Albumin-Binding Evans Blue Derivatives for Diagnostic Imaging and

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Albumin-Binding Evans Blue Derivatives for Diagnostic Imaging and Production of Long-Acting Therapeutics Orit Jacobson, Dale O. Kiesewetter, and Xiaoyuan Chen Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00487 • Publication Date (Web): 28 Sep 2016 Downloaded from http://pubs.acs.org on September 30, 2016

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Bioconjugate Chemistry

Albumin-Binding Evans Blue Derivatives for Diagnostic Imaging and Production of Long-Acting Therapeutics Orit Jacobson, Dale O. Kiesewetter, Xiaoyuan Chen* Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, MD 20892 *Corresponding author: [email protected]

Abstract One of the major design considerations for a drug is its pharmacokinetics: a drug with short blood half-life is less available at a target organ which in turn dictates treatment with either high or more frequent doses, and increases the likelihood of undesirable side effects. One method to improve drug pharmacokinetics is adding functional chemical groups to the drug molecule that can increase the half-life in the blood, hopefully, without significantly affecting its desired biological activity. Evans Blue (EB) dye reversibly binds to serum albumin with moderate affinity and has a long blood half-life. The binding of EB to albumin has been exploited to quantify protein leakage as an indicator of increased vascular permeability. Design of new chemical entities based on EB structure and coupling them to drugs, enables the usage of albumin as a reversible carrier in the blood and improves drug’s half-life. This review summarizes the recent developments of various EB derivatives for molecular imaging and therapy applications.

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Introduction Drug development is the process of converting a lead candidate into an approved drug through modification of the chemical structure. The process involves the optimization of biological activity at a desired druggable target and achieving appropriate bioavailability, pharmacokinetics, pharmacodynamics and metabolism 1-3. In the development of therapeutics, pharmacokinetics determines dosing schedule. For drugs that have relatively fast pharmacokinetics, administration of large or frequently repeated doses is required to achieve and maintain therapeutic blood concentration, which can, in turn, increase the probability of undesired side effects 1-3. Pharmacokinetics is also important for the development of diagnostic agents, where contrast can be highly dependent on the blood clearance rate. A great deal of research has been devoted to the topic of enhancing blood retention. This review will focus on methods to increase blood half-life of diagnostic and therapeutic drugs via attachment of Evans Blue analogs.

Approaches to extending half-life in blood Various approaches have been used to reduce the rate of clearance of the drugs, and thereby increase the half-life of pharmaceutical compounds in the body. For example, the drug can be directly modified by chemical synthesis to obviate a non-desired clearance process, or by co-administration of another drug that inhibits the clearance pathway. One example is the coadministration of β-lactamase inhibitors with penicillin antibiotics, so the lactam ring is able to reach the penicillin binding proteins, the target of β-lactam antibiotics 4, 5. Reduction of clearance is particularly desired for small peptides, small molecules, and oligonucleotides, as they are cleared rapidly via the renal pathway and are also prone to


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degradation 3, 6-8. One method to reduce the clearance rate that has been effective for some proteinaceous drugs, such as interferon-alpha (IFN-α), is the attachment of polyethylene glycol (PEG) 9, 10. PEGs are commercially available in a variety of molecular sizes that contain functional groups allowing chemical attachment to drug molecules in order to adjust the pharmacokinetics and reduce renal clearance 1. Another notable advantage of the attachment of PEG to a drug or therapeutic protein is that it can mask the agent from the host's immune system to reduce immunogenicity and antigenicity 1, 11. Recent studies have revealed, however, that PEGylation of drugs has lesser known disadvantages, including immunogenicity caused by the development of anti-PEG antibodies, heterogeneity of the PEGylated drugs, and decreased biological activity and bioavailability of the drug 12, 13. Another common method for improving pharmacokinetics is attachment of drugs, either by covalent binding or fusion, to blood components that have long half-life such as albumin or the Fc domain of antibodies. In particular, attachment of drugs to either albumin or the Fc of IgG isotype antibodies was recently shown to be highly beneficial because these proteins are recycled back into the blood by the neonatal Fc receptor (FcRn), further extending the blood half-life of the drug 3, 14-16.

