Patent Application
20100015058
The present disclosure relates to heterodimeric compositions for delivery of radiolabeled and other ligands to a cell or tissue. The disclosure further relates to methods of ligand delivery to, and imaging of, cells and tissues expressing an integrin and gastrin-releasing peptide receptor, in particular prostate cancer cells.
The present disclosure includes a sequence listing incorporated herein by reference in its entirety.
Prostate cancer remains one of the leading causes of cancer-related deaths in the United States and Europe (di Sant'Agnese P. A., Urology. (1998) 51: 121-124). As population life expectancy increases, so will the incidence of this disease, creating what will become an epidemic male health problem.
Over-expression of gastrin-releasing peptide receptor (GRPR) has been discovered in androgen-independent human prostate tissues, (di Sant'Agnese P. A., Urology. (1998) 51: 121-124; Chung et al., Surgery (1992) 112: 1059-1065; Glover et al., Mol. Carcinog. (2003); 37: 5-15; Vashchenko & Abrahamsson Eur. Urol. (2005) 47: 147-155), breast cancer, gastric cancer, etc. Various approaches have been explored for the imaging of in vivo GRPR expression. Bombesin (BBN), which was originally isolated from the skin of a frog, is an analog of the gastrin-releasing peptide (GRP). The truncated peptide BBN(7-14) was considered to be sufficient for the specific binding interaction with GRPR, and also is sufficiently metabolically stable for in vivo application. Several BBN peptides have been labeled with various radioisotopes for diagnosis and treatment of GRPR-positive prostate lesions (Zhang et al., J. Nucl. Med. (2006) 47: 492-501; Varvarigou et al., (2004) 19: 219-229; Zhang et al., Cancer Res. (2004) 64: 6707-6715; Smith et al., Anticancer Res. (2003) 23: 63-70; Rogers et al., Bioconjug. Chem. (2003) 14: 756-763).
18F-Labeled BBN peptides were successfully used for detecting GRPR-positive prostate cancer in vivo (Zhang et al., J. Nucl. Med. (2006) 47: 492-501). However, 18F-labeled tracers derived from monomeric BBN had a relatively low tumor accumulation and retention as well as unfavorable hepatobiliary excretion (Zhang et al., J. Nucl. Med. (2006) 47: 492-501). Therefore, modifications are desirable to obtain a better tumor-targeting effect and imaging quality.
Most solid tumors are angiogenesis-dependent and integrins are key players. In particular, integrin αvβ3 was found to be necessary for the formation, survival, and maturation of new blood vessels (Friedlander et al., Science. (1995) 270: 1500-1502; Horton M. A., Int. J. Biochem. Cell Biol. (1997) 29: 721-725; Bello et al., Neurosurgery (2001) 49: 380-389). Synthetic peptides containing the arginine-glycine-aspartate (RGD) sequence motif are active modulators of cell adhesion and can bind specifically to integrin αvβ3 Excellent tumor integrin-targeting efficacy and favorable in vivo kinetics were obtained for radiolabeled multimeric RGD peptides due to the polyvalency effect (Liu S., Mol. Pharm. (2006) 3: 472-487; Jung et al., J. Nucl. Med. (2006) 47: 2000-2007; Zhang et al., J. Nucl. Med. (2006) 47: 113-121; Dijkgraaf et al., Eur. J. Nucl. Med. Mol. Imaging. (2007) 34: 267-273; Dijkgraaf et al., Nucl. Med. Biol. (2007) 34: 29-35; Tucker G. C., Curr. Opin. Investig. Drugs. (2003) 4: 722-731; Li et al., J. Nucl. Med. (2007) 48:1162-1171). However, RGD peptide-based probes, including multimeric RGD peptides with high affinity for integrin αvβ3, had only moderate uptake in prostate cancer models, presumably because of the insufficient expression of this receptor in prostate cancer tumors.
The present disclosure encompasses heterodimeric compositions for delivering radiolabeled and other ligands to a cell or tissue, and particularly provides compositions and methods of their use for targeting and imaging cells and tissues expressing both an integrin and gastrin-releasing peptide receptor, in particular prostate cancer cells. One aspect of the disclosure, therefore, encompasses compositions that comprise a heterodimeric probe comprising a first peptide domain comprising a moiety capable of selectively binding to an integrin; a second peptide domain comprising a moiety capable of selectively binding to a gastrin-releasing peptide receptor; a linker connecting the first peptide domain and the second peptide domain; and a prosthetic group. The prosthetic group is usefully a detectable label such as radionuclide, and optionally a therapeutically advantageous moiety.
In the embodiments of this aspect of the disclosure, the first peptide domain comprises at least one peptide comprising the amino acid sequence of arginine-glycine-aspartate, such as, for example, but not limited to, cyclo(arginine-glycine-aspartate-D-tyrosine-lysine). The moiety capable of selectively binding to a gastrin-releasing peptide receptor can comprise a fragment of the polypeptide bombesin, the fragment specifically binding to a gastrin-releasing peptide receptor. In one embodiment, the second domain is the peptide bombesin(7-14).
In some embodiments of the disclosure, the linker connecting the first peptide domain and the second peptide domain can be a glutamate residue, or an aspartate residue, and can also comprise a tether between the linker and a prosthetic group.
In the compositions of the disclosure, the prosthetic group can be a detectable label, a therapeutic agent, or a combination thereof, but in particular the prosthetic group can be a detectable label such as, but not limited to, the isotopic labels 18F, 68Ga, 64Cu, 76Br, 86Y, 124I, 89Zr, 111In, 99mTc, 123/131I, a fluorescent dye, a quantum dot, an alpha emitter, a beta emitter, and a gamma emitter. In especially useful embodiments, the prosthetic group comprises the fluoride isotope 18F.
Another aspect of the present disclosure provides for methods of identifying a cell or a population of cells expressing an integrin and/or a gastrin-releasing peptide receptor, comprising: contacting a cell or population of cells with an embodiment of a composition according to present disclosure, where the compositions comprise a heterodimeric probe capable of selectively binding to an integrin and to a gastrin-releasing peptide receptor of a cell; allowing the heterodimeric probe to selectively bind to at least one of an integrin and of a gastrin-releasing peptide receptor of a cell or a population of cells; and detecting the presence of the heterodimeric probe on the cell or population of cells, whereby the presence of the heterodimeric probe on the cell or population of cells indicates that the cell or population of cells has an integrin, a gastrin-releasing peptide receptor, or both an integrin and a gastrin-releasing peptide receptor thereon.
In this aspect of the disclosure, the heterodimeric probe may be, but is not limited to, being detected by positron emission tomography, by single photon emission computed tomography, fluorescent imaging, and the like, depending upon the prosthetic group attached to the heterodimeric probe.
Yet another aspect of the disclosure provides methods of delivering an agent to a cell, comprising contacting a cell or population of mammalian cells with a heterodimeric probe according to present disclosure, where the heterodimeric probe is capable of simultaneously binding to an integrin and to a gastrin-releasing peptide receptor, and where the probe further comprises an agent to be delivered to a target cell or tissue of a mammalian subject; and allowing the heterodimeric probe to bind to an integrin, a gastrin-releasing peptide receptor, or both an integrin and a gastrin-releasing peptide receptor, on the cell or population of mammalian cells, thereby delivering the agent to the cell or cells.
Many aspects of the disclosure can be better understood with reference to the following drawings.
Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
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 this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a support” includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.
As used herein, the following terms have the meanings ascribed to them unless specified otherwise. In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein, but which may contain additional structural groups, composition components or method steps (or analogs or derivatives thereof as discussed above). Such additional structural groups, composition components or method steps, etc., however, do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein. “Consisting essentially of” or “consists essentially” or the like, when applied to methods and compositions encompassed by the present disclosure have the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.
BBN, bombesin; GRPR, gastrin-releasing peptide receptor; GRP, gastrin-releasing peptide; RGD, argine-glycine-aspartate; FB, fluorobenzoate; PET, positron emission tomography; SPECT, single photon emission computed tomography; GP, glycoprotein; TFA, trifluoroacetic acid; ACN, acetonitrile. NOTA, 1,4,7-triazacyclononane-1,4,7-triacetic acid; DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetracetate; PEG3, 11-amino-3,6,9-trioxaundecanoic acid; Aca-BBN(7-14), Aca-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2; c(RGDyK), cyclo(Arg-Gly-Asp-D-Tyr-Lys); RGD-BBN, cyclo(Arg-Gly-Asp-D-Tyr-Lys)-Glu-(Aca-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2); SFB, N-succinimidyl-4-fluorobenzoate.
In describing and claiming the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below.
The terms “agent” and “therapeutic agent’ as used herein refer to a compound which is desired to be delivered to a target cell or tissues that have the GPRP and integrin ligands to which the heterodimeric compositions of the present disclosure can selectively bind. Such agents would be useful in modulating the proliferation of cells such as cancer cells, and may be useful in destroying such cells. It is further contemplated that an agent or therapeutic agent may be included in a heterodimeric construct of the present disclosure that further comprises a radiolabeled or otherwise tagged prosthetic group for monitoring the location of the construct on a cell or in the tissues of a treated animal or human host.
The term “cell or population of cells” as used herein refers to an isolated cell or plurality of cells excised from a tissue or grown in vitro by tissue culture techniques. In the alternative, a population of cells may also be a plurality of cells in vivo in a tissue of an animal or human host.
The term “contacting a cell or population of cells” as used herein refers to delivering a composition such as, for example, a heterodimeric probe composition according to the present disclosure with or without a pharmaceutically or physiologically acceptable carrier to an isolated or cultured cell or population of cells, or administering the probe in a suitable pharmaceutically acceptable carrier to an animal or human host. Thereupon, it may be systemically delivered to the target and other tissues of the host, or delivered to a localized target area of the host. Administration may be, but is not limited to, intravenous delivery, intraperitoneal delivery, intramuscularly, subcutaneously or by any other method known in the art. One method is to deliver the composition directly into a blood vessel leading immediately into a target organ or tissue such as a prostate, thereby reducing dilution of the probe in the general circulatory system.
The term “fluorophore” as used herein refers to a component of a molecule that causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength. The amount and wavelength of the emitted energy depend on both the fluorophore and the chemical environment of the fluorophore. Fluorescein isothiocyanate (FITC), a reactive derivative of fluorescein, has been one of the most common fluorophores chemically attached to other, non-fluorescent, molecules to create new fluorescent molecules for a variety of applications. Other historically common fluorophores are derivatives of rhodamine (TRITC), coumarin, and cyanine. Newer generations of fluorophores such as the ALEXA FLUORS™ and the DYLIGHT FLUORS™ are generally more photostable, brighter, and less pH-sensitive than other standard dyes of comparable excitation and emission.
The term “heterodimer” as used herein refers to a molecule comprising two identifiable domains or regions having different functions, amino acid sequences, or other properties. The heterodimers of the present disclosure may comprise a first domain that includes the tri-amino acid sequence arginine-glycine-aspartate (RGD; SEQ ID NO.: 1) that is capable of selectively binding to an integrin, and a second domain comprising a fragment of the polypeptide bombesin, and which is capable of selectively binding to a gastrin-releasing peptide receptor. The first and second domains may be contiguous, or connected by a linker molecule, wherein the first domain may be linked to the amino or the carboxyl end of the bombesin fragment. An especially advantageous heterodimer of this disclosure comprises a first domain linked directly, or by a linker moiety, to the amino end of the second domain.
The term “host” as used herein refers to a mammalian or non-mammalian animal, or human, subject or patient in receipt of a composition according to the present disclosure.
The term “integrin” as used herein refers to a widely expressed family of calcium or magnesium dependent α or β heterodimeric cell surface receptors that bind to extracellular matrix adhesive proteins such as fibrinogen, fibronectin, vitronectin, and osteopontin. The integrin receptors are transmembrane glycoproteins (GP's) known for their large extracellular domains and are classified by at least 8 known β subunits and 14α subunits. For example, the β1 subfamily has the largest number of integrins, where the various a subunits associate with various β subunits: β3, β5, β6, and β8. Some of the disease states that have a strong αvβ3, αvβ5, and αIIbβ3 (also referred to as GPIIb/IIIa) integrin component in their etiologies are unstable angina, thromboembolic disorders or atherosclerosis (GPIIb/IIIa); thrombosis or restenosis (GPIIb/IIIa or αvβ3); restenosis (dual αvβ3/GPIIb/IIIa); rheumatoid arthritis, vascular disorders or osteoporosis (αvβ3); tumor angiogenesis, tumor metastasis, tumor growth, multiple sclerosis, neurological disorders, asthma, vascular injury or diabetic retinopathy (αvβ3 or αvβ5); and, angiogenesis (dual αvβ3/αvβ5).
The term “linker” as used herein refers to any molecular structure that connects the two functionally dissimilar domains that together constitute the single heterodimeric construct of the present disclosure. A particularly useful linker is, for example, a glutamate moiety, the carboxyl groups of which may form peptide bonds with amine groups on each of the two domains to be joined. It is also contemplated that a prosthetic group such as, but not limited to, a fluorobenzoate may attach to a glutamate linker, for example, through the α-amino group of a glutamate linker.
It is further contemplated that a “linker’ may refer to a molecular structure that conjugates two similarly functioning domains, such as, but not limited to, the multimeric domains comprising at least one tripeptide structure comprising the amino acid sequence arginine-glycine-aspartate (SEQ ID NO.: 1). It is also contemplated that a linker molecule suitable for use in the heterodimeric compositions of the present disclosure can be, but is not limited to, a dicarboxylic acid that further includes at least one available group, such as an amine group, for conjugating to a prosthetic group. However, it is also contemplated that other functional side groups may substitute for the amine group to allow for the linking to selected prosthetic groups. Exemplary dicarboxylic acids include, but are not limited to, aspartate, glutamate, and the like, and can have the general formula (HOOC)—(CH2)n—(CHNH2+)—(CH2)m—(COOH), where n and m are each independently 0, or an integer from 1 to about 10. It is further considered within the scope of the disclosure for the linker to be a multimer, or a combination, of at least two such dicarboxylic acids. For example, such linker molecules may include, but are not limited to, (aspartate)x, (glutamate)y, or a combination thereof, where adjacent amino acids can be joined by peptide bonds, and the like. The subscripts x and y are each independently 0, or an integer from 1 to about 12.
