinicians because they are easy to administer and provide T1-weighted, positive contrast. With signal amplification strategies, MR might offer a sensitive modality for molecular imaging complementary to radionuclide-based techniques. Combining a receptor-specific high affinity ligand with multimeric Gd agents for detection has been described as one solution for enabling MR-based receptor imaging. PSMA is overexpressed in primary and metastatic prostate cancer, particularly with respect to the castration resistant form. Furthermore, PSMA is expressed by most solid tumors and tumor neovasculature. We hypothesized that PSMA would be a suitable biomarker for MR molecular imaging. We plan to synthesize three PSMA-targeted MR contrast agent with 1 (PSMA-Cys(Cy5.5)-C6-Lys-(DOTA-Gd)), 2 (PSMA-Cys(Cy5.5)-C6-Lys-(DOTA-Gd)2) and 4 Gd (PSMA-Cys(Cy5.5)-C6-Lys-(DOTA-Gd)4) (
Synthesis of PSMA-1-Cys(Trt)-C6-Lys(NH2)-MBHA Rink-Amide Resin
[0366] PSMA-1-Cys(Trt)-C6-Lys(Mmt) on MBHA Rink-amide resin was synthesized manually based on Fmoc chemistry using the same method as the synthesis of PSMA-1. After all the sequence was assembled on Rink-Amide resin, the resin was exposed to 2% TFA in dichloromethane to selectively remove the Mmt group to get PSMA-1-Cys(Trt)-C6-Lys(NH2)-MBHA Rink-amide resin.
Synthesis of PSMA-1-Cys(Cy5.5)-C6-Lys(DOTA-Gd)
[0367] To PSMA-1-Cys(Trt)-C6-Lys(NH2)-MBHA Rink-amide resin in DMF, 3.3 equiv of DOTA-tris-(t-Bu ester) in DMF activated with 3.3 equiv of HCTU and 5 equiv of diisopropylethylamine (DIPEA) was added. After the reaction was complete, the resin was washed with DMF and then dichloromethane and dried. The peptide was cleaved from resin by TFA/water/triisopropylsilane (950:25:25). The cleaved peptide PSMA-1-Cys-C6-Lys(DOTA) was purified by preparative HPLC and lyophilized. The lyophilized PSMA-1-Cys-C6-Lys(DOTA) was dissolved in water, to which 5-fold excess amount of GdCl.sub.3 in water was added. The reaction mixture was stirred at room temperature for 1 day and then purified by HPLC to get pure PSMA-1-Cys-C6-Lys(DOTA-Gd). Finally, PSMA-1-Cys-C6-Lys(DOTA-Gd) in PBS solution was reaction with 1.5-fold excess amount of Cy5.5 maleimide for 2 hours at room temperature. The crude was then purified by HPLC to get PSMA-1-Cys(Cy5.5)-C6-Lys(DOTA-Gd). (The compounds in this series haven't been synthesized yet).
Synthesis of PSMA-1-Cys(Cy5.5)-C6-Lys(DOTA-Gd)2
[0368] To PSMA-1-Cys(Trt)-C6-Lys(NH2)-MBHA Rink-amide resin in DMF, 3.3 equiv of Fmoc-Lys(Fmoc)-OH activated with 3.3 equiv of HCTU and 5 equiv of diisopropylethylamine (DIPEA) was added. After the reaction was completed, the resin was washed 5 times. The two Fmoc groups of N-terminal amino acid lysine were deprotected by 20% piperidine in DMF. The resin was washed 5 times with DMF, to which 3.3 equiv of DOTA-tris-(t-Bu ester) in DMF activated with 3.3 equiv of HCTU and 5 equiv of diisopropylethylamine (DIPEA) in DMF was added. After the reaction was complete, the resin was washed with DMF and then dichloromethane and dried. The peptide was cleaved from resin by TFA/water/triisopropylsilane (950:25:25). The cleaved peptide PSMA-1-Cys-C6-Lys(DOTA)2 was purified by preparative HPLC and lyophilized. MALDI-MS: C.sub.93H.sub.153N.sub.22O.sub.37S, 2204.1 (found); 2204.3 (calculated). PSMA-1-Cys-C6-Lys(DOTA)2 was then reacted with 5-fold excess amount of GdCl.sub.3 in water for 1 day. The crude was purified by HPLC to get pure PSMA-1-Cys-C6-Lys(DOTA-Gd)2. MALDI-MS: C.sub.93H.sub.147Gd.sub.2N.sub.22O.sub.37S, 2511.8 (found); 2511.8 (calculated). PSMA-1-Cys-C6-Lys(DOTA-Gd)2 in PBS solution was reaction with 1.5-fold excess amount of Cy5.5 maleimide for 2 hours at room temperature. The crude was then purified by HPLC to get PSMA-1-Cys(Cy5.5)-C6-Lys(DOTA-Gd)2. MALDI-MS: C.sub.139H.sub.196Gd.sub.2N.sub.26O.sub.40S, 3217.1 (found); 3217.2 (calculated).