Albumin as universal carrier Albumin is the most abundant protein in human blood and has a crucial role as a carrier protein for various biomolecules and nutrients such as long chain fatty acids and heavy metal ions. The natural role of albumin as carrier protein was harnessed to target drugs to inflamed or malignant tissues or for extending their half-lives 17. The main technologies that use albumin as a carrier are either to combine albumin with lipophilic drugs ex vivo by chemical modification



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methods, or to develop albumin-binding peptides or prodrugs that bind the abundant albumin in the blood immediately after intravenous injection. The albumin binding technology can be designed to either covalently bind the protein or bind by physical interaction. However, physical binding is preferential because it allows for a slow release of the drug 17-21. Covalent binding is traditionally done using the thiol group of cysteine-34 position of albumin because it accounts for approximately 90% of the thiol concentration in blood plasma. One example of a prodrug that reacts with the thiol of albumin’s cysteine-34 is the (6maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO-EMCH). Because the prodrug binds albumin covalently, it was also engineered to have an acid-sensitive linker, which releases the drug in acidic environment such as tumor tissue or intracellular acidic compartments after uptake by target cells 17-19. An additional approach to employ albumin as drug carrier is chemical formulation of albumin and drug as part of drug manufacturing. This method might complicate drug manufacturing, but it is independent of any reaction in vivo and takes advantage of endogenous albumin transport pathways that are discussed below. An example of this approach is the oncologic drug Abraxane®, which is a nanoparticle composed of the drug paclitaxel and albumin. Abraxane’s bioavailability, therapeutic index, and tumor tissue penetration are significantly improved in comparison to paclitaxel alone. Abraxane was approved for the treatment of several types of solid tumors in the USA and is under clinical evaluation of other solid tumors and metastatic lesions 22, 23. A third approach uses natural interaction of long chain fatty acids with albumin as carrier protein. Such interaction was used to improve treatment of diabetes by extending the blood halflife of insulin and the peptide hormone GLP-1-(7-37). The insulin was attached to myristic acid


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(tetradecanoic acid), and GLP-1-(7-37) was conjugated to palmitic acid. In both cases the attachment of fatty acid successfully improved their circulation time from a few minutes to several hours, and in the case of GLP-1-(7-37) it also protected the peptide from degradation 2426

. The FDA approved, magnetic resonance imaging (MRI) contrast agent gadofosveset

trisodium (Ablavar®) also takes advantage of albumin as a carrier and is used for intravascular blood pool measurement in patients with peripheral vascular disease. It consists of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) complex, lipophilic moiety (4diphenylcyclohexyl group) and hydrophilic phosphodiester group that enables it to reversibly bind serum albumin, resulting in significantly longer blood circulation than other gadolinium based contrast agents 27-29. The binding to serum albumin decreases the relaxation time (T1) of water protons resulting in an enhancement in signal intensity of blood and high-resolution angiography 27-30. Other albumin binding molecules have been utilized to modulate pharmacokinetics for cancer therapy purposes 31. Müller et al. reported on the improvement of folate receptor binding tracer/radiotherapy agent by addition of an albumin binding small molecule, identified by screening a chemical library 32. The albumin binding moiety is based on 4-(p-iodophenyl)butyric acid. Subsequent labeling with 177Lu for radiotherapy of tumor-bearing mice gave promising results 32, 33. Application of albumin binding motifs in anti-cancer drugs was shown to have additional therapeutic benefits. As mentioned above, albumin is a macromolecule, and as such it accumulates in the tumor microenvironment due to leaky, abnormal blood vessels as part of the enhanced permeability and retention (EPR) effect. Moreover, it was recently shown that the



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tumor microenvironment over-expresses proteins that bind albumin, such as gp60 receptor and SPARC 34, which further contribute to albumin retention in the tumor vicinity. Hence, employment of albumin as a carrier for anti-cancer drugs not only improves the half-life of these drugs, but also improves the delivery to the tumor and retention therein. Interestingly, the same carrier capabilities of albumin in the circulation provide lymphatic targeting outside the blood circulation. Injection of various albumin binding dyes, vaccines and tracers via non-intravenous routes, such as intra-tumor, subcutaneous, or intramuscular, leads to accumulation of the injected substances in the draining lymph nodes. Such dyes and tracers are currently used as part of lymph node biopsy and during tumor resection to identify and remove draining lymph nodes that might have metastatic disease 35-37. Employment of albumin binding motifs as part of a vaccine was shown to significantly improve nodal delivery of the antigen and significantly enhance antigen specific immune response 38.

