**1. Introduction**

The use of biologically active molecules such as peptides and antibodies continues to increase for both diagnosis and therapy [1–3]. Peptides are attractive platforms for diagnostics due to their ability to achieve high target binding affinity and in part due to their small size which results in short biological half-life and rapid clearance from nontarget tissues, producing good target-to-non-target contrast, low toxicity, and generally low or absent immunogenicity [1]. Synthetic advantages of peptides include simple preparation and easy, flexible functionalization or chemical modification to further improve affinity, stability, selectivity, and overall pharmacokinetic properties [1,4]. However, some of the properties that are desirable for a diagnostic agent can hamper the translation to a therapeutic, which relies on a prolonged circulation for high and persistent uptake in the targeted tissue. Too rapid clearance can render the therapeutic ineffective, and poor clearance from non-target tissue can lead to off-target toxicity. Thus, peptides typically

**Citation:** Davis, R.A.; Hausner, S.H.; Harris, R.; Sutcliffe, J.L. A Comparison of Evans Blue and 4-(*p*-Iodophenyl)butyryl Albumin Binding Moieties on an Integrin αvβ<sup>6</sup> Binding Peptide. *Pharmaceutics* **2022**, *14*, 745. https://doi.org/ 10.3390/pharmaceutics14040745

Academic Editor: Katona Gábor

Received: 1 March 2022 Accepted: 25 March 2022 Published: 30 March 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

require fine-tuning for therapeutic applications to balance circulation time and provide high target accumulation with sufficient clearance from non-target tissues [5–7].

Chemical modifications of peptides offer a route to improving these pharmacokinetic properties; this includes incorporation of polyethylene glycol (PEG; PEGylation), glycosylation, or the formation of protein conjugates (e.g., with serum albumin) [4,8–11]. PEGylation is a convenient approach as PEGs are commercially available in a variety of molecular sizes, including mono-disperse PEGs with various functional groups for synthetic orthogonality [1,9]. PEGylation increases hydrophilicity (reducing kidney, lung, and liver accumulation) [12,13], provides increased stability (by protection from proteases), and reduces immunogenicity (by masking the peptide) [9,13]. The size and placement of the PEG on the peptides can significantly affect the pharmacokinetics and tumor accumulation [12–14]. Stability, circulation time, and tumor uptake of peptides can also be increased by chemical ligation ex vivo to serum albumin (taking advantage of albumin's size, long circulation time, and renal recycling) [8,15–17]. Alternatively, the same benefit can be achieved by direct attachment of a small albumin binding moiety (ABM) onto the peptide without substantially increasing the size. The ABM binds reversibly to albumin in the blood, thereby increasing circulation time and facilitating renal recycling, which, in turn, increases target tissue accumulation [8,15,16,18]. Several ABMs have been employed to modify pharmaceuticals currently used in the clinic, with some being used on their own, primarily for measuring plasma volume [16]; among the first ABMs used to modify pharmaceuticals were long-chain fatty acids, such as myristic and palmitic acid [5], and later other lipophilic molecules including benoxaprofen, phenytoin, ibuprofen, and naproxen [16].

More recently, two ABMs in particular, a fragment of Evans blue (EB) dye and the 4-(*p*-iodophenyl)butytryl (IP) group, have also been used to modify the pharmacokinetic profile of radiopharmaceuticals, in particular small molecules (folic acid and prostate specific membrane antigen (PSMA) agents) and peptides (octreotide, exendin-4, and cRGDfK) [10,11,16,18–20]. The EB-ABM was derived from Evans blue dye, a dye which has been used clinically for over 90 years to measure plasma volume and determine blood-brain barrier integrity [16,17,21]. The EB-ABM fragment was first used in 2004 as an MRI contrast agent for imaging blood vessels [22] and has since been used for a variety of applications, including determining blood volume, vascular permeability, and as a conjugate to enhance receptor targeting agents (small molecules and peptides) for both cancer imaging and therapy [9,17,23–26]. The IP-ABM has also been studied extensively to enhance radiopharmaceuticals (small molecules and peptides), where the group at the *para*-position of the aromatic ring of the IP-ABM can be tuned to adjust serum albumin affinity [15,27,28], and a neighboring aspartate residue (D) has been shown to provide a more sustained tumor retention [29]. Numerous preclinical studies have evaluated both ABMs and noted prolonged blood circulation, with an increase in tumor uptake that can also lead to a reduction of kidney accumulation [7,11,18].

