Advancing Cancer Theranostics Through Integrin αVβ3-Targeted Peptidomimetic IAC: From Bench to Bedside

Abstract

Introduction:

The expression of alpha-five beta-three (αVβ3) integrins is upregulated in various malignancies undergoing angiogenesis. The development of integrin antagonists as diagnostic probes makes the αVβ3 integrin a suitable candidate for targeting tumor angiogenesis. The goal of this study was to optimize the radiolabeling and evaluate the potential of conjugated integrin antagonist carbamate (IAC), a peptidomimetic, as a theranostic radiopharmaceutical for targeting tumor angiogenesis.

Methodology:

Radiolabeling of DOTAGA [2,2′,2”–{10-(2,6-dioxotetrahydro-2H-pyran-3-yl)−1,4,7,10-tetraazacyclododecane-1,4,7-triyl} triacetic-acid]-IAC with [⁶⁸Ga]Ga, [¹⁷⁷Lu]Lu, and [²²⁵Ac]Ac was optimized. The binding affinity (K_d) of DOTAGA-IAC for the αVβ3 receptor and cancer cell lines was quantified. The biodistribution studies were conducted in healthy Wistar rats. Dosimetry analysis was performed on [¹⁷⁷Lu]Lu-DOTAGA-IAC distribution data. A pilot study of [⁶⁸Ga]Ga-DOTAGA-IAC and [¹⁸F]FDG Positron Emission Tomography (PET/CT) imaging was performed in five patients with histopathologically confirmed breast cancer. PET/CT findings were compared between [⁶⁸Ga]Ga-DOTAGA-IAC and [¹⁸F]FDG in these patients.

Results:

Radiopharmaceuticals were prepared with high radiochemical purity (>99.9%). K_d and B_max measurements were 15.02 nM and 417 fmol for αVβ3 receptor protein: 115.7 nM and 295.3 fmol for C6 glioma cells. Biodistribution studies in rats suggested the excretion via kidneys and partially through the hepatobiliary route. The effective dose of [¹⁷⁷Lu]Lu-DOTAGA-IAC was found to be 0.17 mSv/MBq. The dynamic study in patients revealed the optimal imaging time to be 30–35 mins postadministration. Out of the cohort, [⁶⁸Ga]Ga-DOTAGA-IAC detected the primary lesions in all five patients with a mean standard uptake value (SUV_max) of 3.94 ± 0.58 compared with [¹⁸F]FDG (SUV_max 13.8 ± 6.53).

Conclusion:

The study demonstrates that DOTAGA-IAC exhibits strong binding to αVβ3 integrin, positioning it as a promising PET agent for assessing primary and metastatic cancers. The outcomes from the pilot study suggest the potential of [⁶⁸Ga]Ga-DOTAGA-IAC PET/CT in breast carcinoma diagnosis. While recognizing the theranostic potential of DOTAGA-IAC for αVβ3 integrin-expressing tumors, further clinical investigations are warranted to comprehensively assess therapeutic efficacy.

Introduction

Angiogenesis plays a pivotal role not only in physiological processes but also in tumor growth, invasion, and metastasis. Tumor expansion beyond 2 mm³ results in heightened interstitial pressure, leading to reduced nutrient and metabolite supply that results in halting tumor growth.¹ This hypoxic state triggers the angiogenic switch by upregulating proangiogenic proteins like vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and tumor necrotic factor-α (TNF-α). Subsequent steps involve the removal of pericytes and degradation of the extracellular matrix leading to angiogenesis. Mesenchymal stem cells differentiate into pericytes, conferring stability to the newly formed blood vessels and promote tumor growth.^2,3

Various classes of cell adhesion molecules (CAMs) such as integrins, cadherins, selectins, immunoglobulins, vitronectin, fibronectin, and tenascin play a key role in tumor angiogenesis.⁴ Among CAMs, αVβ3 integrins are key promoters of tumor angiogenesis, binding to various extracellular matrix proteins, immunoglobulins, and growth factor receptors (insulin receptor, PDGF, and VEGF) via the recognition of the short peptide sequence Arg-Gly-Asp (RGD).^5,6 Overexpression of αVβ3 integrin has been noted in breast cancer, glioblastoma, pancreatic tumor, prostate carcinoma, and cervical cancer.^7

–12 The nonexpression in normal or nonproliferating cells makes αVβ3 integrin an attractive therapeutic target.¹³

Various RGD-based ligands such as [¹⁸F]FGalacto-RGD, [¹⁸F]F-AH111585, [⁶⁸Ga]Ga-NOTA-RGD, cyclic pentapeptide (RGDfV), and [⁶⁸Ga]Ga-DOTA-RGD₂ have been explored for their utility in targeting angiogenesis.^14

–18 These RGD ligands show promise in diagnosing αVβ3 integrin-expressing tumors. However, their peptide nature may limit their therapeutic potential due to susceptibility to chemical and proteolytic degradation, leading to short physiological half-lives.

To address this limitation, peptidomimetics have gained attention due to their higher receptor affinity, enhanced chemical and proteolytic degradation resistance, and longer physiological half-lives.^6,19 Integrin Antagonist Carbamate Derivative (IAC) is a promising peptidomimetic agent with high affinity for αVβ3 integrin, indicating its potential in detecting αVβ3 integrin-expressing tumors.^20

–23 However, the therapeutic potential of previously developed 1,4,7-triazacyclononane-N-glutamic acid-N’,N”-diacetic acid (NODAGA)-IAC is limited due to the absence of a suitable bifunctional chelator for Lutetium-177 (¹⁷⁷Lu) and Actinium-225 (²²⁵Ac).²⁴

This study explored the theranostic potential of IAC conjugated with DOTAGA [2,2′,2”–{10-(2,6-dioxotetrahydro-2H-pyran-3-yl)−1,4,7,10-tetraazacyclododecane-1,4,7-triyl} triacetic-acid].

The DOTAGA-IAC radiolabeling was optimized and characterized with ⁶⁸Ga, ¹⁷⁷Lu, and ²²⁵Ac followed by their quality control assessment. The biodistribution and dosimetry study was performed for [¹⁷⁷Lu]Lu–DOTAGA-IAC. Whereas, with [⁶⁸Ga]Ga-DOTAGA-IAC, along with in vitro receptor binding assays and preclinical biodistribution study, a pilot study was conducted in patients with breast cancer to explore its diagnostic potential.

Materials and Methods

Radiolabeling with [⁶⁸Ga]GaCl₃

The radiolabeling was optimized for the ligand concentration, reaction pH, temperature, and incubation time. Reactions 1–13 (Supplementary Table S1) were performed using [⁶⁸Ga]GaCl₃ (370 MBq) eluted from a commercially available ⁶⁸Ge/⁶⁸Ga generator (ITM Germany) using 0.05 M HCl and incubated with DOTAGA-IAC (2–20 µg) at 95°C (or room temperature) for 10–25 min. The reaction pH was maintained between 3.5 and 7.5 using 0.25 M sodium acetate buffer. The radiochemical yield (RCY) and radiochemical purity (RCP) were determined through instant thin-layer chromatography (radio-ITLC). Purification was performed using a preconditioned C-18 cartridge, as detailed in section purification by solid-phase extraction. To verify the absence of [⁶⁸Ga]Ga-DOTAGA in the final product, DOTAGA was also radiolabeled with [⁶⁸Ga]GaCl₃. To differentiate between [⁶⁸Ga]GaCl₃, [⁶⁸Ga]Ga-DOTAGA, and [⁶⁸Ga]Ga-DOTAGA-IAC, various stationary and mobile phases were assessed for radio-ITLC. The corresponding retention factors (R _f ) were recorded (Supplementary Table S2). For, radio-ITLC assessment, a 100 mm TLC pate was used and the origin front and solvent front were marked at 10 and 90 mm, respectively.