Evans Blue Analogs The remainder of this review will focus on Evans blue (EB), an albumin binding dye and its derivatives, and the current and potential applications of these reagents in biomedicine. EB displays reversible binding to serum albumin with IC50 in the µM range; however, as each albumin can bind up to 14 molecules of EB and because of the abundance of albumin in the blood, almost all the injected EB is retained in the blood after intravenous injection 31, 39-42 . This attribute is highly valuable to quantify total plasma volume of test subjects and can also be used as a means to show permeability to macromolecules 41, 43-48. For example, intravenous EB injection into healthy volunteers resulted in plasma concentration that increased to a plateau within 20 min that persisted an additional 40 min; subsequently, the concentration diminished at


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a rate of about 5 % per hour 49. Interestingly, EB concentration in patients with shock were observed to exhibit anomalies (lower concentrations and rapid decreases) during the observation window. Bowler et al. studied EB concentration in patients undergoing a major operation 43 and observed similar anomalous results. This anomaly was unrelated to the severity of the operation or the anesthesia. However, the anomaly was associated with the administration of hyoscinemorphine-atropine mixture to the patients. Subsequent studies in normal subjects showed that morphine and hyoscine both elicited the effect, when injected prior to EB administration. Moreover, after the phase of abnormally rapid elimination, EB concentration became relatively constant 43. If the EB injection preceded administration of morphine or hyoscine by more than 20 min, normal flat concentration curves were observed. The authors concluded that at least two factors can be responsible for this anomaly: unequal distribution of EB in the circulation and abnormally rapid elimination of EB from the circulation. EB has been used in a viability assay, as the EB bound to albumin will enter damaged or non-viable cells, but not healthy cells 42, 50. EB can be used to assess permeability of barriers such as of the blood-brain barrier (BBB), which under normal conditions is not permeable to macromolecules such as albumin 42. Infiltration by EB of the brain following a disruption in the blood brain barrier due to injury, tumor, or stroke has been exploited to delineate these lesions in preclinical research. EB’s low propensity to cross the intact BBB provides the advantage that it will not carry attached toxins into the brain and the disadvantage that it cannot be used as a carrier to deliver drugs to the brain.

The chemical structure of EB is not easily conducive to derivatization. Consequently, derivatization requires the design of intermediates that can be functionalized with drug, dyes, and



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other molecular imaging components. Several EB analogues/derivatives have been reported in the literature 51-55 (Fig. 1).

Maleimide-EB

Evans Blue (EB)

tEB MEB-C3-Mal EB-DTPA

NEB

Chelator-Maleimide-EB (CMEB) Chelator = NOTA (NMEB) Chelator = DOTA (DMEB)

Figure 1. Chemical structures of Evans Blue (EB) and its derivatives. The pink circle represents truncated EB (tEB).

More than a decade ago, Yamamoto et al. reported on a functionalized EB derivative as a T1-weighted MRI contrast agent for imaging of blood vessels 51. The EB analogue described contained a single azo link on the o-tolidine of EB (truncated EB, tEB, Fig. 1); the toluidine amine was conjugated to diethylenetriaminepentaacetic acid and denoted as EB-DTPA for gadolinium(III) complexation (Fig. 1). MRI signal intensity was gradually elevated in regions of endothelium damage, but not in areas with normal, intact endothelium, reflecting the higher concentration of EB-DTPA-Gd in areas of vascular damage 51. These authors noted the initial serum protein binding, but their discussion of the contrast mechanism did not include enhanced


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blood retention. The same research group evaluated EB-DTPA-Gd in vivo in rats after balloon injury to the carotid artery 56. They first injected EB-DTPA-Gd into the rats via jugular vein and took samples from the right and left common carotid arteries at different time points after the injury. The arteries were opened, fixed on glass plate, and imaged using T1-weighted MRI. The carotid arteries with and without endothelial injury were clearly distinguished by the presence and absence of EB-DTPA-Gd accumulation 56. EB-DTPA-Gd signal intensity of the extracted left common carotid artery was high at early time point after the injection (10 min) but decreased over time and after 2 h, the signal was almost the same as that of the right (intact) carotid artery. In vivo, EB-DTPA-Gd showed enhanced T1-weighted MRI signals in the injured left common artery but not the right one. In contrast with the ex vivo evaluation, EB-DTPA-Gd had the highest signal intensity at 2 h after the injection. The authors assumed that these differences may be caused by the infiltration of the contrast agent into the tissue through the injured blood vessel surface. EB-DTPA-Gd images were superior to Gd-DTPA, which did not enhance the MRI signals of either injured or intact artery 56. EB-DTPA-Gd was also injected into apolipoprotein-E-knockout (ApoE–/–) mice that spontaneously develop atherosclerotic plaques 57. While wild-type mice showed no abnormal MRI signals in the aorta, the atherosclerotic aorta of the ApoE–/– mice showed significant enhancement in the T1-weighted MRI 57. Ex vivo Sudan Red staining, a specific stain for atherosclerotic plaques, showed high correlation with enhancements on MRI T1 images, supporting the ability of EB-DTPA-Gd to non-invasively image atherosclerotic plaques in vivo 57

