Rhenium-186-labeled ortho-hydroxythiobenzhydrazide: a potential theranostic agent for use in metastatic breast cancer

Abstract

In this study, the aromatic thiobenzhydrazide derivative o-hydroxythiobenzhydrazide [H(htbh)] was radiolabeled with Rhenium-186 (¹⁸⁶Re) [t_1/2: 3.77 days, β_max: 1.07 MeV (71%), γ: 137 keV (9.47%)], produced in an in-house research reactor, yielding the [¹⁸⁶Re]Re-(htbh)₂ complex. The radiochemical purity (RCP) of the complex was found to be (98.07 ± 0.40)%. Mass spectroscopic analyses were used to elucidate the probable structure of the complex. The tumour targeting specificity of [¹⁸⁶Re]Re-(htbh)₂ was preliminarily assessed through in-vitro and in-vivo studies in preclinical models. While in-vitro studies included cell binding and internalization assays using MCF-7 cell lines, in vivo studies involved biodistribution analyses in SCID mice bearing MCF-7 induced human breast cancer xenografts. The pharmacokinetics revealed significant and prolonged tumour uptake, with SPECT/CT imaging showing plasma clearance within 24 hours and minimal retention in the liver and bladder.

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1 Introduction

Metastatic breast carcinoma (MBC) remains one of the most common malignancies in women worldwide. It is typically managed with surgery and chemotherapy,¹ while radiotherapy serves as an additional option. Radiotherapy may involve either external beam radiotherapy (EBRT) or internalized sources of radiation. The latter, referred to as radionuclide therapy or radiopharmaceutical therapy (RPT), employs molecular vectors tagged with therapeutic radioisotopes emitting β⁻ or α particles. RPT, has particularly shown promise in MBC patients with overexpression of the Human Epidermal Growth Factor Receptor 2 (HER-2).²

RPT offers several advantages over EBRT, primarily due to its systemic or locoregional delivery of cytotoxic radiation to cancer cells or their microenvironment, either directly or by using delivery vehicles.³ Among RPT agents, β⁻ emitting radiopharmaceuticals are more widely applied in MBC than those tagged with α emitting particles.^4,5 Radioimmunotherapy (RIT) with radiolabeled monoclonal antibodies (MAbs), such as trastuzumab, has been explored using ¹³¹I, ¹⁷⁷Lu, and ⁹⁰Y. Although ¹³¹I (β_max: 606 keV, γ_max: 364 keV, t_½ 8.02 d) and ¹⁷⁷Lu (β_max: 497 keV, γ_max: 208 keV, t_½ 6.73 d) possess favourable nuclear decay properties, their relatively low β⁻ energies limit efficacy against HER-2 positive lesions.^6–8 Conversely, the use of ⁹⁰Y (β_max: 2.27 MeV, t_½ 64.1 h), a pure β⁻ emitter without γ photons poses limitations towards post-therapy imaging with [⁹⁰Y]Y-trastuzumab.⁹ The short half-life of ⁹⁰Y and the high molecular weight of trastuzumab (142.5 kDa), which slows the clearance kinetics, further reduce the therapeutic efficacy in MBC radiotherapy.^10,11

While radiolabeled monoclonal antibody fragments (MAbs) such as [¹⁷⁷Lu]Lu-(Fab)₂-trastuzumab exhibit reduced residence times,¹² their preparation requires cumbersome Fc-fragment removal from (Fab)₂, prior to coupling with the chelator moiety.¹³ These agents have also remained largely confined only to preclinical evaluation.¹⁴

Although ongoing studies with radiolabeled nanobodies or affibodies show promise due to their reduced residence time and faster in vivo clearance, extensive preclinical studies are mandatory pre-requisites for their clinical translation.^15,16 An important consideration that limits the use of radiolabeled MAbs, (Fab)₂-MAbs, nanobodies, or affibodies, is the requirement of a bifunctional chelator molecule^17,18 for complexation with ¹⁷⁷Lu and ⁹⁰Y and also size exclusion chromatography-based purification, which are time consuming and cumbersome processes.⁶ In the case of ¹³¹I radiolabeled MAbs, radiolabeling requires a time consuming and complex iodination procedures.^19–21 More recently, the fragmented antibody NM-02 radiolabeled with [¹⁸⁸Re]ReO₄⁻ has reached clinical trials in HER-2 positive breast cancer patients, though significant renal and hepatic accumulation limits its therapeutic profile.²²

To overcome such challenges, small molecules have emerged as attractive targeting agents in RPT for MBC patients. Heterocyclic scaffolds such as thiosemicarbazones and thiosemicarbazides are well-studied for their diverse biological activities,²³ including anticancer effects in leukemia,²⁴ cervical carcinoma, and histiocytic lymphoma.²⁵ Substituted derivatives bearing aryl, aroyl, and heteroaryl groups around the thiosemicarbazide scaffold, inhibit DNA topoisomerase IIα (DTII) and indoleamine-2,3-dioxygenase-1 (IDO1),²⁶ which are both upregulated in breast tumours. By disrupting DTII and IDO1, these derivatives induce apoptosis and autophagy in cancer cells.²⁷ The well-documented anticancer activity of heteroaryl compounds bearing a thiosemicarbazide scaffold provided the impetus to investigate the therapeutic potential of Rhenium-186 and Rhenium188 labelled aromatic thiohydrazide-based compounds [R–C(=S)–NH–NH₂][R–C(=S)–NH–NH₂][R–C(=S)–NH–NH₂]. These radionuclides were selected in view of their favourable β⁻ emission characteristics, with maximum β energies of 1.07 MeV for ¹⁸⁶Re and 2.12 MeV²⁷ for ¹⁸⁸Re, rendering them suitable candidates for targeted radiopharmaceutical therapy. The syntheses and characterization of the aromatic thiohydrazide compounds thiobenzhydrazide [H(tbh)], o-hydroxythiobenzhydrazide [H(htbh)], thiophen-2-thiohydrazide [H(tth)], and furan-2-thiohydrazide [H(fth)] have been reported earlier.²⁸ The spectral properties of the metal complexes formed by all four aromatic thiohydrazides with Fe(III), Co(III) and Mo(VI) have also been studied^28–30 and the antibacterial properties of H(tbh) and H(tth) have also been documented earlier.²⁹

Inspired by these findings, we focused on aromatic thiohydrazides, owing to their structural similarity with thiosemicarbazides. Among them, o-hydroxythiobenzhydrazide (H(htbh)) has not yet been explored for its anticancer activity. The ligand H(htbh) possesses a phenolic hydroxyl group and a hydrazide moiety, enabling stable chelation with transition metals, a prerequisite for in-vivo stability and tumour targeting. Hydrazide derivatives also exhibit selective interactions with tumour-associated biomolecules, possibly via nucleophilic binding or receptor-mediated recognition.

The selection of Rhenium (Rhenium-186 or Rhenium-188) as the radioisotope was driven by the favourable disposition of the array of donor atoms present in the H(htbh) ligand, which are well-suited for complexation with Re(V). Incorporating a radioactive Re isotope into the H(htbh) framework could yield a stable radiometal complex with inherent tumour-targeting capability, enabling simultaneous imaging and targeted radiotherapy of metastatic breast cancer lesions. Given the lipophilic and chelating characteristics of H(htbh) derivatives, such complexes may demonstrate favourable pharmacokinetics, including enhanced tumour uptake and rapid clearance from non-target organs, reducing systemic toxicity. The radiolabeled H(htbh) complexes with NS-donor ligands are advantageous due to their lower molecular weight compared to radiolabeled monoclonal antibodies (MAbs), resulting in faster clearance kinetics.³¹ The complexes with N and S donor groups are known to exhibit lipophilic properties.³² Consequently, the rhenium labeled H(htbh) complex is expected to possess suitable lipophilicity, which is crucial for effective distribution and localization in overexpressed DTII and IDO1 enzymes in MBC patients.^27,33

Pertinent to the field of radiopharmaceutical chemistry, Rhenium (Re) complexation of H(htbh) can be possible using either the precursor, [¹⁸⁶Re]ReO₄⁻ or [¹⁸⁸Re]ReO₄⁻. While Re-186 is available as a radiochemical typically in the form of [¹⁸⁶Re]ReO₄⁻, produced in the in-house research reactor (DHRUVA), at a thermal neutron flux of 3 × 10¹³ n cm⁻² s⁻¹, [¹⁸⁸Re]ReO₄⁻ is available as a generator eluate from the commercial ¹⁸⁸W/¹⁸⁸Re generator. Both these Re radioisotopes possess nuclear decay characteristics consisting of γ photons enabling non-invasive monitoring through nuclear imaging modalities and a medium energy (Re-186) and high energy (Re-188) β⁻ particulate emission, offering cytotoxic effects on cancerous cells.^34–37 Thus, the development of a radioactive [^186/188Re]Re-(htbh)₂ complex holds promise as a dual-function theranostic agent for managing metastatic breast cancer, combining the possibility of targeted radiotherapy and diagnostic imaging within a single molecular platform.