To enhance readability and avoid repetition in the following sections, DOTAGA-IAC will be referred to as simply IAC.

Radiolabeling with high specific activity (SA) [¹⁷⁷Lu]LuCl₃

The high SA [¹⁷⁷Lu]LuCl₃ (SA: 3388 MBq/µg) was sourced from ITM Germany. IAC (5 µg) in a 0.5 M sodium ascorbate buffer was incubated with 370 MBq of [¹⁷⁷Lu]LuCl₃ at 100°C for 30 min. As a radio stabilizer, ascorbic acid was added to the buffer solution. The final reaction pH was 4.5. Radiochemical yield and purity were determined by radio-ITLC with sodium citrate (0.5 M, pH 3.0) as the mobile phase and Instant thin layer chromatography with silica gel (ITLC-SG) as the stationary phase. In addition, acetonitrile: water (1:1, v/v) and Whatman No. 3 paper were used as another combination of mobile and stationary phases.

Radiolabeling with low SA [¹⁷⁷Lu]LuCl₃

The low SA [¹⁷⁷Lu]LuCl₃ (SA: 740 MBq/µg) was procured from BRIT, India. DOTAGA-IAC (10 µg) in 0.5 M sodium ascorbate buffer was incubated with 370 MBq of [¹⁷⁷Lu]LuCl₃ at 100°C for 30 min. Gentisic acid (0.25 M) was added to the buffer solution as a radio stabilizer. The final reaction pH was 4.5. The radiochemical yield and purity were assessed using the same method, as described in the previous section.

Radiolabeling with [²²⁵Ac] AcCl₃

The [²²⁵Ac]AcCl₃ was procured from ITM Germany. IAC (20 µg) in 1 M sodium ascorbate buffer (pH 5.0) was incubated with [²²⁵Ac]AcCl₃ (481–999 KBq) at 90°C for 30 min, with final reaction pH toward 5.0. The radiochemical yield and purity were assessed by radio-ITLC. The combination of mobile and stationary phases included sodium citrate and ITLC-SG, as well as acetonitrile: water (1:1, v/v) and Whatman paper 3.

Purification by solid-phase extraction

Purification was performed using sep-pak C-18 cartridges. To condition the cartridge, 70% ethanol (5 mL) was passed through at 1–2 mL/min for activation. Followed by a 10 mL water wash at the same rate for equilibration. The radiolabeled mixture was then passed through the cartridge, trapping radiolabeled IAC via hydrophobic interaction. The product eluted with 50% ethanol (1 mL) at a flow rate of 1 mL/min.

HPLC characterization

⁶⁸Ga and ¹⁷⁷Lu labeled IAC were identified via HPLC analysis with nonradioactive surrogates, ⁶⁹Ga and ¹⁷⁵Lu. The analysis was done on a Shimadzu LC-2030 system equipped with a Photodiode Array (PDA) detector and a Purosphere STAR RP18 end-capped (5.0 µm) RT 125-4 column (Darmstadt, Germany), operating at a flow rate of 1 mL/min. PDA detection ranged from 190 to 800 nm. Before HPLC analysis, nat[⁶⁹Ga]Ga-IAC and nat[¹⁷⁵Lu]Lu-IAC samples were purified using a C-18 cartridge to remove free metals and buffer solutions, followed by elution with 50% ethanol (Supplementary Table S3).

Quality control

Radionuclide purity was evaluated using a thallium-doped sodium iodide [NaI (Tl)]-based γ-ray spectrometer. Characteristic gamma photo peaks were observed for each radiometal: ⁶⁸Ga (511 and 1022 KeV), ¹⁷⁷Lu (113 and 208 KeV), and ²²⁵Ac (78, 213, and 470 KeV). The Endpoint Chromogenic Limulus Amebocyte Lysate test was conducted to determine endotoxin levels in accordance with the U.S. Pharmacopeia (USP), Chapter 85 guidelines. Sterility testing was performed following the procedures specified in USP chapter 71, using two growth media: fluid thioglycollate medium (FTM) and tryptic soy broth (TSB). The shelf life and stabilities of [⁶⁸Ga]Ga-IAC, [¹⁷⁷Lu]Lu-IAC, and [²²⁵Ac]Ac-IAC were assessed over different time points in normal saline at room temperature and in human serum at 37°C.

Determination of partition coefficient (log P)

The log p-values of [⁶⁸Ga]Ga-IAC and [¹⁷⁷Lu]Lu-IAC were determined using the n-octanol/saline model. Equal volumes of the radioactive drug solution and n-octanol solution were mixed vigorously for 10 min, followed by centrifugation at 1509 Relative Centrifugal Force (RCF) for 10 min to separate the phases. Radioactivity in each fraction was measured, and log p-values were calculated using the following formula: $Log Po / w = Log (Radioactivity in Octanol / Radioactivity in Water)$

Plasma protein binding in human blood

To assess plasma protein binding, we used the trichloroacetic acid (TCA) precipitation method. Briefly, 0.1 mL of each radiocomplex was mixed with 0.9 mL of plasma, incubated at 37°C for an hour, treated with 10% TCA for 2–3 min, and then centrifuged at 671 RCF for 5 min. The radioactivity in the pellet and supernatant was measured to calculate % Plasma Protein Binding (PPB). $% PPB = Radioactivity in Pellet / Total Radioactivity$

In vitro radioligand binding study

The binding experiment was performed in MCF-7 (human breast cancer cells), Hep-G2 (human liver cancer cells), and C6 rat glioma cells. The cell lines were procured from the National Center for Cell Sciences, Pune, India. The cells were cultured in RPMI-1640 and Dulbecco's Modified Eagle Medium (DMEM) media supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL) antibiotic solutions in a 5% CO₂ environment at 37°C. Subsequently, 24-well plates were seeded, and the saturation binding experiment was performed to determine the binding affinity (K_d) and the number of binding sites (B_max) of [⁶⁸Ga]Ga-IAC for αVβ3 integrin receptor. See Supplementary Section 1.0. for a detailed experiment design. The saturation binding experiment was also conducted with recombinant human αVβ3 integrin receptor protein purchased from RD (CAT: 3050-AV). A 96-well ELISA plate was coated with the receptor (2.5 µg/mL) in 50 mM carbonate/bicarbonate buffer (pH 9.5) overnight at 4°C. The subsequent steps were the same as followed for the cell lines. The data were processed using Prism software to determine K_d and B_max.