. Following the design of tEB-chelator conjugate, our group developed a positron emission

tomography (PET) tracer for in vivo labeling of serum albumin 52. tEB was conjugated to 1,4,7-



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triazacyclononane-N, N′,N″-triacetic acid (NOTA) to give a new EB analogue, which we named NEB (Fig. 1) 52. The NOTA chelator provides a facile radiolabeling route that allows labeling NEB with different PET isotopes such as 68Ga, 64Cu and Al18F 52, 53, 58, 59. The labeled NEB was initially evaluated in vivo in mice as a blood pool imaging agent under various pathological conditions, including myocardial infarction (MI) and serum leakage from permeable or abnormal blood vessels using inflammation and malignancy models, respectively 52. As mentioned above, upon intravenous injection, the labeled NEB bound the abundant albumin in the blood and remained in circulation for a long time. Using gated imaging protocols, the PET images were analyzed to demonstrate that mice that underwent MI had much lower left ventricular ejection fraction than the control mice (Fig. 2).

Figure 2. (A) Electrocardiography (ECG)-gated transaxial PET images of 8 intervals of 1 cardiac cycle in control versus myocardial infarction (MI) mice, injected with 18F-AlF-NEB. (B) PET quantification of the left ventricular volume. (C) ECG-gated PET analysis of left ventricular


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ejection fraction. *P < 0.05. The image was adapted from J. Nucl. Med.. 2014; 55(7): 1150– 1156. Used with permission.

When injected into mice with a muscular inflammation model, NEB, radiolabeled with [18F]AlF, continuously accumulated over time at the site of inflammation, due to leakage of serum albumin into surrounding interstitial tissues, whereas no apparent changes were detected to the contralateral muscles (Fig. 3) 52. The labeled NEB also accumulated in malignant tumors, reaching a plateau at 4 h post-injection. The accumulation can be explained by both specific retention of albumin in the tumor tissue as well as the EPR effect (Fig. 4).

Figure 3. (A) Hematoxylin and eosin staining of acute inflammatory muscle, 24 h post intramuscular injection of turpentine. (B) Representative transaxial PET images of mice inflammatory lesions at different time points after injection of 18F-AlF-NEB. The inflamed lesions are represented by white arrows. (C) Time–activity curves based on dynamic PET studies



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of control versus inflamed muscle using 18F-AlF-NEB. (D) PET quantification of 18F-AlF-NEB uptake of healthy and inflamed muscles. The image was adapted from J. Nucl. Med.. 2014; 55(7): 1150–1156. Used with permission.

Figure 4. (A) Representative maximum-intensity-projection (MIP) PET images of a mouse bearing a subcutaneous implanted UM22B tumor, injected with 64Cu-NEB. White arrows indicated tumor location. (B) Time activity curve of heart (used for blood radioactivity) and tumor after injection of 64Cu-NEB. The image was adapted from J. Nucl. Med.. 2014; 55(7): 1150–1156. Used with permission.


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We further evaluated Al18F-NEB for visualization and detection of sentinel lymph nodes (SLNs). Subsequent to local injection, Al18F-NEB quickly formed a complex with endogenous albumin within the interstitial fluid and allowed detection of SLNs with high signal to noise ratio (Fig. 5) 53. The accumulation of Al18F-NEB in the SLNs was also verified by co-injection of Al18F-NEB with EB, then excising the lymph nodes and observing the strong blue color of EB 53.

