Dual targeting ligand for cancer diagnosis and treatment
11167048 · 2021-11-09
Assignee
Inventors
Cpc classification
A61K51/088
HUMAN NECESSITIES
A61K51/0497
HUMAN NECESSITIES
A61K51/08
HUMAN NECESSITIES
International classification
A61P35/00
HUMAN NECESSITIES
Abstract
Disclosed are compositions and methods relating to prostate cancer. In particular, disclosed are bivalent targeting ligands that specifically bind prostate specific membrane antigen and gastrin-releasing peptide receptor. Bivalent binding agents disclosed herein can be used to image a tissue in a subject in need thereof and to diagnose prostate cancer in a subject in need thereof. Bivalent binding agents disclosed herein can be used to treat prostate cancer in a subject in need thereof.
Claims
1. A bivalent binding agent of formula (I)
[DUPA-6-Ahx-Lys(DOTA)-X-RM2] (I) wherein X is selected from the group consisting of 5-aminovaleric acid (5-Ava), 6-amino hexanoic acid (6-Ahx), 8-aminooctanoic acid) (8Aoc), and paraamino benzoic acid (AMBA).
2. A bivalent binding agent of formula (II)
[DUPA-6-Ahx-Lys(M-DOTA)-X-RM2] (II) wherein X is selected from 5-aminovaleric acid (5-Ava), 6-amino hexanoic acid (6-Ahx), 8-aminooctanoic acid) (8Aoc), and paraamino benzoic acid (AMBA); and wherein M is selected from Gallium (Ga), Indium (In), Lutetium (Lu), Yttrium (Y), Samarium (Sm), Promethium (Pm), .sup.67Ga, .sup.68Ga, .sup.111In, .sup.177Lu, .sup.90Y, .sup.86Y, .sup.153Sm, and .sup.149Pm.
3. A method of imaging a tissue in a subject in need thereof having or suspected of having prostate cancer, the method comprising: administering to the subject having or suspected of having prostate cancer a bivalent binding agent of formula (II)
[DUPA-6-Ahx-Lys(M-DOTA)-X-RM2] (II) wherein X is selected from 5-aminovaleric acid (5-Ava), 6-amino hexanoic acid (6-Ahx), 8-aminooctanoic acid) (8Aoc), and paraamino benzoic acid (AMBA); and wherein M is selected from .sup.67Ga, .sup.68Ga, .sup.111In, .sup.86Y, and .sup.177Lu; and applying an imaging technique to detect emitted gamma rays.
4. The method of claim 3, wherein the imaging technique is selected from the group consisting of positron-emission tomography (PET) and single photon emission computed tomography (SPECT).
5. A method of diagnosing prostate cancer in a subject having or suspected of having prostate cancer, the method comprising: administering to the subject a bivalent binding agent of formula (II)
[DUPA-6-Ahx-Lys(M-DOTA)-X-RM2] (II) wherein X is selected from 5-aminovaleric acid (5-Ava), 6-amino hexanoic acid (6-Ahx), 8-aminooctanoic acid) (8Aoc), and paraamino benzoic acid (AMBA); and wherein M is selected from .sup.67Ga, .sup.68Ga, .sup.111In, .sup.86Y, and .sup.177Lu; applying an imaging technique to detect emitted gamma rays; and diagnosing the subject as having prostate cancer based on uptake of the bivalent binding agent as detected in the imaging of the subject.
6. The method of claim 5, wherein the imaging technique is selected from the group consisting of positron-emission tomography (PET) and single photon emission computed tomography (SPECT).
7. A method of treating prostate cancer in a subject having or suspected of having prostate cancer, the method comprising: administering to the subject a bivalent binding agent of formula (II)
[DUPA-6-Ahx-Lys(M-DOTA)-X-RM2] (II) wherein X is selected from 5-aminovaleric acid (5-Ava), 6-amino hexanoic acid (6-Ahx), 8-aminooctanoic acid) (8Aoc), and paraamino benzoic acid (AMBA); and wherein M is selected from .sup.177Lu, .sup.90Y, .sup.153Sm, and .sup.149Pm.
8. The method of claim 7, wherein the subject is administered about 100 μg/70 kg of the bivalent binding agent of formula (II).
9. The method of claim 7, wherein the subject is administered less than 100 μg/70 kg of the bivalent binding agent of formula (II).
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The disclosure will be better understood, and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings wherein:
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DETAILED DESCRIPTION
(8) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described below.
(9) As used in this application, including the appended claims, the singular forms “a,” “an,” and “the” include plural references, unless the content clearly dictates otherwise, and are used interchangeably with “at least one” and “one or more.”
(10) Disclosed are compositions and methods relating to prostate cancer. Bivalent binding agents disclosed herein specifically bind prostate specific membrane antigen and gastrin-releasing peptide receptor. Bivalent binding agents disclosed herein can be used to image a tissue in a subject in need thereof. Bivalent binding agents disclosed herein can also be used to diagnose prostate cancer in a subject having or suspected of having prostate cancer.
(11) In one aspect, the present disclosure is directed to a bivalent binding agent of formula (I):
[DUPA-6-Ahx-Lys(DOTA)-X-RM2] (I)
(12) wherein X is selected from 5-aminovaleric acid (5-Ava), 6-amino hexanoic acid (6-Ahx), 8-aminooctanoic acid) (8Aoc), and paraamino benzoic acid (AMBA).
(13) DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)) is used as a complexing agent with high affinity for di- and trivalent cations. DUPA (2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid) is a small-molecule that specifically binds to prostate specific membrane antigen (PSMA). RM2 (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) (SEQ ID NO:1) is an antagonist analogue of bombesin (BBN) peptide that specifically binds to gastrin-releasing peptide receptor (GRPR).
(14) In one aspect, the present disclosure is directed to a bivalent binding agent of formula (II):
[DUPA-6-Ahx-Lys(M-DOTA)-X-RM2] (II).
(15) Suitably, X is selected from 5-Ava, 6-Ahx, 8Aoc, and AMB. Suitably, M is selected from Gallium (Ga), Indium (In), Lutetium (Lu), Yttrium (Y), Samarium (Sm), Promethium (Pm), .sup.67Ga, .sup.68Ga, .sup.111In, .sup.177Lu, .sup.90Y, .sup.86Y, .sup.153Sm, and .sup.149Pm.