The radiochemical precursor [^186/188Re]ReO₄⁻, is expected to form stable complexes with [H(htbh)] in the (+5) oxidation state.³⁸ Our primary objective was to develop a stable formulation of [^186/188Re]Re-(htbh)₂ complex and carry out its characterization (physico-chemical, in-vitro and in-vivo, stability) as well as preclinical evaluation, in assessing its potential as a theranostic agent. In line with the envisaged strategy, aromatic thiohydrazide, [H(htbh)] was radiolabeled with medium specific activity, carrier added [¹⁸⁶Re]ReO₄⁻ with a view to optimizing the radiochemical synthesis of [¹⁸⁶Re]Re-(htbh)₂ for obtaining consistent and reliable radiochemical purity (RCP) of the complex. The quality control parameters of the [¹⁸⁶Re]Re-(htbh)₂ complex were thoroughly validated. Various in-vitro pharmacokinetic studies involving cell binding, cell internalization, flow cytometry and [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide)] assay (MTT assay) of the complex were carried out in breast cancer cell lines (MCF7). Its in-vivo pharmacological behaviour was studied in female SCID mice with MCF7 tumour xenografts. The results conclusively established the effectiveness of the [¹⁸⁶Re]Re-(htbh)₂ in targeting breast cancer. The specificity of the [¹⁸⁶Re]Re-(htbh)₂ complex in MBC was evaluated by carrying out various in-vitro pharmacokinetic and in-vivo pharmacological studies. The results reveal that [¹⁸⁶Re]Re-(htbh)₂ could be effectively used to target high-capacity receptor overexpression, particularly in cases of metastatic breast carcinoma.²²

The in-vivo distribution was examined in female SCID mice with MCF7 tumour xenografts; using SPECT/CT based static imaging, at 1 h post injection (p.i). The preclinical study of the [¹⁸⁶Re]Re-(htbh)₂ complex reported herein, provides confirmatory indications towards its potential as a theranostic agent for targeting breast cancers. The strategy envisaged is to extrapolate the present findings towards the development of a [¹⁸⁸Re]ReO₄⁻ based therapeutic agent having comparatively higher β_max (2.12 MeV) compared to that of [¹⁸⁶Re]ReO₄⁻, with potential application in targeted radiotherapy of MBC.

2 Experimental

2.1 Materials

2-hydroxy thiobenzhydrazide [H(htbh)] used in this studies was custom synthesized as per reported procedure.^28,29 Enriched target (¹⁸⁵Re metallic powder, enrichment > 95%, purity > 99.999%) used for irradiation, was obtained from Isoflex, Russia. Stannous chloride dihydrate (SnCl₂·2H₂O) was purchased from Sigma-Aldrich, USA. HCl (30%, Ultrapur®), ethanol (Emsure), 2,5- dihydroxybenzoic acid (Gentisic acid, purity > 98%) and ammonium perrhenate (purity: 99.999% trace-metal basis) were procured from Merck, Germany. Polyethersulfone (PES, pore size: 0.22 µm) membrane syringe filter was procured from Millipore-Merck, Germany. Non-bleeding type pH indicator strips (range: 0–14) were from Merck, India. Sterile, pyrogen-free saline was procured from Nirlife Healthcare, India. Bio-safety cabinet (ISO Class 5) was purchased from Microfilt, India, while the fume hood (lead shielded) was from Labguard, India. Analytical radio-high performance liquid chromatography (radio-HPLC) was performed using HPLC system from Knauer, Germany, equipped with UV and radioactive detectors, connected in series. Radio-thin layer chromatography (radio-TLC) and radio paper chromatography (radio-PC) were performed using miniGita equipped with NaI(Tl) radioactive detector from Bioscan, USA. Radionuclide purity (RNP) of [¹⁸⁶Re]ReO₄⁻ obtained after radiochemical processing of reactor-irradiated targets was determined by gamma ray spectroscopy, using high-purity germanium (HPGe) detector (Baltic Scientific Instruments, Russia), coupled to 4 k and 64 k multi-channel analyzer (MCA) (ITECH instruments, France). Dose calibrator (CRC 25R) was from Capintec, USA. Endotoxin limit (EL) was quantified by gel-clot BET assay method using LAL reagent from Charles River Laboratories Inc, USA, while the sterility test was performed by direct inoculation method using soya bean casein digest and fluid thioglycolate media from Himedia, India. In-vitro cell binding and internalization studies were carried out using human breast cancer cells MCF7 (ATCC®, HTB-22™). Media (IMDM), fetal bovine serum (FBS) and bovine serum albumin (BSA) for growing MCF7 cells were obtained from Gibco, Thermo Fisher Scientific USA. In-vivo distribution studies of [¹⁸⁶Re]Re-(htbh)₂ were carried out in female SCID mice aged 3 to 4 weeks [SCID-Beige mice (C.B-Igh-1b/GbmsTac-Prkdc^scid-Lyst^bg N7)] were procured from Vivo Biotech, India. Monkey kidney cell line (Vero cells) were procured from National Centre for Cell Science (NCCS), Pune, India. NaI(Tl) γ-counter was procured from Para Electronics, India. SPECT/CT imaging for SCID mice bearing tumour xenografts was carried out using NM/CT 870 DR from GE, Sweden.

2.2 Methods

2.2.1 Production and quality control of [¹⁸⁶Re]ReO₄⁻. Enriched rhenium metallic powder (∼10 mg, enrichment: >95%) was irradiated in the DHRUVA reactor, at a thermal neutron flux of 3 × 10¹³ n cm⁻² s⁻¹, for 7 days via ¹⁸⁵Re(n,γ)¹⁸⁶Re nuclear reaction. The irradiated target after cooling for 4 days, was dissolved in 5 mL of 2 M HNO₃. The radiochemical separation and purification was carried out to obtain clinical grade Na[¹⁸⁶Re]ReO₄, as per our earlier reported procedure.³⁹ The specific activity of the clinical grade product, Na[¹⁸⁶Re]ReO₄ varied in the range of 9.99–10.28 GBq mg⁻¹.

The radionuclide purity (RNP) of the produced ¹⁸⁶Re was estimated by gamma ray spectroscopy. The assay of radionuclidic (RN) purity and radioactivity of ^188/186Re formed were estimated by gamma ray spectrometry, using HPGe detector connected to a 4 K multichannel analyzer. ¹⁵²Eu standard source obtained from Amersham International (United Kingdom) was used for energy calibration of the instrument. An aliquot of the sample (37–74 KBq) was counted to estimate the percentages of ¹⁸⁸Re and ¹⁸⁶Re in the samples as well as any other RN impurities. The ¹⁸⁸Re and ¹⁸⁶Re were analysed using the 155 and 137 keV γ photo peaks, respectively.

The radiochemical purity (RCP) of the Na[¹⁸⁶Re]ReO₄ was assessed by radio-TLC and radio-PC using saline and radio-HPLC. The pH of the Na[¹⁸⁶Re]ReO₄ was determined by narrow range pH strip.