Figure 5. (A) Representative coronal PET images of popliteal lymph nodes (upper row) and sciatic lymph nodes (lower row) in mice injected with 18F-AlF-NEB. Mice had been injected with turpentine oil to induce limb inflammation on the left side. Lymph nodes on inflamed sided are indicated by white arrows. (B) T2-weighted magnetic resonance images of inflamed popliteal lymph node (white arrow). (C) Fusion of PET and 2D X-ray image. The lymph node location is indicated by a white arrow and the injection site represented by a white arrowhead. (D) PET



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analysis of inflamed v. normal popliteal lymph nodes and (E) sciatic lymph nodes at different time points, after injection of 18F-AlF-NEB. *P < 0.05. The image was adapted from Proc. Natl. Acad. Sci. U. S. A. 2015; 112(1):208-13. Used with permission.

These successful demonstrations of enhanced uptake in inflammation and other vascular disruptions encouraged us to evaluate NEB in patients 59. NEB was labeled with 68Ga and evaluated in healthy volunteers to study its normal pharmacokinetics and calculate the radiation effective dose. Similar to our findings in the mice study, upon intravenous injection, 68Ga-NEB was retained in the blood circulation and reached equilibrium within a few minutes. A slow but steady clearance of the radioactivity from the blood was observed, which was mainly caused by the turnover of albumin from blood circulation to the interstitial space and slow dissociation of 68

Ga-NEB from albumin. The mean radiation effective dose was calculated to be 0.0151–0.0159

mSv/MBq which is comparable to the effective dose of the clinical PET gold standard, 18F-FDG 59, 60

68

. Since the liver has a considerable blood supply, it had relatively high accumulation of

Ga-NEB (Fig. 6).


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Figure 6. MIP PET images of health volunteer injected with 68Ga-NEB, at different time points. The image was adapted from J. Nucl. Med. 2015; 56:1609–1614. Used with permission.

Then, 68Ga-NEB was injected intravenously into patients with focal hepatic lesions and subcutaneously into patients with different suspected lymphatic drainage abnormalities 58. As a blood volume imaging agent, 68Ga-NEB imaging clearly differentiated hepatic hemangioma from other benign or malignant focal hepatic lesions, whereas 18F-FDG could not distinguish these (Fig. 7) 59. When 68Ga-NEB was injected subcutaneously into patients, it visualized lymphatic vessels and lymph nodes and provided information on lymphatic disorders that was



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more accurate than the nuclear medicine procedure using 99mTc-sulfur colloid (SC) lymphoscintigraphy 58.

Figure 7. Comparison of transaxial PET images of 68Ga-NEB (left column) and 18F-FDG (middle column) with CT transaxial images (right column) of patients. The patients had different focal hepatic lesions as follows: A- hepatic hemangioma; B - hepatic carcinoma; C neuroendocrine tumor liver metastasis; D - hepatic cysts. Right column represents CT transaxial images. The image was adapted from J. Nucl. Med. 2015; 56:1609–1614. Used with permission.


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Albumin-based therapeutics have a broad potential and there is a need for improved drugalbumin conjugates. In order to approach this goal, our group developed new tEB derivatives which contain a maleimide functional group for conjugation with free thiols (Fig. 1) 54, 55. One of these tEB derivatives, EB-maleimide, was conjugated to anti-diabetic peptide drug exendin-4 (denoted as Abextide) 54. Exendin-4 is a glucagon-like protein-1 receptor (GLP-1R) agonist that induces insulin release and β cell proliferation, while suppressing glucagon secretion, and is approved for the treatment of type 2 diabetes and obesity 54, 61, 62. In mice, intravenous injection of 18F-Abextide showed higher blood circulation and lower renal clearance than [18F]fluorobenzoyl-exendin-4. Abextide was also evaluated as a therapeutic drug in hypoglycemic type-2 diabetic mice. Upon subcutaneous injection, Abextide’s biological half-life was extended from 5 h to 36 h and it sustained normal glycemia 3.6 times longer than the native exendin-4 (Fig. 8) 54. This general technology platform can be applied to other small molecules and biologics for the development of long-acting therapeutics.



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Figure 8. (A) Plasma concentration of exendin-4 versus Abextide over time after subcutaneous injection. (B) Glucose levels of exendin-4 and Abextide over time measured after subcutaneous injection and compared to control. (C) Same as B but addition of cut-off line that was set to 8.35 mM (150 mg/dL) which is consider being an indicator value of the potential for antidiabetic treatment. The image was adapted from Theranostics 2016; 6(2):243-53. Used with permission.