The Sutcliffe laboratory has spent over a decade developing and optimizing an integrin αvβ6-binding peptide (αvβ6-BP) [30] to selectively target integrin αvβ6, an epithelium cell surface receptor that is absent or expressed in low levels in healthy adult epithelia, but is highly expressed in numerous challenging cancers, where it is associated with angiogenesis, proliferation, invasion, metastasis, and chemoresistance [31–41]. Thus, the integrin αvβ<sup>6</sup> has been recognized as negative prognostic indicator with the expression levels correlating to poor prognosis and overall survival in many cancers [31–41]. During the optimization of the αvβ6-BP, the bi-terminal PEGylation with monodispersed PEG<sup>28</sup> of the 20 amino acid A20FMDV2-peptide (NAVPNLRGDLQVLAQKVART) derived from the integrin αvβ6 targeting foot and mouth disease virus, showed greatly improved integrin αvβ<sup>6</sup> affinity and selectivity, and improved on the peptide's stability and tumor accumulation and retention [14]. Since then, further modifications have been tested in numerous preclinical models with an advancement of the peptide to >10-fold increase in tumor accumulation and the successful translation of the 4-[18F]fluorobenzoyl labeled [18F]αvβ6-BP into the

clinic for PET imaging of a variety of cancers, including pancreatic adenocarcinoma [30]. Further optimization of αvβ6-BP continues towards an integrin αvβ<sup>6</sup> targeted peptide receptor radionuclide therapy (PRRT).

Recently, Hausner et al. described the IP-ABM modified αvβ6-BP radiolabeled using 1,4,7-triazacyclo-nonane-*N*,*N*',*N*"-triacetic acid (NOTA) for aluminum [18F]fluoride chelation, with the goals of improving the biodistributions and simplifying the fluorine-18 radiochemistry [42]. The [18F]AlF NOTA-IP-ABM-αvβ6-BP had increased blood circulation and tumor accumulation that allowed for high-contrast PET imaging at 6 h post-injection (p.i.) [42], and >3.5-fold lower kidney retention than the very early generation [18F]AlF NOTA-A20FMDV2-peptide [43]. Building on these data and to extend the imaging window beyond that of fluorine-18 (t1/2 = 109.7 min), a copper-64 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA) IP-ABM-αvβ6-BP (t1/2 = 12.7 h) was prepared, which again resulted in an increased tumor accumulation that allowed PET imaging up to 72 h p.i. [44].

In the present study, we describe a head-to-head comparison of the αvβ6-BP modified with either EB-ABM or IP-ABM, with the goal to examine if fine tuning of the ABM could further increase tumor accumulation. Copper-64 radiolabeled [64Cu]Cu DOTA-EB-αvβ6-BP ([64Cu]**1**) and [64Cu]Cu DOTA-IP-αvβ6-BP ([64Cu]**2**), along with the non-αvβ6-targeting ABM controls [64Cu]Cu DOTA-EB ([64Cu]**3**) and [64Cu]Cu DOTA-IP ([64Cu]**4**; Figure 1) were synthesized. Peptides [64Cu]**1** and [64Cu]**2** were evaluated in vitro by competitive ELISA, serum stability, albumin binding assays, and cell binding and internalization assays with DX3puroβ6 (αvβ6+), DX3puro (αvβ6−), and BxPC-3 (αvβ6+) cells (against controls [ <sup>64</sup>Cu]**3** and [64Cu]**4**), and in vivo by PET/CT imaging and biodistribution studies in mice bearing BxPC-3 xenograft tumors (4–72 h, p.i., against controls [64Cu]**3** and [64Cu]**4** at 4 h, p.i.).

**Figure 1.** (**A**) Chemical structures of <sup>64</sup>Cu-radiolabeled-ABM-αvβ<sup>6</sup> -BP: [64Cu]Cu DOTA-EB-αvβ<sup>6</sup> -BP and [64Cu]Cu DOTA-IP-αvβ<sup>6</sup> -BP ([64Cu]**1** and [64Cu]**2**). (**B**) Chemical structures of <sup>64</sup>Cu-radiolabeled non-targeting-ABM controls: [64Cu]Cu DOTA-EB and [64Cu]Cu DOTA-IP ([64Cu]**3** and [64Cu]**4**). [αvβ<sup>6</sup> -BP = PEG28-NAVPNLRGDLQVLAQRVART-PEG28-CONH<sup>2</sup> ].