2.2.2 Radiolabeling of H(htbh) with Na[¹⁸⁶Re]ReO₄. Non-carrier-free Na[¹⁸⁶Re]ReO₄ (600 µL, specific activity: 10 GBq mg⁻¹, pH: 6.0) solution (1.6 GBq of [¹⁸⁶Re]ReO₄⁻, 159 µg, 0.855 µmol Re) was acidified with 1 M HCl (150 µL), the resultant pH of [¹⁸⁶Re]ReO₄⁻ was 2.0. Acidified [¹⁸⁶Re]ReO₄⁻ (1.6 GBq) was then added to 1436 µL of H(htbh) ligand (1436 µg) (concentration: 1 µg µL⁻¹ of solution in ethanol, 8.55 µmol, 10 equivalent of Re), containing 20 µL (1.96 mg, 8.67 µmole) of stannous chloride (SnCl₂·2H₂O dissolved in 30% HCl). The reaction was carried out in a boiling water bath (100 °C) for 4–5 minutes till the colorless reaction mixture changes to faint green color. The reaction mixture was cooled to room temperature (25 °C). On cooling, 5 mg of 2,5-dihydroxybenzoic acid (gentisic acid as a stabilizer for preventing radiolytic dissociation) in 1.0 mL of saline was added to the reaction mixture. The reaction mixture was further diluted with 11.8 mL of sterile, pyrogen-free saline (0.9% NaCl), in order that the radioactive concentration (RAC) and ethanol concentration was maintained at 107 MBq mL⁻¹ and <10% v/v respectively in the final formulation. The concentration of 2,5-dihydroxybenzoic acid in [¹⁸⁶Re]Re-(htbh)₂ was found to be 3.1 mg GBq⁻¹. The final product [¹⁸⁶Re]Re-(htbh)₂, volume: 15 mL was filtered using sterile 0.22 µm PES membrane syringe filter.

2.2.3 Synthesis and chemical characterization of [¹⁸⁵Re]Re-(htbh)₂ complex. An aqueous solution of 0.5 mL of ammonium perrhenate (concentration: 20 mg mL⁻¹ of solution in ethanol) containing 10 mg of ammonium perrhenate (54 µmole) was acidified with 1 M HCl, the resultant pH of NH₄[¹⁸⁵Re]ReO₄ was 1.0–2.0. The acidified NH₄[¹⁸⁵Re]ReO₄ was added to 2.28 mL (91 mg) of H(htbh) ligand (concentration: 40 mg mL⁻¹ of solution in ethanol, 541 µmol, 10 equivalent of ammonium perrhenate) containing 60 µL (5.88 mg, 26.01 µmole) of stannous chloride (SnCl₂.2H₂O dissolved in 30% HCl). The reaction was carried out in a boiling water bath (100 °C) for 4–5 minutes till a faint green color was observed. The non-radioactive reference complex i.e. [¹⁸⁵Re]Re-(htbh)₂ was used in ethanol medium. The UV-HPLC of the non-radioactive [¹⁸⁵Re]Re- (htbh)₂ complex was carried out using Eurosphere C-18 reversed phase column {Dimension: 300 mm (Length) × 4 mm (Diameter), particle size: 5 µm}, equipped with diode array detector (DAD)-UV (wavelength set at 310 nm), using 0.1% TFA in water and acetonitrile as solvent system. The flow-rate was maintained at 0.5 mL min⁻¹, in gradient mode (0–1 min 95% water, 1–2 min 95% to 60% water, 2–5 min 60% to 75% water, 5–8 min 75% to 85% water, 8–12 min 85% to 95% water and 12–16 min 95% water). The non-radioactive reference complex [¹⁸⁵Re]Re-(htbh)₂ was synthesized in ethanol and characterized by UV-HPLC. The UV-HPLC chromatogram exhibited a single, well-defined peak at a retention time (R_t) of 3.20 min, confirming the formation of a single complex without detectable impurities. This retention time closely matched that of the radioactive analogue [¹⁸⁶Re]Re-(htbh)₂ (R_t = 3.33 min, radio-HPLC), thereby establishing the chemical equivalence of the non-radioactive and radioactive complexes. Ethanol was removed by rotary evaporation to obtain the complex in powder form for further characterization, by FT-IR, ¹H-NMR spectroscopy and high-resolution mass spectrometry (HRMS).

Mass spectroscopy analyses of [¹⁸⁵Re]Re-H(htbh)₂ complex was carried out in Agilent LCMS Spectrometer (LCMSQTOF-G6545B). ¹H-NMR spectroscopy was performed using Bruker Advance Neo (400 MHz) instrument. Atteneuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy for [¹⁸⁵Re]Re-(htbh)₂ complex was carried out in Bruker Platinum ATR Tensor-II FT-IR spectrometer.

2.2.4 Quality control studies of [¹⁸⁶Re]Re-(htbh)₂. The pH of [¹⁸⁶Re]Re-(htbh)₂ was measured by observing the colour change (using narrow range pH strips on spotting 1–2 µL of the final product.

The radiochemical purity (RCP) of the [¹⁸⁶Re]Re-(htbh)₂ complex was initially assessed by radio-PC (Whatman 3 MM, thickness: 0.34 mm) using an ethanol/chloroform/benzene (1.5/2/1.5, v/v/v) solvent system. In addition, RCP was also evaluated by radio-TLC using saline and acetone as mobile phases. This additional assessment was undertaken because the ethanol/chloroform/benzene system did not provide clear separation of potential impurities such as [¹⁸⁶Re]ReO₄⁻ and [¹⁸⁶Re]ReO₂ from the desired product, if present.

The RCP of [¹⁸⁶Re]Re-(htbh)₂ was also ascertained by radio-HPLC using Eurosphere C-18 Reversed phase column {Dimension: 300 mm (Length) × 4 mm (Diameter), particle size: 5 µm} equipped with diode array detector (DAD)-UV (wavelength set at 310 nm) and radioactive detectors [NaI(Tl)], connected in series, using 0.1% TFA in water and acetonitrile as solvent system. The flow-rate was maintained at 0.5 mL min⁻¹ in gradient mode (0–1 min 95% water, 1–2 min 95% to 60% water, 2–5 min 60% to 75% water, 5–8 min 75% to 85% water, 8–12 min 85% to 95% water and 12–16 min 95% water).

2.2.5 Stability and transchelation studies of [¹⁸⁶Re]Re-(htbh)₂.
2.2.5.1 Saline stability. The stability of the [¹⁸⁶Re]Re-(htbh)₂ complex, in saline. on storage at room temperature (25 °C), was evaluated by radio-HPLC up to 3 h post-radiolabeling.
2.2.5.2 Phosphate buffered saline (PBS) stability. In-vitro stability of the [¹⁸⁶Re]Re-(htbh)₂ complex was evaluated in PBS (pH: 7.4) by radio-HPLC. Towards this, [¹⁸⁶Re]Re-(htbh)₂ complex (∼0.2 GBq in 100 µL) was incubated with 400 µL of PBS at 37 °C for 24 h. The stability of the [¹⁸⁶Re]Re-(htbh)₂ complex was evaluated by radio-HPLC, post 24 h of incubaton at 37 °C.
2.2.5.3 Serum stability. Towards assessing the serum stability, the [¹⁸⁶Re]Re-(htbh)₂ complex was incubated with the serum from healthy volunteers, at 37 °C for 3 h. Post incubation, serum proteins were precipitated (acetonitrile, RCF 3500 g), while serum samples were stored at −20 °C up to 18 h. The RCP of the serum samples was evaluated by radio-PC, post 18 h on storage at −20 °C.
2.2.5.4 Transchelation studies. Transchelation or ligand exchange studies of the [¹⁸⁶Re]Re-(htbh)₂ was carried out using diethylenetriaminepentaacetic acid (DTPA).
2.2.5.5 Control samples. The [¹⁸⁶Re]Re-(htbh)₂ complex (∼0.2 GBq in 100 µL) was incubated with 400 µL of PBS, at 37 °C for 24 h.
2.2.5.6 Test samples. The [¹⁸⁶Re]Re-(htbh)₂ complex (∼0.18 GBq, ¹⁸⁶Re: ∼120 µg, ∼0.64 µmoles) along with DTPA (25 mg, ∼63.55 µmoles, ∼100 equivalent of Re) was incubated with 400 µL of PBS at 37 °C for 24 h. Experiments were performed in triplicate.

Post-incubation at 37 °C for 24 h, RCP for the control and test samples were assessed by radio-HPLC.

Endotoxin limit (EL) was quantified by gel-clot BET assay method using lysate, with sensitivity 0.125 EU mL⁻¹, at 200 maximum valid dilution (MVD). The sterility testing of the [¹⁸⁶Re]Re-(htbh)₂ complex was carried out by direct inoculation method.