Our next step was to improve this technology into a functionalized “add-on” that transforms any drug into a theranostic agent. To do so, we designed a novel derivative that contains a lysine that was conjugated to a maleimide on the ε-amine in order to provide easy 18 ACS Paragon Plus Environment

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coupling to drug of choice, and to a chelator on the α amine, for easy labeling with radioactive metal isotopes for imaging and/or radiotherapy (CMEB, Fig. 1). As a proof of concept we chose RGD peptide as the targeting biomolecule. This cyclic peptide is a ligand of integrin αvβ3 with high affinity (nM range) that undergoes internalization upon receptor binding; however, its pharmacokinetics are extremely poor resulting in rapid clearance from the blood within a few minutes 63, 64. Previous attempts to improve the pharmacokinetics and binding of RGD, including PEGylation or formulation into liposomes and nanoparticles, were suboptimal, and reduced RGD binding to its target, reduced the accumulation of RGD in αvβ3 expressing tumors, or reduced tumor to background contrast 65-68. CMEB (Fig. 1), prepared with two different chelators, was conjugated with an RGD derivative via the maleimide and denoted as NMEB-RGD and DMEB-RGD, where the chelators are NOTA and DOTA, respectively. NMEB-RGD, was labeled with 64Cu to determine its pharmacokinetics in vivo. 64Cu-NMEB-RGD had 10-fold increase in accumulation of radioactivity in the tumor when compared to 64Cu-RGD and, at 4 h post-injection, it was virtually cleared from the blood and metabolic organs and provided an excellent tumor-tobackground contrast. Moreover, we proved that this higher uptake of 64Cu-NMEB-RGD was mediated by the slower pharmacokinetics, afforded by albumin binding, and resulted in significantly increased radionuclide uptake and better imaging contrast. The high accumulation of 64Cu-NMEB-RGD in the tumor provided enhanced sensitivity, and detected tumors that expressed lower amounts of αvβ3 integrin, which might have been stratified as “αvβ3-negative” otherwise. We further conjugated a DOTA chelator instead of NOTA to allow labeling with the therapeutic radionuclide 90Y. The 90Y-DMEB-RGD was evaluated as a radiotherapeutic agent in



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mouse model of glioblastoma. One injection of 7.4 MBq 90Y-DMEB-RGD resulted in tumor shrinkage, but was not enough to cure the mice; however, when we gave two injections of 7.4 MBq, one when the tumors were palpable and another 14 days later, we eliminated the subcutaneous tumors.

Conclusions Pharmacokinetics of drugs can be modified by including an albumin-binding moiety that prolongs half-life in the blood. The ability of EB derivatives to bind serum albumin has been employed by us and others in the development of novel agents for different imaging modalities. We have already demonstrated effectiveness of EB conjugation in improving contrast with diagnostic imaging agents, improving effective drug half-life of exendin-4, and radiotherapy of RGD targeted tumors. The first EB derivative that was developed in our lab for blood volume measurements, NEB, has been evaluated in a small number of human subjects. The early results suggest that NEB may be helpful for patient pre-selection and therapy response monitoring by quantitative evaluation of tumor vasculature and permeability that often differ in tumor types and between patients. Conjugation of EB derivatives onto a therapeutic small drug-like molecule, bioactive peptide, or an oligonucleotide aptamer would be expected to improve the half-life in the blood, provide slow release, and offer better therapeutic effects. Our “add-on” NMEB is a new prosthetic group that contains reactive groups for easy covalent binding of drugs or targeting agents and, because of the metal chelator, adds theranostic potential. Development of novel theranostic EB derivatives, which contain functional groups for easy coupling to drugs and a


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chelator for imaging/radiotherapy, proved to increase tumor uptake and provide high contrast for diagnostic imaging purposes and increase radiotherapeutic dose delivery. To date, there have been no direct comparison studies of the various chemical entities for albumin binding. The EB derivatives provide a robust enhancement in the blood half-life and lead to increases in tumor to background ratios in models we have tested. This is just a starting point as the ability to create other EB derivatives containing moieties with differential selectivity can be envisioned. We see NMEB as a lead to the design of improved prosthetic groups for albumin-binding theranostic agents. Some of the analogs already prepared are very promising and will be further investigated in pre-clinical and clinical studies.

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Albumin-Binding Evans Blue Derivatives for Diagnostic Imaging and

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