Biodistribution in healthy Wistar rats

Animal biodistribution studies were conducted with prior ethical clearance from the Institutional Animal Ethics Committee (IAEC-724/IAEC/107/105) of PGIMER, Chandigarh, India. Eighteen healthy female Wistar rats, 16 weeks old, weighing 286 ± 21 g, received intravenous injections of [⁶⁸Ga]Ga-IAC (7.55 ± 1.3 MBq) and [¹⁷⁷Lu]Lu-IAC (4.07 ± 0.1 MBq) via the lateral tail vein. All rats were anesthetized with a mixture of ketamine (40 mg/kg) and xylazine (4 mg/kg) and euthanized through cervical dislocation. For [⁶⁸Ga]Ga-IAC, groups of rats (n = 3 at each time point) were sacrificed at 0.5, 1, and 2 h postinjection. As for [¹⁷⁷Lu]Lu-IAC, rats were sacrificed at 1, 24, and 48 h postinjection. The counts in each organ were recorded using a NaI (Tl)-based gamma-ray spectrometer (CAPTUS 4000). The percentage of injected dose per gram of tissue (%ID/g) was determined using the following formula: $ID / g of tissue = \frac{Counts i n tissue}{Injected Activity Counts \times Weight o f tissue} \times 100$ Where: $Injected activity counts = Total counts - (Counts at Injection site + Residual counts)$

Dosimetry for [¹⁷⁷Lu]Lu-IAC Preclinical Biodistribution Data

The radiation dose estimates of [177Lu]Lu-IAC were calculated for human organs from the animal distribution data. As the animal data were presented in concentration (% injected dose/g of tissue), extrapolation to humans was performed using a mass-based extrapolation. In the mass extrapolation method, the animal concentration was normalized by the ratio of the total body weight of animals and humans.²⁵ Extrapolated data were fit using the SAAM II software.²⁶ Time integrals of activity were entered into the OLINDA/EXM software to compute organ effective dose, using the adult male model.^27,28 As no excretion data were available, the decay in the remainder of the body was assumed to be solely by radioactive decay.

Patient imaging

[⁶⁸Ga]Ga-IAC PET/CT imaging was conducted with PGIMER Institutional Ethics Committee clearance (IEC-03/2020-1545). The inclusion criteria included age >18 years, patient consent, histopathologically confirmed breast cancer, and prior [¹⁸F]FDG PET/CT, while exclusions included refusal, pregnancy, lactation, or significant illness. The patients were closely monitored for vital parameters and instructed to self-report any adverse events at home.

[⁶⁸Ga]Ga-IAC dynamic and whole-body PET/CT imaging

A dynamic PET/CT study was conducted with a cohort of three patients to determine the optimal time of maximum uptake of [⁶⁸Ga]Ga-IAC in the lesion of interest. The lesion was positioned within the scanner’s field of view, and [⁶⁸Ga]Ga-IAC (111.37 ± 10.81 MBq) was administered. The injected mean mass of IAC per patient was 2.5 ± 0.34 µg. PET data were acquired for up to 60 min using a hybrid 3D PET/CT scanner (Siemens Healthineers Biograph mCT S-64, Germany). A series of 10 PET images were retro-reconstructed at different time intervals, ranging from 5 to 60 min postadministration. A region of interest was drawn around the lesion, and the maximum standard uptake value (SUV_max) was calculated. The SUV_max of the lesion and the ratio of SUV_max of the lesion to SUV_max of the aorta (representing blood uptake) were determined for all reconstructed images. Both parameters were plotted over time to assess dynamic behavior.

In addition to dynamic imaging, three patients also underwent whole-body (WB) PET/CT imaging at 60 min. For the remaining two patients, whole-body PET/CT imaging was acquired at 30 min, as determined by the dynamic study. The PET/CT parameters are referenced in the Supplementary Section 3.0. The [⁶⁸Ga]Ga-IAC PET/CT findings were then compared with the results obtained from [¹⁸F]FDG PET/CT scans (263.44 ± 37.31 MBq) for comprehensive evaluation.

Results

Radiolabeling and quality control of [⁶⁸Ga]Ga-IAC

Among the different combinations tested, sodium citrate (0.5M, pH 3.0) with ITLC-SG, trifluoracetic acid (TFA): acetonitrile (ACN) (1:1, v/v) with ITLC-SG and ACN: Water (1:1, v/v) with W#3 proved effective in distinguishing the three radiochemical species (Supplementary Fig. S1). The sodium citrate with ITLC-SG combination showed radio peaks at the origin for [⁶⁸Ga]Ga-DOTAGA (R _f 0.0) and [⁶⁸Ga]Ga-IAC (R _f 0.0) and a radio peak at the near solvent front for ⁶⁸Ga (R _f 0.8). This combination was able to identify the presence of free ⁶⁸Ga in the final product. TFA-ACN with ITLC-SG showed a radio peak at the origin for [⁶⁸Ga]Ga-IAC (R _f 0.0) and a radio peak at the near solvent front for [⁶⁸Ga]Ga-DOTAGA (R _f 0.8), enabling the detection of [⁶⁸Ga]Ga-DOTAGA in the final product. ACN: Water with W#3 showed a radio peak at R _f 0.6 for [⁶⁸Ga]Ga-IAC, confirming the absence of hydrolyzed ⁶⁸Ga in the final product, as hydrolyzed species would remain at the origin in any combination of mobile and stationary phases.

The effect of temperature, pH, and IAC concentration was also assessed. Only a 7.5% RCY was achieved when the reactions (1–3) were performed at room temperature for up to 25 min (Supplementary Table S1). The yield was increased to 95% on heating at 95°C within 10 min of reaction time. The RCY was reduced at pH 3.5 and 5.5% to 5% and 18%, respectively, indicating pH and temperature as the major factors in radiolabeling of IAC with ⁶⁸Ga (Supplementary Fig. S12). Furthermore, the highest labeling yield (> 95%) was noted at ratios of IAC to ⁶⁸Ga (µg: MBq) of 0.5:37 and 1:37 (Supplementary Fig. S12). Radio-ITLC analysis of reactions displayed >95% radiochemical yield. After purification with a sep-pak C18 cartridge, >99% radiochemical purity was achieved (Fig. 1A). The specific activity for [⁶⁸Ga]Ga-IAC was achieved up to 74 MBq/µg.

FIG. 1.

Radio-ITLC chromatograms of [⁶⁸Ga]Ga-DOTAGA–IAC (A), [¹⁷⁷Lu]Lu-DOTAGA (B), and [²²⁵Ac]Ac-DOTAGA–IAC (C) developed using sodium citrate and ITLC-SG strip indicating more than 99% radiochemical purity. DOTAGA, [2,2′,2” –{10-(2,6-dioxotetrahydro-2H-pyran-3-yl)−1,4,7,10-tetraazacyclododecane-1,4,7-triyl} triacetic-acid]; IAC, integrin antagonist carbamate.

Gamma-ray spectra of ⁶⁸Ga showed energy peaks at 511 KeV and 1022 KeV, indicating >99% radionuclide purity (Supplementary Fig. S2A). No photopeak was observed after 10 half-lives of ⁶⁸Ga ruling out the presence of ⁶⁸Ga breakthrough (Supplementary Fig. S2B). A single radio peak at R _f 0.0 was consistently observed at each time point for up to 4 h, indicating the high shelf-life of [⁶⁸Ga]Ga-IAC at room temperature. Similarly, a single radio peak at R _f 0.0 for [⁶⁸Ga]Ga-IAC in human serum at 37°C demonstrated no evidence of decomplexation in serum for up to 4 h (Supplementary Table S4 and Supplementary Figs. S3 and Figs. S4). No observable growth in TSB and FTM medium at 25°C and 30°C, respectively, for up to 14 days indicated the sterility of the in-house synthesized [⁶⁸Ga]Ga-IAC. Endotoxin levels for [⁶⁸Ga]Ga-IAC samples were calculated to be 7.2 ± 0.7 EU/V (Supplementary Table S5).