The term “peptide” as used herein refers to short polymers formed from the linking, in a defined order, of α-amino acids. The link between one amino acid residue and the next is known as an amide bond or a peptide bond. Proteins are polypeptide molecules (or consist of multiple polypeptide subunits). The distinction is that peptides are short and polypeptides/proteins are long. There are several different conventions to determine these. Peptide chains that are short enough to be made synthetically from the constituent amino acids are called peptides, rather than proteins, with one dividing line at about 50 amino acids in length.
Modifications and changes can be made in the structure of the peptides of this disclosure and still result in a molecule having similar characteristics as the peptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a peptide that defines that peptide's biological functional activity, certain amino acid sequence substitutions can be made in a peptide sequence and nevertheless obtain a peptide with like properties.
In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a peptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a peptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant peptide, which in turn defines the interaction of the peptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent peptide. In such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biologically functional equivalent peptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (−0.5±1); threonine (−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent peptide. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.
As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take one or more of the foregoing characteristics into consideration are well known to those of skill in the art and include, but are not limited to (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a peptide as set forth above. In particular, embodiments of the peptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the peptide of interest.
The term “pharmaceutically acceptable carrier” as used herein refers to a diluent, adjuvant, excipient, or vehicle with which a heterodimeric probe of the disclosure is administered and which is approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. When administered to a patient, the heterodimeric probes and pharmaceutically acceptable carriers preferably should be sterile. Water is a useful carrier when the heterodimeric probe is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as glucose, lactose, sucrose, glycerol monostearate, sodium chloride, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The present compositions advantageously may take the form of solutions, emulsion, sustained-release formulations, or any other form suitable for use.
The term “physiologically acceptable” as used herein refers to a composition that, in contact with a cell, isolated from a natural source or in culture, or a tissue of a host, has no toxic effect on the cell or tissue.
The term “positron emission tomography” as used herein refers to a nuclear medicine imaging technique that produces a three-dimensional image or map of functional processes in the body. The system detects pairs of gamma rays emitted indirectly by a positron-emitting radioisotope, which is introduced into the body on a metabolically active molecule. Images of metabolic activity in space are then reconstructed by computer analysis. Using statistics collected from tens-of-thousands of coincidence events, a set of simultaneous equations for the total activity of each parcel of tissue can be solved by a number of techniques, and a map of radioactivities as a function of location for parcels or bits of tissue may be constructed and plotted. The resulting map shows the tissues in which the molecular probe has become concentrated. Radioisotopes used in PET scanning are typically isotopes with short half lives such as carbon-11 (about 20 min), nitrogen-13 (about 10 min), oxygen-15 (about 2 min), and fluorine-18 (about 10 min). PET technology can be used to trace the biologic pathway of any compound in living humans (and many other species as well), provided it can be radiolabeled with a PET isotope. The half life of fluorine-18 is long enough such that fluorine-18 labeled radiotracers can be manufactured commercially at an offsite location.
The term “prosthetic group’ as used herein refers to a chemical moiety conjugated to a region of the heterodimeric constructs of the present disclosure. The prosthetic group may include a “tether” and a detectable moiety such as, but not limited to a radiolabel, a fluorescent dye, and the like. In addition, or in place of, the detectable moiety, the prosthetic group may be an agent such as a therapeutic agent required to be targeted to a cell bearing the GPRP and integrin ligands to which the heterodimeric compositions of the present disclosure can selectively bind.
The term “radiolabel prosthetic group” as used herein refers to a moiety conjugated to a heterodimer of the present disclosure, where the moiety includes a radiolabel. Most advantageous for the heterodimers of the disclosure are moieties that may be attached to a tether such as, but not limited to, a benzoate derivative. The prosthetic group may have a radiolabel attached thereto. For example, one useful prosthetic group is fluorobenzoate, where the carboxyl group of the benzoate may be conjugated to the α-amino group of a glutamate linker, and the fluoride is the isotope 18F detectable by such as PET.
The heterodimer constructs according to the present disclosure can be labeled with a radionuclide suitable for imaging by such as, but not limited to, Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT), or for the detection of, or the therapeutic use of, alpha-(α), beta-(β), and gamma (γ)-emitting isotopes. Some exemplary embodiments of elements that can be used as labels in the present disclosure include, but are not limited to, F-19 (F-18), C-12 (C-11), 1-127 (1-125, 1-124, 1-131, 1-123), CI-36 (CI-32, CI-33, CI-34), Br-80 (Br-74, Br-75, Br-76, Br-77, Br-78), Re-185/187 (Re-186, Re-188), Y-89 (Y-90, Y-86), Lu-177, and Sm-153, as well as those described in the figures. Imaging probes for use in the probes of the present disclosure can be labeled with one or more radioisotopes, preferably including, but not limited to, 11C, 18F, 76Br, 123I, 124I, or 131I, and are suitable for use in peripheral medical facilities and PET clinics. In particular embodiments, for example, the PET isotope can include, but is not limited to, 64/61Cu, 124I, 76/77Br, 88Y, 89Zr, and 68Ga.
The term “target” as used herein can refer to a polypeptide for which it is desired to detect. The target polypeptide for use in the methods herein disclosed may be an isolated polypeptide, a polypeptide immobilized on a solid support or in free solution. Alternatively, the target polypeptide may be on a cell surface, the cell being isolated from an animal host, a cultured cell or a cell or population of cells in a tissue of an animal.
The term “tether” as used herein refers to a linker joining a prosthetic group such as, but not limited to, a radioactive label to a molecular structure. For example, and not intended to be limiting, a tether moiety can be an N-succinimidyl benzoate that can conjugate to an amine side group of another moiety such as a linker connecting the two heteropeptide domains. In one embodiment, a tether such as a benzoate may be substituted at the 4-position, for example, with the radiolabel 18-fluoride prosthetic group.
Molecular imaging of cancer is a fast growing research field. Molecular imaging technologies have demonstrated great benefits for better understanding cancer biology, as well as for facilitating cancer drug development and cancer early detection. Development of novel imaging methods and molecularly targeted probes will allow not only to locate a tumor, but also to visualize the expression and activity of specific molecular targets and biological processes in a tumor.
In recent years, it has been learned that some cancer cells contain gastrin releasing peptide (GRP) receptors (GRP-R) of which there are a number of subtypes. In particular, it has been shown that several types of cancer cells have over-expressed or uniquely expressed GRP receptors. GRP and GRP analogues can selectively bind to the GRP receptor family. One GRP analogue is bombesin (BBN), (i.e., tetradecapeptide) isolated from frog skin that can bind to GRP receptors with high specificity and with an affinity similar to GRP.
GRP receptors have been shown to be over-expressed or uniquely expressed on several types of cancer cells. In addition to being seen in prostate cancers, GRPR is also expressed in almost 60% of primary breast carcinoma cases and in almost all infiltrated lymph nodes. Extremely high numbers of GRPRs have also been detected in gastrointestinal stromal tumors. Binding of GRP receptor agonists (also autocrine factors) increases the rate of cell division of these cancer cells. The fragments of bombesin useful in the embodiments of the heterodimers of the present disclosure contain either the same primary structure of the bombesin GPR binding region, i.e. bombesin(7-14) (SEQ ID NO.: 7) or bombesin(8-14) (SEQ ID NO.: 6), or similar primary structures, with specific amino acid substitutions, that will specifically bind to GRP receptors. Compounds containing this bombesin GPR binding region (or binding moiety), when covalently linked to other groups may also be referred to as bombesin conjugates.
Integrin αvβ3 is expressed in GRPR-positive cancers, as well as many other cancer types. The application of BBN-RGD heterodimers according to the present disclosure for tumor targeting will thus be applicable to many cancer types that express both GRPR and integrin, GRPR only, or integrin only.
A dual GRPR-integrin αvβ3-targeting approach according to the present disclosure provides improved imaging probes over those that recognize only a single receptor type. Accordingly, the heterodimeric probe construct 18F-FB-BBN-RGD comprising a BBN peptide motif for GRPR targeting, and an RGD peptide motif for integrin αvβ3 targeting, was synthesized and radiolabeled. The receptor-binding assay data demonstrated that BBN-RGD heterodimeric construct is similar to Aca-BBN(7-14) for GRPR binding, and is similar to c(RGDyK) for integrin αvβ3 binding.
As shown in
As PC-3 tumor cells express both GRPR and integrin αvβ3, the imaging quality of 18F-FB-BBN-RGD was tested in a PC-3 xenograft model. Compared with 18F-FB-BBN and 18F-FB-RGD, the PC-3 tumor uptake of 18F-FB-BBN-RGD was much higher than the sum of the monomeric tracers at all time points examined, as illustrated in
18F-FB-BBN-RGD also had the highest tumor to non-tumor ratios when compared with 18F-FB-BBN and 18F-FB-RGD. Overall, a synergistic effect has been observed for 18F-FB-BBN-RGD compared to probes comprising just one binding domain, and significantly higher tumor uptake and contrast have been obtained in the PC-3 tumor model. In the blocking experiment, neither non-radioactive BBN peptide nor non-radioactive RGD peptide could totally inhibit the uptake of 18F-FB-BBN-RGD in PC-3 tumor, as the tracer could still bind to the unblocked receptors. The BBN and RGD double blocking could further reduce the tumor uptake, which strongly supports the dual-receptor specificity of 18F-FB-BBN-RGD in vivo. For 18F-FB-BBN-RGD, the RGD blocking resulted in a slightly higher tumor uptake than BBN blocking, which may be due to the PC-3 cells expressing a high level of GRPR but only a medium level of integrin αvβ3. Moreover, the advantage of this heterodimer tracer is apparent when only one receptor type is over-expressed. For example, the DU-145 tumor expresses a moderate level of integrin αvβ3, but expresses a low level of GRPR (Cooper et al., Neoplasia (2002) 4: 191-194; Markwalder & Reubi, Cancer Res. (1999) 59: 1152-1159; Haywood-Reid et al., Prostate (1997) 31: 1-8). 18F-FB-BBN that binds to GRPR but not to integrin αvβ3 is unable to provide enough tumor uptake and tumor-to-background contrast. 18F-FB-BBN-RGD, on the other hand, had a tumor uptake similar to that of 18F-FB-RGD, but had a significantly lower background.
In the metabolic stability study, the metabolites of 18F-FB-BBN-RGD may be determined primarily by the FB unit and BBN sequence as the cyclic RGD-containing pentapeptide is highly stable in vivo. The structure of the BBN-RGD heterodimer, as illustrated in
If, however, the heterodimer is initially bound to integrin instead, the dissociation of the RGD motif from integrin will reorient the BBN-RGD to bind to GRPR, resulting in an apparent low off-rate of the ligand binding. Both the increased number of binding sites and the apparent low off-rate of the dual-receptor-targeting ligand may be expected to have enhanced tumor uptake and retention as compared with those single-receptor-recognizing ligands. The added molecular size and change of overall molecular charge and hydrophilicity can also have effects on the in vivo kinetics of the resulting probes.
For integrin binding, multimeric RGD peptides can be advantageous over monomeric counterparts in terms of receptor-binding affinity in vitro and tumor-targeting efficacy in vivo, most likely due to the so-called “multivalency effect” (Liu S. Mol. Pharm. 2006; 3:472-487; Li et al., J. Nucl. Med. 2007; 48:1162-1171; Wu et al., J. Nucl. Med. 2007; 48:1536-1544). It is contemplated, therefore, that in the various embodiments of the present disclosure, BBN analogs may be linked with dimeric or oligomeric RGD tripeptide units through a linker molecule such as, but not limited to, a glutamate linker.
Embodiments of the heterodimeric compositions of the disclosure connected with glutamate are likely to be mixtures of Glu-BBN-RGD (where RGD is on the side-chain 8-position) and Glu-RGD-BBN (where BBN is on the side-chain 8-position), as shown in
The radiolabeling yield for the heterodimeric BBN-RGD peptide was found to be lower than that for the monomeric BBN or RGD peptides when using 18F-SFB as the synthon. There was also a reduced 18F-labeling yield for an RGD homodimer and a homotetramer in which 18F-SFB was reacted with the glutamate amine group. While not wishing to be limited to any one theory, the reduced labeling yield may be due to steric hindrance and relatively low reactivity of the glutamate α-amino group. In one embodiment of the disclosure, therefore, and to improve the radiolabeling yield, a mini-PEG linker, 11-amino-3,6,9-trioxaundecanoic acid (NH-mini-PEG-COOH) was introduced to the glutamate residue.
The recent introduction of 68Ga into clinical practice represents the beginning of the development of a PET imaging probe that is not dependent on the availability of a medical cyclotron. 68Ga has the physical property of high positron yield reaching 89% of all disintegrations, which is suitable for PET imaging. Its short physical half-life of 68 min matches the biological half-lives of many peptides and other small molecules owing to their fast blood clearance, quick penetration and rapid target localization. In this study, the in vitro and in vivo characteristics of 68Ga-labeled RGD-BBN heterodimeric peptide in a dual integrin- and GRPR-positive PC-3 tumor model were investigated.