2.2.6 In-vitro pharmacokinetic studies of [¹⁸⁶Re]Re-(htbh)₂.
2.2.6.1 Cell binding. Human breast cancer cell line MCF7 (ATCC®, HTB-22™) was used for in-vitro cell binding study for the evaluation of [¹⁸⁶Re]Re-(htbh)₂ complex. The cell line was grown in 6 well, tissue culture dish using IMDM medium supplemented with 10% FBS in 5% CO₂ at 37 °C, under humid condition. MCF7 cells (1 × 10⁶ cells each) were subjected to [¹⁸⁶Re]Re-(htbh)₂ (0.1 pmole). The cells were incubated with the [¹⁸⁶Re]Re-(htbh)₂ complex, in cell binding buffer (IMDM with 0.2% BSA), for different time intervals (15, 30, 60 and 120 min). The incubation was followed by washing twice with ice cold 0.05 M phosphate buffered saline (PBS, pH 7.4) and centrifugation at 4000 rpm for 5 minutes. The cell pellet was then analyzed in a well-type NaI(Tl) scintillation gamma counter for determining the extent of total cell bound activity. The extent of non-specific binding (NSB) was confirmed by incubating the same number of cells and [¹⁸⁶Re]Re-(htbh)₂ with 100 nM of cold H(htbh). In brief, NSB was determined by performing the binding assay in the presence of an excess concentration (100 nM) of cold H(htbh), which competitively blocks the specific binding. Under these conditions, the remaining cell-associated radioactivity was considered to be non-specific binding. This competitive binding approach was employed to evaluate the specificity of interaction of H(htbh) with the cell line. Additionally, binding studies with monkey kidney cell line, Vero cells were carried out under identical condition, in order to further ascertain the specificity of the [¹⁸⁶Re]Re-(htbh)₂. Each treatment was completed in triplicate (n = 3), with each experiment being repeated at least twice.
2.2.6.2 Cell internalization. For internalization studies, cells were incubated with [¹⁸⁶Re]Re-(htbh)₂ as described earlier. Following incubation, the culture medium was removed and the cells were washed twice with ice-cold PBS. The adherent MCF7 cells were detached using a high-concentration EDTA solution, as per standard protocol. To remove the surface-bound radioactivity, cells were subjected to an acid wash while remaining adherent to the culture plate, using 500 µL of acidic buffer (50 mM glycine, 100 mM NaCl, pH 2.8), for 5 min at 4 °C. The acidic buffer was then removed, and the cells were washed again with cold PBS to ensure complete removal of membrane-bound activity.

Subsequently, the cells were lysed using 500 µL of 1 N NaOH to release internalized radioactivity. All washing and lysis steps were performed at 4 °C to minimize membrane disruption and prevent artifactual internalization. The radioactivity associated with the cell lysate was measured using a NaI(Tl) gamma counter and considered as the internalized fraction.

2.2.6.3 MTT assay for assessment of cytotoxicity. The cytotoxic potential of [¹⁸⁶Re]Re-(htbh)₂ and cold H(htbh) were assessed using MTT assay⁴⁰ in MCF7 cell lines. Briefly, 1 × 10⁴ cells per well were seeded in 96-well plates and incubated overnight. Cells were then exposed to increasing concentrations (5–40 µg mL⁻¹, expressed as ligand-equivalent mass) of H(htbh) and [¹⁸⁶Re]Re-(htbh)₂ for 2 h. Following exposure, the treatment medium was removed, cells were washed with PBS to eliminate unbound activity, and fresh culture medium was added. Cells were then incubated for an additional 24 h, after which cytotoxicity was assessed by the MTT assay. For radiolabeled samples, the corresponding radioactivity of ¹⁸⁶Re in [¹⁸⁶Re]Re-(htbh)₂ ranged from 0.1–0.2 MBq per well.

As [¹⁸⁶Re]Re-(htbh)₂ was dissolved in labeling buffer, the same buffer in the treatment groups was used as vehicle control. Cells were washed with PBS and incubated further for 3 h with 100 µL of 0.5 mg mL⁻¹ of MTT. The formazan crystals formed were dissolved in 100 µL of 10% SDS for 2 h and absorbance was measured at 570 nm using a micro-plate spectrophotometer (iMax™, BioRad, USA). Each treatment was carried out in triplicate with each experiment being repeated at least twice. For the MTT assay, the relative cell viability (%) compared to the control cells was calculated using the following equation:

Cell viability (% of sample) = (A_sample/A_control) × 100,

where A_sample and A_control are the absorbance values obtained for cells treated with cold H(htbh) and [¹⁸⁶Re]Re-(htbh)₂ formulations and for cells incubated with the culture medium, respectively. Histogram was plotted as different formulations versus percent cell viability.

2.2.6.4 Flow cytometry analysis. The distribution of necrotic/apoptotic and live cells was tested using flow cytometry (CyFlow® Space, Sysmex, USA) method. Towards this, MCF7 cells were exposed to bare H(htbh) for 24 h with equivalent concentration (10 and 20 µg mL⁻¹). The cells were harvested and washed with cold PBS two times. The cells were fixed thereafter by addition of 80% ethanol and incubated at −20 °C for 24 h. After incubation the cells were removed and washed with ice cold PBS (1X). For determination of Sub G1 i.e. apoptotic cell population, cell pellets were collected by centrifugation and stained with 2 mL PI staining solution for 1 h, in dark (PI stain: D/W, containing 0.1% Triton X 100, 0.1% sodium citrate, 50 µg mL⁻¹ PI and 50 µg mL⁻¹ RNase A). For each experiment, at least 10 000 cells were counted. The DNA contents were analyzed by the FlowJo 5 Express software (De Novo Software, USA).^41,42

2.2.7 In-vivo distribution studies of [¹⁸⁶Re]Re-(htbh)₂. Female SCID mice aged 3 to 4 weeks were used for the evaluation of in-vivo pharmacokinetics and bio-distribution [SCID-Beige mice (C.B-Igh-1b/GbmsTac-Prkdc^scid-Lyst^bgN7)]. MCF7 cells (2 × 10⁶ cells per mice) were injected subcutaneously into the flank region of mice. The xenografts were allowed to grow for 6 weeks until the tumour attains the necessary size (volume: 1–1.5 cm³). [¹⁸⁶Re]Re-(htbh)₂ (3.0–3.7 MBq per animal) were injected in xenografted mice through the tail vein. Post-injection, the in-vivo localization of the [¹⁸⁶Re]Re-(htbh)₂ were assessed at 0.5 h, 2 h and 24 h time intervals. Each experimental group consisted of three mice (n = 3). The accumulated radioactivity in the vital organs of the animals was measured, using a flat-bed NaI(Tl) gamma counter. Biodistribution data of accumulated activity for the organs were plotted as %ID g⁻¹. The organ/tumour ratio were plotted for major organs. The in-vivo distribution, 1 h post injection (p.i.) was examined by static imaging using a SPECT/CT camera (GE Medical, Sweden). All the biodistribution studies were carried out under Isoflurane-oxygen anesthesia, following the ethical guidelines for animal experimentation.⁶ Studies were performed in SCID mice after obtaining regulatory clearance from the Institutional Animal Ethics Committee (IAEC) and in accordance with the guidelines set for conducting experimentation in laboratory animals.

2.2.8 Regulatory clearance aspects. All animal experiments were carried out with the prior approval of the Institutional Animal Ethics Committee (IAEC), Bhabha Atomic Research Centre (BARC), Department of Atomic Energy (DAE), Government of India, following the principle of good laboratory practices (GLP).

The protocol followed for carrying out serum stability study was approved by the Institutional Scientific and Medical Ethics Committee of the Radiation Medicine Centre, BARC and was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. Blood samples, required for serum extraction, were collected from healthy volunteers after obtaining their prior written informed consent.

3 Results

3.1 Radiolabeling of [¹⁸⁶Re]Re-(htbh)₂

The radiochemical precursor [¹⁸⁶Re]ReO₄⁻, produced and processed in house, was used for radiolabeling of H(htbh) and 1.6 GBq of the complex [¹⁸⁶Re]Re-(htbh)₂ was prepared, (n = 7) with RCY (97.04 ± 0.49)%. The [¹⁸⁶Re]Re-(htbh)₂ complex was found to be clear being faint green in color, with pH in the range of 4.0–5.0, and RAC between ∼107 MBq mL⁻¹. The maximum RCY was observed at a metal (M) : ligand (L) molar ratio of 1 : 10, where M is [¹⁸⁶Re]Re and the ligand is H(htbh).