Radiolabeling and quality control of [¹⁷⁷Lu]Lu-IAC

The radio-ITLC chromatograms obtained using sodium citrate and ITLC-SG revealed R _f values of 0.8 for free ¹⁷⁷Lu and R _f 0.0 for [¹⁷⁷Lu]Lu-IAC. ACN: Water (1:1, v/v) and W#3, R _f values were 0.0 for ¹⁷⁷Lu and R _f 0.6 for [¹⁷⁷Lu]Lu-IAC (Supplementary Fig. S6). The absence of a radio peak at the origin in ACN: Water indicated the absence of hydrolyzed ¹⁷⁷Lu in the final product. Similar patterns were observed for both low and high specific activities [¹⁷⁷Lu]Lu-IAC. Radio-ITLC chromatograms of both low and high specific activities [¹⁷⁷Lu]Lu-IAC demonstrated >99% radiochemical purity (Fig. 1B), and the specific activities of both the radio constructs were 37 MBq/µg and 74 MBq/µg, respectively.

The multichannel acquisition spectrum displayed characteristic energy peaks of ¹⁷⁷Lu at 113 KeV and 208 KeV, indicating >99% radionuclide purity (Supplementary Fig. S2C). A single radio peak at R _f 0.0 for [¹⁷⁷Lu]Lu-IAC was observed for up to 14 days when incubated at room temperature in normal saline (0.9%), at 37°C in human serum, demonstrating good stability (Supplementary Table S6 and Supplementary Figs. S7 and Figs. S8). Both culture mediums remained free from visible microbial growth for up to 14 days, confirming the sterile production of [¹⁷⁷Lu]Lu-IAC. The endotoxin levels obtained from pyrogenicity experiments were 22.2 ± 2.0 EU/V (Supplementary Table S5).

Radiolabeling and quality control of [²²⁵Ac]Ac-IAC

More than 99% RCY was achieved for [²²⁵Ac]Ac-IAC (Fig. 1C). Radio-ITLC of free ²²⁵Ac showed two peaks at R _f 0.7 and 1.0 (Supplementary Fig. S5). The same ITLC-SG strip was reread after 2 h and showed only a single radio peak at R _f 0.7 for ²²⁵Ac; a radio peak at R _f 1.0 diminished. The reaction showed a single peak at R _f = 0.0 indicating >99% [²²⁵Ac]Ac-IAC. After 15 min of synthesis, a major peak of [²²⁵Ac]Ac-IAC at R _f = 0.0 (with noise at R _f of 0.2–0.5) was observed. However, rescanning the same ITLC-SG strip after 2 h, only a single radio peak at the origin (R _f 0.0) was obtained for [²²⁵Ac]Ac-IAC. At 1 h of end of synthesis (EOS), multiple peaks were observed (R _f 0.0, 0.3, and 0.5), and peaks at R _f 0.3 and 0.5 diminished when the same plate was rescanned after 2 h. Radiochromatogram appeared using 2 M HCl: acetone showed a radio peak at R _f = 0.5; no peak was observed at the origin. (Supplementary Fig. S9). The [²²⁵Ac]Ac-IAC was prepared with a specific activity of up to 50 KBq/µg.

The ²²⁵Ac radionuclidic purity was greater than 99.9% (Supplementary Fig. S2D). [²²⁵Ac]Ac-IAC incubated in human serum and normal saline demonstrated a single radio peak at R _f 0.0 for up to 14 days indicating high stability (Supplementary Table S7). No visible microbial growth for up to 14 days in both culture mediums confirmed the sterility of the prepared drug solution. Endotoxin levels in the final product solution were 0.4 ± 0.0 EU/V (Supplementary Table S5).

HPLC characterization

The HPLC analysis of IAC revealed a purity of 100% with a retention time (tR) of 8.3 min. The labeled nat[⁶⁹Ga]Ga-IAC and nat[¹⁷⁵Lu]Lu-IAC were identified at tR = 8.6 min, along with the unlabeled IAC (tR = 8.3 min) in the reaction mixture (Supplementary Figs. S10 and Figs. S11).

Partition coefficient and plasma protein binding

The log p-values for [⁶⁸Ga]Ga-IAC and [¹⁷⁷Lu]Lu-IAC were found to be −2.23 ± 0.07 and −2.35 ± 0.06, respectively. The plasma protein binding values for [⁶⁸Ga]Ga-IAC and [¹⁷⁷Lu]Lu-IAC were found to be 34.45% and 36.12%, respectively.

In vitro radioligand binding study

The nonlinear regression between bound radioligand (fmol) on the Y-axis and radioligand concentration (nM) on the X-axis estimated the K_d of 15.02 nM and a B_max of 417 fmol for recombinant human α_vβ₃ integrin receptor (Fig. 2A, B). For the C6 cell line, the K_d and B_max values were found to be 115.7 nM and 295.3 fmol, respectively (Fig. 2C, D). However, both Hep-G2 and MCF-7 cells did not exhibit any specific binding for the [⁶⁸Ga]Ga-IAC radioligand (Fig. 2E).

FIG. 2.

[⁶⁸Ga]Ga-IAC total, nonspecific, and specific binding with αvβ3 integrin receptor (A, B) and C6 glioma cells (C, D). Specific and nonspecific binging with MCF-7 and Hep-G2 cells (E). The experiments were performed in triplicates and repeated twice. MCF-7, human breast cancer cells; Hep-G2, human liver cancer cells; IAC, integrin antagonist carbamate.

Biodistribution studies in healthy Wistar rats

The elevated levels of radioactivity detected in the kidneys for both radiotracers indicated that this is the main route of excretion. In addition, moderate levels of radioactivity, observed in the stomach, small intestine, and large intestine, suggested partial excretion through the hepatobiliary route. A significant decrease in blood pool radioactivity was observed at 1 h ([⁶⁸Ga]Ga-IAC: 0.11 ± 0.05; [¹⁷⁷Lu]Lu IAC: 0.15 ± 0.03) compared with 0.5 h ([⁶⁸Ga]Ga-IAC: 0.28 ± 0.04), indicating rapid clearance of both radiotracers from the bloodstream. Relatively low radioactivity was found in the salivary glands (0.17 ± 0.02), bone (0.09 ± 0.05), and heart (0.07 ± 0.02) at 1 h after administration of the radiotracers. Few counts were also detected in the brain (% ID/g 0.01 ± 0.00). Notably, [¹⁷⁷Lu]Lu-IAC was effectively cleared from all organs within 24 h (Fig. 3).

FIG. 3.

Biodistribution of [⁶⁸Ga]Ga-IAC in Wistar rats at 0.5 h, 1 h, and 2 h (A). Biodistribution of [¹⁷⁷Lu]Lu-IAC in Wistar rats at 1, 24, and 48 h (B). n = 3 at each time point. IAC, integrin antagonist carbamate.

Dosimetry analysis

The total-body dose was calculated to be 0.28 mSv/MBq, and the effective dose was 0.174 mSv/MBq. The equivalent doses in organs were 0.114 mSv/MBq for kidneys, 0.097 mSv/MBq for liver, 0.082 mSv/MBq for spleen, and 0.041 mSv/MBq for the lungs. The highest radiation dose was found for osteogenic cells (0.313 mSv/MBq) followed by almost similar doses in the intestine, pancreas, prostate, and bladder wall (∼0.284 mSv/MBq). The values of equivalent doses for various organs are summarized in Table 1.

Table 1.