#### **2. Materials and Methods**

#### *2.1. Materials and General Information*

Amino acids *N*-terminally protected with a fluorenylmethyloxycarbonyl (Fmoc) protecting group and acid labile side chain protecting groups (trityl, Pbf, *tert*-butyl, or Boc) were purchased from Novabiochem (MA, USA) or GL Biochem (Shanghai, China). The orthogonally protected lysine with a 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl

(ivDde) sidechain protecting group and an *N*-terminal Fmoc protecting group, Fmoc-Lys(ivDde)-OH was purchased from ChemPep (Wellington, FL, USA) and the reverse ivDde-Lys(Fmoc)-OH was purchased from EMD (MA, USA). The Fmoc-NH-PEG<sup>28</sup> carboxylic acid was purchased from Polypure (Oslo, Norway) and the chelator DOTA-tris(*tert*butyl ester) was purchased from CheMatech (Dijon, France) and Macrocyclics (Plano, TX, USA). The coupling reagent 1-[bis(dimethylamino)methylene]-1*H*-1,2,3-triazolo[4,5 b]pyridinium 3-oxid hexafluorophosphate (HATU) was purchased from GL Biochem, and benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate (PyBOP) was purchased from Novabiochem. Ethylenediaminetetraacetic acid (EDTA), manganese chloride (MnCl2), and Tris were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tween 20 and sodium chloride (NaCl) were purchased from Fisher (Hampton, NH, USA). The non-fat dry milk powder was purchased from Raley's (West Sacramento, CA, USA). Anhydrous *N*,*N*diisopropylethylamine (DIPEA) and hydrazine were purchased from Sigma-Aldrich and used without additional purification. Solvents *N*,*N*-dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetonitrile (ACN), methanol (MeOH), dichloromethane (DCM), ethyl acetate (EtOAc), *n*-hexanes, and pyridine were purchased from EMD or Acros (NJ, USA). Water used was purified with a Millipore Integral 5 Milli-Q water system at 18.2 MΩ/cm resistivity through a 0.22 µm filter. All solid phase couplings were carried out by rotation in a fritted polypropylene reactor. Thin-layered chromatography (TLC) plates (silica gel 60 with 254 nm fluorescent indicator) from EMD were visualized by UV lamp at 254 nm and/or iodine staining (for the synthesis of **6**). Purification of compound **6** was carried out by normal phase flash column chromatography with silica gel (40–63 µm; Silicycle, QC, Canada). Characterization, purity, and stability were assessed by analytical C12-reverse-phase (RP) high-pressure liquid chromatography (HPLC) column (Jupiter Proteo, 250 mm × 4.6 mm × 4 µm; Phenomenex, Torrance, CA, USA). A Semi-preparative C18-RP-column (Proteo-Jupiter, 250 mm × 10 mm × 10 µm; Phenomenex) was used for purification as described in the Supporting Information (Table S3). All RP-HPLC were carried out on a Dionex Ultimate 3000 HPLC system or a Beckman Coulter Gold HPLC with the latter being used for all radio-RP-HPLC analysis. RP-HPLC were monitored by a UV detector at a wavelength of 220 nm; a serially connected gamma detector was used to monitor radioactivity. [64Cu]CuCl<sup>2</sup> was from the University of Wisconsin Medical Physics Department (WIMR Cyclotron Labs, Madison, WI, USA). Tissue culture and cellular assays used Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), bovine serum albumin (BSA), penicillin-streptomycin-glutamine (PSG), puromycin, and phosphate buffered saline (PBS; all: Gibco/Thermo Fisher). The DX3puroβ6 and DX3puro cells were a gift from Dr. John Marshall. The DX3puroβ6 and DX3puro cell lines were maintained in DMEM medium, supplemented with 10% FBS, 1% penicillin-streptomycin-glutamine, and 2 mg/mL-puromycin. The BxPC-3 cells were purchased from American Type Culture collection (ATCC, Manassas, VA, USA) and maintained in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin-glutamine. Cells were kept in a humidified incubator at 37 ◦C under a 5%-carbon dioxide atmosphere. A Wizard 1470 or Wizard<sup>2</sup> 2470 automatic γcounter (Perkin-Elmer, Waltham, MA, USA) was used to measure radioactivity samples. Mass spectrometry analysis was performed at the UC Davis Mass Spectrometry Facility using either a matrix assisted laser desorption ionization time of flight (MALDI- TOF) spectrometer (UltraFlextreme; Bruker, Billerica, MA, USA) in positive ionization mode with a sinapic acid matrix (Sigma-Aldrich), or with electrospray ionization (ESI) using a quadrupole ion-trap mass spectrometer (Orbitrap; ThermoFisher). Nuclear magnetic resonance (NMR) spectra were collected at the UC Davis NMR Facility on an 800 MHz Bruker instrument with the chemical shifts referenced to the residual solvent of deuterium oxide (D2O, HOD 4.79 ppm).