To be a dual functional tracer, each binding motif of the heterodimer must maintain its own function. The receptor binding assay data demonstrated that the binding affinities of RGD-BBN and NOTA-RGD-BBN were similar to that of Aca-BBN(7-14) for GRPR binding and c(RGDyK) for integrin αvβ3 binding, indicating that the RGD-BBN heterodimer can bind both integrin and GRPR in vitro. 68Ga-NOTA-RGD-BBN showed lower uptake than 68Ga-NOTA-BBN, but higher uptake than 68Ga-NOTA-RGD in PC-3 tumor cells. This may be due to the facts that PC-3 cells have higher numbers of GRPR than integrin, and that the RGD-integrin complex does not tend to internalize into the cells. The internalization of BBN in the RGD-BBN heterodimer was significantly hampered by the recognition of the RGD motif with the cell-surface integrin receptor. The in vivo behavior of 68Ga-NOTA-RGD-BBN was tested in a PC-3 tumor model using small-animal PET. The PC-3 tumor uptake of 68Ga-NOTA-RGD-BBN was slightly higher than that of 68Ga-NOTA-BBN, but much higher than that of 68Ga-NOTA-RGD at all time points examined (
As the binding affinity of RGD monomer is relatively low, the tracer accumulated around the tumor vessel may dissociate from the loosely bound integrin receptors, diffuse into the extracellular matrix and rebind to the tumor cells that express both GRPR and integrin αvβ3. One of the main drawbacks of BBN-based radiotracers is their unfavorable hepatobiliary excretion, which usually results in high intestinal uptake. For example, in this study, as shown in
Overall, the heterodimeric tracer significantly reduced the intestinal accumulation of radioactivity, making the tracer more suitable for imaging of abdominal cancer than BBN analogs. The production of 68Ga-NOTA-RGD-BBN is easy and does not need an onsite cyclotron, which allows possible kit formulation and widespread availability. The PC-3 tumor uptake of 68Ga-NOTA-RGD-BBN (6.55±0.83, 5.26±0.32, and 4.04±0.28% ID/g at 30, 60, and 120 min, respectively) was significantly higher than that of 18F-FB-PEG3-RGD-BBN (6.35±2.52, 4.41±0.71, and 2.47±0.81% ID/g at 30, 60, and 120 min, respectively) at 60 min after injection (p<0.05). The higher tumor uptake of the 68Ga-labeled RGD-BBN is likely due to the internalization and effective trapping of radiometal inside the tumor cells as compared to 18F-labeled tracers, which is supported by cell efflux studies.
After allowing efflux for 1 h, the efflux ratio was about 40% for 18F-labeled RGD-BBN, but only about 20% for 68Ga-NOTA-RGD-BBN. The cellular uptake of 68Ga-NOTA-BBN was much higher than that of 68Ga-NOTA-RGD-BBN (
The advantage of dual receptor binding of the heterodimer tracer is apparent when only one receptor type is over-expressed in a tumor model. For example, in the MDA-MB435 tumor model, which expresses a moderate level of integrin αvβ3 but no GRPR, 68Ga-NOTA-BBN was unable to detect the tumors because it only recognizes GRPR. In contrast, 68Ga-NOTA-RGD and 68Ga-NOTA-RGD-BBN had a clear tumor uptake due to the function of RGD (
The MDA-MB-435 tumor uptake of 68Ga-NOTA-RGD-BBN was even higher than that of 68Ga-NOTA-RGD tumor, which may have resulted from the improved in vivo kinetics and increased circulation half-life of 68Ga-NOTA-RGD-BBN over 68Ga-NOTA-RGD. In the 68Ga-NOTA-RGD-BBN heterodimeric peptide, the RGD and BBN motifs were linked through a glutamic acid. Due to the short length of the linker, it is impossible for the RGD and BBN motifs to bind both integrin and GRPR simultaneously. Therefore, in future it would be interesting to investigate the effects of linkers of different lengths, solubility, lipophilicity, and flexibility on the in vitro and in vivo behaviors of the heterodimeric peptides. The design of heteromultimeric tracers that recognize other tumor targets is also worth further investigation for tumor-targeted imaging and therapy. In conclusion, we have described the design and synthesis of 68Ga-labeled RGD-BBN heterodimer peptide containing both RGD and BBN motifs for dual integrin and GRPR-targeted tumor imaging. 68Ga-NOTA-RGD-BBN exhibited dual receptor targeting properties both in vitro and in vivo. The high affinity and specificity and improved pharmacokinetics of the 68Ga-labeled RGD-BBN heterodimer make it a promising agent for molecular imaging of tumors with both or either receptor expression pattern. The heterodimer and heteromultimer strategy may also provide general methods of developing tumor-targeted imaging probes and therapeutic agents.
64Cu-Labeled RGD-BBN heterodimeric Peptide
1,4,7,10-Tetraazacyclododecane-N,N9,N99,N999-tetraacetic acid (DOTA) is a known bifunctional chelators for 64Cu labeling. However, the relatively low thermodynamic and kinetic stability of 64Cu-DOTA in vivo is well documented (Wadas et al., (2008). J. Nucl. Med.; 49: 1819-1827; Prasanphanich et al., (2007) Proc. Natl. Acad. Sci. USA 104: 12462-12467; Boswell et al., (2004) J. Med. Chem. 47: 1465-1474; Garrison et al., (2007) J. Nucl. Med. 48: 1327-1337). The instability of the 64Cu-DOTA conjugates results in demetallation and subsequent accumulation in non-target tissues such as liver (Prasanphanich et al., (2007) Proc. Natl. Acad. Sci. USA 104: 12462-12467). Prasanphanich et al. recently reported 64Cu-labeled BBN analogs using 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) as a chelator. The results suggested high in vivo kinetic stability of 64Cu-NOTA-BBN vectors with little or no dissociation of 64Cu from NOTA (Prasanphanich et al., (2007) Proc. Natl. Acad. Sci. USA 104: 12462-12467). The present disclosure provides data for the advantages of 64Cu-labeled NOTA-RGD-BBN heterodimer over its monomeric counterparts NOTA-RGD and NOTA-BBN for imaging GRPR-positive tumors, and also compare the in vitro and in vivo characteristics of 64Cu-labeled RGD-BBN heterodimer using NOTA as a chelator with those using DOTA as a chelator. The present disclosure further provides methods for using 64Cu-NOTARGD-BBN to image tumors that express integrin but not GRPR (e.g., 4T1 murine mammary carcinoma). The synergistic effects of the heterodimer, RGD-BBN was shown with labeling 64Cu (t1/2=12.7 h) using DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) as the chelator, respectively. The in vitro and in vivo characteristics of 64Cu-NOTA-RGD-BBN were compared with 64Cu-NOTA-RGD, 64Cu-NOTA-BBN, and 64Cu-DOTA-RGD-BBN. The dual receptor targeting properties of 64Cu-NOTA-RGD-BBN was also investigated in tumor models.
64Cu-NOTA-RGD-BBN and 64Cu-DOTA-RGD-BBN had comparable dual integrin αvβ3 and GRPR-binding affinities, but their affinities were both slightly lower than that of RGD and BBN respectively. 64Cu-NOTA-RGD-BBN possessed significantly higher tumor uptake compared with 64Cu-NOTA-RGD, 64Cu-NOTA-BBN, the mixture of 64Cu-NOTA-RGD+64Cu-NOTA-BBN, and also 64Cu-DOTA-RGD-BBN. 64Cu-NOTA-RGD-BBN also showed improved in vivo kinetics such as lower liver and intestine activity accumulation than the BBN tracers. The synergistic effects of 64Cu-NOTA-RGD-BBN were observed in both the dual receptor-positive PC-3 tumor model and one receptor-positive 4T1 tumor model.
64Cu has favorable decay characteristics (half-life, 12.7 h; b1, 17.8%; b2, 38.4%), making it useful for both PET and internal radiotherapy. 64Cu can be produced in high yield and at high specific activity on a small biomedical cyclotron and is labeled with biomolecules through macrocyclic chelators, which allow possible kit formulation and wide availability. More important, the longer half-life of 64Cu among all the positron emitters allows imaging at late time points to acquire more in vivo information than is possible for 18F (half-life, 109.7 min).
Both DOTA and NOTA can be used as bifunctional chelators for 64Cu labeling. The 64Cu-DOTA conjugates usually exhibit a high accumulation of liver radioactivity because of the dissociation of 64Cu in vivo from DOTA, followed by metabolism and transchelation to other proteins. NOTA is most commonly used for 68Ga (half-life, 68 min) labeling because the rapid reaction kinetics of NOTA match the short half-life of 68Ga. NOTA was also reported to be labeled with 64Cu, with reduced liver accumulation. In the present disclosure, DOTA-RGD-BBN and NOTA-RGD-BBN were synthesized and labeled both conjugates with 64Cu. Compared with DOTA-RGD-BBN, NOTA-RGD-BBN can be more easily labeled with 64Cu, as shown in
64Cu-CB-TE2A-8-AOC-BBN(7-14)NH2 showed significant improvement in clearance because of its improved in vivo stability, compared with the DOTA conjugates. Most important, the liver uptake of the CB-TE2A conjugate was also significantly lower than that of the DOTA conjugates. Compared with 64Cu-CB-TE2A-8-AOC-BBN(7-14)NH2, 64Cu-NOTA-RGD-BBN showed slightly higher liver uptake, but the tumor uptake of 64Cu-NOTA-RGD-BBN was also higher than that of 64Cu-CB-TE2A-8-AOC-BBN(7-14)NH2. Radiolabeling of CB-TE2A conjugates requires harsher reaction conditions than does radiolabeling of DOTA and NOTA conjugates. The high temperature and high pH required for CB-TE2A labeling may not be suitable for peptides such as RGD-BBN. In contrast, the fast reaction kinetics of NOTA-conjugates would be more suitable for clinical translation. The in vivo behaviors of the 64Cu-labeled NOTA conjugates were compared in the PC-3 tumor model. Because of the high GRPR and low integrin αvβ3 expression of the PC-3 tumor, tumor uptake of 64Cu-NOTA-RGD was low and the 64Cu-NOTA-BBN showed relatively high tumor contrast. However, high accumulation of radioactivity in the abdominal region, especially in the intestines, was observed in the mice receiving 64Cu-NOTA-BBN and other reported BBN tracers, suggesting hepatobiliary excretion of 64Cu-NOTA-BBN. In contrast, 64Cu-NOTA-RGD-BBN showed much lower intestinal accumulation, and the tumor uptake of 64Cu-NOTA-RGDBBN was also significantly higher than that of 64Cu-NOTA-RGD, 64Cu-NOTA-BBN, and 64Cu-NOTA-RGD plus 64Cu-NOTA-RGD. The high lipophilicity of 64Cu-NOTA-BBN resulted in rapid hepatobiliary excretion, which led to a short circulation half-life for the tracer, and thus insufficient time for the tracer to extravasate from the tumor blood vessels, diffuse in the extracellular space, and bind with GRPR expressed on the tumor cells. In contrast, 64Cu-NOTA-RGD-BBN had longer blood retention and predominantly renal clearance.
Another major reason for the higher tumor uptake of 64Cu-NOTA-RGD-BBN than of 64Cu-NOTA-BBN is the dual GRPR- and integrin-targeting properties of the RGD-BBN heterodimer (
The dual-receptor targeting of 64Cu-NOTA-RGD-BBN may also contribute to prolonged tumor retention of the tracer. For example, at 24 h after injection, tumor uptake of 64Cu-NOTA-RGD-BBN was 2.04±0.35% ID/g, which is significantly higher than that of the corresponding 64Cu-NOTA-BBN (0.44±0.39% ID/g).
Tumor retention of 64Cu-NOTA-RGD-BBN was also higher than that of the 64Cu-labeled NOTA conjugated BBN tracers reported by Prasanphanich et al. The prolonged tumor retention of 64Cu-NOTA-RGD-BBN, compared with that of 64Cu-NOTA-BBN, was consistent with the in vitro findings that the efflux ratio of 64Cu-NOTA-RGD-BBN was much lower than that of the BBN tracer (
64Cu-NOTA-RGD-BBN had blood and kidney clearance curves comparable to those of 18F-PEG3-RGD-BBN tracer in the first hour after injection, but between 1 and 2 h, the 18F tracer cleared more rapidly, possibly because of the higher hydrophilicity of 18F-PEG3-RGD-BBN. The PC-3 tumor uptake of 18F-PEG3-RGD-BBN was also significantly higher than that of 64Cu-NOTA-RGD-BBN at 30 min (6.35±2.52 vs. 3.06±0.11% ID/g) and 1 h (4.41±0.71 vs. 2.78±0.56% ID/g).
Although liver uptake of 64Cu-NOTARGD-BBN was relatively low (3.5% ID/g at any time point tested), it was still higher than that of 18F-PEG3-RGD-BBN. Taken together, 18F-PEG3-RGD-BBN is better than 64Cu-NOTA-RGD-BBN for tumor imaging within 2 h after injection. However, because of the short half-life of 18F, the absolute tumor signal of 18F-PEG3-RGD-BBN was low after 2 h. In contrast, the plateau in tumor uptake of 64Cu-NOTA-RGD-BBN from 4 to 20 h allows a persistent imaging signal. More important, because of the decay characteristics of 64Cu, the longer tumor retention of 64Cu-NOTA-RGD-BBN makes possible GRPR-positive tumor-targeted therapy.
Breast cancers can be sorted into two categories, estrogen dependent (ER+) and estrogen-independent (ER−), based on the presence or absence of estrogen receptors (Vaïk et al., (2009) Proteomics Clin Appl 3: 41-50). Nowadays, many ER+ and ER− tumor cells are being used for breast cancer research in animal studies. We screened the GRPR and integrin αvβ3 expression in both the ER+ (T47D, BT474, MCF-7) and ER− (MDA-MB-231, MDA-MB-435, MDA-MB468, BT20) breast cancer cells (Cassoni et al., (2001) J. Clin. Endocrinol. Metab. 86: 1738-1745; Bajo et al., (2002) Proc. Natl. Acad. Sci. USA 99: 3836-3841; Anzick et al., (1997) Science 277: 965-968; Brandi et al., (2003) Cancer Res 63: 40284036).
The GRPR expression on estrogen-dependent tumor cells such as T47D, BT474 was high, but the integrin αvβ3 expression was relatively low or moderate. However, the estrogen-independent tumor cells such as MDA-MB435, MDA-MB-231, MDA-MB-468 expressed higher integrin αvβ3, but their GRPR expression was undetectable (
The in vivo behaviors of the three tracers were tested by microPET in T47D and MDA-MB435 orthotopic breast cancer models. All the tracers showed contrast tumor imaging in the two tumor models from 30 min p.i. The radiolabeled BBN was also tested in the MDA-MB-435 tumor model that did not express GRPR for control studies. The much higher tumor uptake of the 18F, 64Cu, or 68Ga labeled RGD-BBN tracer than that of the corresponding BBN tracer indicated that the RGD-BBN tracers were useful to detect the tumor with only one receptor positive, but the BBN tracers can only be used for GRPR-positive tumor imaging.