3.2 Quality control studies of [¹⁸⁶Re]Re-(htbh)₂

The RCP of [¹⁸⁶Re]Re-(htbh)₂ (n = 7) derived by radio-HPLC was (98.77 ± 0.40)% with R_t of 3.33 minutes (Fig. 1A). The RCP was also assessed by radio-PC using ethanol/chloroform/benzene (1.5/2/1.5, v/v/v) and was found to be (99.10 ± 0.66)%, having R_f of 0.59 (Fig. 1B). No activity was found in the solvent front (SF) and point of spotting (POS) ruling out the presence of [¹⁸⁶Re]ReO₄⁻ and [¹⁸⁶Re]ReO₂.

Fig. 1 Chromatograms of [¹⁸⁶Re]Re-(htbh)₂; (A) radio-HPLC (R_t: 3.33 min); (B) radio-PC (R_f: 0.59); (C) radio-HPLC (R_t: 3.52 min) at 3 h post radiolabeling, with stabilizer, on storage, at room temperature i.e. 25 °C in saline; (D) radio-PC (R_f: 0.52) after 3 h incubation (in healthy human serum, at 37 °C) & on further storage up to 18 h at −20 °C; (E) UV-HPLC chromatogram of non-radioactive [¹⁸⁵Re]Re-(htbh)₂ complex (R_t: 3.20 min); (F) radio-HPLC of [¹⁸⁶Re]Re-(htbh)₂ (R_t: 3.52 min) co-injected with Na[¹⁸⁶Re]ReO₄ (R_t: 2.62 min); (G) radio-HPLC (R_t: 3.33 min) at 24 h post incubation in PBS; (H) radio-HPLC of [¹⁸⁶Re]Re-(htbh)₂ (R_t: 3.42 min) at 24 h post incubation (37 °C) with 100 fold molar excess of DTPA.

A comparative analysis of the radio- and UV-HPLC chromatograms of [¹⁸⁶Re]Re-(htbh)₂ and its non-radioactive analogue [¹⁸⁵Re]Re-(htbh)₂ confirms the formation of the ¹⁸⁶Re-labeled complex. This is evidenced by the close retention times of the radioactive peak at 3.33 minutes (Fig. 1A) and the UV-detected peak of the non-radioactive complex at 3.20 minutes (Fig. 1E). For validating the radio-HPLC method, the [¹⁸⁶Re]Re-(htbh)₂ was spiked with known concentration of [¹⁸⁶Re]ReO₄⁻. Radio-HPLC chromatogram (Fig. 1F) exhibits two peaks with R_t 3.52 and 2.62 minutes corresponding to [¹⁸⁶Re]Re-(htbh)₂ and [¹⁸⁶Re]ReO₄⁻ respectively.

3.3 Stability and transchelation studies results of [¹⁸⁶Re]Re-(htbh)₂ complex

3.3.1 Saline stability. The retention of RCP values of (98.01 ± 0.47)%, up to 3 h, on storage, at 25 °C (room temperature), using 2,5-dihydroxybenzoic acid as stabilizer, with RAC of ∼107 MBq mL⁻¹ as observed from radio-HPLC chromatogram (R_t: 3.52 minutes, Fig. 1C, n = 7), were indicative of the in-vitro saline stability of the [¹⁸⁶Re]Re-(htbh)₂ complex.

3.3.2 PBS stability. The RCP of the [¹⁸⁶Re]Re-(htbh)₂ complex was found to be (98.27 ± 0.37)% up to 24 h, post-incubation in PBS at 37 °C using radio-HPLC. The complex stability was further confirmed by radio-HPLC of the complex, post incubation in PBS showing distinct single radioactive peak (Fig. 1G, n = 3) having R_t of 3.33 minutes.

3.3.3 Serum stability. Radio-PC chromatogram of the complex in serum samples, post-storage (at −20 °C up to 18 h) exhibit a clear and distinct single radioactive peak (Fig. 1D, n = 7) with R_f of 0.52, (solvent system: ethanol/chloroform/benzene: 1.5/2/1.5, v/v/v) thus establishing the RCP to be > 98%.

3.3.4 Transchelation/ligand exchange. Transchelation studies, were carried out post 24 h incubation of the [¹⁸⁶Re]Re-(htbh)₂ complex, at 37 °C along with DTPA (100 fold molar excess of Re). The RCP of the complex was found to be (98.09 ± 0.11)% using radio-HPLC. The stability was further confirmed by radio-HPLC of [¹⁸⁶Re]Re-(htbh)₂, post incubation, with excess DTPA wherein a distinct single radioactive peak (Fig. 1H, n = 3) at R_t of 3.42 minutes was observed. This confirmed the stability of the complex [¹⁸⁶Re]Re-(htbh)₂ in the presence of challenging ligands.

The specific activity of [¹⁸⁶Re]Re-(htbh)₂ complex (n = 7) was found to be 187.11 MBq µmole⁻¹. The EL of [¹⁸⁶Re]Re-(htbh)₂ was found to be < 25 EU mL⁻¹ (n = 7) with sterility remaining intact for all the produced batches.

3.4 Characterization studies of [¹⁸⁵Re]Re-(htbh)₂ complex

3.4.1 HRMS. The formation of the complex was confirmed by HRMS using LCMS-QTOF method. The HRMS spectrum exhibits a characteristic isotopic cluster at m/z 516.9941, 518.9960, 519.9989, 520.9924, and 521.9921 corresponding to the calculated mass of C₁₄H₁₄N₄O₂¹⁸⁵Re₁S_2.

3.4.2 FT-IR data (powder form, ν, cm⁻¹). 3100–3300 (br, νO–H), 3055 (br, νN–H), 1611 (νC = N), 1577 (δN–H), 1455 (νC–N), 1153 (νC = S), 746 (νC–S), 564 (νRe–S), 445 (νRe–N). Notably, no absorption band is observed in the 950–1000 cm⁻¹ region corresponding to ν(Re=O).

3.4.3 ¹ H NMR (400 MHz, CDCl₃ δ ppm). 11.15 (s, phenolic OH), 7.56 (d, J = 7.8 Hz Aromatic), 7.43 (t, J = 7.9 Hz, Aromatic), 7.15 (d, J = 8.4 Hz, Aromatic), 7.00 (t, J = 7.5 Hz, Aromatic).

3.5 In-vitro pharmacokinetic studies of [¹⁸⁶Re]Re-(htbh)₂

3.5.1 Cell binding and internalization studies. In-vitro cell binding and cell internalization analyses of [¹⁸⁶Re]Re-(htbh)₂ were carried out in MCF7 cells. The results showed a specific binding of (32.01 ± 3.66)% (Fig. 2A). A sizeable fraction of the cell-bound [¹⁸⁶Re]Re-(htbh)₂ (20.59 ± 3.07)% gets internalized into the MCF7 cells, upon 2 h of incubation (Fig. 2A). A significant reduction in specific bindings were observed on pre-incubation with 100 nM of cold H(htbh) (0.82 ± 0.33)%. This ascertains the specificity of [¹⁸⁶Re]Re-(htbh)₂ for the cell surface antigens. Non-specific binding studies performed using Vero cells (non-cancerous African green monkey kidney cells) showed minimal and weakly time-dependent binding, with total cell binding remaining below (2.14 ± 0.42)% even at 120 min (Fig. 2A), indicating negligible non-specific interaction.

Fig. 2 (A): Cell binding, cell internalization and surface bound–[¹⁸⁶Re]Re-(htbh)₂ in MCF7 and Vero cell line; (B): MTT-based Cytotoxicity of H(htbh) and [¹⁸⁶Re]Re-(htbh)₂ in MCF7 and Vero cell line, showing comparative cell viability following pulsed treatment; Flow Cytometry-based detection of percentage sub G1 or dead cell population (DC) in (C): Control MCF 7 cells; and in treatment groups including (D): H(htbh); (E): [¹⁸⁶Re]Re-(htbh)₂–Group 1; (F): [¹⁸⁶Re]Re-(htbh)₂–Group 2. (G): Setting for live and dead cell selection using FSC and SSC plot; and (H): histogram showing percentage of dead cell population (DC) in different treatment groups.