Synthesis of PSMA-1-Cys(Cy5.5)-C6-Lys(DOTA-Gd)4
[0369] To PSMA-1-Cys(Trt)-C6-Lys(NH2)-MBHA Rink-amide resin in DMF, 3.3 equiv of Fmoc-Lys(Fmoc)-OH activated with 3.3 equiv of HCTU and 5 equiv of diisopropylethylamine (DIPEA) in DMF was added. After the reaction was completed, the resin was washed 5 times. The two Fmoc groups of N-terminal amino acid lysine were deprotected by 20% piperidine in DMF. The resin was washed 5 times with DMF, then 3.3 equiv of Fmoc-Lys(Fmoc)-OH activated with 3.3 equiv of HCTU and 5 equiv of diisopropylethylamine (DIPEA) in DMF was added. After the reaction was completed, the four Fmoc groups were removed by 20% piperidine. The resin was washed 5 times with DMF, to which 3.3 equiv of DOTA-tris-(t-Bu ester) in DMF activated with 3.3 equiv of HCTU and 5 equiv of diisopropylethylamine (DIPEA) in DMF was added. After the reaction was complete, the resin was washed with DMF and then dichloromethane and dried. The peptide was cleaved from resin by TFA/water/triisopropylsilane (950:25:25). The cleaved peptide PSMA-1-Cys-C6-Lys(DOTA)4 was purified by preparative HPLC and lyophilized. MALDI-MS: C.sub.137H.sub.230N.sub.34O.sub.53S, 3232.5 (found); 3232.5 (calculated). PSMA-1-Cys-C6-Lys(DOTA)4 was then reacted 5-fold excess amount of GdCl.sub.3 in water for 1 day. The crude was purified by HPLC to get pure PSMA-1-Cys-C6-Lys(DOTA-Gd)4. MALDI-MS: C.sub.137H.sub.218Gd.sub.4N.sub.34O.sub.53S, 3851.2 (found); 3851.2 (calculated). PSMA-1-Cys-C6-Lys(DOTA-Gd)4 in PBS solution was reaction with 1.5-fold excess amount of Cy5.5 maleimide for 2 hours at room temperature. The crude was then purified by HPLC to get PSMA-1-Cys(Cy5.5)-C6-Lys(DOTA-Gd)4. MALDI-MS: C.sub.183H.sub.267Gd.sub.4N.sub.38O.sub.56S, 3217.1 (found); 4556.6 (calculated).
In Vivo Imaging
[0370] For in vivo experiments, we first tested if the new probes can selectively bind to PSMA expressing PC3pip tumors using Maestro In Vivo imaging system. Mice bearing PSMA-positive PC3pip and PSMA-negative PC3flu tumors received 1 nmol of PSMA-1-Cys(Cy5.5)-C6-Lys(DOTA-Gd)2 through tail vein injection; images were taken at different time points post injection. At 2 hours post injection, selective accumulation in PC3pip was observed and the signal reached highest at 4 hours post injection, then cleared out from the body gradually (
[0371] From the above description of the invention, those skilled in the art will perceive improvements, changes and modifications. Such improvements, changes and modifications within the skill of the art are intended to be covered by the appended claims. All references, publications, and patents cited in the present application are herein incorporated by reference in their entirety.