(16) Gallium- (Ga), Indium- (In), Yttrium- (Y), and Lutetium (Lu)-labeled radio-pharmaceuticals each have a half-life, decay mode and emission profile suitable for imaging. The beta emission of .sup.177Lu also renders it useful as a therapeutic radionuclide. .sup.111In is a cyclotron-produced radionuclide via the (p,2n) reaction and decays mainly by electron capture, producing two gamma photons (171 keV and 245 keV) which fall into a suitable range for imaging purposes. A physical half-life of 2.8 days also makes .sup.111In suitable for molecular imaging. For example, since it is readily available, it can be procured within a reasonable timeframe to allow for drug preparation, quality control, drug delivery, and molecular imaging investigations (preclinical or clinical). As a reactor-produced product, .sup.177Lu can be obtained in a high specific activity/carrier-free state from a .sup.176Yb target via indirect neutron capture (gamma) as to avoid the presence of long-lived, high-energy, metastable isotopes. However, it is most regularly synthesized in moderate specific activity via direct neutron capture using a .sup.176Lu-enriched target. In addition to the two gamma photons that are suitable for imaging (113 keV and 208 keV), .sup.177Lu also emits a beta particle with an energy of 498 keV that can achieve a tissue penetration depth of ˜2 mm, making it suitable for use with smaller size tumors. The 6.7 day physical half-life makes it suitable for use as a diagnostic or therapeutic peptide-based cell targeting agent with a longer in vivo biological half-life. .sup.68Ga also possesses ideal nuclear characteristics for PET molecular imaging. As a radionuclide produced by the elution of parent .sup.68Ge in a radionuclide generator, .sup.68Ga decays by positron emission [Eβ+max=1.899 MeV (89%)]. It has a half-life of 68 minutes, which is also sufficiently long for drug preparation, quality control, drug delivery, drug clearance, and patient imaging. However, the half-life of .sup.68Ga may not be sufficiently long enough for in vivo investigations at later time-points. As a result, .sup.67Ga can used as a substitute due to its extended half-life of 78.26 hours. .sup.67Ga is also produced via a cyclotron. Charged particle bombardment of enriched .sup.68Zn is used to produce .sup.67Ga. The half-life of .sup.67Ga is 78 hours. It decays by electron capture, then emits de-excitation gamma rays (93, 185, 288, 394 KeV energy) that are detected by a gamma camera. .sup.177Lu, .sup.111In, and .sup.67Ga lend themselves well to successful, inert chelation within the DOTA bifunctional chelator as a result of their inherent chemical properties, such as 3+ oxidation states, hard metal centers, and larger-sized ionic radii. .sup.86Y is suitable for imaging uses and .sup.90Y is suitable for therapeutic uses.
(17) In one aspect, the present disclosure is directed to a method of imaging a tissue in a subject in need thereof. The method includes administering to a subject in need thereof a bivalent binding agent of formula (II)
[DUPA-6-Ahx-Lys(M-DOTA)-X-RM2] (II).
(18) Suitably, X is selected from 5-Ava, 6-Ahx, 8Aoc, and AMB. Suitably, M is selected from .sup.67Ga, .sup.68Ga, .sup.111In, .sup.86Y, and .sup.177Lu. The method further includes applying an imaging technique to detect emitted gamma rays.
(19) Suitable imaging techniques include positron-emission tomography (PET) and single photon emission computed tomography (SPECT). Positron-emission tomography (PET) is a nuclear medicine imaging technique that detects pairs of gamma rays emitted indirectly by a positron-emitting radioligand that is introduced into the body of a subject. Three-dimensional images of tracer concentration within the body are then constructed by computer analysis. Singlephoton emission computed tomography (SPECT, or less commonly, SPET) is a nuclear medicine tomographic imaging technique directly detecting gamma rays emitted by a radionuclide that is introduced into the body of a subject. SPECT imaging is performed by using a gamma camera to acquire multiple 2-D images (also called projections), from multiple angles. A computer is then used to apply a tomographic reconstruction algorithm to the multiple projections, yielding a 3-D data set. This data set may then be manipulated to show thin slices along any chosen axis of the body.
(20) As used herein, “subject in need thereof” (also used interchangeably herein with “a patient in need thereof”) refers to a subject susceptible to or at risk of a specified disease, disorder, or condition. The methods disclosed herein can be used with a subset of subjects who are susceptible to or at elevated risk for prostate cancer. Because some of the method embodiments of the present disclosure are directed to specific subsets or subclasses of identified subjects (that is, the subset or subclass of subjects “in need” of assistance in addressing one or more specific conditions noted herein), not all subjects will fall within the subset or subclass of subjects as described herein for certain diseases, disorders or conditions. Formulations of the present disclosure can be administered to “a subject in need thereof”. As used herein, “a subject” (also interchangeably referred to as “an individual” and “a patient”) refers to animals including humans and non-human animals. Accordingly, the compositions and methods disclosed herein can be used for human and veterinary medical applications. Suitable subjects include warm-blooded mammalian hosts, including humans, companion animals (e.g., dogs, cats), cows, horses, mice, rats, rabbits, primates, and pigs.
(21) Suitable methods for administration of formulations of the present disclosure are by parenteral (e.g., intravenous (IV)) routes or orally, and the formulations administered ordinarily include effective amounts of product in combination with acceptable diluents, carriers and/or adjuvants.
(22) In one aspect, the present disclosure is directed to a method of diagnosing prostate cancer in a subject having or suspected of having prostate cancer. The method includes administering to a subject having, or suspected of having, prostate cancer a bivalent binding agent of formula (II)
[DUPA-6-Ahx-Lys(M-DOTA)-X-RM2] (II).
(23) Suitably, X is selected from 5-Ava, 6-Ahx, 8Aoc, and AMBA. Suitably, M is selected from .sup.67Ga, .sup.68Ga, .sup.111In, .sup.86Y, and .sup.177Lu. The method further includes applying an imaging technique to detect emitted gamma rays. A subject is diagnosed as having prostate cancer based on uptake of the bivalent binding agent as detected in PET or SPECT imaging of the subject. Further, a positive correlation of the bivalent binding agent uptake in a tumor by physiological PET or SPECT investigations and anatomical computerized tomography (CT) scan or magnetic resonance imaging (MRI) scan can further confirm the diagnosis.
(24) Suitable imaging techniques include positron-emission tomography (PET) and single photon emission computed tomography (SPECT), as described herein.
(25) In one aspect, the present disclosure is directed to a method of treating prostate cancer in a subject having or suspected of having prostate cancer, the method comprising: administering to the subject a bivalent binding agent of formula (II) [DUPA-6-Ahx-Lys(M-DOTA)-X-RM2] (II) wherein X is selected from 5-aminovaleric acid (5-Ava), 6-amino hexanoic acid (6-Ahx), 8-aminooctanoic acid) (8Aoc), and paraamino benzoic acid (AMBA); and wherein M is selected from .sup.177Lu, .sup.90Y, .sup.153Sm, and .sup.149Pm.