3.5.2 In vitro cytotoxic potency of [¹⁸⁶Re]Re-(htbh)₂. The cytotoxic potential of free H(htbh) and [¹⁸⁶Re]Re-(htbh)₂ was evaluated in MCF-7 cells using a colorimetric MTT assay, with vehicle-treated cells as controls. As shown in Fig. 2B, treatment of MCF-7 cells with [¹⁸⁶Re]Re-(htbh)₂ resulted in a clear concentration-dependent reduction in metabolically active viable cells, corresponding to increasing ¹⁸⁶Re-associated radioactive dose at higher ligand-equivalent concentrations.

Following 24 h incubation, [¹⁸⁶Re]Re-(htbh)₂ produced pronounced cytotoxicity, with cell viability decreasing from 71.9 ± 7.3% at 1 µg mL⁻¹ (∼1.1 MBq mL⁻¹) to 11.8 ± 0.6% at 30 µg mL⁻¹ (∼33.5 MBq mL⁻¹). A significant loss of viability was observed at 10 µg mL⁻¹ (∼11.1 MBq mL⁻¹) (39.2 ± 1.2%) and 20 µg mL⁻¹ (∼22.3 MBq mL⁻¹) (26.2 ± 0.4%), confirming a strong dose–response relationship (P < 0.001).

The free H(htbh) ligand showed minimal cytotoxicity, with cell viability remaining 96.8 ± 11.7% at 1 µg mL⁻¹ and 28.7 ± 9.1% at 30 µg mL⁻¹.

To assess non-specific toxicity, the MTT assay was also performed in Vero (non-cancerous) cells, where [¹⁸⁶Re]Re-(htbh)₂ maintained high cell viability (>83%) even at 30 µg mL⁻¹ (∼33.42 MBq mL⁻¹), indicating low off-target cytotoxicity. Collectively, these results demonstrate that the observed dose-dependent cytotoxicity in MCF-7 cells is predominantly driven by ¹⁸⁶Re-associated radioactivity, with minimal contribution from the ligand alone. Results are expressed as mean ± SD (n = 3).

3.5.3 Flow cytometry based cytotoxicity studies of [¹⁸⁶Re]Re-(htbh)₂. Flow cytometry-based analysis was carried out to determine the efficacy of [¹⁸⁶Re]Re-(htbh)₂ on inducing apoptotic/necrotic cell death in breast cancer cell line MCF-7, as compared with unlabeled H(htbh) at an equivalent dose. In our study, the necrotic and apoptotic cell subset with fragmented DNA were depicted as Sub-G1 cell population in Propidium iodide-based flow cytometry analysis. From Fig. 2C–F, it was observed that the percentage of Sub G1 cells were significantly higher in [¹⁸⁶Re]Re-(htbh)₂ 10 and 20 µg mL⁻¹ treatment group (58.1% and 66.18% respectively) as compared to H(htbh) 10 and 20 µg mL⁻¹ (12.05%). The percentage of Sub G1 cells in vehicle control group was 6.42%. This is apparent from the flow histogram that [¹⁸⁶Re]Re-(htbh)₂ has higher potential to induce cytotoxicity and apoptotic cell death in breast cancer cell line, MCF7, as compared to an equivalent dose of the unlabeled H(htbh).

Fig. 2G and H depicts the graphical representation of flow cytometry-based cell population selected for analysis and histogram of cell cytotoxicity analysis in MCF7 cell line by PI staining respectively. Cells were incubated for 24 h with different H(htbh) formulations including H(htbh) (10 µg mL⁻¹ and 20 µg mL⁻¹), [¹⁸⁶Re]Re-(htbh)₂ (10 µg mL⁻¹ and 20 µg mL⁻¹), and vehicle control. The “Y” axis represents the cell number and the “X” axis represents the cellular DNA content as fluorescence intensity of the PI dye.

3.6 In-vivo distribution studies of [¹⁸⁶Re]Re-(htbh)₂

A significant degree of tumour-specific uptake of [¹⁸⁶Re]Re-(htbh)₂ was observed in in-vivo distribution study, using tumour-xenografted mice model (Fig. 3A). Considerable retention of the complex was seen in the tumour induced with MCF7 cell lines. Maximum accumulation in MCF7 xenograft tumour (25.99 ± 3.55)% ID g⁻¹ was observed at 2 h p.i, with nominal depletion at latter time points. There were no reported studies on the uptake pattern of [¹⁸⁶Re]Re-(htbh)₂ in cell line xenograft. Moderate accumulation of the complex was observed in other major organs including in the kidneys (1.27 ± 0.21)% ID/g and liver (4.76 ± 0.26)% ID g⁻¹, at 2 h post injection, which showed progressive clearance with time via the renal route. Minimal accumulation of activity was seen in other organs, at 2 h post-administration which gradually reduced with time (24 h). Notably, a measurable degree of gastric uptake was observed, which is consistent with the known physiological behavior of perrhenate {[¹⁸⁶Re]ReO₄⁻), a substrate of the sodium–iodide symporter (NIS) expressed in gastric mucosa. Literature reports indicate that perrhenate exhibits prominent early uptake in stomach and salivary tissues, followed by partial washout over time. In the present study, although stomach uptake was evident, it did not show a sustained increasing trend at later time points. Furthermore, thyroid uptake remained low and non-progressive across time points.⁴³ Together, these findings suggest that any in vivo reoxidation of [¹⁸⁶Re]Re-(htbh)₂ is limited and transient, and does not lead to significant systemic release of free perrhenate.

Fig. 3 (A): Biodistribution of [¹⁸⁶Re]Re-(htbh)₂ in MCF-7 tumour xenografted female SCID mice; (B): tumour to organ ratio, 2 h post injection; (C and D): SPECT/CT images of female SCID mice with MCF7 tumour xenograft.

The radioactivity in blood was high at initial time points (4.02 ± 0.08)% ID g⁻¹ at 2 h which cleared with time (2.55 ± 0.09%) ID g⁻¹ at 24 h. Relatively less uptake in other major organs with respect to HER-2 expressing tumour is also presented in Fig. 3A. Histogram of tumour-to-organ ratios in tumour-xenografted mice, 2 h post injections are depicted in Fig. 3B.

The SPECT/CT analyses revealed uptake of the [¹⁸⁶Re]Re-(htbh)₂ complex in the MCF7 xenografted tumour of female SCID mice at 2 h p.i. as well as clearance from the plasma by 24 h (Fig. 3C and D). Moderately low amount of radioactivity was present in the liver and bladder, at longer time points. The findings from the imaging studies are in agreement with the in-vivo distribution study in mice.

3.7 Optimization of the radiolabeling process

3.7.1 Metal to ligand ratios. In order to maximize the RCY of the final product, [¹⁸⁶Re]Re-(htbh)₂, an optimization of metal to ligand molar ratios is one of the important requirement, while using non-carrier free, medium specific activity [¹⁸⁶Re]ReO₄⁻ (specific activity ranging from 9.99–10.28 GBq mg⁻¹). Hence, the radiolabeling was carried out with three different metal {[¹⁸⁶Re]Re(VII)} to ligand [H(htbh)] molar ratios, viz. (1 : 6, 1 : 8 and 1 : 10). The highest RCY and RCP of [¹⁸⁶Re]Re-(htbh)₂ was observed (Fig. 4A) on carrying out the radiolabeling at 1 : 10 metal to ligand ratios.

Fig. 4 Influence of (A): metal (Re): ligand [H(htbh)] molar ratio; (B): [¹⁸⁶Re]ReO₄⁻ pH on radiochemical purity (RCP) and radiochemical yield (RCY) of [¹⁸⁶Re]Re-(htbh)₂; (C): radio-PC of [¹⁸⁶Re]Re-(htbh)₂ in ethanol/chloroform/benzene (1.5/2/1.5, v/v/v) solvent system, showing the degradation of the product (shift in R_f from 0.59 to 0.85) under the influence of prolonged incubation time (10 min) at 100 °C; and (D): influence of storage temperature on RCP of [¹⁸⁶Re]Re-(htbh)₂ up to 6 h post formulation without gentisic acid.