#### *2.2. Synthesis of EB-ABM 8*

The synthesis of the Evans blue fragment (EB-ABM **8**) was based on previously described methods [7,17,45] (Scheme 1). In brief, *o*-tolidine **5** (531 mg, 2.5 mmol; TCI America, Inc., OR, USA) was dissolved in anhydrous pyridine (1 mL) followed by the addition of succinic anhydride (300 mg, 3.0 mmol) in DMF (1 mL) and allowed to react overnight at room temperature. The crude reaction mixture was concentrated under vacuum and purified by silica-gel column chromatography using a four solvent gradient system beginning with EtOAc/n-hexanes (1/1, *v*/*v*) to remove unreacted *o*-tolidine (**5**, yellow band). The solvent was then changed to 100% EtOAc before switching to MeOH/DCM (1/9, *v*/*v*) and gradually ramping to 3/7 (*v*/*v*) to obtain **6** (648 mg, rt = 0.13, 1:1 hexanes:EtOAc) as a white solid in 83% yield. Compound 6 was analyzed by analytical RP-HPLC and ESI mass spectrometry (Figure S17).

**Scheme 1.** (**A**) Synthetic route to modified EB-ABM **8**. (**B**) Radiochemical Synthesis of [64Cu]**1**–**4**. a. Succinic anhydride, DMF, pyridine (1:1), b. NaNO<sup>2</sup> , MeOH, HCl/H2O, 0 ◦C, c. 1-amino-8-napthol-2,4-disulfonic acid, NaHCO<sup>3</sup> , H2O, 0 ◦C, d. [64Cu]CuCl<sup>2</sup> , NH4OAc, 37 ◦C.

Compound **6** (300 mg, 0.96 mmol) was added to a 25 mL round bottom flask with stir bar containing MeOH (7 mL) and water (5 mL). The contents were cooled to 0 ◦C (ice/brine solution) and allowed to stir for 15 min prior to addition of concentrated hydrochloric acid 240 µL (HCl, 12.1 N; EMD). The diazonium formation of **7** was most successful when the addition of sodium nitrite was done in two portions; the first portion of sodium nitrite (NaNO2, 70 mg, 1.01 mmol; Sigma-Aldrich) was allowed to react for 5 min before the addition of the second portion (NaNO2, 70 mg, 1.01 mmol), after which the reaction was stirred an additional 30 min to generate **7** in situ, which was produced in better yields using the methanol co-solvent than water alone [46]. During in situ formation of **7**, sodium bicarbonate (350 mg, 4.17 mmol; EMD) was dissolved in water (4 mL) with 1-amino-8 napthol-2,4-disulfonic acid (377 mg, 1.18 mmol; TCI America, Inc.) in a separate 25 mL round bottom flask and the contents cooled in an ice/brine solution (~20 min). Next, the diazonium **7** reaction mixture (yellow) was cannulated into the 1-amino-8-napthol-2,4-disulfonic acid (brown-purple) solution by drop-wise addition over 20 min while maintaining both solutions at 0 ◦C. Upon complete addition of **7**, the reaction contents were allowed to stir for 3 h at 0 ◦C, and the crude reaction mixture was lyophilized and purified by semi-preparative RP-HPLC, and the collected material lyophilized. The EB-ABM **8** was

afforded as a fluffy purple solid (480 mg, 78%) and was analyzed by analytical RP-HPLC, ESI mass spectrometry, and NMR (Figures S18 and S19).