(26) As used herein, “a subject in need thereof” refers to a subject susceptible to or at risk of a specified disease, disorder, or condition. More particularly, in the present disclosure the methods of treating prostate cancer is to be used with a subset of subjects who are susceptible to or at elevated risk for experiencing prostate cancer. Such subjects may include, but are not limited to, subjects susceptible to or at elevated risk of prostate cancer. Subjects may be susceptible to or at elevated risk for prostate cancer due to family history, age, environment, and/or lifestyle. Based on the foregoing, because some of the method embodiments of the present disclosure are directed to specific subsets or subclasses of identified subjects (that is, the subset or subclass of subjects “in need” of assistance in addressing one or more specific conditions noted herein), not all subjects will fall within the subset or subclass of subjects as described herein for certain diseases, disorders or conditions. As used herein, “susceptible” and “at risk” refer to having little resistance to a certain disease, disorder or condition, including being genetically predisposed, having a family history of, and/or having symptoms of the disease, disorder or condition.
(27) As used herein, the term “composition” generally refers to any product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts. It is to be understood that the compositions described herein may be prepared from isolated compounds described herein or from salts, solutions, hydrates, solvates, and other forms of the compounds described herein. It is also to be understood that the compositions may be prepared from various amorphous, non-amorphous, partially crystalline, crystalline, and/or other morphological forms of the compounds described herein. It is also to be understood that the compositions may be prepared from various hydrates and/or solvates of the compounds described herein. Accordingly, such pharmaceutical compositions that recite compounds described herein are to be understood to include each of, or any combination of, the various morphological forms and/or solvate or hydrate forms of the compounds described herein. Illustratively, compositions may include one or more carriers, diluents, and/or excipients. The compounds described herein, or compositions containing them, may be formulated in a therapeutically effective amount in any conventional dosage forms appropriate for the methods described herein. The compounds described herein, or compositions containing them, including such formulations, may be administered by a wide variety of conventional routes for the methods described herein, and in a wide variety of dosage formats, utilizing known procedures (see generally, Remington: The Science and Practice of Pharmacy, (21.sup.st ed., 2005)).
(28) The term “therapeutically effective amount” as used herein, refers to that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated. In one aspect, the therapeutically effective amount is that which may treat or alleviate the disease or symptoms of the disease at a reasonable benefit/risk ratio applicable to any medical treatment. However, it is to be understood that the total daily usage of the compounds and compositions described herein may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically-effective dose level for any particular patient will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, gender and diet of the patient: the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidentally with the specific compound employed; and like factors well known to the researcher, veterinarian, medical doctor or other clinician of ordinary skill.
(29) The dosage of each compound of the claimed combinations depends on several factors, including: the administration method, the condition to be treated, the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the person to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular patient may affect the dosage used.
(30) The term “administering” as used herein includes all means of introducing the compounds and compositions described herein to the patient, including, but are not limited to, oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal, and the like. The compounds and compositions described herein may be administered in unit dosage forms and/or formulations containing conventional nontoxic pharmaceutically-acceptable carriers, adjuvants, and vehicles. Illustrative formats for oral administration include tablets, capsules, elixirs, syrups, and the like. Illustrative routes for parenteral administration include intravenous, intraarterial, intraperitoneal, epidurial, intraurethral, intrasternal, intramuscular and subcutaneous, as well as any other art recognized route of parenteral administration.
(31) Depending upon the disease as described herein, the route of administration and/or whether the compounds and/or compositions are administered locally or systemically, a wide range of permissible dosages are contemplated herein, including doses of about 100 μg/70 kg or less. The dosages may be single or divided, and may be administered according to a wide variety of protocols, including q.d., b.i.d., t.i.d., or even every other day, once a week, once a month, once a quarter, and the like. In each of these cases it is understood that the therapeutically effective amounts described herein correspond to the instance of administration, or alternatively to the total daily, weekly, month, or quarterly dose, as determined by the dosing protocol.
(32) In making the pharmaceutical compositions of the compounds described herein, a therapeutically effective amount of one or more compounds in any of the various forms described herein may be mixed with one or more excipients, diluted by one or more excipients, or enclosed within such a carrier which can be in the form of a capsule, sachet, paper, or other container. Excipients may serve as a diluent, and can be solid, semi-solid, or liquid materials, which act as a vehicle, carrier or medium for the active ingredient. Thus, the formulation compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. The compositions may contain anywhere from about 0.1% to about 99.9% active ingredients, depending upon the selected dose and dosage form.
(33) The effective use of the compounds, compositions, and methods described herein for treating or ameliorating one or more effects of prostate cancer using one or more compounds described herein may be based upon animal models, such as murine, canine, porcine, and non-human primate animal models of disease. For example, it is understood that prostate cancer in humans may be characterized by a loss of function, and/or the development of symptoms, each of which may be elicited in animals, such as mice, and other surrogate test animals. In particular the mouse model described herein, may be used to evaluate the methods of treatment and the pharmaceutical compositions described herein to determine the therapeutically effective amounts described herein.
(34) The following examples further illustrate specific embodiments of the invention; however, the following illustrative examples should not be interpreted in any way to limit the invention.
EXAMPLES
Example 1
(35) Commercially-available chemical reagents were purchased from Chem-Impex International (Wood Dale, Ill.) and Fisher Scientific (Waltham, Mass.) and used without further purification. Amino acid residues and resins for solid-phase and manual peptide synthesis were purchased from Novabiochem/EMD Biosciences, Inc. (La Jolla, Calif.) and Advanced ChemTech (Louisville, Ky.). Electrospray-ionization (ESI-MS) and Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrometry, for characterization of the peptide precursor, conjugate, and metallated conjugates were performed at the MS Facility, University of Missouri (Columbia, Mo.). PC-3 and LNCaP cells were obtained from American Type Culture Collection and were maintained by the University of Missouri Cell and Immunobiology Core Facility (Columbia, Mo.). Reversed-phase High-Performance Liquid Chromatography (RP-HPLC) analyses of compounds were performed on a Shimadzu SCL-10A system (Shimadzu, Kyoto, Japan) equipped with a Shimadzu SPD-10A UV-vis tunable absorbance detector (λ=280 nm), an Eppendorf TC-50 column heater (Eppendorf, Hamburg, Germany).
(36) Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC)
(37) Purification of [DUPA-6-Ahx-Lys-6-Ahx-RM2] and [DUPA-6-Ahx-Lys(DOTA)-6-Ahx-RM2] conjugate was performed on a semi-preparative, reversed-phase C18 column (Phenomenex Jupiter Proteo, 250×10.00 mm, 10 μm; Phenomenex, Torrance, Calif.). Purification of [DUPA-6-Ahx-Lys(.sup.nat/*M-DOTA)-6-Ahx-RM2], where M=.sup.nat/67Ga, .sup.nat/111In, .sup.nat/177Lu=natural/radioisotopic gallium, indium, and lutetium conjugates, was performed on an analytical, reversed-phase C18 column (Phenomenex Jupiter Proteo, 250×4.60 mm, 5 μm). The solvent system consisted of ultrapure water containing 0.1% trifluoroacetic acid (Solvent A) and acetonitrile containing 0.1% trifluoroacetic acid (Solvent B). A linear gradient of 70:30A/B to 40:60A/B gradient over 15 min (followed by an additional 10 min at 5:95A/B) was used to purify the peptides and metallated constructs. Flow rates of 5 mL/min for the semipreparative and 1.5 mL/min for the analytical RP-HPLC were used during purification procedures. Purified peptide conjugates were lyophilized in a CentriVap system (Labconco, Kansas City, Mo., USA).
