A modular strategy to prepare multivalent inhibitors of prostate-specific membrane antigen (PSMA).

We have developed a modular scaffold for preparing high-affinity, homo-multivalent inhibitors of the prostate-specific membrane antigen (PSMA) for imaging and therapy of prostate cancer (PCa). Our system contains a lysine-based (µ-, e-) dialkyne residue for incorporating a PSMA binding Lys-Glu urea motif exploiting click chemistry and a second lysine residue for subsequent modification with an imaging or therapeutic moiety. The utility of the multivalent scaffold was examined by synthesizing bivalent compounds 2 and 3 and comparing them with the monovalent analog 1. Determination of inhibition constants (Ki) revealed that bivalent 2 (0.2 nM) and 3 (0.08 nM) are significantly more potent (~ 5 fold and ~ 11 fold, respectively) inhibitors of PSMA than monovalent 1 (0.9 nM). A single photon emission computed tomography (SPECT)-CT imaging study of [111In]3 demonstrated high and specific uptake in PSMA+ PC-3 PIP tumor until at least 48 h post-injection, with rapid clearance from non-target tissues, including kidney. A biodistribution study revealed that [111In]3 demonstrated 34.0 ± 7.5 percent injected dose per gram of tissue in PSMA+ tumor at 24 h post-injection and was capable of generating target-to-non-target ratios of ~ 379 in PSMA+ PC-3 PIP tumors vs. isogenic PSMA-negative PC3-flu tumors in vivo. The click chemistry approach affords a convenient strategy toward multivalent PSMA inhibitors of enhanced affinity and superior pharmacokinetics for imaging.


INTRODUCTION
Prostate cancer (PCa) will kill an estimated 33,720 men in the US alone this year [1]. The integral membrane protein prostate-specific membrane antigen (PSMA) is becoming increasingly recognized as a viable target for imaging and therapy of prostate and other forms of cancer [2][3][4]. PSMA is significantly over-expressed in PCa and metastases, particularly with respect to the castrationresistant form [5]. Accordingly, PSMA may provide a negative prognostic indicator for PCa -enabling distinction of indolent from aggressive disease. Imaging PSMA has also provided insight into androgen signaling [6] and information on response to taxane therapy [7].
Recently we and others have demonstrated successful PSMA-targeted radionuclide imaging in experimental models of PCa using cysteine-glutamate or lysine-glutamate ureas. With those agents the radionuclide ( 11 C, 125 I, 18 F) is attached to the cysteine or lysine moiety via a small prosthetic group [8][9][10][11][12]. For large molecular fragments, such as radiometal ( 99m Tc, 68 Ga, 111 In) chelators, organic fluorescent molecules, and nanoparticles, we have determined that a linking moiety of at least 20 Å (long-linker) between the large molecule and the lysine moiety facilitates productive binding [13][14][15]. We have also developed a PSMA-targeted, dual (radionuclide and optical) modality imaging platform that enables sequential, dual modality imaging [16]. As an extension of this program, here we prepare bivalent ligands with a view to improving the affinity and pharmacokinetic properties of the urea class of PSMA inhibitors. The strategy we employ can be generalized to multivalent compounds. Because they present multiple copies of the pharmacophore, multivalent ligands can bind to receptors with high avidity and affinity, thereby serving as powerful inhibitors [17,18]. Various approaches have been reported to exploit multivalent scaffolds for the construction of molecular imaging probes [19][20][21][22]. However, the chemistry used to produce them can become complicated, even more so when a bifunctional chelator must be attached to a separately multimerized construct to introduce a radionuclide, for example, for imaging. Although, the concept of multimerization for PSMA targeted, near-infrared imaging agents has been proffered for in vitro cell binding studies [22], to our knowledge a multivalent PSMA-binding agent has not yet been shown to image PSMA successfully in vivo. Here we use click chemistry [23,24] with our long-linker platform as a convenient route to build a modular scaffold for multimeric presentation of PSMA targeting species and demonstrate the enhanced ability of the bivalent form, over the corresponding monomer, to target PSMA in vivo.