Radiation Dose Estimates (Adult Male Model) for [¹⁷⁷Lu]Lu-IAC

Organ	Equivalent dose (mSv/MBq)	Organ	Equivalent dose (mSv/MBq)
Adrenals	0.279	Lungs	0.041
Brain	0.274	Pancreas	0.284
Esophagus	0.275	Prostate	0.284
Eyes	0.274	Salivary Glands	0.279
Gallbladder	0.28	Red Marrow	0.214
Left colon	0.284	Osteogenic Cells	0.313
Small Intestine	0.285	Spleen	0.082
Stomach Wall	0.279	Testes	0.275
Right colon	0.283	Thymus	0.276
Rectum	0.285	Thyroid	0.277
Heart Wall	0.279	Urinary Bladder Wall	0.284
Kidneys	0.114	Total Body	0.28
Liver	0.097	Effective Dose	0.174

PET/CT imaging

Dynamic and whole-body PET/CT imaging

For patient 1, [¹⁸F]FDG PET/CT revealed a well-defined, heterogeneously enhancing soft tissue lesion (SUV_max 7.6) in the retroareolar area of the right breast and mild uptake in perilesional fat stranding. No significant hypermetabolic lesion was observed in other parts of the body (Fig. 4M–O). The dynamic [⁶⁸Ga]Ga-IAC PET/CT of the same breast lesion showed varying SUV_max values, which were 3.77 (at 5 min) and 3.75 (at 60 min), with a peak value of 5.61 at 15 min (Fig. 4A–K). The tumor and blood (aorta) SUV_max ratios ranged from 0.37 (at 5 min) to 1.82 (at 60 min), peaking at 2.08 between 30 and 35 min (Fig. 4L). The normal right breast tissue showed low background activity (SUV_max 0.9), resulting in a good tumor-to-background ratio of 4.2 (Fig. 4P). On whole-body [⁶⁸Ga]Ga-IAC PET/CT (Fig. 4Q), normal organ distribution demonstrated physiological uptake in the thyroid (SUV_max 2.6), lungs (SUV_max 1.3), liver (SUV_max 3.62), and muscles (SUV_max 1.67). The excretion of [⁶⁸Ga]Ga-IAC was observed from the kidney (SUV_max 10.2). Compared to [¹⁸F]FDG (SUV_max 20.9), [⁶⁸Ga]Ga-IAC showed minimal to negligible uptake in the brain (SUV_max 0.19).

FIG. 4.

A 41-year-old woman (patient 1) was diagnosed with right breast cancer (T4b N1 M0), biopsy showed invasive ductal carcinoma grade III and immunohistochemistry confirmed the receptor status as Estrogen Receptor Positive (ER+), Progesterone Receptor Positive (PR+), Human Epidermal Growth Factor Receptor 2 (HER2) - with Ki67 value of 20%. Day 1: [¹⁸F]FDG (305.25 MBq) WB PET/CT revealed a primary lesion on the right breast (M, N). Day 2: [⁶⁸Ga]Ga-DOTAGA-IAC (124.32 MBq) dynamic transaxial PET images (A–J) demonstrated variation in SUV_max for 0 to 60 min in the upper right breast quadrant. Graphs illustrate how a tumor’s SUV_max evolves over time (K). The tumor-to-aorta ratio over time (L). [⁶⁸Ga]Ga-DOTAGA-IAC PET/CT localized the tracer avidity in a primary lesion on the right breast (O–Q). DOTAGA, [2,2′,2”–{10-(2,6-dioxotetrahydro-2H-pyran-3-yl)−1,4,7,10-tetraazacyclododecane-1,4,7-triyl} triacetic-acid]; IAC, integrin antagonist carbamate; SUV_max, standard uptake value.

[¹⁸F]FDG and [⁶⁸Ga]Ga-IAC detected primary lesions in all five patients, with mean SUV_max values of 13.8 ± 6.53 and 3.94 ± 0.58, respectively. The detailed patient analysis is summarized in Table 2. Patient 4 presented with extensive metastases, [⁶⁸Ga]Ga-IAC detected all the primary and metastatic lesions which were revealed on the [¹⁸F]FDG (Fig. 5). The [¹⁸F]FDG and [⁶⁸Ga]Ga-IAC demonstrated multiple soft tissue lesions in the right breast as primary (SUV_max FDG vs. IAC: 16.7 vs. 4.7). In addition, multiple right axillary (SUV_max 11.0 vs 3.9), internal mammary (SUV_max 5.0 vs. 2.2), and abdominal (SUV_max 7.7 vs. 4.5) lymph nodes were also noted with liver (SUV_max 15.1 vs. 4.2), marrow-based, and lytic skeletal lesions (SUV_max 13.2 vs. 5.3). Owing to the physiological uptake of [¹⁸F]FDG in the brain, skull lesions were not clearly visible. However, the lack of avidity of [⁶⁸Ga]Ga-IAC in the normal brain enabled the successful identification of multiple tracer-avid skull metastatic lesions (SUV_max 3.7) (Fig. 5G).

FIG. 5.

A 50-year-old female (patient 4) presented with disease progression and bone metastasis post 7 cycles of neoadjuvant chemotherapy. [¹⁸F]FDG (236.8 MBq) WB PET/CT was performed for disease evaluation post 45 min i.v. administration and [⁶⁸Ga]Ga-DOTAGA-IAC (96.2 MBq) WB PET/CT was performed post 30 min i.v. administration. The MIP, corresponding transaxial CT (C) and fused PET/CT (B, D) images localized the tracer uptake in the right breast (SUV_max FDG vs. IAC: 16.7 vs. 4.7) as a primary lesion. The MIP (A, H) also demonstrated multiple metastatic lesions. [⁶⁸Ga]Ga-DOTAGA-IAC detected multiple metastatic lytic lesions (SUV_max 3.7) on the skull (F, G), unlike [¹⁸F]FDG (E). DOTAGA, [2,2′,2”–{10-(2,6-dioxotetrahydro-2H-pyran-3-yl)−1,4,7,10-tetraazacyclododecane-1,4,7-triyl} triacetic-acid]; IAC, integrin antagonist carbamate; SUV_max, standard uptake value.

Table 2.

Patient Details

		Dose (MBq)					Lesions detected		Primary lesion (SUV_max)		Metastatic lesion (SUV_max)
S. No	Age/ sex	¹⁸F-FDG	⁶⁸Ga-IAC	Histology	TNM staging	Receptor status	¹⁸F-FDG	⁶⁸Ga-IAC	¹⁸F-FDG	⁶⁸Ga-IAC	¹⁸F-FDG	⁶⁸Ga-IAC
1	41/ F	290	116	IDC II	T_4bN₁M₀	ER⁺, PR⁺, HER2⁻	1	1	7.26	3.75	—	—
2	59/ F	215	106	IDC II	T_4bN₁M₀	ER⁺, PR⁺, HER2⁻	3	2	11.6	3.2	20.7	9.88
3	52/ M	307	128	IDC II	T_4bN₁M₀	ER⁺, PR⁺, HER2 equivocal	3	3	9.7	3.71	5.74	2.2
4	50/ F	237	107	IDC III	T_4bN3M1	ER⁻, PR⁻, HER2⁺	Multiple	Multiple	16.7	4.7	11.1	3.7
5	58/ F	274	78	IDC II	T_4bN_3cM₀	ER⁻, PR⁻, HER2⁻	3	3	23.7	4.34	15.0	7.95

F, female; M, male; SUVmax, Maximum Standardized Uptake Value; FDG, fluorodeoxyglucose; IAC, integrin antagonist carbamate; IDC, Invasive ductal carcinoma; TNM, Tumor, Nodes, and Metastasis; ER+, Estrogen Receptor Positive; PR+, Progesterone Receptor Positive; Her2+, Human Epidermal Growth Factor Receptor 2 Positive; Her2−, Human Epidermal Growth Factor Receptor 2 Negative.