(38) Chemistry
(39) Synthesis of the PSMA-Targeting, Small-Molecule, DUPA
(40) Synthesis of 2-[3-(1,3-Bis-tert-butoxycarbonylpropyl)-ureido]pentanedioic acid 1-tert-butyl ester (DUPA precursor) was prepared according to a published procedure with only slight modification [57]. Triethyl amine (2.0 mL, 16.38 mmol) was added to a solution of L-glutamate di-tert-butyl ester hydrochloride (2.0 g, 6.78 mmol) and triphosgene (659.6 mg, 2.24 mmol) in methylene chloride (55.0 mL) and stirred for 2 h at −78° C. under a nitrogen atmosphere. After stirring, a methylene chloride solution (10.0 mL) containing L-Glu (OBn-OtBu) (2.4 g, 7.44 mmol) and triethyl amine (1.2 mL, 9.82 mmol) was added. The reaction mixture was allowed to come to room temperature over a one hour time-period, and the solution continued to stir overnight. The reaction was quenched with 1.0 M HCl, and the organic layer was washed with brine and dried over Na.sub.2SO.sub.4. The crude product was purified using flash chromatography (Hexane:Ethyl Acetate, 1:1) to produce a colorless oil that was recrystallized from a mixture of hexane and methylene chloride. Compounds 2-[3-(3-Benzyloxycarbonyl-1-tert-butoxycarbonyl-propyl)-ureido]pentanedioic acid) di-tert-butyl ester were characterized by .sup.1H and .sup.13C nuclear magnetic resonance (NMR) spectroscopy and characterized by ESI-MS (C.sub.30H.sub.47N.sub.2O.sub.9: Calculated, 578.3184; Found, 579.3289). Deprotection of the benzyl group was performed by hydrogenation. Ten percent palladium on carbon (Pd/C) was added to a solution of 2-[3-(3-Benzyloxycarbonyl-1-tert-butoxycarbonyl-propyl)-ureido]pentanedioic acid) di-tert-butyl ester (2 g, 3.45 mmol) in methylene chloride. The reaction mixture was hydrogenated at 1 atm for a period of 24 h at room temperature, after which the Pd/C was filtered through a celite pad and washed with methylene chloride. The crude product was purified using flash chromatography (Hexane:Ethyl Acetate, 0.4:0.6) to produce 2-[3-(1,3-Bis-tert-butoxycarbonylpropyl)-ureido]pentanedioic acid 1-tert-butyl ester as a colorless oil (1.35 g, 80.2%). The purified product was characterized by .sup.1H and .sup.13C NMR spectroscopy and characterized by ESI-MS (C.sub.23H.sub.41N.sub.2O.sub.9: Calculated, 488.2812; Found, 489.2808).
(41) Direct Synthesis of [DUPA-6-Ahx-Lys(DOTA)-6-Ahx-RM2] Conjugate
(42) The bivalent, PSMA/GRPR targeting precursor [DUPA-6-Ahx-Lys-6-Ahx-RM2], was prepared by direct stepwise synthesis using a manual, resin-based solid phase peptide synthesis employing traditional F-moc chemistry as shown in Scheme 1 (
(43) TABLE-US-00001 TABLE 1 Matrix assisted laser desorption ionization (MALDI)-TOF mass spectrometry values for DUPA-6-Ahx-Lys-6-Ahx-RM2, DUPA-6-Ahx-Lys(DOTA)-6-Ahx-RM2 and metallated DUPA-6-Ahx-Lys(M-DOTA)-6-Ahx-RM2 conjugates. M is defined as .sup.nat/67Ga, .sup.nat/111In, .sup.nat/177Lu. HPLC tr Analog Mol. Formula Calculated Observed (min) DUPA-6-Ahx-Lys-6-Ahx- C.sub.84H.sub.128N.sub.20O.sub.22 1768.95 1769.97 9.0 RM2 DUPA-6-Ahx-Lys(DOTA)- C.sub.100H.sub.154N.sub.24O.sub.29 2155.11 2156.13 9.8 6-Ahx-RM2 DUPA-6-Ahx-Lys(.sup.natGa- C.sub.100H.sub.154N.sub.24O.sub.29.sup.natGa 2223.03 2224.01 9.2 DOTA)-6-Ahx-RM2 DUPA-6-Ahx-Lys(.sup.natIn- C.sub.100H.sub.154N.sub.24O.sub.29.sup.natIn 2267.88 2268.74 9.3 DOTA)-6-Ahx-RM2 DUPA-6-Ahx-Lys(.sup.natLu- C.sub.100H.sub.154N.sub.24O.sub.29.sup.natLu 2327.60 2328.58 9.3 DOTA)-6-Ahx-RM2 DUPA-6-Ahx-Lys(.sup.67Ga- 9.2 DOTA)-6-Ahx-RM2 DUPA-6-Ahx-Lys(.sup.111In- 9.3 DOTA)-6-Ahx-RM2 DUPA-6-Ahx-Lys(.sup.177Lu- 9.3 DOTA)-6-Ahx-RM2
(44) DOTA-NHS ester, 2,2′,2″-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, was conjugated as the final step in the synthetic procedure via an active ester onto the ε-amine of the lysine residue of the bivalent peptide precursor [DUPA-6-Ahx-Lys-6-Ahx-RM2] to produce [DUPA-6-Ahx-Lys(DOTA)-6-Ahx-RM2], using a slightly-modified procedure that is similar to what has previously been described [49]. Briefly, DOTA-NHS (29 μmol) was stirred at room temperature in 200 μL of 0.1 M sodium phosphate buffer (pH=7.0). [DUPA-6-Ahx-Lys-6-Ahx-RM2] (2.9 μmol) was dissolved in 0.1 M sodium phosphate buffer and the pH was adjusted to 7.4 using 10% NaOH. The reaction mixture was allowed to stir for 6 h at 5-10° C., after which it was allowed to stir overnight at ambient temperature. The bivalent DOTA conjugate was purified by RP-HPLC and obtained in ˜35% yield. MALDI-TOF mass spectrometry was used to confirm the identity of the new bivalent conjugate (Table 1).