The present disclosure provides data that demonstrate that 18F, 64Cu, and 68Ga labeled RGD-BBN heterodimeric peptides can be used to detect both the GRPR+/(integrin αvβ3 low expression) and GRPR−/integrin αvβ3+ breast cancers by microPET imaging. Although 18F-labeled RGD-BBN showed lower tumor uptake than 64Cu-NOTA-RGD-BBN and 68Ga-NOTA-RGD-BBN, it was able to detect breast cancer tumors in xenograft models with high contrast and low background. However, the preparation of the 18F-FB-PEG3-RGD-BBN was more complex and time-consuming. Synthesis of 64Cu-NOTA-RGD-BBN and 68Ga-NOTA-RGD-BBN is faster, which allows kit formulation and wide availability.
64Cu-NOTA-RGD-BBN showed prolonged tumor uptake, but also higher liver retention and kidney uptake. Modification of the 64Cu-chelator system would be the future focus to develop a superior RGD-BBN radiotracer for GRPR and integrin targeting and possible internal radiotherapy. 68Ga-NOTA-RGD-BBN possessed high tumor signals, but also high background uptake. The insertion of hydrophilic linkers such as PEG3 between the RGD-BBN and NOTA may be applied for future development of 68Ga labeled RGD-BBN tracer with low background signals for breast cancer imaging.
It is further contemplated that the heterodimeric compositions according to the present disclosure may be suitable as carriers to transport a non-labeling agent, such as a therapeutic agent, to a target cell having a combination of cell-surface exposed GPRP and integrin molecules. Embodiments of the heterodimeric probes of the disclosure may, therefore, further comprise covalently bound agents including, but not limited to, cytotoxic agents, cell proliferation modulating agents and the like that may be attached to an exposed side-group of the linker of a domain of the probe construct. It is further contemplated that embodiments of the heterodimeric probes of the present disclosure may comprise both a labeled prosthetic group and a non-labeled prosthetic group such as a therapeutic agent, a radionuclide, or the like such that the site of delivery of the non-labeled group may be imaged. Furthermore, it is contemplated that embodiments of the heterodimeric probes of the present disclosure may comprise more than one labeled prosthetic group, whereby more than one detection technique may be used to determine the location of the probe within a cell or whole animal. For example, but not limiting, an F-18 label for PET scanning and a fluorescent fluorophore may be tethered to the heterodimeric probe.
One aspect of the disclosure, therefore, provides compositions that can comprise a heterodimeric probe, where the heterodimeric probe comprises: a first peptide domain comprising a moiety capable of selectively binding to an integrin; a second peptide domain comprising a moiety capable of selectively binding to a gastrin-releasing peptide receptor; a linker connecting the first peptide domain and the second peptide domain; and a prosthetic group.
In embodiments of this aspect of the disclosure, the first peptide domain may comprise at least one tripeptide comprising the amino acid sequence arginine-glycine-aspartate (Arg-Gly-Asp).
In embodiments of this aspect of the disclosure, the moiety capable of selectively binding to an integrin may comprise at least one peptide selected from the group consisting of: cyclo(Arg-Ala-Asp-D-Phe-Lys), cyclo(Arg-Ala-Asp-D-Phe-Val), cyclo(Arg-Ala-Asp-D-Tyr-Cys), cyclo(Arg-Ala-Asp-D-Tyr-Lys), cyclo(Arg-Gly-Asp-D-Phe-Cys), cyclo(Arg-Gly-Asp-D-Phe-Glu), cyclo(Arg-Gly-Asp-D-Phe-Lys), cyclo(Arg-Gly-Asp-D-Tyr-Cys), cyclo(Arg-Gly-Asp-D-Tyr-Glu), cyclo(Arg-Gly-Asp-D-Tyr-Lys), cyclo[Arg-Gly-Asp-D-Phe-Lys(Ac-SCH2CO)], cyclo[Arg-Gly-Asp-D-Phe-Lys(H-Ser)], cyclo[Arg-Gly-Asp-D-Phe-Lys(PEG-PEG)], H-Glu[cyclo (Arg-Gly-Asp-D-Phe-Lys)]2, H-Glu[cyclo(Arg-Gly-Asp-D-Phe-Lys)]2, H-Glu[cyclo(Arg-Gly-Asp-D-Tyr-Lys)]2, H-Gly-Arg-Ala-Asp-Ser-Pro-OH (SEQ ID NO.: 1), H-Gly-Arg-Gly-Asp-Asn-Pro-OH (SEQ ID NO.: 2), H-Gly-Arg-Gly-Glu-Ser-OH (SEQ ID NO.: 3), cyclo(Arg-Gly-Asp-D-Phe-Lys), H-Arg-Gly-Asp-Ser-Lys-OH (SEQ ID NO.: 4), H-Arg-Ala-Asp-Ser-Lys-OH (SEQ ID NO.: 5), Ac-Gly-D-Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-(Gly)-4-Ser-D-Arg-(Leu)-6-D-Arg-NH2, cyclo(Arg-Gly-Glu-D-Phe-Lys), and cyclo(Arg-Gly-Asp-D-Phe-Val).
In some embodiments of this aspect of the disclosure, the moiety capable of selectively binding to an integrin may comprise cyclo(arginine-glycine-aspartate-D-tyrosine-lysine).
In embodiments of this aspect of the disclosure, the first peptide domain may comprise a multimer of conjugated peptides, wherein at least one peptide of the multimer of peptides comprises the amino acid sequence arginine-glycine-aspartate.
In some embodiments of this aspect of the disclosure, the amino acid sequence of each peptide of the multimer of peptides may comprise the amino acid sequence of arginine-glycine-aspartate.
In one embodiment of this aspect of the disclosure, at least one peptide of the multimer of peptides comprises cyclo(arginine-glycine-aspartate-D-tyrosine-lysine).
In embodiments of this aspect of the disclosure, the moiety capable of selectively binding to a gastrin-releasing peptide receptor may comprise a fragment of the polypeptide bombesin, wherein the fragment has an affinity for a gastrin-releasing peptide receptor.
In embodiments of this aspect of the disclosure, the moiety capable of selectively binding to a gastrin-releasing peptide receptor can be selected from the group consisting of: bombesin(7-14) having the amino acid sequence of glutamine-tryptophan-alanine-valine-glycine-histidine-leucine-methionine (SEQ ID NO: 6), bombesin(8-14) having the amino acid sequence of asparagine-glutamine-tryptophan-alanine-valine-glycine-histidine-leucine-methionine (SEQ ID NO: 7), [Lys3]BBN (SEQ ID NO.: 8), [(D)Phe6, Leu-NHEt13, des-Met14]BN(6-14), (H-(D)Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt, or substituted variants thereof, wherein the substituted variants have an affinity for a GRPR.
In embodiments of this aspect of the disclosure, the second domain is bombesin(7-14) and comprises the amino acid sequence of glutamine-tryptophan-alanine-valine-glycine-histidine-leucine-methionine (SEQ ID NO.: 6). In these embodiments of this aspect of the disclosure, the heterodimer probe selectively binds to the integrin (αvβ3.
In embodiments of this aspect of the disclosure, the heterodimer probe may selectively bind to the integrin αvβ3 and gastrin-releasing peptide receptor.
In embodiments of this aspect of the disclosure, the linker connecting the first peptide domain and the second peptide domain may comprise the formula (HOOC)—(CH2)n—(CHNH2.)—(CH2)m—(COOH)a, wherein n and m are each independently 0, or an integer from 1 to about 10, and a is an integer from 1 to about 10.
In embodiments of this aspect of the disclosure, the linker connecting the first peptide domain and the second peptide domain can be selected from the group consisting of (aspartate)x, (glutamate)y, wherein x and y are each independently integers from 1 to about 10, or any combination thereof.
In some embodiments of this aspect of the disclosure, the linker connecting the first peptide domain and the second peptide domain is a glutamate residue or an aspartate residue.
In embodiments of this aspect of the disclosure, the linker may further comprise a tether covalently bound thereto, and wherein the tether is between the linker and the prosthetic group. In these embodiments of this aspect of the disclosure, the tether between the linker comprises (Gly)n, wherein n is an integer from 1 to about 12.
In some embodiments of this aspect of the disclosure, the tether may further comprise at least one polyethylene glycol moiety, and wherein the polyethylene glycol moiety has a molecular weight of about 200 to about 5000 daltons.
In one embodiment of this aspect of the disclosure, the tether is a polyethylene glycol-3 (11-amino-3,6,9,-trioxaundecanoate moiety.
In embodiments of this aspect of the disclosure, the prosthetic group comprises one of: a detectable label, a therapeutic agent, a reactive group capable of covalently bonding to a detectable label or a therapeutic agent, or a combination thereof.
In embodiments thereof, the prosthetic group may comprise a detectable label, or a group capable of bonding to a detectable label.
In embodiments of this aspect of the disclosure, the group capable of bonding to a detectable label can be selected from an amine group, a carboxyl group, and metal chelating group.
In some embodiments of this aspect of the disclosure, the metal chelating group is NOTA (1,4,7-triazacyclononane-1,4,7-triacetate) or DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetracetate).
In embodiments of this aspect of the disclosure, the prosthetic group may comprise a radiolabel, an optical label, or a radiolabel suitable for radiotherapy. In these embodiments of this aspect of the disclosure, the prosthetic group may comprise a detectable label selected from the group consisting of: the fluoride isotope 18F, 68Ga, 64Cu, 86Y, 124I, 111In, 99mTc, 123/131I, a fluorescent dye, a quantum dot, an alpha emitter, a beta emitter, and a gamma emitter.
In some embodiments of this aspect of the disclosure, the prosthetic group comprises a radionuclide selected from the group consisting of 18F, 68Ga, and 64Cu.
In one embodiment of this aspect of the disclosure, the prosthetic group is 18F-fluorobenzoate.
In some embodiments of this aspect of the disclosure, the heterodimer has a formula selected from the group consisting of: I, II, III, IV, V, VI, VII, VIII, VIIIa, and IX, as shown in
In embodiments of this aspect of the disclosure, the compositions may further comprise a pharmaceutically acceptable carrier.
Another aspect of the present disclosure encompasses methods of identifying a cell or a population of cells expressing an integrin and a gastrin-releasing peptide receptor, comprising: contacting a cell or population of cells with a composition according to any of the above embodiments, the composition comprising a heterodimeric probe capable of selectively binding to an integrin and to a gastrin-releasing peptide receptor of a cell; allowing the heterodimeric polypeptide probe to selectively bind to at least one of an integrin and to a gastrin-releasing peptide receptor of a cell or a population of cells; and detecting the presence of the heterodimeric probe on the cell or population of cells, whereby the presence of the heterodimeric probe on the cell or population of cells indicates that the cell or population of cells has an integrin, a gastrin-releasing peptide receptor, or both an integrin and a gastrin-releasing peptide receptor thereon.
In embodiments of this aspect of the disclosure, the cell or population of cells may be mammalian cells, and the cells or population of cells may be isolated cells.
In other embodiments of this aspect of the disclosure, the cell or population of cells comprise mammalian cells, and wherein the cells or population of cells are in a tissue of a human or animal host.
In embodiments of this aspect of the disclosure, the heterodimer probe may bind to the integrin αvβ3, the gastrin-releasing peptide receptor, or the combination thereof.
In embodiments of this aspect of the disclosure, the composition may comprise the heterodimeric probe is administered to an animal or human host.
In embodiments of this aspect of the disclosure, the heterodimer has a formula selected from the group consisting of: I, II, IV, VII, VIIIa, and IX, wherein, M+ can be a radionuclide selected from 68Ga and 64Cu, and as shown in
In embodiments of this aspect of the disclosure, the heterodimeric probe can be detected by positron emission tomography or by single photon emission computed tomography.
In embodiments of this aspect of the disclosure, the heterodimeric probe may be admixed with a pharmaceutically acceptable carrier.
Yet another aspect of the present disclosure provides methods of imaging a tissue in an animal or human host comprising the steps of: administering to an animal or human host a heterodimeric probe according to any of claims 1-30, wherein the probe has a detectable label thereon; detecting the presence of the detectable label in the animal or human host; and identifying a tissue in the animal or human host wherein the amount of the detectable label in the tissue is greater than in other tissues of the host, thereby determining the position of a tissue binding to the heterodimeric probe within the animal or human host.
In embodiments of this aspect of the disclosure, the heterodimeric probe is selected from the group consisting of: formula I, II, IV, VII, VIIIa, and IX, wherein, M+ can be a radionuclide selected from 68Ga and 64Cu, and as shown in
In embodiments of this aspect of the disclosure, the heterodimeric probe may be detected by positron emission tomography or by single photon emission computed tomography.
In embodiments of this aspect of the disclosure, the heterodimeric probe selectively binds to a tumor in the animal or human host, wherein the tumor comprises cells expressing αvβ3 and/or GRPR.
In some embodiments of this aspect of the disclosure, the tumor may be a tumor of the breast, the prostate, a malignant melanoma, an ovarian carcinoma, a gastro-intestinal carcinoma, or a glioblastoma.
Still another aspect of the present disclosure encompasses methods of delivering an agent to a cell, comprising contacting a cell or population of mammalian cells with a heterodimeric probe according to claims 1-30 capable of simultaneously binding to two an integrin and to a gastrin-releasing peptide receptor, and wherein the probe further comprises an agent to be delivered to a target cell or tissue of a mammalian subject; and allowing the heterodimeric probe to bind to an integrin, a gastrin-releasing peptide receptor, or both an integrin and a gastrin-releasing peptide receptor, on the cell or population of mammalian cells, thereby delivering the agent to the cell or cells.
In some embodiments of this aspect of the disclosure, the cell or population of cells comprise mammalian cells, and wherein the cells or population of cells are isolated cells.
In some embodiments of this aspect of the disclosure, the cell or population of cells comprise mammalian cells, and wherein the cells or population of cells are in a tissue of a human or animal host.