3.7.2 pH of [¹⁸⁶Re]ReO₄⁻. Though rhenium and technetium have similar chemical properties by virtue of being in the same group of the periodic table, reduction of rhenium complexes are comparatively difficult, since these complexes are kinetically inert for ligand substitution.⁴⁴ While radiolabeling with [^99mTc]TcO₄⁻ are generally carried out at pH between 5-6, the radiolabeling of ligands with ^186/188Re must be achieved at a lower pH of [^188/186Re]ReO₄⁻ in the range of 1–2.⁴⁴ In the present study, the radiolabeling of H(htbh) with [¹⁸⁶Re]ReO₄⁻ was carried out at pH 2, which resulted in highest RCY and RCP of (97.04 ± 0.49)% and (98.77 ± 0.40)% respectively. However, on carrying out the radiolabeling at pH 5–6, the RCY and RCP of the [¹⁸⁶Re]Re-(htbh)₂ complex was < 70% as seen from Fig. 4B. The low RCP could be attributed to the higher pH, wherein the redox potential of Sn²⁺/Sn⁴⁺ ion is low. However, at pH of ∼2, the reduction of [¹⁸⁶Re]ReO₄⁻ increases thereby facilitating the maximum complexation, resulting in > 98% RCP of the [¹⁸⁶Re]Re-(htbh)₂ complex.^45,46

3.7.3 Incubation temperature and time. During the initial optimization of the radiolabeling protocol, reactions were carried out at 100 °C for 5 min. In addition, radiolabeling was also attempted at lower temperatures (25 °C and 50 °C) to evaluate the effect of temperature on complex formation.

At 100 °C (5 min), the formation of [¹⁸⁶Re]Re-(htbh)₂ was confirmed by radio-PC and radio-HPLC analyses. The radio-PC chromatogram exhibited a single distinct peak with an R_f value of 0.59 (Fig. 1B), while radio-HPLC showed a single radioactive peak at R_t = 3.33 min (Fig. 1A). Further confirmation was obtained from the UV-HPLC chromatogram of the non-radioactive analogue {[¹⁸⁵Re]Re-(htbh)₂}, which displayed a corresponding UV-active peak at R = 3.20 min (Fig. 1E).

In addition to chromatographic characterization, both radioactive and non-radioactive complexes exhibited a faint green colour; however, such visual observations were regarded as supplementary and interpreted in conjunction with radio-PC and radio-HPLC data.

Radiolabeling at 25 °C and 50 °C also resulted in formation of the desired complex, as indicated by radio-PC (R_f ≈ 0.50–0.60; data not shown), along with similar faint green colouration. However, significantly longer incubation times (∼90 min at 25 °C and ∼40 min at 50 °C) were required, indicating slower reaction kinetics. These conditions were therefore not considered suitable for standardization of the radiolabeling protocol.

To investigate the stability of the complex under prolonged heating, the reaction was also carried out at 100 °C for 10 min. Under these conditions, the radio-PC chromatogram exhibited a single peak with a shifted R_f value of 0.85 (Fig. 4C), indicating the formation of a different radiochemical species. Concurrently, the solution became colourless, suggesting transformation or degradation of the initially formed complex.

This observation is consistent with literature reports that Re-186 labelled thiohydrazide-based complexes generally retain their core structure at elevated temperatures for short durations; however, prolonged heating can lead to decomposition, possibly through loss of coordinated or associated water molecules and subsequent weakening or cleavage of ligand coordination bonds.⁴⁷

3.7.4 In vitro stability of [¹⁸⁶Re]Re-(htbh)₂. The [¹⁸⁶Re]Re-(htbh)₂ (RAC of the complex was ∼107 MBq mL⁻¹) was found to be stable up to 24 h post formulation on incubation at 37 °C in PBS, in presence of gentisic acid (3.1 mg GBq⁻¹). The in-vitro saline stability of [¹⁸⁶Re]Re-(htbh)₂ complex was also investigated at three different time points (1 h, 2 h and 3 h), post formulation, at room temperature without any stabilizing agent. The radio-PC (solvent: ethanol/chloroform/benzene, 1.5/2/1.5, v/v/v) chromatogram showed the formation of [¹⁸⁶Re]ReO₄⁻ via reoxidation of Re(III) and also the presence of reduced/hydrolyzed Re in the form of [¹⁸⁶Re]ReO₂ in the product. The amount of [¹⁸⁶Re]ReO₄⁻ and [¹⁸⁶Re]ReO₂ species in the product was found to increase with time in absence of any stabilizer (Fig. 4D) due to radiolysis.⁴⁸

The observations indicate that in absence of a stabilizer, the Re(III) centre in the [¹⁸⁶Re]Re-(htbh)₂ complex is not stabilized by complexation with the H(htbh) ligand and reoxidation takes place leading to the formation of [¹⁸⁶Re]ReO₄⁻ and [¹⁸⁶Re]ReO₂ species. This can be attributed to possible multiple bonds formation between nitrogen and oxygen in its +3 oxidation state.⁴⁹ However, the formation of such multiple bonds could be restricted by adding a suitable stabilizer (gentisic acid), in appropriate concentration.

4 Discussion

4.1 Quality control of the radiochemical precursor [¹⁸⁶Re]ReO₄⁻ used for radiolabeling

Despite the similarities in chemical properties of [^99mTc]TcO₄⁻ and [¹⁸⁶Re]ReO₄⁻ by virtue of their periodic analogies, an inherent challenge involved in the development of ¹⁸⁶Re based radiopharmaceuticals is that the rhenium complexes require more stringent conditions for their preparation and that the Re complexes have a higher tendency to get reoxidized to perrhenate than the analogous technetium complexes.⁵⁰ Hence it becomes essential to use [¹⁸⁶Re]ReO₄⁻ sourced from the reactor, in its purest form, free from any oxidizing impurities which are generated from different radiochemical species, if any, present in the [¹⁸⁶Re]ReO₄⁻. Generally different radiochemical species in the [¹⁸⁶Re]ReO₄⁻ are generated during the 4 d cooling period of ^186/188Re, post irradiation in the reactor. However such radiochemical species in the [¹⁸⁶Re]ReO₄⁻ are eliminated by suitable methods of optimization, radiochemical separation and purification.^39,50

The enriched ¹⁸⁵Re target (enrichment: >95%) on irradiation, produces ¹⁸⁶Re (t_1/2: 90.64 h) via ¹⁸⁵Re(n,γ)¹⁸⁶Re nuclear reaction. However, small quantity of ¹⁸⁸Re (t_1/2: 16.98 h) was also co-produced via ¹⁸⁷Re(n,γ)¹⁸⁸Re nuclear reaction during the irradiation of enriched ¹⁸⁵Re, due to existence of ¹⁸⁷Re in the target material. The presence of ¹⁸⁸Re in the [¹⁸⁶Re]ReO₄⁻ form, therefore constitutes a major radionuclide impurity which cannot be eliminated by any optimized radiochemical separation and purification process. Hence, a 4 d cooling of the irradiated target was required to eliminate the radionuclide impurity of ¹⁸⁸Re < 1% in the produced [¹⁸⁶Re]ReO₄.^50,51

Prior to radiolabeling of H(htbh) ligand with [¹⁸⁶Re]ReO₄⁻, the RCP of the precursor radiochemical viz. [¹⁸⁶Re]ReO₄⁻ was evaluated. This was necessary since the irradiated target was subjected to 4 d cooling before radiochemical separation and purification. In order to rule out the presence of any other radiochemical species in [¹⁸⁶Re]ReO₄⁻ which might interfere during radiolabeling with H(htbh) ligand, the RCP evaluation was carried out by radio-TLC in saline. The RCP of the [¹⁸⁶Re]ReO₄⁻ precursor was estimated to be ∼99% (observed activity at R_f 0.98) with insignificant activity at the POS.

4.2 Characterization of [¹⁸⁵Re]Re-(htbh)₂ complex

On the basis of the combined FT-IR, NMR and HRMS data, the ligand ortho-hydroxythiobenzhydrazide provides a suitable multidentate coordination environment capable of stabilising reduced rhenium centers, leading to the formulation of Re complex coordinated by two ligand molecules.