Adverse events

Following the injection of [⁶⁸Ga]Ga-IAC and during the hospital stay, no adverse events or drug-related side effects were observed in any of the patients enrolled in the clinical study. The vital parameters of the patients remained stable, and no adverse events were observed in patients after their hospital stay.

Discussion

The radio-ITLC analyses revealed more than 95% RCY for [⁶⁸Ga]Ga-IAC when 740 MBq [⁶⁸Ga]GaCl₃ was incubated with 10–20 µg IAC at 95°C for 10 min and reaction pH 4.5. The pH and temperature were the major factors in the radiolabeling of IAC with ⁶⁸Ga (Supplementary Fig. S12). The optimization of IAC concentration on the radiochemical yield was important for the judicious utilization of the IAC, particularly in scenarios where the patient pool is limited. In addition, the higher ratio of IAC to ⁶⁸Ga during radiolabeling can reduce the specific activity and excess of unlabeled IAC and may cause receptor saturation that could lower the PET image resolution. High specific activity and >99% RCP are preferred for high-resolution PET images and to reduce an unnecessary radiation burden to patients. The incorporation of C-18 cartridge purification in the preparation procedure resulted in more than 99.9% RCP. A peak at 8.6 min in HPLC validated the complexation of gallium with IAC.

The [¹⁷⁷Lu]Lu-IAC was prepared with a high RCY (>99.9%), allowing the direct administration of the reaction mixture without a purification step. The high SA ¹⁷⁷Lu contains only radioactive lutetium atoms in contrast to the low SA ¹⁷⁷Lu (carrier added). Therefore, the radiolabeling of high SA ¹⁷⁷Lu was achieved at a reasonably low IAC concentration compared with low SA ¹⁷⁷Lu. Clinically, the high SA [¹⁷⁷Lu]Lu-IAC is advantageous in tumors exhibiting high receptor expression and low receptor expression. However, in the case of low SA ¹⁷⁷Lu, the IAC gets labeled with nonradioactive lutetium atoms that may consequently saturate the receptor sites, potentially blocking [¹⁷⁷Lu]Lu-IAC binding. The impact of low SA ¹⁷⁷Lu is more prominent when the receptor expression is low.

The [²²⁵Ac]Ac-IAC was prepared with a high radiochemical yield (>99%). The stability assessment was challenging. When the radio-ITLC was performed immediately after synthesis, a single peak at the origin for [²²⁵Ac]Ac-IAC was observed, with a radiochemical purity >99%. However, the delayed ITLC assessment resulted in multiple peaks at R _f 0.0–0.5, and the intensity of these peaks was increasing over time (Supplementary Fig. S5).

This phenomenon could be understood as follows: (1) at the end of synthesis, all ²²⁵Ac and daughters were bound with IAC, (2) post-synthesis, the recoil effect from α decay caused the daughters to detach from IAC, and (3) the accumulation of recoiled daughters increased over time and appeared as multiple peaks in the radio-chromatogram which eventually decreased the purity to 76%. This finding emphasizes the importance of injecting [²²⁵Ac]Ac-IAC immediately after synthesis to avoid undesired localization and toxicity by the recoiled daughter radionuclides. If there is a time gap between synthesis and injection of [²²⁵Ac]Ac-IAC, incorporation of a purification step using a C-18 cartridge may effectively remove recoiled daughter radionuclides.

To confirm that multiple peaks were due to free daughter radionuclides, the same strips (developed at 15 min and 60 min post-synthesis) were rescanned after 2 h and showed a single peak at R _f 0.0.

The other peaks were diminished due to the short half-lives (in min) of the recoiled daughter radionuclides. To validate that multiple peaks were due to free daughter radionuclides, the same strips (developed at 15 and 60 min post-synthesis) were rescanned after 2 h. The resulting single peak at Rf 0.0 confirms the decay of the short-lived recoiled daughter radionuclides over time. Our findings are in concordance with the available literature.²⁹

Another interesting question that could arise is the absence of recoiled daughter peaks in the radio-chromatogram obtained immediately at the end of synthesis. This could be attributed to the possibility of a low concentration of recoiled daughters present in the formulation, rendering them undetectable by the radio-ITLC detector. However, as time passes, the accumulation of daughters increases to the detectable range.

The low K_d (15.02 nM) demonstrates a high binding affinity of IAC to αvβ3 integrin receptor. The two known FDA-approved radiopharmaceuticals such as [⁶⁸Ga]Ga-DOTATATE and [⁶⁸Ga]Ga-PSMA-11 also have nanomolar binding affinities for their corresponding receptors, 7.36 and 4.3 nM, respectively.^30,31 The nanomolar affinities of IAC for αvβ3 integrin receptor suggest the strong binding between the radioligand and receptor. The binding of [⁶⁸Ga]Ga-IAC with the C6 glioma cells line indicates its potential application in glioblastoma (Fig. 2). Although C6 rat glioma cells are considered safe, and effective, and simulate human glioblastoma, there still might be an interspecies variation in the binding of IAC with rat and human integrin. Therefore, additional studies are warranted in human glioblastoma cells (U87 cells) to establish the application of IAC in glioblastoma.

The log p-values of [⁶⁸Ga]Ga-IAC (−2.35 ± 0.06) and [¹⁷⁷Lu]Lu-IAC (−2.23 ± 0.07) indicate the hydrophilic nature of both radiocomplexes. The difference in lipophilicity arises from structural dissimilarities in the [⁶⁸Ga]Ga-DOTAGA and [¹⁷⁷Lu]Lu-DOTAGA complexes. Ga(III) and Lu(III) share similar chemistry but differ in ionic radii: Lu³⁺ (0.861 Å) forms 8-coordination with 4 amine nitrogens, 4 carboxylate oxygens, and Ga³⁺ (0.62 Å) forms 6-coordination with 4 amine nitrogens, 2 carboxylate oxygens, leaving 2 unbound carboxylates, hence increasing hydrophilicity. Routinely used DOTANOC/DOTATATE exhibits a similar lipophilicity of −2.88 ± 0.12.³² The relatively lower hydrophilicity of IAC indicates that it will prefer the renal clearance route and may also show a partial hepatobiliary clearance route.

Fast blood pool clearance of [⁶⁸Ga]Ga-IAC at 60 min and negligible blood pool activity of [¹⁷⁷Lu]Lu-IAC at 24 h makes this radioligand a potential theranostic agent. The biodistribution of [¹¹¹In]In-DOTA-Bz-SCN-IAC, as reported by Jang et al. in 2007 shows significantly higher values in all organs compared to our findings for [⁶⁸Ga]Ga/[¹⁷⁷Lu]Lu-IAC.²⁰ The difference in %ID/g for [⁶⁸Ga]Ga-IAC versus [¹¹¹In]In-DOTA-Bz-SCN-IAC at 60 min postinjection were as follows: Blood: 0.11 ± 0.05 vs. 1.22 ± 0.40; spleen: 0.39 ± 0.13 vs. 3.17 ± 0.31; bone; 0.09 ± 0.05 vs. 2.20 ± 0.14; liver: 0.20 ± 0.03 vs. 3.01 ± 0.27; kidney: 0.58 ± 0.23 vs. 14.43 ± 1.56.²⁰ This variation in physiological distribution could be due to the different chelator, linker, and radioisotope used in both studies. Furthermore, our preclinical distribution pattern was in concordance with clinical uptake findings. The clinical studies performed using [⁶⁸Ga]Ga-IAC show minimal physiological distribution in the whole body.