(45) Modified Synthesis of [DUPA-6-Ahx-Lys(DOTA)-6-Ahx-RM2] Conjugate
(46) Due to the relatively poor synthetic yields of [DUPA-6-Ahx-Lys(DOTA)-6-Ahx-RM2] under the direct synthetic procedure, the new bivalent agent was also prepared using a (IvDde) protected ε-amine of lysine [Lys-(IvDde)] under a modified synthetic procedure as illustrated in Scheme 2 (
(47) Preparation of [DUPA-6-Ahx-Lys(.sup.nat/*M-DOTA)-6-Ahx-RM2]
(48) The protocol for metallation of each conjugate was based upon a previously published procedure [49] with only minor modifications. Briefly, either natural GaCl.sub.3 3H.sub.2O or InCl.sub.3 3H.sub.2O, or LuCl.sub.3 3H.sub.2O in 0.05 N HCl (90 nmol) was added to purified [DUPA-6-Ahx-Lys(DOTA)-6-Ahx-RM2] peptide conjugate (90 nmol) dissolved in 250 μl 0.4 M ammonium acetate. Immediately following a 1 hour incubation at 80° C., 50 μL of a 10 mM diethylene-triaminepentaacetic acid (DTPA) was added to the mixture to scavenge remaining unbound metal. The resulting, metallated compounds were purified by RP-HPLC and submitted for MALDI-TOF mass spectrometry characterization prior to in vitro competitive binding assays. Similarly, synthesis of radio conjugates was achieved by the reaction of 50 μg of purified [DUPA-6-Ahx-Lys(DOTA)-6-Ahx-RM2] in 200 μl 0.4 M ammonium acetate with .sup.67GaCl.sub.3 3H.sub.2O (˜489 MBq, 1.32×104 μCi), .sup.111InCl.sub.3 3H.sub.2O (˜436 MBq, 1.18×104 μCi), or .sup.177LuCl.sub.3 3H.sub.2O (˜410 MBq, 1.11×104 μCi) in 0.05 N HCl for 1 h at 80° C. This was followed by the addition of 50 μL of 10 mM DTPA solution to scavenge the remaining unbound metal. The resulting radio conjugates were purified using RP-HPLC and collected into 10 mg of ascorbic acid dissolved in 100 μL of 1 mg/mL bovine serum albumin (BSA) prior to in vitro stability assays. Acetonitrile was removed under a steady stream of nitrogen, and the radiochemical purity was assessed by RP-HPLC.
(49) In Vitro Assays
(50) In Vitro RP-HPLC Stability Assays
(51) RP-HPLC purified radioconjugates [DUPA-6-Ahx-Lys[.sub.67Ga-DOTA]-6-Ahx-RM2], [DUPA-6-Ahx-Lys[.sup.111In-DOTA]-6-Ahx-RM2], and [DUPA-6-Ahx-Lys[.sup.77Lu-DOTA]-6-Ahx-RM2] were incubated in phosphate-buffered saline and were analyzed by RP-HPLC in order to assess the degree of product degradation due to radiolysis or radionuclide dissociation from the DOTA bifunctional chelating agent. Time-points were assessed at 2, 12, 24, and 48 hours.
(52) In Vitro Competitive Displacement Binding Assays
(53) A competitive displacement binding affinity assay (IC.sub.50) with purified [DUPA-6-Ahx-Lys-6-Ahx-RM2] and [DUPA-6-Ahx-Lys(DOTA)-6-Ahx-RM2] was performed in GRPR-positive PC-3 cells and PSMA-positive homogenized cell membranes using [.sup.125I-(Tyr4)-BBN] and [N-acetyl aspartyl .sup.3H-glutamate] (NAAG) as the radioligands. For the GRPR-positive assay, 3×10.sup.4 PC-3 cells (in D-MEM/F-12 K media containing 0.01 M MEM and 2% BSA, pH=5.5) were incubated with 20,000 counts per minute of [.sup.125I-(Tyr4)-BBN] and increasing concentrations (1×10.sup.−13-1×10.sup.−5 M) of the metallated targeting vector (1 h, 37° C., 5% CO.sub.2-enriched atmosphere). After incubation, the reaction medium was aspirated and the cells were rinsed three times with cold media. Cell-associated radioactivity was determined using a Packard Riastar gamma counter. The percent of bound radioligand was plotted against the increasing concentrations of the metallated conjugate to determine the IC.sub.50 value. IC.sub.50 values were determined by curve fitting using Prism Software (version 6.0). For the PSMA-positive assay, the binding affinity was measured using the N-acetylated-α-linked acidic dipeptidase (NAALADase) assay with only minor modification [58]. Briefly, [DUPA-6-Ahx-Lys-6-Ahx-RM2] and [DUPA-6-Ahx-Lys(DOTA)-6-Ahx-RM2] (increasing concentrations 1×10.sup.−13-1×10.sup.−5 M) in 50 μL of Tris-HCl buffer (50 mM, pH=7.4) were incubated with LNCaP tissue culture homogenized cell membranes for a period of 45 min. Then, [N-acetyl aspartyl .sup.3H-glutamate] was added to the reaction mixture and the solution was allowed to incubate for an additional 15 min at 37° C. The enzymatic reaction was stopped by addition of 50 μL of cold sodium phosphate buffer (0.1 M, pH=7.4). [N-acetyl aspartyl .sup.3H-glutamate] and [.sup.3H-glutamate] were resolved by cation exchange chromatography using AG 50 W-X8 columns (200-400 mesh). Columns were preequilibrated with 0.2 M HCl prior to loading of the reaction mixture. Fractions containing [.sup.3H glutamate] were eluted using 6 mL of 2 M HCl. Scintillation cocktail was added to each fraction and the amount of radioactivity in each was determined by liquid scintillation counting. The percent of bound radioligand was plotted against the increasing concentrations of the conjugate to determine the IC.sub.50 value. IC.sub.50 values were determined by curve fitting using Prism Software (version 6.0).
Example 2
(54) In this Example, biodistribution studies were conducted.
(55) Biodistribution Studies in Normal Mice
(56) The in vivo behavior of the .sup.111In-labeled DUPA-6-Ahx-Lys(.sup.111In-DOTA)-X-RM2 of (X=5-Ava, 6-Ahx, 8-Aoc and AMBA) was determined in CF-1 normal mice after intravenous (i.v.) administration of each compound (100 μL, 3-4 MBq, 0.1-0.2 nmol/mouse). The biodistribution (1 hour post-injection (p.i.)) is summarized in Table 2. DUPA-6-Ahx-Lys(.sup.111In-DOTA)-5-Ava-RM2 presented high kidneys (4.92±0.87% ID/g) and pancreas (1.02±37% ID/g) uptake, whereas for DUPA-6-Ahx-Lys(.sup.111In-DOTA)-8-Aoc-RM2 the high uptake was demonstrated in pancreas (5.45±0.47% ID/g) and small intestines (2.20±3.37% ID/g) respectively.