## *2.3. Synthesis of DOTA-ABM-αvβ6-BPs* **1** *and* **2**

The αvβ6-BP (PEG28-NAVPNLRGDLQVLAQRVART-PEG28) was synthesized on NovaSyn TGR resin (NovaBiochem) and PEGylation was done using monodisperse Fmoc-amino-PEG-propionic acid (Fmoc-PEG28-CO2H; FW = 1544.8 g/mol) as previously described [30] using standard Fmoc-chemistry. After each coupling or deprotection the resin was rinsed with DMF (3×), MeOH (3×), and DMF (3×). The αvβ6-BP-resin was split in equal portions (100 mg, 0.0088 mmol) and further modified at the *N*-terminus for the synthesis of peptides **1** and **2**. The DOTA-EB-αvβ6-BP **1** was generated by first removing the *N*-terminal Fmoc of the αvβ6-BP with 20% piperidine (Sigma-Aldrich) in DMF (2 × 10 min) followed by the addition of Fmoc-Lys(ivDde)-OH (50.6 mg, 0.088 mmol) using HATU (32.3 mg, 0.085 mmol) and DI-PEA (30 µL, 0.172 mmol) in DMF (1 mL) for 2 h. The Fmoc was subsequently removed with 20% piperidine in DMF (2 × 10 min) and DOTA-tris(*tert*-butyl ester) (50.3 mg, 0.088 mmol) was coupled for 2 h to the *N*-terminus with HATU (32.3 mg, 0.085 mmol) and DIPEA (30 µL, 0.172 mmol) in DMF (1 mL). The removal of the ivDde lysine-sidechain protecting group was done with hydrazine (50 µL) in DMF (1 mL, 2 × 30 min) and the resin dried under vacuum. The EB-ABM **8** (60 mg, 0.093 mmol) was then coupled to the ε-amine of the sidechain of the DOTA-lysine on the αvβ6-BP-resin using PyBOP (125 mg, 0.24 mmol) and DIPEA (50 µL, 0.287 mmol) for 6 h to yield DOTA-EB-αvβ6-BP-resin **1** (Figure S4). The DOTA-EB-αvβ6-BP **1** was cleaved off the resin with concomitant removal of the protecting groups using trifluoroacetic acid (TFA, 2 mL; EMD), triisopropylsilane (TIPS, 50 µL; Alfa Aesar, Haverhill, MA, USA) and water (50 µL), concentrated, purified, and characterized by analytical RP-HPLC and MALDI-TOF (Figure S5). The IP-ABM containing-αvβ6-BP **2** was prepared as previously described [42,44] were upon removal the *N*-terminal Fmoc of the αvβ6-BP-resin, a ivDde-Lys(Fmoc)-OH was coupled. Completion of DOTA-IP-αvβ6-BP **2** was done by sequential coupling/deFmocing of (1) Fmoc-Asp(OtBu)-OH, (2) *N*-γ-Fmoc-γ-aminobutyric acid, and (3) 4-(*p*-iodophenyl)butyric acid using HATU and DIPEA for each coupling. Completion of DOTA-IP-αvβ6-BP **2** was achieved by removal of the *N*-terminal ivDde protecting group with 5%-hydrazine in DMF followed by attachment of DOTA-tris(*tert*-butyl ester) [44]. The completed DOTA-IP-αvβ6-BP **2** was cleaved, purified, and analyzed as described above for the DOTA-EB-αvβ6-BP **1** (Figure S8) [44].

#### *2.4. Synthesis of Non-Targeting ABMs* **3** *and* **4**

Using Fmoc-chemistry with Rink AM resin (200 mg, 0.114 mmol; GL Biochem), DOTA-ABM non-targeting compounds **3** and **4** were synthesized by first coupling Fmoc-Lys(ivDde)-OH (196.6 mg, 0.342 mmol) using HATU (123.5 mg, 0.325 mmol) and DIPEA (100 µL, 0.574 mmol) in DMF (1 mL). The Fmoc was removed with 20% (*v*/*v*) piperidine in DMF (1 mL, 2 × 10 min) and DOTA-tris(*tert*-butyl ester) (80 mg, 0.140 mmol) was coupled for 2 h with HATU (50 mg, 0.132 mmol) and DIPEA (50 µL, 0.287 mmol). Following the ivDde protecting group removal with hydrazine (50 µL) in DMF (1 mL, 2 × 30 min), the resin was dried under vacuum and split into equal portions for synthesis of **3** and **4**. For **3**, the EB-ABM **8** (165 mg, 0.256 mmol) was coupled using PyBOP (166.5 mg, 0.32 mmol) and DIPEA (100 µL, 0.574 mmol) for 6 h. The EB-ABM **3** was cleaved off the resin, purified, and characterized by analytical RP-HPLC and MALDI-TOF (Figure S11). IP-ABM **4** was prepared by sequential coupling/deFmocing of (1) Fmoc-Asp(OtBu)-OH (90 mg, 0.219 mmol), (2) *N*-γ-Fmoc-γ-aminobutyric acid (70 mg, 0.215 mmol), and (3) 4-(*p*-iodophenyl)butyric acid (65 mg, 0.224 mmol) using HATU (78 mg, 0.205 mmol) and DIPEA (100 µL, 0.574 mmol) for each coupling. The IP-ABM **4** was then cleaved off the resin, purified, and characterized by analytical RP-HPLC and MALDI-TOF (Figure S14).