Notably, slow renal elimination was observed for both [⁶⁸Ga]Ga-IAC and [¹⁷⁷Lu]Lu-IAC in animals. However, therapeutic radionuclide-induced radiation nephrotoxicity is avoidable by appropriate hydration and using renal protection agents. Nowadays, pretargeted radioimmunodiagnosis (PRID) and radioimmunotherapy (PRIT) strategies are also gaining attention for their potential to enhance therapeutic efficacy and reduce toxicity.³³

The effective dose for [¹⁷⁷Lu]Lu-IAC in humans, as estimated using animal biodistribution data, is of 0.17 mSv/MBq. The value indicates favorable dosimetry as it is significantly lower than other established ¹⁷⁷Lu radiopharmaceuticals.³⁴ The dose-limiting tissue was found to be osteogenic cells with a dose of 0.31 mSv/MBq. However, the dose appears to be safe in view of the reported permissible cumulative dose of 2 Gy for bone.^35,36

The dynamic imaging in patients revealed peak [⁶⁸Ga]Ga-IAC uptake at 10–15 min. However, a favorably low blood pool with the highest tumor-to-blood ratio was achieved at 30–35 min. At 60 min, the blood pool activity was minimal, but the tumor uptake was also decreased due to the physical decay of ⁶⁸Ga. Therefore, 30–35 min is the optimum imaging time for whole-body [⁶⁸Ga]Ga-IAC PET/CT (Fig. 4).

In patients, [⁶⁸Ga]Ga-IAC effectively detected all the primary and metastatic lesions positively revealed by [¹⁸F]FDG PET/CT (Table 2). [⁶⁸Ga]Ga-IAC may offer a considerable advantage over [¹⁸F]FDG for detecting brain lesions. The high physiological uptake of [¹⁸F]FDG in the brain makes it challenging to delineate skull lesions in the [¹⁸F]FDG PET/CT imaging. However, for [⁶⁸Ga]Ga-IAC, the absence of physiological brain uptake makes it a better agent for delineating skull lesions. Besides these findings, minimal physiological distribution in healthy organs and a good tumor-to-background ratio makes IAC a potential diagnostic and therapeutic agent for the αVβ3 expressing tumors. The chemotherapy-resistant, refractory, and metastatic patients may benefit from the IAC when chelated with ¹⁷⁷Lu and ²²⁵Ac.

Conclusions

This study provides radiolabeling, quality controls, and clinical evidence that [⁶⁸Ga]Ga-IAC is a useful tracer for the visualization of angiogenic breast cancer tumors and was found to be safe and effective. The bifunctional chelator, DOTAGA, is suitable for radiolabeling with ¹⁷⁷Lu and ²²⁵Ac as well. The probe was evaluated clinically and demonstrated selective binding and rapid accumulation in the tumor neovasculature. The high receptor binding affinity, significant tumor-to-background ratio, minimal physiological uptake in body organs, and favorable dosimetry suggest theranostic agent potential of IAC. Peptide Receptor Radionuclide Therapy and Targeted alpha-therapy using [¹⁷⁷Lu]Lu-IAC and [²²⁵Ac]Ac-IAC may have the potential for treating a broad range of early and late-stage cancers expressing αVβ3 integrin. Further clinical investigations with [¹⁷⁷Lu]Lu/[²²⁵Ac]Ac-IAC are warranted to establish their therapeutic efficacy.

Footnotes

Acknowledgment

This research and development were supported in part by Advanced Innovative Partners, Inc. (AIP), Miami, Florida.

Authors’ Contributions

S.P.: radiolabeling optimization, quality control assessment, cell culture, animal studies, patient recruitment, data analysis, and article writing; G.K.: radiopharmaceutical preparation, cell culture and animal studies; N.R.: patient imaging, image reconstruction and determination of optimal PET/CT imaging time; S.C.: radiopharmaceutical preparation and patient imaging; I.R.: animal studies; R.K.: interpretation of patients image; I.L.: patients evaluation and recruitment; V.D.C.: animal studies; S.S.: conceptualization of application of IAC; M.G.S.: Dosimetry Study; B.R.M.: interpretation of patients image; J.S.: study design, data interpretation, article review and proofreading.

Disclosure Statement

Dr. Stanley Satz receives compensation from Advanced Innovative Partners, Inc. No other author has any potential conflict of interest.

Funding Information

Advanced Innovative Partners, Inc. (AIP), Miami, Florida

Supplementary Material

Supplementary Section

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4

Supplementary Figure S5

Supplementary Figure S6

Supplementary Figure S7

Supplementary Figure S8

Supplementary Figure S9

Supplementary Figure S10

Supplementary Figure S11

Supplementary Figure S12

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Table S4

Supplementary Table S5

Supplementary Table S6

Supplementary Table S7

References

Folkman

. Tumor angiogenesis: Therapeutic implications. N Engl J Med, 1971; 285(21):1182–1186.

Carmeliet

. Mechanisms of angiogenesis and arteriogenesis. Nat Med, 2000; 6(4):389–395.

Nussenbaum

, Herman

. Tumor angiogenesis: Insights and innovations. J Oncol, 2010; 2010:132641.

Francavilla

, Maddaluno

, Cavallaro

. The functional role of cell adhesion molecules in tumor angiogenesis. Semin Cancer Biol, 2009; 19(5):298–309.

Desgrosellier

, Cheresh

. Integrins in cancer: Biological implications and therapeutic opportunities. Nat Rev Cancer, 2010; 10(1):9–22.

Danhier

, Le Breton

, Préat

. RGD-based strategies to target alpha (v) beta (3) integrin in cancer therapy and diagnosis. Mol Pharmaceutics, 2012; 9(11):2961–2973.

Brooks

, Strömblad

, Klemke

, et al. Antiintegrin alpha v beta 3 blocks human breast cancer growth and angiogenesis in human skin. J Clin Invest, 1995; 96(4):1815–1822.

Takayama

, Ishii

, Ikeda

, et al. The relationship between bone metastasis from human breast cancer and integrin alpha(v)beta3 expression. Anticancer Res, 2005; 25(1A):79−–83.

Uhm

, Dooley

, Kyritsis

, et al. Vitronectin, a Glioma-derived extracellular Matrix Protein, protects tumor cells from apoptotic death. Clin Cancer Res, 1999; 5(6):1587–1594.

10.

Hosotani

, Kawaguchi

, Masui

, et al. Expression of integrin alphaVbeta3 in pancreatic carcinoma: Relation to MMP-2 activation and lymph node metastasis. Pancreas, 2002; 25(2):e30–e35.

11.

Cooper

, Chay

, Pienta

. The Role of alpha (v) beta (3) in Prostate Cancer Progression. Neoplasia, 2002; 4(3):191–194.

12.

Chattopadhyay

, Chatterjee

. Studies on the Expression of alpha v beta 3 Integrin Receptors in Non-malignant and Malignant Human Cervical Tumor Tissues. J Exp Clin Cancer Res, 2001; 20(2):269–275.

13.

Beer

, Schwaiger

. Imaging of integrin αvβ3 expression. Cancer Metastasis Rev, 2008; 27(4):631–644.

14.

Debordeaux

, Chansel-Debordeaux

, Pinaquy

, et al. What about αvβ3 integrins in molecular imaging in oncology? Nucl Med Biol, 2018; 62-63:31–46.

15.

Knetsch

, Petrik

, Griessinger

, et al. [68 Ga] NODAGA-RGD for imaging αvβ3 integrin expression. Eur J Nucl Med Mol Imaging, 2011; 38(7):1303–1312.