(57) Biodistribution Studies in PC-3 and PC-3PIP Tumor-Bearing Mice
(58) The in vivo behavior of the .sup.111In-labeled DUPA-6-Ahx-Lys(.sup.111In-DOTA)-8-Aoc-RM2 heterodimer was evaluated in ICR-SCID male mice bearing PSMA positive PC-3PIP and GRPr positive PC-3 tumors (after intravenous (i.v.) administration of each compound (100 μL, 3-4 MBq, 0.1-0.2 nmol/mouse). The results of the respective tumor uptakes for both PC-3 and PC-3PIP tumor models are presented in Table 3, 4 (for the full organ distribution tables), whereas Table 2 summarizes the normal tissue ratios for muscle, kidney, spleen, and liver and Table 3 and 4 summarizes the tumor tissue ratios for muscle, kidney, spleen, and liver. The tumor uptake of DUPA-6-Ahx-Lys(.sup.111In-DOTA)-8-Aoc-RM2 (in tumor-bearing PC-3 mice was 1 hr: 4.74±0.90; 4 hr: 4.70±1.01 and 24 hr: 2.64±1.07% ID/g) whereas accumulation and retention in pancreas was significantly lower (4 hr: 0.58±0.10 and 24 hr: 0.14±0.01% ID/g)).
(59) The uptake/accumulation for DUPA-6-Ahx-Lys(.sup.111In-DOTA)-8-Aoc-RM2 in PC-3PIP tumor-bearing mice was 1 hr: 5.38±1.07; 4 hr: 4.43±0.51 and 24 hr: 1.61±0.50% ID/g, whereas accumulation and retention in pancreas was significantly lower (4 hr: 0.51±0.24 and 24 hr: 0.27±0.08% ID/g). For PC-3 tumor-bearing mice and PC-3PIP tumor-bearing mice, DUPA-6-Ahx-Lys(.sup.111In-DOTA)-8-Aoc-RM2 showed high tumor uptake. The tumor accumulation rate was rather similar in both cases (PC-3 tumor-bearing mice and PC-3PIP tumor-bearing mice), but kidney and pancreas uptake was reduced for PC-3 tumor-bearing mice.
(60) TABLE-US-00002 TABLE 2 Biodistribution studies of [DUPA-6-Ahx-[.sup.111In-DOTA]-X-RM2] in CF-1 normal mice at 1 hr p.i. (% ID/g ± SD, n = 5). X = 5-Ava, 6-Ahx, 8-Aoc, AMBA. 5-Ava 6-Ahx 8-Aoc AMBA 1 h 1 h 1 h 1 h Heart 0.11 ± 0.06 0.16 ± 0.22 0.20 ± 0.14 0.12 ± 0.05 Lung 0.68 ± 0.25 0.86 ± 0.70 0.37 ± 0.03 1.01 ± 1.15 Liver 0.15 ± 0.05 0.23 ± 0.14 0.38 ± 0.04 0.28 ± 0.04 Kidneys 4.92 ± 0.84 2.84 ± 0.64 1.37 ± 0.31 1.37 ± 0.31 Spleen 0.13 ± 0.05 0.12 ± 0.13 0.49 ± 0.34 0.15 ± 0.04 Stomach 0.23 ± 0.18 0.14 ± 0.11 1.73 ± 2.49 0.15 ± 0.06 S. Intestine 0.26 ± 0.09 0.22 ± 0.13 2.20 ± 3.37 0.20 ± 0.04 L. Intestine 0.12 ± 0.05 0.14 ± 0.12 0.49 ± 0.25 0.09 ± 0.02 Muscle 0.06 ± 0.04 0.07 ± 0.07 0.10 ± 0.01 0.08 ± 0.02 Bone 0.09 ± 0.06 0.08 ± 0.06 0.14 ± 0.04 0.06 ± 0.04 Brain 0.02 ± 0.01 0.03 ± 0.04 0.02 ± 0.00 0.02 ± 0.01 Pancreas 1.02 ± 0.37 0.19 ± 0.19 5.45 ± 0.47 0.24 ± 0.06 Blood* 0.24 ± 0.17 0.43 ± 0.73 0.38 ± 0.14 0.26 ± 0.13 Urine* 91.53 ± 2.22 91.00 ± 5.58 79.74 ± 6.05 88.72 ± 1.96 *Data presented as % ID
(61) TABLE-US-00003 TABLE 3 Biodistribution studies of [DUPA-6-Ahx-[.sup.111In-DOTA]-8-Aoc-RM2] in ICR-SCID PC-3 tumor mice at 1 hr, 4 hr and 24 hr p.i. (% ID/g ± SD). 8-Aoc 8-Aoc 8-Aoc 1 h 4 h 24 h Heart 0.31 ± 0.02 0.06 ± 0.03 0.03 ± 0.01 Lung 0.67 ± 0.08 0.12 ± 0.03 0.04 ± 0.01 Liver 0.56 ± 0.11 0.14 ± 0.04 0.10 ± 0.02 Kidneys 8.90 ± 1.40 2.62 ± 0.88 0.40 ± 0.20 Spleen 1.16 ± 0.39 0.13 ± 0.04 0.10 ± 0.03 Stomach 0.66 ± 0.12 0.19 ± 0.03 0.10 ± 0.14 S. Intestine 0.94 ± 0.16 0.27 ± 0.04 0.10 ± 0.07 L. Intestine 0.40 ± 0.11 0.64 ± 0.08 0.19 ± 0.17 Muscle 0.13 ± 0.04 0.02 ± 0.00 0.01 ± 0.00 Bone 0.17 ± 0.08 0.07 ± 0.03 0.03 ± 0.01 Brain 0.04 ± 0.01 0.01 ± 0.00 0.00 ± 0.00 Pancreas 9.08 ± 1.88 0.58 ± 0.10 0.14 ± 0.02 Blood* 1.19 ± 0.22 0.13 ± 0.05 0.02 ± 0.00 Urine* 70.27 ± 11.30 93.57 ± 1.19 94.09 ± 4.00 Tumor 4.74 ± 0.90 4.70 ± 1.01 2.64 ± 1.07 *Data presented as % ID
(62) TABLE-US-00004 TABLE 4 Biodistribution studies of [DUPA-6-Ahx-[.sup.111In-DOTA]-8-Aoc-RM2] in ICR-SCID PC-3PIP tumor mice at 1 hr, 4 hr and 24 hr p.i. (% ID/g ± SD). 8-Aoc 8-Aoc 8-Aoc 1 h 4 h 24 h Heart 0.85 ± 0.33 0.16 ± 0.07 0.04 ± 0.01 Lung 1.77 ± 0.52 0.35 ± 0.16 0.13 ± 0.04 Liver 1.11 ± 0.33 0.39 ± 0.15 0.28 ± 0.04 Kidneys 21.71 ± 7.61 3.30 ± 0.69 0.93 ± 0.22 Spleen 2.94 ± 1.14 0.41 ± 0.16 0.27 ± 0.08 Stomach 0.87 ± 0.31 0.64 ± 0.28 0.12 ± 0.06 S. Intestine 2.33 ± 0.97 2.35 ± 3.80 0.18 ± 0.09 L. Intestine 0.77 ± 0.22 0.93 ± 0.07 0.70 ± 0.40 Muscle 0.23 ± 0.06 0.05 ± 0.02 0.02 ± 0.01 Bone 0.39 ± 0.17 0.15 ± 0.11 0.10 ± 0.02 Brain 0.09 ± 0.03 0.03 ± 0.01 0.01 ± 0.00 Pancreas 18.02 ± 6.54 1.19 ± 0.56 0.27 ± 0.08 Blood* 3.18 ± 0.83 0.51 ± 0.24 0.07 ± 0.02 Urine* 60.11 ± 9.83 87.72 ± 6.90 91.91 ± 3.17 Tumor 5.38 ± 1.07 4.43 ± 0.51 1.61 ± 0.50 *Data presented as % ID
Example 3
(63) In this Example, in vitro competitive displacement binding assays were conducted.