In some embodiments of this aspect of the disclosure, the agent is a therapeutic agent or a detectable agent,
The above discussion is meant to be illustrative of the principles and various embodiments of the present disclosure. Numerous variations and modifications will become apparent to those skilled in the art once the above disclosure is fully appreciated. It is intended that the following claims be interpreted to embrace all such variations and modifications.
Now having described the embodiments of the disclosure, in general, the example describes some additional embodiments. While embodiments of present disclosure are described in connection with the example and the corresponding text and figures, there is no intent to limit embodiments of the disclosure to these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.
All chemicals obtained commercially were of analytic grade and used without further purification. ‘No-carrier-added’-18F-F− was obtained from an in-house PET trace cyclotron (GE Healthcare). Reversed-phase extraction CI8 Sep-Pak cartridges were obtained from Waters and were pretreated with ethanol and water before use. The syringe filter and polyethersulfone membranes (pore size, 0.22 □m; diameter, 13 mm) were obtained from Nalge Nunc International. 125I-Echistatin, labeled by the lactoperoxidase method to a specific activity of 74 TBq/mmol (2,000 Ci/mmol) and 125I-[Tyr4]BBN (74 TBq/mmol (2,000 Ci/mmol)) were purchased from GE Healthcare. Na125I was purchased from Perkin-Elmer (Waltham, Mass.). 64Cu was obtained from University of Wisconsin (Madison, Wis.).
The peptides Aca-BBN(7-14) and c(RGDyK) (as shown in
Analytic as well as semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) were performed on a Dionex 680 chromatography system with a UVD 170U absorbance detector and model 105S single-channel radiation detector (Carroll & Ramsey Associates). The recorded data were processed using Chromeleon version 6.50 software. Isolation of peptides and 18F-labeled peptides was performed using a Vydac protein and peptide column (218TP510; 5 μm, 250×10 mm). The flow was set at 5 mL/min using a gradient system starting from 95% solvent A (0.1% trifluoroacetic acid (TFA) in water) and 5% solvent B (0.1% TFA in acetonitrile (ACN)) (0-2 min) and ramped to 35% solvent A, 65% solvent B, at 32 min. The analytic HPLC was performed using the same gradient system, but with a Vydac column (218TP54, 5 fxm, 250×4.6 mm) and a flow of 1 mL/min. The ultraviolet (UV) absorbance was monitored at 218 nm, and the identification of the peptides was confirmed based on the UV spectrum acquired using a photodiode array detector.
The NOTA conjugation and radiolabeling procedures were all performed under metal-free conditions.
The Boc-protected glutamic acid activated ester Boc-E(OSu)2 was prepared as previously reported by Wu et al., J. Nucl Med. 2005; 46: 1707-1718 incorporated herein by reference in its entirety. To a solution of Boc-E(OSu)2 (4.4 mg, 10 μmol) in 2 mL anhydrous N,N-dimethylformamide (DMF), 0.8 eq. Aca-BBN(7-14) (8.4 mg, 8 μmol) was added. The pH of the resulting mixture was adjusted to 8.5-9.0 with diisopropylethyl amine (DIPEA). After stirring at room temperature for 2 hr, 1.2 eq. c(RGDyK) (7.6 mg, 12 μmol) was added. The desired product Boc-BBN-RGD was isolated by preparative HPLC. The Boc-group was then removed by anhydrous TFA, and the crude product was again purified by HPLC. A total of 6.9 mg BBN-RGD was obtained as white powder in 48.4% overall yield. Analytic HPLC (retention time [Rt]=16.8 min) and mass spectrometry (MALDI-TOF-MS [matrix-assisted laser desorption/ionization time-of-flight mass spectrometry]: m/z 1,783.03 for [MH]+ (C8iH123N24O20S, calculated molecular weight [MW] 1,783.90)) confirmed the identity of the purified product.
N-Succinimidyl-4-fluorobenzoate (SFB) (4 mg, 16.8 Sμmol) and BBN-RGD (2 mg, 1.12 μmol) were mixed in 0.05 M borate buffer (pH 8.5) at room temperature. After 2 hr, the desired product FB-BBN-RGD was isolated by semi-preparative HPLC in 62% yield. Analytic HPLC (Rt=18.1 min) and mass spectrometry (MALDI-TOF-MS: m/z 1,905.90 for [MH]+ (C88H126FN24C21S, calculated [MW] 1,905.92)) analyses confirmed the product identification.
N-Succinimidyl-4-18F-fluorobenzoate (18F-SFB) was synthesized according to a previously reported procedure (Wu et al., J. Nuc. Med. (2007) 48: 1536-1544, incorporated herein by reference in its entirety). This procedure has been adapted into a commercially available synthesis module (GE TRACERlab FX
The peptide mixture was incubated at 60° C. for 30 min. After dilution with 700 μL of 1% TFA, the mixture was purified by semi-preparative HPLC. The desired fractions were combined and rotary evaporated to remove the solvent. The 18F-labeled peptides were then formulated in normal saline and passed through an 0.22-μm Millipore filter into a sterile multidose vial for in vitro and in vivo experiments.
The synthesis of BBN-RGD heterodimer was performed through an active ester method by coupling Boc-Glu(OSu)2 with BBN and RGD peptides sequentially. After TFA deprotection, BBN-RGD was obtained as a fluffy white powder with a yield of 48.4%. Four products were generated from the coupling reaction [Boc-Glu(BBN)2, Boc-Glu(RGD)2, Boc-Glu-BBN-RGD (RGD on the side-chain 8-position), and Boc-Glu-RGD-BBN (BBN on the side-chain 5-position)]. The Boc-Glu(BBN)2 and Boc-Glu(RGD)2 impurities could be efficiently removed. However, there was no observed difference in HPLC retention time between Boc-Glu-BBN-RGD and Boc-Glu-RGD-BBN. Therefore, as shown in
The total synthesis time for 18F-SFB was about 100 min, and the decay-corrected yield was 67%±11% (n=10) using the modified GE synthetic module (TRACERlab FX
Tert-butyl 2-aminoethylcarbamate (1.6 g, 10 mmol) and benzyl acrylate (16.2 g, 100 mmol) were heated to 70° C. under nitrogen for 7 days. Excess benzyl acrylate was distilled off at 60° C. and the residue was purified by column chromatography to yield 3,3′-(2-(tert-butoxycarbonylamino)ethylazanediyl)dipropanoate as a colorless oil (4.3 g, 89%). 1H NMR (CDCl3) δ 7.26-7.36 (m, 10H), 5.11 (s, 1H), 5.04 (bs, 4H), 3.14-3.15 (m, 2H), 2.76 (t, J=6.93 HZ, 4H), 2.51 (t, J=5.73 Hz, 2H), 2.44 (t, J=6.93 Hz, 4H).
Pd/C (50 mg, 10%) was added to a solution of benzyl 3,3′-(2-(tert-butoxycarbonylamino)ethylazanediyl)dipropanoate (1.5 g, 3.1 mmol) in EtOH (100 ml). The reaction mixture was bubbled with hydrogen overnight. The reaction mixture was filtered through a plug of celite, washed with EtOH (5 mL) and evaporated to dryness to give the crude product as a colorless oil. To the residue was added CH2Cl2 (5 mL). The solvent was evaporated to yield 3,3′-(2-(tert-butoxycarbonylamino)ethylazanediyl)dipropanoic acid as a white solid (0.7 g, 74%). 1H NMR (DMSO) δ 2.93 (m, 2H), 2.65 (t, J=7.1 Hz, 4H), 2.41 (t, J=7.1 Hz, 2H), 2.29 (t, J=9.93 Hz, 4H), 1.38 (s, 9H).
To a solution of 3,3′-(2-(tert-butoxycarbonylamino)ethylazanediyl)dipropanoic acid (3.04 mg, 10 μmol) in DMF (300 μL) was added a solution of TSTU (6.02 mg, 20 μmol) in DMF (600 μL) followed by DIPEA (20 μL). The reaction mixture was stirred for 30 min at room temperature. BBN (10.5 mg, 10 μmol) in DMF (1.0 mL) was added and the reaction mixture was stirred for 20 min at room temperature. After adding RGD (12.4 mg, 20 μmol) in DMF (1.2 mL), the reaction mixture was heated at 60° C. for 1 h. The reaction progress was monitored by analytical HPLC. Once the reaction reached completion, HOAc solution (2 mL, 5%) was added to quench the reaction. The Boc protected crude product was purified by preparative HPLC and lyophilized to yield a white powder. The Boc protection group was removed by dissolve the product in 3 mL TFA and stirred for 10 min at room temperature. After removing excess TFA under reduced pressure, the final product was purified by preparative HPLC and lyophilized to afford AEADP-RGD-BBN hetero dimer as a white powder (5.9 mg, 32% overall yield, two steps). Analytical HPLC (Rt=18.1 min) and mass spectrometry (MALDITOF-MS: m/z 1841.76 for [MH]+ (C84H129N25O20S, calculated [MW] 1840.96)) analyses confirmed the product identification.
N-Succinimidyl-4-fluorobenzoate (SFB) (2 mg, 8.4 μmol) in DMF (200 μL), AEADP-RGD-BBN (1.8 mg, 1.0 μmol) in DMF (200 μL) and DIPEA (20 μL) were mixed. The reaction mixture was heated at 60° C. for 30 min. The reaction mixture was quenched with 2 mL 5% HOAc. The crude product FB-AEADP-RGD-BBN was purified by preparative HPLC and lyophilized to give a white powder in 85% yield. Analytic HPLC (Rt=20.3 min) and mass spectrometry (MALDI-TOF-MS: m/z 1961.95 for [MH]+ (C91H13218FN25O21 S, calculated [MW] 1961.98)) analyses confirmed the product identification.
To a mixture of peptides (200 μg) in DMSO (20 μL) and DIPEA (20 μL) was added 18F-SFB. The reaction mixture was heated for 15 min at 90° C. The reaction was quenched with 800 μL of 5% HOAc. The 18F labeled peptide was purified by semi-preparative HPLC. The desired fractions were combined and the solvent was removed under reduced pressure. The 18F-labeled peptide was then formulated in normal saline and passed through an 0.22-μm Millipore filter into a sterile multidose vial for in vitro and in vivo experiments.
Approximately 111 kBq of 18F-FB-BBN, 18F-FB-RGD, or 18F-FB-BBN-RGD in 500 μL of PBS (pH 7.4) were added to 500 μL of octanol in an Eppendorf microcentrifuge tube. The mixture was vigorously vortexed for 1 min at room temperature. After centrifugation at 12,500 rpm for 5 min in an Eppendorf microcentrifuge (model 5415R; Brinkman), 200-μL aliquots of both layers were measured using a 7-counter (Packard Instruments). The experiment was performed in triplicate.
The c(RGDyK) (RGD), Aca-BBN(7-14) (BBN) and RGD-BBN peptides were conjugated with NOTA under standard SCN-amine reaction conditions as previously described (Li et al., (2008) Eur. J. Med. Mol. Imaging. 35: 1100-1108, incorporated herein by reference in its entirety). Briefly, a solution of 2 μmol of peptide (RGD, BBN, or RGD-BBN) was mixed with 6 μmol of p-SCN-Bn-NOTA in sodium bicarbonate buffer (pH 9.0). After stirring at room temperature overnight, the NOTA-conjugated peptides were isolated by semi-preparative HPLC. The desired fractions were combined and lyophilized to afford the final product as a white powder.
NOTA-c(RGDyK) (NOTA-RGD) was obtained in 61% yield with a 13.4 min retention time on analytical HPLC. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) was m/z 1,070.4 for [MH]+ (C47H68N13O14S, calculated molecular weight 1,070.5 Da). NOTA-BBN was obtained in 72% yield with a 22.05 min retention time on analytical HPLC. MALDITOF-MS was m/z 1504.0 for [MH]+ (C69H102N18O16S2, calculated molecular weight 1503.8).
NOTA-RGD-BBN (VII), as shown in
RGD-bombesin was also conjugated with DOTA. Briefly, DOTA was activated by 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide and N-hydroxysulfonosuccinimide for 30 min with a molar ratio of 10:5:4 for DOTA: 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide: N-hydroxysulfonosuccinimide. The DOTA-OSSu (6 mmol, calculated on the basis of N-hydroxysulfonosuccinimide) was added to RGD-bombesin (2 mmol) in 0.1N NaHCO3 solution (pH 9.0). After being stirred at 4° C. overnight, the DOTA conjugate was isolated by semi-preparative HPLC. DOTA-RGD-bombesin was obtained in 60% yield with more than 95% HPLC purity (Rt, 20.63 min). MALDI-TOF-MS: m/z, 2,171.2 for [MH]1 (C97H148N28O27S, calculated molecular weight, 2,170.4).
On the analytic HPLC, no significant difference in retention time was observed between 64Cu-labeled tracers and the unlabeled NOTA and DOTA conjugates. NOTA-RGD-bombesin was more easily labeled with 64Cu than was DOTARGD-bombesin as determined by the labeling condition studies, the results of which are shown in
The 68Ga labeling was performed according to methods previously described (Li et al., (2008) Eur. J. Med. Mol. Imaging. 35: 1100-1108, incorporated herein by reference in its entirety). Briefly, 10 nmol of NOTA-RGD, NOTA-BBN, or NOTA-RGD-BBN peptide, was dissolved in 500 μl of 0.1 M sodium acetate buffer and incubated with 185 MBq of 68Ga for 10 min at 40° C. 68Ga-NOTA-RGD, 68Ga-NOTA-BBN, or 68Ga-NOTA-RGD-BBN was then purified by analytical HPLC and the radioactive peak containing the desired product was collected. After removing the solvent by rotary evaporation, the activity was reconstituted in PBS and passed through a 0.22-μm Millipore filter into a sterile multidose vial for in vitro and in vivo experiments. The labeling was done with a 92% decay-corrected yield for NOTA-RGD (Rt 12.9 min), 95% for NOTA-BBN (Rt 21.8 min), and 90% for NOTA-RGD-BBN (Rt 19.9 min).