16.

Dijkgraaf

, Yim

, Franssen

, et al. PET imaging of α_vβ₃ integrin expression in tumours with ⁶⁸Ga-labelled mono-, di-and tetrameric RGD peptides. Eur J Nucl Med Mol Imaging, 2011; 38(1):128–137.

17.

Pohle

, Notni

, Bussemer

, et al. ⁶⁸Ga-NODAGA-RGD is a suitable substitute for ¹⁸F-Galacto-RGD and can be produced with high specific activity in a cGMP/GRP compliant automated process. Nucl Med Biol, 2012; 39(6):777–784.

18.

Vatsa

, Shukla

, Kumar

, et al. Effect of Macro-Cyclic Bifunctional Chelators DOTA and NODAGA on Radiolabeling and In Vivo Biodistribution of Ga-68 Cyclic RGD Dimer. Cancer Biother Radiopharm, 2019; 34(7):427–435.

19.

Hsu

, Veeravagu

, Cai

, et al. Integrin αvβ3 antagonists for anti-angiogenic cancer treatment. Recent Pat Anticancer Drug Discov, 2007; 2(2):143–158.

20.

Jang

, Lim

, Park

, et al. Radiolabeled high affinity peptidomimetic antagonist selectively targets αvβ3 receptor-positive tumor in mice. Nucl Med Biol, 2007; 34(4):363–370.

21.

Shin

, Maeng

, Jang

, et al. 99mTc-labeling of peptidomimetic antagonist to selectively target αvβ3 receptor-positive tumor: Comparison of PDA (Parenteral Drug Association)and EDDA as co-ligands. Curr Radiopharm, 2010; 3(1):1–8.

22.

Kim

, Nwe

, Milenic

, et al. Synthesis and characterization of αvβ3-targeting peptidomimetic chelate conjugates for PET and SPECT imaging. Bioorg Med Chem Lett, 2012; 22(17):5517–5522.

23.

Baum

, Kulkarni

, Müller

, et al. First-in-human study demonstrating tumor-angiogenesis by PET/CT imaging with 68Ga-NODAGA-THERANOST, a high-affinity peptidomimetic for αvβ3 integrin receptor targeting. Cancer Biother Radiopharm, 2015; 30(4):152–159.

24.

Price

, Orvig

. Matching chelators to radiometals for radiopharmaceuticals. Chem Soc Rev, 2014; 43(1):260–290.

25.

Kirschner

, Ice

, Beierwaltes

. Radiation dosimetry of 131I-19-iodocholesterol: The pitfalls of using tissue concentration data, the author’s reply. J Nucl Med, 1975; 16(3):248–249.

26.

Foster

, Barrett

. Developing and testing integrated multicompartment models to describe a single-input multiple-output study using the SAAM II software system. In: Proc. Sixth International Radiopharmaceutical Dosimetry Symposium, Oak Ridge Institute for Science and Education, in press, 1998.

27.

Stabin

, Siegel

. Physical models and dose factors for use in internal dose assessment. Health Phys, 2003; 85(3):294–310.

28.

Stabin

, Sparks

, Crowe

. OLINDA/EXM: The Second-Generation personal computer software for internal dose assessment in nuclear medicine. J Nucl Med, 2005; 46(6):1023–1027.

29.

Kelly

, Amor-Coarasa

, Sweeney

, et al. A suitable time point for quantifying the radiochemical purity of 225Ac-labeled radiopharmaceuticals. EJNMMI Radiopharm Chem, 2021; 6(1):38–34.

30.

Xie

, Liu

, Ding

, et al. Synthesis, preclinical evaluation, and a pilot clinical imaging study of [18F] AlF-NOTA-JR11 for neuroendocrine neoplasms compared with [68Ga] Ga-DOTA-TATE. Eur J Nucl Med Mol Imaging, 2021; 48(10):3129–3140.

31.

Wang

, Shao

, Wu

, et al. Preparation of 68Ga-PSMA-11 with a synthesis module for micro-PET-CT imaging of PSMA expression during prostate cancer progression. Contrast Media Mol Imaging, 2018; 2018:1–9.

32.

Antunes

, Ginj

, Zhang

, et al. Are radiogallium-labelled DOTA-conjugated somatostatin analogues superior to those labelled with other radiometals? Eur J Nucl Med Mol Imaging, 2007; 34(7):982–993.

33.

Cheal

, Chung

, Vaughn

, et al. Pretargeting: A path forward for radioimmunotherapy. J Nucl Med, 2022; 63(9):1302–1315.

34.

Okamoto

, Thieme

, Allmann

, et al. Radiation dosimetry for 177Lu-PSMA IT in metastatic castration-resistant prostate cancer: Absorbed dose in normal organs and tumor lesions. J Nucl Med, 2017; 58(3):445–450.

35.

Kind

, Michalski

, Yousefzadeh-Nowshahr

, et al. Bone marrow impairment during early [177Lu] PSMA-617 radioligand therapy: Haematotoxicity or tumour progression? EJNMMI Res, 2022; 12(1):20.

36.

Wahl

, Sgouros

, Iravani

, et al. Normal-tissue tolerance to radiopharmaceutical therapies, the knowns and the unknowns. J Nucl Med, 2021; 62(Suppl 3):23S–35S.

Supplementary Material

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Advancing Cancer Theranostics Through Integrin αVβ3-Targeted Peptidomimetic IAC: From Bench to Bedside

Abstract

Introduction:

Methodology:

Results:

Conclusion:

Introduction

Materials and Methods

Radiolabeling with [68Ga]GaCl3

Radiolabeling with high specific activity (SA) [177Lu]LuCl3

Radiolabeling with low SA [177Lu]LuCl3

Radiolabeling with [225Ac] AcCl3

Purification by solid-phase extraction

HPLC characterization

Quality control

Determination of partition coefficient (log P)

Plasma protein binding in human blood

In vitro radioligand binding study

Biodistribution in healthy Wistar rats

Dosimetry for [177Lu]Lu-IAC Preclinical Biodistribution Data

Patient imaging

[68Ga]Ga-IAC dynamic and whole-body PET/CT imaging

Results

Radiolabeling and quality control of [68Ga]Ga-IAC

Radiolabeling and quality control of [177Lu]Lu-IAC

Radiolabeling and quality control of [225Ac]Ac-IAC

HPLC characterization

Partition coefficient and plasma protein binding

In vitro radioligand binding study

Biodistribution studies in healthy Wistar rats

Dosimetry analysis

PET/CT imaging

Dynamic and whole-body PET/CT imaging

Adverse events

Discussion

Conclusions

Footnotes

Acknowledgment

Authors’ Contributions

Disclosure Statement

Funding Information

Supplementary Material

References

Supplementary Material

Radiolabeling with [⁶⁸Ga]GaCl₃

Radiolabeling with high specific activity (SA) [¹⁷⁷Lu]LuCl₃

Radiolabeling with low SA [¹⁷⁷Lu]LuCl₃

Radiolabeling with [²²⁵Ac] AcCl₃

Dosimetry for [¹⁷⁷Lu]Lu-IAC Preclinical Biodistribution Data

[⁶⁸Ga]Ga-IAC dynamic and whole-body PET/CT imaging

Radiolabeling and quality control of [⁶⁸Ga]Ga-IAC

Radiolabeling and quality control of [¹⁷⁷Lu]Lu-IAC

Radiolabeling and quality control of [²²⁵Ac]Ac-IAC