RESULTS AND DISCUSSION
Our modular multivalent scaffold contains a lysinebased (∝-, ε-) dialkyne residue for incorporating PSMA binding Lys-Glu urea moieties exploiting click chemistry [23,24] and a second lysine residue for subsequent modification with an imaging and/or therapeutic nuclide or a cytotoxic ligand for tumor cell kill. The divalent agent was anticipated to have a prolonged biological half-life and enhanced specific binding and retention in tissues expressing PSMA. To evaluate the anticipated multivalent effect, a versatile Lys-Glu-urea-based azide intermediate (1) was prepared to serve as a monovalent control Chart 1: www.impactjournals.com/oncotarget compound (Chart 1) against the bivalent compound 2 and the DOTA-chelated bivalent urea analog, 3 to examine the effect of adding a chelating agent to bivalent urea 2. Compounds 2 and 3 were conveniently prepared by employing simple peptide coupling and click chemistry [23,24] as shown in Scheme 1.
Starting with commercially available Fmoc-Lys(Boc)-Wang resin and using standard Fmoc-based solid phase peptide chemistry, 1 -4 were prepared in suitable yields. In brief, Fmoc-Lys(Boc)-Wang was treated with 20% piperidine/DMF to remove the Fmoc group followed by coupling with commercially available Fmoc-Lys(Fmoc)-OH in the presence of benzotriazol-1yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) and N,N-diisopropylethylamine (DIEA). In the next step, a microwave-assisted coupling reaction was performed using propiolic acid in presence of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) in DMF to improve the yield and purity of the desired product. Finally, 4 was isolated in ~ 40% yield after treating the resin with a cocktail of TFA/H 2 O/TES (95/2.5/2.5) at ambient temperature for 0.5 h. Compound 4, a Lys-based (∝-, ε-) dialkyne peptide, served as the key intermediate to introduce multimerization. Copper catalyzed click chemistry was employed using the azide intermediate 1 and dialkyne peptide 4 to produce multivalent compound 2 in moderate yield after purification by high-performance liquid chromatography (HPLC). Compound 3 was prepared by coupling the free amine of the lysine residue of 2 with the N-hydroxysuccinimide ester of DOTAtris-acid using DIEA as a base in DMSO at ambient temperature for 4 h. Compound 3 was purified by HPLC and obtained in ~15% overall yield. Compound 3 was labeled with 111 In at 95°C in 0.3 M NaOAc buffer within 20 min in ~70 -90% yield and specific radioactivity of ~ 8.4 -204.4 GB/µmol. PSMA inhibition constant (K i ) values for 1 -3 were determined using a fluorescence-based PSMA inhibition assay [8]. The data are presented in Table 1. As revealed from the K i values, the binding affinity was found to increase 5-fold from monovalent 1 to bivalent 2. Interestingly, there was an 11-fold increase to the DOTA-chelated bivalent compound 3 compared to 1 leading to subnanomolar binding affinity for 3. Under the same experimental conditions, the K i value of the known PSMA inhibitor ZJ-43 [25] was 1.16 nM, indicating the Scheme 1: high inhibitory potency of 3. The inhibition curves of 1 -3, which are expressed with respect to the amount of glutamate released from hydrolysis of the natural PSMA substrate, N-acetylaspartylglutamate (NAAG), are shown in Figure 1. A structurally similar triazole version of 1, compound 6 (Chart 1, Table 1) was also tested for PSMA inhibitory activity in vitro in a previous experiment [34]. The K i value of 6 was 0.92 nM in that experiment, in which ZJ-43 demonstrated a K i value of 0.35 (95% CI, 0.2 -0.6 nM), suggesting that the affinity of 6 is likely significantly less than the bivalent compounds 2 or 3. Compound 6 was radiolabeled with 99m Tc and its biological properties were tested in vivo [34]. A manuscript describing those biological data is in preparation. Figure 2 shows the pharmacokinetic behavior of [ 111 In]3 in vivo in SCID mice bearing both PSMA+ PC3-PIP and PSMA-PC3-flu xenografts [26]. We prefer to use the isogenic PSMA+ PIP vs PSMA-flu comparison as the two cell lines are phenotypically identical, differing only in PSMA expression. In this experiment 44.4 MBq (1.2 mCi) of [ 111 In]3 was administered intravenously and the animal was imaged repeatedly over an eight day period. Intense radiotracer uptake was seen only in the PSMA+ PIP tumors and in the kidneys. Kidney uptake of the radiotracer is partially due to its route of excretion    as well as to specific uptake from the expression of PSMA in mouse kidneys [27]. Clearance of radioactivity from kidney and non-target tissues was more rapid than from target tumor such that by 48 h post-injection (p.i.) a high tumor/background ratio was observed ( Figure 2). Significantly, PSMA+ tumor was possible to image out to eight days p.i. To validate the in vivo imaging data, [ 111 In]3 was also assessed for its pharmacokinetics ex vivo. Table  2 [10,13,14,16,22,[28][29][30][31] [15], which has recently been administered to human subjects [30]. We also compared the in vivo properties of the bivalent compound [ 111 In]3, with that of one of our lead DOTA-chelated monovalent compounds, [ 111 In]5 ( Figure 3 and Table 3). The synthesis and characterization of 5 [32] 5 in tumors reflects the advantages of the multimeric design of the former, which affords improved retention in vivo in addition to the anticipated multivalent effects on target binding affinity. One explanation for those results could be that the binding of one PSMA-targeting moiety would significantly enhance the local concentration of the other PSMA-targeting moiety of the homodimer in the vicinity of the active site of PSMA, which may lead to a faster rate of receptor binding or a slower rate of dissociation   and translate into higher uptake and longer retention time in the tumor. The apparent increase in molecular size may also prolong circulation time of the dimer and consequently reduce the tumor washout rate.
The technique described is able to be generalized to other modalities and for molecular radiotherapy. Since DOTA is a general chelating agent, 3 may also be used with other radiometals such as 68 Ga, 64 Cu or 86 Y for positron emission tomography (PET) or 90 Y and 177 Lu for therapy. Technetium-99m can also be incorporated by replacing DOTA with standard peptide-based chelating agents containing nitrogen and sulfur donors (N 3 S, N 2 S 2 ), the HYNIC chelator or by use of single amino acid chelating (SAAC) agents [33]. Further attesting to its utility, bivalent 2 can also be used as a versatile intermediate for medically important nonmetals, such as the radiohalogenated imaging isotopes 18 F, 123 I or 211 At/ 131 I for radiotherapy. Other fluorophores/chelating agents/ radiometals/nonmetals/cytotoxic agent combinations can also be envisioned using this approach. Another significant aspect of the multivalent scaffold is that it will enable us to generate systematically at least 4-and 8-valent urea compounds from the lysine-diamine intermediate 4 upon repeated conjugation of 4 with Fmoc-Lys(Fmoc-OH) to produce a lysine-based multimeric urea dendron.