For in vitro and in vivo studies, 5-10 nmol of NOTA-RGD, NOTA-BBN, NOTA-RGD-BBN, or DOTA-RGD-BBN dissolved in NaOAc buffer was labeled with 64Cu in the conditions of 42° C. 1 h for DOTA conjugates, and room temperature 15 min for NOTA-conjugates. The labeled peptides were then purified by analytical HPLC. The radioactive peak containing the desired product was collected and rotary evaporated to remove the solvent. The products were then formulated in phosphate-buffered saline (PBS), and passed through a 0.22-μm Millipore filter into a sterile multidose vial for in vitro and in vivo experiments.
The NOTA conjugates of BBN and RGD-BBN were analyzed by both HPLC and mass spectroscopy to confirm the identity of the products. The characterizations of 18F-FB-PEG3-RGD-BBN, 64Cu-NOTA-RGD-BBN, and 68Ga-NOTA-RGD-BBN are listed in Table 1.
68Ga-NOTA-RGD-BBN.
18F-FB-PEG3-
64Cu-NOTA-
68Ga-NOTA-
The decay-corrected labeling yield of 18F-FB-PEG3-RGD-BBN was 40-50% based on 18F-SFB. The decay-corrected labeling yields of 64Cu-NOTA-RGD-BBN, and 68Ga-NOTA-RGD-BBN were all higher than 90% under the condition of reaction at 40° C. for 15 min. After the purification with HPLC, the radiochemical purity of the tracers were all higher than 98%. The overall preparation time was approximately 180 min for 18F-FB-PEG3-RGD-BBN from 18F-F, approximately 40 min for 64Cu-NOTA-RGD-BBN from 64CuCl2, and approximately 45 min for 68Ga-NOTA-RGD-BBN from 68Ga3+ elution.
The PC-3 and DU-145 human prostate carcinoma cell lines were purchased from American Type Culture Collection. PC-3 cells were grown in F-12K nutrient mixture (Kaighn's modification) (Invitrogen Corp.), and DU-145 cells were grown in minimum essential medium (Eagle) mixture supplemented with 10% (v/v) fetal bovine serum (Invitrogen) at 37° C. with 5% CO2. The PC-3 and DU-145 tumor models were generated by subcutaneous injection of 5×106 tumor cells into the front flank of male athymic nude mice (Harlan). The mice were subjected to micro-PET studies when the tumor volume reached 100-300 mm3 (3-4 wk after inoculation).
The PC-3 human prostate carcinoma cell line and MDAMB-435 human melanoma cell line (Lacroix M. (2009) Cancer Chemother. Pharmacol. 63: 567; Rae et al., (2007) Breast Cancer Res. Treat.; 104:1) were purchased from the American Type Culture Collection (ATCC, Manassas, Va.).
The 4T1 murine breast cancer cell line was purchased from American Type Culture Collection and grown in RMPI 1640 medium (Invitrogen Corp.) supplemented with 10% (v/v) fetal bovine serum (Invitrogen) at 37° C. with 5% CO2. The 4T1 tumor model was generated by subcutaneous injection of 5×106 tumor cells into the left front flank of female normal BALB/c mice (Harlan). The mice were used for micro-PET studies when the tumor volume reached 100˜300 mm3 (about 1-2 weeks for 4T1 tumor model).
The MDA-MB-231, MDA-MB468, BT474, BT-20, T47D, MCF-7 and MDA-MB435 human breast cancer cell lines were all obtained from the American Type Culture Collection (ATCC) and maintained under standard conditions according to ATCC. The MDA-MB435 tumor model was established by orthotopic injections of 5×106 cells into the right mammary fat pad of female athymic nude mice. For T47D tumor model establishment, the female nude mice were first subcutaneously implanted with 60-day release 17β-estradiol pellets (Innovative Research of America, Sarasota, Fla.) in the left neck. One day after the estradiol implantation, 1×107 T47D cells were orthotopically injected into the right mammary fat pad of the nude mice. The mice were subjected to microPET studies when the tumor volume reached 100-300 mm3 (2-3 weeks for MDA-MB-435, and 4-5 wk for T47D).
In vitro integrin αvβ3-binding affinities and specificities of RGD, BBN-RGD, and FB-BBN-RGD were assessed via displacement cell-binding assays using 1251-echistatin as the radioligand. Experiments were performed on U87MG human glioblastoma cells by a previously described method (Wu et al., J. Nucl. Med. (2005) 46: 1707-1718, incorporated herein by reference in its entirety). In vitro GRPR-binding affinities and specificities of BBN, BBN-RGD, and FB-BBN-RGD were assessed via displacement cell-binding assays using 125I-[Tyr4]BBN as the radioligand. Experiments were performed on PC-3 human prostate carcinoma cells by a previously described method (Chen et al., J. Nucl. Med. (2004) 45: 1390-1397, incorporated herein by reference in its entirety). The best-fit 50% inhibitory concentration (IC50) values were calculated by fitting the data with nonlinear regression using Graph-Pad Prism (GraphPad Software, Inc.). Experiments were performed with triplicate samples.
The binding affinities of Aca-BBN(7-14), BBN-RGD, and FB-BBN-RGD for GRPR were evaluated for PC-3 cells. Results of the cell-binding assay were plotted in sigmoid curves for the displacement of 1251-[Tyr4]BBN from PC-3 cells as a function of increasing concentration of BBN analogs. The IC50 values were determined to be 20.7±3.2 nM for BBN monomer, 35.7±4.4 nM for heterodimer BBN-RGD, and 32.0±1.9 nM for FB-BBN-RGD on 105 PC-3 cells, as shown in
In vitro integrin αvβ3-binding affinities and specificities of RGD-BBN, NOTA-RGD-BBN, and BBN were compared with those of RGD via displacement cell-binding assays using 125I-c[RGDyK] as the radioligand. 125I-c(RGDyK) was prepared by labeling c(RGDyK) with Na125I at high specific activity (about 44.4 TBq/mmol) (Chen et al., Mol Imaging Biol. (2004) 6: 350-359, incorporated herein by reference in its entirety).
Experiments were performed on U87MG human glioma cells expressing integrin αvβ3 as previously described (Wu et al., (2007) Eur. J. Nucl. Med. Mol. Imaging. 34: 1823-1831; Cai et al., (2006) J. Nuc. Med. 47: 1172-1180, incorporated herein by reference in their entireties). In vitro GRPR binding affinities and specificities of RGD-BBN, NOTA-RGD-BBN and RGD were compared with those of BBN via displacement cell-binding assays using 125I-[Tyr4] BBN as the radioligand. Experiments were performed on GRPR-expressing PC-3 cells following our previously described procedure (Chen et al., Mol Imaging Biol. (2004) δ: 350-359; Cai et al., (2006) J. Nuc. Med. 47: 1172-1180, incorporated herein by reference in their entireties). The best-fit 50% inhibitory concentration (IC50) values were calculated by fitting the data with nonlinear regression using GraphPad Prism (GraphPad Software). Experiments were performed twice with triplicate samples.
The integrin αvβ3 receptor-binding affinities of RGD-BBN, NOTA-RGD-BBN were determined by performing competitive binding assay with 125I-c(RGDyK) as the radioligand. Cyclic RGD peptide c(RGDyK) and BBN were also added for comparison. RGD-BBN and NOTA-RGD-BBN inhibited the binding of 125I-c(RGDyK) to integrin-expressing U87MG cells in a concentration-dependent manner. The IC50 values for RGD-BBN, NOTA-RGD-BBN and c(RGDyK) were 17.91±5.70 nM, 22.57±6.68 nM, and 11.19±4.21 nM, respectively (
The results of the cell-binding assay were plotted as sigmoid curves for the displacement of 125I-[Tyr4]BBN from PC-3 cells as a function of increasing concentration of BBN analogs. The IC50 values were determined to be 67.92±4.97 nM for RGD-BBN, 55.89±4.23 nM for NOTA-RGD-BBN, and 78.96±4.86 nM for BBN on PC-3 cells. RGD did not show significant binding inhibition of 125I-[Tyr4]BBN with GRPR (
The integrin αvβ3 receptor-binding affinities of DOTA-RGD-bombesin and NOTA-RGD-bombesin were compared with c(RGDyK) by conducting a competitive binding assay on U87MG cells using 125I-c(RGDyK) as the radioligand. The inhibitory concentrations of 50% for DOTA-RGD-BBN, NOTA-RGD-BBN, and c(RGDyK) were 21.55 6 2.19 nM, 16.15 6 2.77 nM, and 10.84 6 2.55 nM, respectively, as shown in
GRPR and Integrin αvβ3 Expression on Breast Cancer Cells
The expression of GRPR and integrin αvβ3 on various breast cancer cells were determined by radioligand receptor-binding assay using 125I-[Tyr4]BBN and 125I-c(RGDyK), respectively. As shown in
Cell Uptake and Efflux Studies
(i) Uptake and Efflux of 18F-FB-BBN, 18F-FB-RGD, and 18F-FB-BBN-RGD into PC-3 Cells
Uptake and efflux of 18F-FB-BBN, 18F-FB-RGD, and 18F-FB-BBN-RGD into PC-3 cells were examined according to the following protocol. In the cell uptake experiment, PC-3 cells were seeded into 12-well plates at a density of 5×105 cells per well for overnight incubation. Cells were rinsed 3 times with phosphate-buffered saline (PBS), followed by the addition of 18F-FB-RGD, 18F-FB-BBN, or 18F-BBN-RGD to the cultured wells in triplicate (about 2 μCi/well). After incubation at 37° C. for 5, 15, 30, 60, and 120 min, cells were rinsed 3 times with PBS and lysed with NaOH-sodium dodecyl sulfate (SDS) (0.2 M NaOH, 1% SDS). The cell lysate was collected in measurement tubes for counting. The cell uptake was normalized in terms of added radioactivity.
In the cell efflux experiment, PC-3 cells were seeded into 12-well plates at a density of 5×105 cells per well for overnight incubation. Cells were rinsed 3 times with PBS and then the appropriate 18F-labeled peptide tracer was added. The cells were incubated at 37° C. for 2 hr, washed with PBS, and then reincubated with serum-free medium. The cells were washed at different time points (0, 15, 30, 60, 120, 180 min) with PBS and lysed with NaOH-SDS (0.2 M NaOH, 1% SDS). The cell lysate was collected in measurement tubes for counting. Efflux values at different time points were calculated by subtracting retention from 0-min retention, and normalized by dividing the total counts at 0 min.
Due to the relative low receptor-binding affinity of the monomeric RGD peptides and moderate integrin receptor density of the PC-3 cells, 18F-FB-RGD had relatively low cell uptake (<0.5%). On the other hand, PC-3 cells express a high level of GRPR, 18F-FB-BBN binding to GRPR facilitates effective internalization of this radioligand, and the uptake of 18F-FB-BBN is thus rapid and high, reaching about 7% within 30 min of incubation, and plateaus afterward. The cell uptake behavior of 18F-FB-BBN-RGD is similar to that of 18F-FB-BBN but the uptake value is slightly lower (
The cell uptake studies were performed as we have previously described with some modifications (Chen et al., Mol Imaging Biol. (2004) δ: 350-359; Cai et al., (2006) J. Nuc. Med. 47: 1172-1180, incorporated herein by reference in their entireties). Briefly, PC-3 cells were seeded into 12-well plates at a density of 5×105 cells per well and incubated (about 18 kBq/well) with 68Ga-labeled tracers at 37° C. for 15, 30, 60, and 120 min. Tumor cells were then washed three times with chilled PBS and harvested by trypsinization with 0.25% trypsin/0.02% EDTA (Invitrogen). The cell suspensions were collected and measured in a y counter (Packard, Meriden, Conn.). The cell uptake was expressed as the percent added dose (% AD) after decay correction. Experiments were performed twice with triplicate wells. For efflux studies, 68Ga-labeled tracers (about 18 kBq/well) were first incubated with PC-3 cells in 12-well plates for 1 h at 37° C. to allow internalization. Then cells were washed twice with PBS, and incubated with cell culture medium for 15, 30, and 60 min. After washing three times with PBS, cells were harvested by trypsinization with 0.25% trypsin/0.02% EDTA. The cell suspensions were collected and measured in a γ counter. Experiments were performed twice with triplicate wells. Data are expressed as percent added dose after decay correction.
The cell uptake and efflux of 68Ga-NOTA-RGD-BBN were evaluated in studies in PC-3 tumor cells that express high levels of GRPR and moderate levels of integrin αvβ3 (2.7×106 GRPRs per cell and 2.76×103 integrins per cell, as described by Zhang et al., (2006) J. Nucl. Med.; 47: 113-121, and Cai et al., (2006) Cancer Res. 66: 9673-9681).
68Ga-NOTA-BBN had rapid and high cell uptake (
(iii) Uptake and Efflux of 64Cu-NOTA-RGD-BBN and 64Cu-DOTA-RGD-BBN
64Cu-NOTA-BBN showed a rapid and high uptake in the PC-3 tumor cells, whereas 64Cu-NOTA-RGD had low cell uptake, as shown in
(iv) T47D and MDA-MB435 tumor cells were seeded into 12-well plates at a density of 5×105 cells per well one day before experiment to allow adherence. Cells were incubated with 18F-FB-PEG3-RGD-BBN, 64Cu-NOTA-RGD-BBN or 68Ga-NOTA-RGD-BBN (approximately 18 kBq/well) at 37° C. for 15, 30, 60, and 120 min. Tumor cells were then washed three times with chilled PBS and harvested by trypsinization with 0.25% trypsin/0.02% EDTA (Invitrogen, Carlsbad, Calif.). The cells suspensions were collected and measured in a y counter (Packard, Meriden, Conn.). The cell uptake was expressed as the percent added dose (% AD) after decay correction. Experiments were performed twice with triplicate wells.