(64) A competitive displacement binding assay (IC.sub.50) of [DUPA-6-Ahx-Lys[DOTA]-X-RM2] and [DUPA-6-Ahx-Lys[nat-In/nat-Lu-DOTA]-X-RM2] where X=5-Ava, 6-Ahx, 8-Aoc and AMBA were determined in GRPR-positive PC-3 cells and PSMA-positive homogenized cell membranes using [.sup.125I-(Tyr4)-BBN] and [N-acetyl aspartyl .sup.3H-glutamate] (NAAG) as the radioligands. For the GRPR-positive assay, 3×10.sup.4 PC-3 cells (in D-MEM/F-12 K media containing 0.01 M MEM and 2% BSA, pH=5.5) were incubated with 20,000 counts per minute of [.sup.125I-(Tyr4)-BBN] and increasing concentrations (1×10.sup.−13-1×10.sup.−5 M) of the metallated targeting vector (1 h, 37° C., 5% CO.sub.2-enriched atmosphere). After incubation, the reaction medium was aspirated and the cells were rinsed three times with cold media. Cell-associated radioactivity was determined using a Packard Riastar gamma counter. The percent of bound radioligand was plotted against the increasing concentrations of the metallated conjugate to determine the IC.sub.50 value. IC.sub.50 values were determined by curve fitting using Prism Software (version 6.0). For the PSMA-positive assay, the binding affinity was measured using the N-acetylated-α-linked acidic dipeptidase (NAALADase) assay. Briefly [DUPA-6-Ahx-Lys[DOTA]-X-RM2] and [DUPA-6-Ahx-Lys[nat-In/nat-Lu-DOTA]-X-RM2] (increasing concentrations 1×10.sup.−13-1×10.sup.−5 M) in 50 μL of Tris-HCl buffer (50 mM, pH=7.4) were incubated with LNCaP tissue culture homogenized cell membranes for a period of 45 min. Then, [N-acetyl aspartyl .sup.3H-glutamate] was added to the reaction mixture and the solution was allowed to incubate for an additional 15 min at 37° C. The enzymatic reaction was stopped by addition of 50 μL of cold sodium phosphate buffer (0.1 M, pH=7.4). [N-acetyl aspartyl .sup.3H-glutamate] and [.sup.3H-glutamate] were resolved by cation exchange chromatography using AG 50 W-X8 columns (200-400 mesh). Columns were pre-equilibrated with 0.2 M HCl prior to loading of the reaction mixture. Fractions containing [.sup.3H-glutamate] were eluted using 6 mL of 2 M HCl. Scintillation cocktail was added to each fraction and the amount of radioactivity in each was determined by liquid scintillation counting. The percent of bound radioligand was plotted against the increasing concentrations of the conjugate to determine the IC.sub.50 value. IC.sub.50 values were determined by curve fitting using Prism Software (version 6.0). Results are summarized in Table 5.
(65) TABLE-US-00005 TABLE 5 Mass spectrometry, IC.sub.50, and RP-HPLC data for [DUPA-6-Ahx-[DOTA]-spacer-RM2] and [DUPA-6-Ahx-[nat-In/nat-Lu-DOTA]-spacer-RM2]. Calculated Observed IC.sub.50 IC.sub.50 Name of molecular molecular PC3 LNCaP HPLC Compound Molecular Formula mass Mass cells cells t.sub.r(min) [DUPA-6-Ahx- C.sub.99H.sub.152N.sub.24O.sub.29 2141.44 2142.45 5.14 ± 1.39 nM 11.21 ± 2.05 nM 11.5 [DOTA]-5-Ava- RM2] [DUPA-6-Ahx- C.sub.100H.sub.154N.sub.24O.sub.29 2156.44 2157.60 3.85 ± 1.12 nM 10.49 ± 2.77 nM 11 .5 [DOTA]-6-Ahx- RM2] [DUPA-6-Ahx- C.sub.102H.sub.158N.sub.24O.sub.29 2184.45 2185.44 5.93 ± 1.09 nM 12.69 ± 2.29 nM 11 .7 [DOTA]-8-Aoc- RM2] [DUPA-6-Ahx- C.sub.101H.sub.148N.sub.24O.sub.29 2157.96 2159.02 5.27 ± 1.31 nM 11.97 ± 2.73 nM 11.5 [DOTA]-AMBA- RM2] [DUPA-6-Ahx- C.sub.99H.sub.149N.sub.24O.sub.29.sup.natIn 2253.00 2254.04 5.74 ± 1.23 nM 14.37 ± 2.53 nM 11 .8 [.sup.natIn-DOTA]-5- Ava- RM2] [DUPA-6-Ahx- C.sub.100H.sub.151N.sub.24O.sub.29.sup.natIn 2267.01 2268.27 4.35 ± 1.47 nM 13.53 ± 2.96 nM 11.8 [.sup.natIn-DOTA]-6- Ahx-RM2] [DUPA-6-Ahx- C.sub.102H.sub.155N.sub.24O.sub.29.sup.natIn 2295.04 2296.32 6.12 ± 1.03 nM 15.69 ± 2.71 nM 12.3 [.sup.natIn-DOTA]-8- Aoc-RM2] [DUPA-6-Ahx- C.sub.101H.sub.145N.sub.24O.sub.29.sup.natIn 2272.96 2274.23 5.73 ± 0.98 nM 14.87 ± 2.45 nM 11.8 [.sup.natIn-DOTA- AMBA-RM2] [DUPA-6-Ahx- C.sub.99H.sub.149N.sub.24O.sub.29.sup.natLu 2313.03 2314.30 5.92 ± 1.11 nM 14.12 ± 2.17 nM 11.8 [.sup.natLu-DOTA]-5- Ava-RM2] [DUPA-6-Ahx- C.sub.100H.sub.151N.sub.24O.sub.29.sup.natLu 2327.05 2328.05 4.83 ± 1.19 nM 13.85 ± 2.53 nM 11.8 [.sup.natLu-DOTA]-6- Ahx-RM2] [DUPA-6-Ahx- C.sub.102H.sub.155N.sub.24O.sub.29.sup.natLu 2355.08 2356.08 6.49 ± 1.26 nM 15.95 ± 2.79 nM 12.3 [.sup.natLu-DOTA]-8- Aoc-RM2] [DUPA-6-Ahx- C.sub.101H.sub.145N.sub.24O.sub.29.sup.natLu 2333.3 2334.38 5.51 ± 1.43 nM 15.09 ± 2.64 nM 11.8 [.sup.natLu-DOTA]- AMBA-RM2
(66) Results