,22-tetracarboxylic acid, Compound 1
This compound was prepared following our previous report [34]. Briefly, commercially available Boc-Lys(Azide)-OH was treated with a solution of TFA/ CH 2 Cl 2 (1:1) at ambient temperature for 4h to remove the Boc group. After solvent removal, the crude product, H-Lys(azide)-OH, was directly used for the next step. To a solution of H-Lys(azide)-OH (50 mg, 0.29 mmol in 500 µL DMSO) was added NHS-ester of Lys-Glu urea (24 mg, 0.43 mmol in 500 µL DMSO) [16] and DIEA (100 µL) and left at ambient temperature for 4 h. Solvent was evaporated to dryness and the residue was dissolved in water and purified by HPLC (Method 1). Retention time

PSMA Inhibition
The PSMA inhibitory activities of 1 -3 and [ 113/115 In]3 were determined using a fluorescence-based assay according to a previously reported procedure [8]. Briefly, lysates of LNCaP cell extracts (25 µL) were incubated with the inhibitor (12.5 µL) in the presence of 4 µM NAAG (12.5 µL) for 120 min. The amount of glutamate released upon hydrolysis of NAAG was measured by incubating with a working solution (50 µL) of the Amplex Red Glutamic Acid Kit (Life Technologies, Grand Island, NY) for 60 min. Fluorescence was measured with a VICTOR3V multilabel plate reader (Perkin Elmer Inc., Waltham, MA) with excitation at 530 nm and emission at 560 nm. Inhibition curves were determined using semi-log plots and IC 50 values were determined at the concentration at which enzyme activity was inhibited by 50%. Assays were performed in triplicate. Enzyme inhibitory constants (K i values) were generated using the Cheng-Prusoff conversion [36]. Data analysis was performed using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA).