The cell uptake studies of 18F-FB-PEG3-RGD-BBN, 64Cu-NOTA-RGD-BBN and 68Ga-NOTA-RGD-BBN were performed on T47D and MDA-MB-435 tumor cells. As shown in
(a) PET scans and image analysis were performed using a micro-PET R4 rodent model scanner (Siemens Medical Solutions) as reported by Li et al., J. Nucl. Med. (2007) 48: 1162-1171 and Wu et al., J Nucl. Med. (2005) 46: 1707-1718, both of which are incorporated herein by reference in their entireties). Tumor-bearing mice were each tail-vein injected with approximately 3.7 MBq (100 μCi) of 18F-FB-RGD, 18F-FB-BBN, or 18F-FB-BBN-RGD under isoflurane anesthesia. Five-minute static PET images were then acquired at 0.5, 1, and 2 h after injection. The images were reconstructed by a 2-dimensional ordered-subsets expectation maximum (OSEM) algorithm. No attenuation or scatter correction was applied. For the receptor-blocking experiment, c(RGDyK) (10 mg/kg), Aca-BBN(7-14) (15 mg/kg), or RGD+BBN (10 mg/kg RGD and 15 mg/kg BBN) were co-injected with 3.7 MBq of 18F-FB-BBN-RGD to PC-3 tumor mice. The 5-min static PET scans were then acquired at 1 hr after injection. For each small-animal PET scan, regions of interest (ROIs) were drawn over the tumor, normal tissue, and major organs by using vendor software (ASI Pro 5.2.4.0) on decay-corrected whole-body coronal images. The average radioactivity concentration (accumulation) within a tumor or an organ was obtained from mean pixel values within the multiple ROI volume, which were converted to counts/mL/min using a conversion factor. Assuming a tissue density of 1 g/mL, the ROIs were converted to counts/g/min, and then divided by the administered activity to obtain an imaging ROI-derived percentage injected dose per gram of tissue (% ID/g).
Static small-animal PET scans were performed on a PC-3 xenograft model (n=3, both GRPR- and integrin αvβ3-positive) (Cooper et al., Neoplasia. (2002) 4: 191-194; Zheng et al., J. Biol. Chem. (2000) 275: 24565-24574; Cai et al., Cancer Res. (2006) 66: 9673-9681; Markwalder & Reubi, Cancer Res. (1999) 59: 1152-1159 incorporated herein be reference in their entireties), and selected coronal images at different time points after injection of 18F-FB-BBN-RGD, 18F-FB-BBN, or 18F-FB-RGD are shown in
18F-FB-BBN-RGD also showed substantially lower liver and significantly decreased renal uptake compared with 18F-FB-BBN (slightly increased kidney uptake compared with 18F-FB-RGD). Because of the enhanced tumor-targeting efficacy and improved in vivo pharmacokinetics, 18F-FB-BBN-RGD had higher tumor-to-organ ratios than 18F-FB-BBN and 18F-FB-RGD, as shown in
The receptor specificity of 18F-FB-BBN-RGD in vivo was confirmed by several blocking experiments, as shown in
(b) Each PC-3 or MDA-MB-435 tumor-bearing mouse was injected in a tail vein with about 3.7 MBq (100 μCi) of 68Ga-NOTA-RGD, 68Ga-NOTA-BBN or 68Ga-NOTA-RGD-BBN under isoflurane anesthesia (n=4 per group). For static PET, 5-min scans were acquired at 30 min, 1 h, and 2 h after injection. For dynamic PET, 30-min scans (1×30 s, 4×1 min, 1×1.5 min, 4×2 min, 5×3 min; total 15 frames) were started 1 min after injection, and two 5-min static PET images were also acquired at 1 h and 2 h after injection. The images were reconstructed using a two-dimensional ordered subsets expectation maximum (OSEM) algorithm and no correction was applied for attenuation or scatter. For the blocking experiment, PC-3 tumor-bearing mice were co-injected with c(RGDyK) (RGD) at 10 mg/kg body weight, Aca-BBN (7-14) (BBN) at 15 mg/kg or RGD at 10 mg/kg+BBN at 15 mg/kg and 3.7 MBq of 68Ga-NOTA-RGD-BBN, and 5-min static PET scans were then acquired at 1 h after injection (n=3 per group). For each small-animal PET scan, regions of interest (ROIs) were drawn over the tumor, normal tissue, and major organs using vendor software ASI Pro 5.2.4.0 on decay-corrected whole-body coronal images. The maximum radioactivity concentrations (accumulation) within a tumor or an organ were obtained from mean pixel values within the multiple ROI volume, and were converted to megabecquerels per milliliter per minute using a conversion factor. These values were then divided by the administered activity to obtain (assuming a tissue density of 1 g/ml) an image ROI-derived percent injected dose per gram (% ID/g).
Representative coronal small-animal PET images of PC-3 tumor-bearing mice (n=4 per group) at different times after intravenous injection of 3.7 MBq (100 μCi) of 68Ga-NOTA-RGD-BBN are shown in
Tumor and major organ activity accumulation in the small-animal PET scans was quantified by measuring the ROIs that encompassed the entire organ on the coronal images. The tumor uptake of 68Ga-NOTA-RGD-BBN was determined to be 6.55±0.83, 5.26±0.32, and 4.04±0.28% ID/g at 30, 60, and 120 min (
The kidney uptake of 68Ga-NOTA-RGD-BBN and 68Ga-NOTA-BBN was significantly higher than that of RGD at all time points (p<0.05), as shown in
The tumor/kidney, tumor/liver and tumor/muscle ratios of the three tracers are shown for 1 h after injection in
The in vivo integrin and GRPR dual receptor binding property of 68Ga-NOTA-RGD-BBN was confirmed by several blocking studies, as shown in (
The in vivo behaviors of 68Ga-NOTA-RGD-BBN, 68Ga-NOTA-BBN, and 68Ga-NOTA-RGD were also tested in a MDA-MB435 tumor model, which expresses moderate levels of integrin αvβ3, but undetectable levels of GRPR (based on radioligand binding assays). As shown in
The tumor targeting property of 68Ga-NOTA-RGD-BBN in PC-3 tumor-bearing mice was also evaluated by a 30-min dynamic small-animal PET scan followed by 5-min static scans at 1 h and 2 h after injection. Representative coronal images and quantified % ID/g by ROI analysis at different time points after injection are shown in
(c) Under isoflurane anesthesia, each PC-3 tumor mouse received an injection via the tail vein of approximately 5.5 MBq (150 mCi) of 64Cu-NOTA-RGD, 64Cu-NOTA-bombesin, 64Cu-NOTA-RGD-bombesin, 64Cu-NOTA-RGD (75 mCi) plus 64Cu-NOTA-bombesin (75 mCi), or 64Cu-DOTA-RGD-bombesin. Five-minute static PET images were acquired at 30 min, 1 h, and 4 h after injection of each tracer (n=4/group), and 10-min static PET images were acquired at 20 h. The images were reconstructed using a 2-dimensional ordered-subsets expectation maximum algorithm without attenuation or scatter correction.
Under isoflurane anesthesia, each 4T1 tumor mouse received an injection via the tail vein of 3.7 MBq (100 mCi) of 64Cu-NOTA-RGD, 64Cu-NOTA-BBN, or 64Cu-NOTA-RGD-BBN. Five-minute static PET images were then acquired at 2 h after injection (n=3/group).
A series of blocking studies was also performed to validate the in vivo dual-receptor binding affinity of 64Cu-NOTA-RGD-BBN in PC-3 tumor-bearing nude mice at 1 h after injection of about 5.5 MBq (150 mCi) of tracer (n=3/group). For each small-animal PET scan, regions of interest were drawn over each tumor, over normal tissue, and over major organs using vendor software (ASI Pro, version 5.2.4.0) on decay-corrected whole-body coronal images. The maximum radioactivity concentration (accumulation) within a tumor or an organ was obtained from the mean pixel values within the multiple-region-of-interest volume, which were converted to MBq/mL/min using a conversion factor. Assuming a tissue density of 1 g/mL, the regions of interest were converted to MBq/g/min and then divided by the administered activity to obtain an imaging region-of-interest-derived percentage injected dose (% ID)/g.
All tumors were clearly visible after injection of the different tracers, with high contrast to contralateral background at all time points measured from 30 min to 20 h, except for 64Cu-NOTA-RGD (
64Cu-NOTA-RGD
64Cu-NOTA-BBN
64Cu-NOTA-RGD plus
64Cu-NOTA-BBN
64Cu-NOTA-RGD-BBN
64Cu-DOTA-RGD-BBN
Injection dose was about 5.5 Mbq (150 mCi) per mouse. Data are % ID/g±SD (n=4/group).
For 64Cu-NOTA-RGD, 64Cu-NOTA-BBN, 64Cu-NOTA-RGD plus 64Cu-NOTA-BBN, and 64Cu-NOTA-RGD-BBN, the tracers cleared rapidly from the blood, with less than 1% ID/g remaining at 30 min after injection. 64Cu-NOTA-RGD-BBN showed slightly lower blood clearance than the other tracers, whereas 64Cu-NOTA-BBN cleared the most rapidly. The tumor uptake of 64Cu-NOTARGD-BBN was determined to be 3.06±0.11, 2.78±0.56, 2.21±0.49, and 2.04±0.35% ID/g at 0.5, 1, 4, and 20 h after injection, respectively-significantly higher than all the other tracers tested (P, 0.01, n=4/group, Table 2).
Tumor uptake was about the same for 64Cu-NOTA-RGD, 64Cu-NOTA-BBN, and 64Cu-NOTA-RGD plus 64Cu-NOTA-BBN from 1 to 20 h after injection. 64Cu-NOTA-RGD-BBN showed higher kidney uptake than the other tracers at any time examined. 64Cu-NOTA-RGD and 64Cu-NOTA-RGD plus 64Cu-NOTA-BBN showed a similar clearance curve in the kidneys, whereas the kidney uptake of 64Cu-NOTA-BBN was the lowest at all time points. 64Cu-NOTA-BBN and 64Cu-NOTA-RGD plus 64Cu-NOTA-BBN exhibited predominantly liver uptake, whereas uptake of 64Cu-NOTA-RGD-BBN and 64Cu-NOTA-RGD in the liver was relatively low. The tumor to non-tumor ratios of 64Cu-NOTA-RGD-BBN were significantly higher than those of the other tracers at 4 h after injection (P, 0.05,
The tumor and major organ uptake and tumor to non-tumor ratios of 64Cu-NOTA-RGD-BBN and 64Cu-DOTA-RGD-BBN are directly compared in
The in vivo behaviors of 64Cu-NOTA-RGD, 64Cu-NOTA-BBN, and 64Cu-NOTA-RGD-BBN were also tested in a murine 4T1 breast tumor model. The 4T1 tumor tissue expresses a moderate level of murine integrin b3 but undetectable GRPR. As shown in
The in vivo integrin and GRPR dual-receptor binding property of 64Cu-NOTA-RGD-BBN was confirmed by several blocking studies (
(c) Representative coronal microPET images of T47D and MDA-MB435 tumor-bearing mice (n=4/group) at different times after intravenous injection of 3.7-5.6 MBq (100-150 μCi) of 18F-FB-PEG3-RGD-BBN, 64Cu-NOTA-RGD-BBN or 68Ga-NOTA-RGD-BBN are shown in
The tumor and major organ uptake comparison of 18F-FB-PEG3-RGD-BBN, 64Cu-NOTA-RGD-BBN and 68Ga-NOTA-RGD-BBN are depicted in
18F-FB-PEG3-RGD-BBN
68Ga-NOTA-RGD-BBN
64Cu-NOTA-RGD-BBN
The T47D or MDA-MB435 tumor uptake is expressed as the average of each tracer in four mice, while the normal organ uptake is expressed as the average of each tracer in eight mice (four T47D tumor-bearing mice and four MDA-MB-435 tumor-bearing mice per tracer). As shown in
The liver uptake of 64Cu-NOTA-RGD-BBN was significantly higher than those of the 18F and 68Ga labeled RGD-BBN tracers at any time points examined (n=8, P<0.05). At 4 h and 24 h p.i, the liver uptake of 64Cu-NOTA-RGD-BBN was still higher than that of the tumor uptake (Table 3). The liver uptake of 18F-FB-PEG3-RGD-BBN was very low at any time with the highest uptake being 1.13±0.43% ID/g at 30 min p.i, indicating the 18F labeled RGD-BBN tracer was almost not excreted from the hepatobiliary route (
18F, 64Cu and 68Ga labeled BBN tracers were also tested in the nude mice bearing MDA-MB-435 tumor, which was integrin αvβ3-positive, but GRPR-negative (
A PC-3 tumor mouse was injected intravenously with 3.7 MBq of 18F-FB-BBN-RGD. At 1 hr after injection, the mouse was sacrificed, the blood, urine, liver, kidneys, and tumor were collected, and metabolite analysis was performed as reported previously (Wu et al., (2007) Eur. J. Nucl. Med. Imaging 34: 1823-1831). In brief, the blood sample was immediately centrifuged for 5 min at 13,200 rpm. Other tissues were homogenized and then centrifuged for 5 min at 13,200 rpm. Each supernatant was passed through a C18 Sep-Pak cartridge. The urine sample was diluted directly with 1 mL of PBS and passed through a C18 Sep-Pak cartridge. The cartridges were each washed with 2 mL of water and eluted with 2 mL of ACN containing 0.1% TFA. The ACN eluent was concentrated and injected onto the analytic HPLC system. The eluant was collected with a fraction collector (0.5 min/fraction), and the radioactivity of each fraction was measured with a 7-counter.
The metabolic stability of 18F-FB-BBN-RGD was determined in mouse blood, urine, liver, kidneys, and tumor homogenates at 60 min after injection. The extraction efficiencies were 91.4% for blood, 73.6% for liver, 95.2% for kidneys, and 94.6% for PC-3 tumor, respectively. The elution efficiencies of the soluble fractions were 93.2% for blood, 68.6% for liver, 89.1% for kidneys, and 90.0% for PC-3 tumor. HPLC analysis results of the ACN-eluted fractions were shown in
Although we did not identify the composition of the metabolites, we found that all metabolites came off the HPLC column earlier than those for the parent compound. No defluoridation of 18F-FB-BBN-RGD was observed, as no visible bone uptake was found on any of the small-animal PET scans. Overall, 18F-FB-BBN-RGD exhibited comparable metabolic stability with 18F-FB-BBN (5).