(67) The novel, dual-biomarker, targeting ligands described herein have high affinity and specificity for PSMA/GRPR. [DUPA-6-Ahx-Lys(DOTA)-6-Ahx-RM2] and its metallated conjugates are the first of their kind for this new family of dual-biomarker targeting agents. In the Examples, 2-[3-(1,3-Bis-tert-butoxycarbonylpropyl)-ureido]pentanedioic acid 1-tert-butyl ester (DUPA precursor) was prepared, purified by flash chromatography, and characterized by ESI-MS and .sup.1H/.sup.13C NMR spectroscopy. Deprotection by hydrogenation afforded 2-[3-(1,3-Bis-tert-butoxycarbonylpropyl)-ureido]pentanedioic acid 1-tert-butyl ester. The PSMA/GRPR dual targeting ligand precursor [DUPA-6-Ahx-Lys-6-Ahx-RM2] conjugate, was first originally prepared by solid-phase peptide synthesis (SPPS) with 2-[3-(1,3-Bis-tert-butoxycarbonylpropyl)-ureido]pentanedioic acid 1-tertbutyl ester being the final addition to the sequence. In the final procedure for preparation of the bivalent agent, DOTA-tris(tBu) NHS active ester was conjugated to [DUPA-6-Ahx-Lys-6-Ahx-RM2]. However, the DOTA-conjugated product [DUPA-6-Ahx-Lys(DOTA)-6-Ahx-RM2] was obtained in poor yield (35%,
Example 4
(68) In this Example, in vitro competitive displacement binding assays were conducted.
(69) A competitive displacement binding assay (IC.sub.50) of [DUPA-6-Ahx-Lys[DOTA]-spacer-RM2] and [DUPA-6-Ahx-Lys[nat-In/nat-Lu-DOTA]-spacer-RM2] where spacer=5-Ava, 6-Ahx, 8-Aoc and AMBA were determined in GRPR-positive PC-3 cells and PSMA-positive homogenized cell membranes using [.sup.125I-(Tyr4)-BBN] and [N-acetyl aspartyl .sup.3H-glutamate](NAAG) as the radioligands. For the GRPR-positive assay, 3×10.sup.4 PC-3 cells (in D-MEM/F-12 K media containing 0.01 M MEM and 2% BSA, pH=5.5) were incubated with 20,000 counts per minute of [.sup.125I-(Tyr4)-BBN] and increasing concentrations (1×10.sup.−13-1×10.sup.−5 M) of the metallated targeting vector (1 h, 37° C., 5% CO.sub.2-enriched atmosphere). After incubation, the reaction medium was aspirated and the cells were rinsed three times with cold media. Cell-associated radioactivity was determined using a Packard Riastar gamma counter. The percent of bound radioligand was plotted against the increasing concentrations of the metallated conjugate to determine the IC.sub.50 value. IC.sub.50 values were determined by curve fitting using Prism Software (version 6.0). For the PSMA-positive assay, the binding affinity was measured using the N-acetylated-α-linked acidic dipeptidase (NAALADase) assay. Briefly [DUPA-6-Ahx-Lys[DOTA]-spacer-RM2] and [DUPA-6-Ahx-Lys[nat-In/nat-Lu-DOTA]-spacer-RM2] (increasing concentrations 1×10.sup.−113-1×10.sup.−5 M) in 50 μL of Tris-HCl buffer (50 mM, pH=7.4) were incubated with LNCaP tissue culture homogenized cell membranes for a period of 45 min. Then, [N-acetyl aspartyl .sup.3H-glutamate] was added to the reaction mixture and the solution was allowed to incubate for an additional 15 min at 37° C. The enzymatic reaction was stopped by addition of 50 μL of cold sodium phosphate buffer (0.1 M, pH=7.4). [N-acetyl aspartyl .sup.3H-glutamate] and [.sup.3H-glutamate] were resolved by cation exchange chromatography using AG 50 W-X8 columns (200-400 mesh). Columns were pre-equilibrated with 0.2 M HCl prior to loading of the reaction mixture. Fractions containing [.sup.3H-glutamate] were eluted using 6 mL of 2 M HCl. Scintillation cocktail was added to each fraction and the amount of radioactivity in each was determined by liquid scintillation counting. The percent of bound radioligand was plotted against the increasing concentrations of the conjugate to determine the IC.sub.50 value. IC.sub.50 values were determined by curve fitting using Prism Software (version 6.0).
(70) In this study, a novel, dual-biomarker, targeting ligand having high affinity and specificity for PSMA/GRPr receptors that are expressed on most prostate cancers was prepared. [DUPA-6-Ahx-Lys(DOTA)-X-RM2] was synthesized and the new conjugate was metallated macroscopically with GaCl.sub.3, InCl.sub.3, and LuCl.sub.3 to form [DUPA-6-Ahx-Lys(M-DOTA)-X-RM2](where M=Ga, In, or Lu). These new agents, when radiolabeled with M=In-111 or Lu-177 hold theranostic potential for patients presenting with prostate cancer disease.
(71) X=5-Ava, 6-Ahx, 8Aoc, AMBA
(72) ##STR00001## ##STR00002##
(73) The results presented herein demonstrate that PSMA and the GRPR can serve as useful biomarkers for bivalent molecular targeting agents for diagnosis of disease via positron-emission tomography (PET) or singlephoton emission computed tomography (SPECT) and radiotherapy. Disclosed herein is the preparation of novel, dual-biomarker, targeting ligands having high affinity and specificity for PSMA/GRPR biomarkers. [DUPA-6-Ahx-Lys(DOTA)-6-Ahx-RM2] was synthesized by manual solid-phase peptide synthesis. The new conjugates were metallated with .sup.nat/67GaCl.sub.3, .sup.nat/111InCl.sub.3, and .sup.nat/177LuCl.sub.3 to form [DUPA-6-Ahx-Lys(.sup.nat/*M-DOTA)-6-Ahx-RM2] (