Cell Culture and Animal Models
Sublines of the androgen independent PC-3 human prostate cancer xenograft originally derived from an advanced androgen independent bone metastasis were used. Those sublines have been modified to express high (PC-3 PIP) and low (PC-3 flu) PSMA levels and were generously provided by Dr. Warren Heston (Cleveland Clinic). Both PSMA-expressing (PC-3 PIP) and nonexpressing (PC-3 flu) prostate cancer cell lines were grown in RPMI 1640 medium (Mediatech Inc., Manassas, VA) containing 10% fetal bovine serum (FBS) (Sigma Aldrich, St.Louis, MO) and 1% Pen-Strep (Mediatech Inc., Manassas, VA) as previously described [12]. All cell cultures were maintained at 5% carbon dioxide (CO 2 ), at 37°C in a humidified incubator. Animal studies were carried out in full compliance with the regulations of the Johns Hopkins Animal Care and Use Committee. Six-to eight-week-old male, non-obese diabetic (NOD)/ severe-combined immunodeficient (SCID) mice (Johns Hopkins Animal Core, Baltimore, MD) were implanted subcutaneously (s.c.) with PC-3 PIP (PSMA+) and PC-3 flu (PSMA-) cells (2 x 10 6 in 100 µL of Matrigel) at the forward right and left flanks, respectively. Mice were imaged or used in biodistribution assays when the xenografts reached 5 to 7 mm in diameter.

Gamma Scintigraphy and SPECT/CT
Compound [ 111 In]3 was imaged using male SCID mice. Xenograft models were generated as described above. Mice were anesthetized using 1% isoflurane in oxygen flowing at 0.6 L/min prior to and during radiochemical injection. Mice were injected via the tail vein with approximately 1.2 mCi (44.4 MBq) of [ 111 In]3 formulated in 100 μL of saline, pH 7. After allowing for 30 -60 min of radiochemical uptake, anesthetized mice were placed on the scanner gantry and secured with medical tape while the anesthetic flow was increased to 0.8 L/min. The body temperature of the mice was maintained by covering them with several layers of absorbent, disposable pads and illumination with a dissection lamp during scanning. Single-pinhole median-energy (PHME) collimators with an aperture size of 1.0 mm, and stepwise rotation for 64 projection angles in a full 360° rotation, 40 s increments were used for SPECT imaging. The radius of rotation (ROR) was set at 7 cm, which provided a field of view of 7.5 cm to cover the mouse body from head to bladder. A CT scan was performed prior to scintigraphy for both anatomical co-registration and attenuation correction. A total of 512 projections were acquired in a 2 min continuous rotation mode covering a full 360° rotation. Data were reconstructed and fused using commercial software from the vendor (Gamma Medica-Ideas, Northridge, CA), which includes a 2D-OSEM algorithm. Data were analyzed and volume-rendered images were generated using AMIDE software (SourceForge, http:// amide.sourceforge.net/). Biodistribution PSMA+ PC-3 PIP and PSMA-PC-3 flu xenograftbearing SCID mice were injected via the tail vein with 0.74 MBq (20 μCi) of [ 111 In]3. Four mice were sacrificed by cervical dislocation at 2 and 24 h p.i. The heart, lungs, liver, stomach, pancreas, spleen, fat, kidney, muscle, small and large intestines, urinary bladder, and PC-3 PIP and flu tumors were quickly removed. A 0.1 mL sample of blood was also collected. Each organ was weighed, and the tissue radioactivity was measured with an automated gamma counter (1282 Compugamma CS, Pharmacia/ LKB Nuclear, Inc., Gaithersburg, MD). The percentage injected dose per gram of tissue (%ID/g) was calculated by comparison with a standard dilution of the initial dose. All measurements were corrected for radioactive decay.