18F-FB-PEG3-RGD-BBN, 64Cu-NOTA-RGD-BBN, or 68Ga-NOTA-RGD-BBN were incubated in fetal bovine serum (FBS) for 2 h at room temperature to test the in vitro serum stability. After passing through a 0.22-μm Millipore filter, the samples were analyzed by radio-HPLC. For metabolism studies, female nude mice (n=2/group) were injected with 18F-FB-PEG3-RGD-BBN, 64Cu-NOTA-RGD-BBN, or 68Ga-NOTA-RGD-BBN at a dose of 7.4 MBq (200 μCi) in 0.2 mL PBS via tail vein. At 60 min p.i., the urine samples were collected and then centrifuged at 8,000 rpm for 5 min. The supernatant was collected, filtered through a 0.22-μm Millipore filter, and then analyzed by radio-HPLC.
The serum stability of 18F-FB-PEG3-RGD-BBN, 64Cu-NOTA-RGD-BBN, and 68Ga-NOTA-RGD-BBN was tested by incubating with FBS for 2 h at room temperature. As shown in
The metabolic stability of the three tracers in mice urine at 60 min after injection was also studied. As shown in
Male athymic nude mice bearing PC-3 xenografts were injected with 0.74 MBq (20 μCi) of 68Ga-NOTA-RGD-BBN to evaluate the distribution of the tracer in the tumor tissues and major organs. At 0.5 h and 1 h after injection of the tracer, the tumor-bearing mice were killed and dissected. Blood, tumor, major organs, and tissues were collected and wet-weighed. The radioactivity in the tissue was measured by y counter (Packard). The results are presented as percentage injected dose per gram of tissue (% ID/g). For each mouse, the radioactivity of the tissue samples was calibrated against a known aliquot of the injectate and normalized to a body mass of 20 g. Values were expressed as mean±SD for groups of four animals (n=4 per group).
The biodistribution study of 68Ga-NOTA-RGD-BBN was performed in nude mice bearing PC-3 tumors. Each mouse was injected with 0.74 MBq (20 μCi) of 68Ga-NOTA-RGD-BBN and then killed at 0.5 h and 1 h after injection (n=4 per group). As shown graphically in
Normal BALB/c mice received an injection via the tail vein of 370 kBq (10 mCi) of 64Cu-NOTA-RGD-bombesin to evaluate the distribution of the tracer. The blocking experiments were also performed by co-injection of 64Cu-NOTA-RGD-bombesin with a saturating dose of c(RGDyK) (10 mg/kg of mouse body weight), bombesin (15 mg/kg), or RGD (10 mg/kg) plus bombesin (15 mg/kg). All mice were sacrificed at 1 h after injection of the tracer. Blood, tumor, and major organs and tissues were collected and wet-weighed. Stomach and intestines were cleaned of their contents in this experiment. The radioactivity in the tissue was measured using a g-counter (Packard). The results were presented as percentage injected dose per gram of tissue (% ID/g). Values were expressed as mean 6 SD (n=4/group).
The biodistribution of 64Cu-NOTA-RGD-BBN (370 kBq/mouse) was examined in normal BALB/c mice. The blocking experiments were also performed by co-injecting 64Cu-NOTA-RGD-BBN with a saturating dose of RGD, BBN, or RGD plus BBN and then sacrificing the mice at 1 h after injection (n=4/group). As shown in
Immunofluorescence staining studies were performed as described in Wu et al., (2007) Eur. J. Nucl. Med. Mol. Imaging. 34: 1823-1831, incorporated herein by reference in its entirety, with some modifications. Briefly, frozen PC-3 tumor and organ tissue slices (5-μm thickness) from the tumor-bearing nude mice were fixed with ice-cold acetone, rinsed with PBS and blocked with 10% goat serum for 30 min at room temperature. The slices were incubated with goat anti-GRPR antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, Calif.), humanized anti-human integrin αvβ3 antibody Abegrin (Wu et al., (2007) Eur. J. Nucl. Med. Mol. Imaging. 34: 1823-1831) (20 μg/ml), or hamster anti-β3 antibody (1:100; BD Biosciences, San Jose, Calif.) for 1 h at room temperature, and then visualized with FITC-conjugated donkey antigoat, Cy3-conjugated donkey antihuman or Cy3-conjugated goat anti-hamster secondary antibodies (1:200; Jackson ImmunoResearch Laboratories, West Grove, Pa.), respectively. For the overlaid staining of CD31 and murine P3, PC-3 tumor slices were incubated with rat anti-mouse CD31 antibody (1:100; BD Biosciences) and hamster anti-β3 antibody (1:100; BD Biosciences) and then visualized with Cy3-conjugated goat anti-rat and FITC-conjugated goat anti-hamster secondary antibody (1:200; Jackson ImmunoResearch Laboratories).
The expression of GRPR and integrin αvβ3 in the PC-3 tumor and normal organs was tested by immunofluorescent staining using anti-GRPR, anti-human αvβ3 and antimurine β3 antibodies. PC-3 tumors were found to be positive for GRPR, human αvβ3 and murine β3 (
The expression of GRPR, human integrin αvβ3 and murine integrin β3 on T47D and MDA-MB435 tumor tissues were detected by immunofluorescent staining. Briefly, frozen T47D and MDA-MB435 tumor slices (5-mm thickness) from the tumor-bearing nude mice were fixed with ice-cold acetone, rinsed with PBS and blocked with 10% goat serum for 30 min at room temperature. The slices were incubated with goat anti-GRPR antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, Calif.), humanized anti-human integrin αvβ3 antibody (ABEGRIN™, 20 μg/mL) (38), or hamster anti-β3 antibody (1:100; BD Biosciences, San Jose, Calif.) for 1 h at room temperature and then visualized with FITC-conjugated donkey anti-goat, Cy3-conjugated donkey anti-human and FITC-conjugated goat anti-hamster secondary antibodies (1:200; Jackson Immuno-Research Laboratories, West Grove, Pa.), respectively.
The expression of GRPR and integrin αvβ3 in the T47D and MDA-MB435 tumor tissues was detected by immunofluorescent staining. As shown in
A Glu-RGD-BBN peptide heterodimer was synthesized step-wise by solid-phase peptide synthesis method, illustrated in
Loading of Fmoc-Met-Rink Amide MBHA resin, synthesis of the bombesin peptide follows standard peptide synthesis protocols. Side chain protection was trityl (Trt) for histidine (His) and glutamine (Gln) and tert-butoxycarbonyl (Boc) for tryptophan (Trp). After loading Fmoc-Glu-OAII onto Aca, the α-allyl ester was then removed by treatment with Pd(Ph3P)4/CHCl3/AcOH/NMM. The α-carboxylate was activated and coupled with cyclic RGD peptide cyclo(Arg-Gly-Asp-DTyr-Lys) (RGD) via the lysine side chain ε-amine group. After removing the Fmoc from Glu, the final peptide cyclo[Arg-Gly-Asp-D-Tyr-Lys(Glu*-Aca-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2)] (Glu-RGD-BBN) was obtained by detaching/deprotecting the Rink Amide-MBHA resin using 95% TFA in dichloromethane (DCM) plus ethandithiol (EDT) and triisopropylsilane (TIS) as scavengers. ES-MS: m/z 1783.9 for [M+H]+ (C81H123N24O20S, calcd. 1783.9). RP-HPLC Rt=18.6 min.
To a solution of Boc-11-amino-3,6,9-trioxaundecanoic acid (Boc-NH-PEG3-COOH, 40 mg, 0.13 mmol) and N,N-diisopropylethylamine (DIPEA, 20 μl) in ACN was added O—(N-succinimidyl)-1,1,3,3-tetramethyl-uronium tetrafluoroborate (TSTU, 27 mg, 0.09 mmol). The reaction mixture was stirred at room temperature for 0.5 h and then added to a solution of Glu-RGD-BBN (36 mg, 0.02 mmol) in N,N′-dimethylformamide (DMF). After being stirred at room temperature for 2 h, the Boc-protected PEG3-Glu-RGD-BBN was isolated by preparative HPLC. The Boc group was then removed with anhydrous TFA and the crude product was again purified by preparative HPLC. The collected fractions were combined and lyophilized to afford 23 mg of PEG3-Glu-RGD-BBN as a white fluffy powder (yield: 58%). (MALDI-TOF MS: m/z 1973.3 for [M+H]+ (C89H138N25O24S, Calcd. 1974.3)). RP-HPLC Rt=18.8 min.
N-Succinimidyl-4-fluorobenzoate (SFB, 4 mg, 16.8 μmol) and PEG3-Glu-RGD-BBN (2 mg, 1.0 μmol) were mixed in 0.05 mol/L borate buffer (pH 8.5) at room temperature. After constant shaking for 2 h, the desired product FB-PEG3-Glu-RGD-BBN was isolated by semi-preparative HPLC (1.6 mg, yield: 76%). Analytical HPLC (RP-HPLC Rt=23.3 min) and mass spectrometry (MALDITOF-MS: m/z 2095.9 for [M+H]+ (C96H141FN25O25S, calcd. 2095.4)) analyses confirmed the product identification.
N-Succinimidyl-4-18F-fluorobenzoate (18F-SFB) was synthesized and purified with HPLC as we previously reported by modifying GE TRACERlab FX-FN module27. The purified 18F-SFB was rotary evaporated to dryness, redissolved in dimethyl sulfoxide (DMSO, 200 μL), and added to a DMSO solution of PEG3-Glu-RGD-BBN peptide (200 μg) and DIPEA (20 μL). The reaction mixture was incubated at 60° C. for 30 min. After dilution with 5% aqueous acetic acid solution (3 mL), the mixture was purified by semi-preparative HPLC. The desired fractions containing 18F-PEG3-RGD-BBN were combined and rotary evaporated to dryness. The activity was then reconstituted in PBS and passed through a 0.22-μm Millipore filter into a sterile multidose vial for in vitro and in vivo experiments.
The integrin αvβ3 receptor-binding affinities of cyclic RGD peptide c(RGDyK), PEG3-Glu-RGD-BBN, and FB-PEG3-Glu-RGD-BBN were determined by performing competitive binding assay with 125I-c(RGDyK) as the radioligand. All peptides inhibited the binding of 125I-c(RGDyK) to integrin expressing U87MG cells in a concentration-dependent manner. The IC50 values for c(RGDyK), PEG3-Glu-RGD-BBN, and FB-PEG3-Glu-RGD-BBN were 11.19±1.44, 10.80±1.46, and 13.77±1.82 nM, respectively, as shown in
The binding affinities of Aca-BBN(7-14), PEG3-Glu-RGD-BBN, and FB-PEG3-Glu-RGD-BBN for GRPR were evaluated using GRPR positive PC-3 cells with 125I-[Tyr4]BBN as the radioligand. Results of the cell-binding assay were plotted in sigmoid curves for the displacement of 125I-[Tyr4]BBN from PC-3 cells as a function of increasing concentration of BBN analogs. The IC50 values were determined to be 78.96±2.12 nM for BBN, 85.45±1.95 nM for PEG3-Glu-RGD-BBN, and 73.28±1.57 nM for FB-PEG3-Glu-RGD-BBN on PC-3 cells (
The cell uptake of 18F-FB-PEG3-Glu-RGD-BBN was evaluated in PC-3 tumor cells that express high GRPR and moderate integrin levels.
The cell uptake of 18F-FB-PEG3-Glu-RGD-BBN was significantly increased when incubated at 37° C. due to both cell-surface receptor binding and receptor mediated internalization. As shown in the
Representative coronal microPET images of PC-3 tumor-bearing mice (n=4) at different times after intravenous injection of 3.7 MBq (100 μCi) of 18F-FB-PEG3-Glu-RGD-BBN are shown in
Quantification of tumor and major organ activity accumulation in microPET scans was realized by measuring the regions of interest (ROIs) that encompassing the entire organ on the coronal images. The tumor uptake of 18F-FB-PEG3-Glu-RGD-BBN was determined to be 6.35±2.52, 4.41±0.71, and 2.47±0.81% ID/g at 30, 60, and 120 min. The liver uptake was very low, the highest of which is less than 2% ID/g at 30 min post-injection (
The integrin and GRPR dual-receptor binding specificity of 18F-FB-PEG3-Glu-RGD-BBN in vivo was conformed by several blocking studies (
The tumor targeting efficacy of 18F-FB-PEG3-Glu-RGD-BBN in PC-3 tumor-bearing nude mice was also evaluated by 30 min dynamic microPET scanning followed by 5-min static scans at 1 h and 2 h postinjection. As shown in
To validate the accuracy of microPET quantification, a biodistribution study was performed in nude mice bearing PC-3 tumors. Each mouse was injected with 0.74 MBq (20 μCi) of 18F-FB-PEG3-Glu-RGD-BBN and then sacrificed at 1 h p.i. (n=4). As shown in
Quantitative data are expressed as mean±SD. Means were compared using 1-way ANOVA and the Student t test. P values of <0.05 were considered statistically significant.
The above discussion is meant to be illustrative of the principles and various embodiments of the present disclosure. Numerous variations and modifications will become apparent to those skilled in the art once the above disclosure is fully appreciated. It is intended that the following claims be interpreted to embrace all such variations and modifications.
This application claims priority to U.S. Provisional Patent Application Ser. No. 61/075,359, entitled “RADIOLABELED BBN-RGD HETERODIMERS FOR CANCER TARGETING” filed on Jun. 25, 2008, the entirety of which is hereby incorporated by reference.
This disclosure was made with government support under National Cancer Institute Grant Nos: R01 CA119053, R21 CA121842, R21 CA102123, P50 CA114747, U54 CA119367, AND R24 CA93862, awarded by the U.S. National Institutes of Health of the United States government, and grant nos: W81XWH-07-1-0374, W81XWH-04-1-0697, W81XWH-06-1-0665, W81XWH-06-1-0042, AND DAMD17-03-1-0143, awarded by the U.S. Department of Defense. The government has certain rights in the disclosure.
| Number | Date | Country | |
|---|---|---|---|
| 61075359 | Jun 2008 | US |