The mode of coordination of H(htbh) in the rhenium complex has been inferred from its known coordination behaviour and from spectroscopic evidence obtained in the present study. Complexation was carried out under acidic conditions, under which deprotonation of the phenolic –OH group is not favoured. Consistent with this, the phenolic –OH proton is clearly observed in the ¹H NMR spectrum at δ 11.15 ppm and the corresponding ν(O–H) stretching vibration (br 3100–3300 cm⁻¹) is retained in the FT-IR spectrum, indicating that the phenolic oxygen does not participate in metal coordination. The ligand therefore coordinates to the rhenium centre predominantly through the hydrazide nitrogen and the thioamide sulphur donor atoms, forming a N,S-chelate. Owing to the equilibrium between the two forms of the donor array R–C(=S)–NH–NH₂ (Form A) ↔ R–C(SH)=N–NH₂ (Form B),²⁸ present in the ligand H(htbh) the coordination of [¹⁸⁵Re]Re metal for the “Form A” (Fig. 5C) takes place involving coordinate bond with = S and covalent bond with NH₂, while for the “Form B” (Fig. 5C) the coordination of [¹⁸⁵Re]Re metal takes place involving a covalent bond with SH and coordinate bond with NH₂.^52,53 The FT-IR spectrum of the complex shows the complete absence of the characteristic ν(Re=O) stretching vibration (expected at 960–1000 cm⁻¹), confirming that the (Re=O) core is absent in the complex. The other bands viz. 1611 (νC = N), 1577 (δN–H), 1455 (νC–N), 1153 (νC = S), 746 (νC–S), 564 (νRe–S), 445 (νRe–N), observed in the FT-IR spectrum also support the speculated mode of coordination. Reduction of perrhenate during radiolabeling in the presence of SnCl₂ is therefore inferred to generate rhenium in a lower oxidation state, most plausibly Re(III), in agreement with reported rhenium chemistry under similar conditions. Comparable Re(III) complexes featuring N,S-donor ligands and solvent or water completed octahedral coordination spheres have been reported^54,55 in the rhenium coordination chemistry literature, supporting the structural assignment proposed here. Such a structure is favourable for both in-vivo stability and target engagement, as chelation through hard-soft donor combinations creates a robust coordination sphere around the metal.

Fig. 5 (A): Two possible bonding modes of Re, with Forms A and B of the ligand H(htbh), resulting in the two forms of [¹⁸⁵Re]Re-(htbh)₂; (B): HRMS spectrum of [¹⁸⁵Re]Re-(htbh)₂ complex, exhibiting prominent molecular ion peaks (m/z 516.9941, 518.9960 and 520.9924).

Based on the Q-TOF HRMS data, the complex is best described as having a coordinated Re(III) centre in which two ligands bind through N and S donor atoms, while the remaining coordination sites are likely occupied by solvent molecules and/or coordinated water. The proposed structure (Fig. 5A) is consistent with this coordination description and is supported by the available spectroscopic data.

The proposed mode of complexation is supported by Q-TOF HRMS analysis of the non-radioactive [¹⁸⁵Re]Re-(htbh)₂ complex (C₁₄H₁₄N₄O₂¹⁸⁵ReS₂). The spectrum exhibits a prominent molecular ion peak at m/z 518.9960, corresponding to [¹⁸⁵M]⁺, along with a peak at m/z 519.9989 assigned to the protonated species [¹⁸⁵M + H]⁺. In addition, a peak at m/z 516.9941, attributed to the doubly deprotonated species [¹⁸⁵M–2H]²⁻, was observed. The formation of such ions in Q-TOF HRMS is commonly influenced by solvent polarity, analyte concentration, and instrumental parameters such as cone voltage.^56,57

Additional peaks at m/z 520.9924 ([¹⁸⁷M]⁺) and 521.9921 ([¹⁸⁷M + H]⁺) were also detected, consistent with the natural isotopic distribution of rhenium (¹⁸⁵Re: 37.40%; ¹⁸⁷Re: 62.60%). The ∼2 unit mass shift relative to the corresponding ¹⁸⁵Re peaks confirms the contribution of the ¹⁸⁷Re isotope.

Overall, the HRMS data are consistent with the formation of [Re(htbh)₂], comprising two coordinated H(htbh) ligands. Although single-crystal X-ray diffraction would be required for definitive structural elucidation, the structure proposed in Fig. 5A represents the most plausible arrangement supported by the available spectroscopic data.

Notably, no solvent- or water-coordinated species were observed in the mass spectrum. This is expected, as HRMS measurements are performed under high vacuum, where weakly coordinated solvent molecules are readily dissociated during ionization.⁵⁸ Furthermore, the orthogonal sampling interface of Q-TOF instruments limits the transmission of solvent clusters into the mass analyser.⁵⁹

4.3 In-vitro pharmacokinetic studies of [¹⁸⁶Re]Re-(htbh)₂

The biological relevance of the structural characteristics of the resulting [¹⁸⁶Re]Re-(htbh)₂ complex, particularly its stability and potential lipophilicity conferred by the aromatic thiobenzyl moiety, may favour its passive accumulation and retention in tumour tissue,⁶⁰ while the presence of hydrazide functionality may also allow for specific interactions with biomolecular targets overexpressed in malignant cells.

The present study highlights the potential of [¹⁸⁶Re]Re-(htbh)₂ as a selective radiolabeled agent for breast cancer targeting. The in-vitro binding and internalization studies in MCF7 cells demonstrated efficient and specific uptake of [¹⁸⁶Re]Re-(htbh)₂, with minimal non-specific interaction in non-cancerous Vero cells. Competitive inhibition with unlabeled H(htbh) confirmed receptor-mediated binding, underscoring its specificity towards receptor overexpression in breast cancer cells. The cytotoxic efficacy of [¹⁸⁶Re]Re-(htbh)₂ was evident from MTT assays, where the radiolabeled formulation was found to significantly reduce MCF7 cell viability, while the unlabeled H(htbh) exhibited negligible cytotoxicity. Flow cytometry-based analysis further revealed substantial apoptotic cell death induced by [¹⁸⁶Re]Re-(htbh)₂, as reflected by the marked increase in Sub-G1 cell population compared to controls. In-vivo biodistribution and SPECT/CT imaging studies corroborated these findings, demonstrating preferential tumour accumulation of [¹⁸⁶Re]Re-(htbh)₂ with sustained retention up to 24 hours. Favourable tumour-to-organ ratios and clearance via renal pathways further support its potential for targeted radiotherapy. Minimal off-target accumulation in non-target organs such as liver and kidneys indicates a promising safety profile. Notably, a measurable degree of gastric uptake was observed, consistent with the known physiological behavior of perrhenate {[¹⁸⁶Re]ReO₄⁻} via sodium–iodide symporter (NIS)-mediated transport in gastric mucosa. However, the absence of a progressive increase in gastric uptake, together with low and non-progressive thyroid accumulation, indicates that any in vivo reoxidation of [¹⁸⁶Re]Re-(htbh)₂ is limited and transient.⁴³ Collectively, these results establish [¹⁸⁶Re]Re-(htbh)₂ as a promising agent for targeted radionuclide therapy of breast cancer, warranting further preclinical evaluation.

It is well documented that high specific activity of [¹⁸⁶Re]ReO₄⁻ (111–148 GBq mg⁻¹) is a pre-requisite for formulation of multiple clinical therapeutic doses (single patient dose: ∼2.8 GBq) of any [¹⁸⁶Re]Re based complexes viz. [¹⁸⁶Re]Re-HEDP, [¹⁸⁶Re]Re-BMEDA, [¹⁸⁶Re]Re-bivatuzumab.²² However, the primary aim of our work was to evaluate the specificity of the complex [¹⁸⁶Re]Re-(htbh)₂ towards targeting the overexpressed receptors in MBC. Hence, we carried out the radiolabeling of H(htbh) with [¹⁸⁶Re]ReO₄⁻ having medium to low specific activity of ∼10 GBq mg⁻¹.

5 Conclusion

In conclusion, we report the successful development of a robust and reproducible radiolabeling strategy for the aromatic thiobenzhydrazide derivative, o-hydroxythiobenzhydrazide, using [¹⁸⁶Re]ReO₄⁻ to yield the stable complex [¹⁸⁶Re]Re-(htbh)₂. The radiocomplex demonstrates high radiochemical purity and stability, along with favourable biological characteristics, including significant cytotoxicity and binding affinity in MCF-7 cells. Notably, in vivo evaluation in MCF7-induced xenograft models revealed sustained and selective tumour accumulation, as confirmed by SPECT/CT imaging.

Collectively, these findings highlight the potential of [¹⁸⁶/¹⁸⁸Re]Re-(htbh)₂ as a promising radiotheranostic agent for metastatic breast cancer, combining diagnostic capability with therapeutic relevance. Importantly, the optimized radiolabeling protocol is readily translatable to [¹⁸⁸Re], thereby facilitating its broader clinical applicability. Overall, this study provides compelling preclinical evidence supporting further investigation and development of this radiocomplex toward targeted radionuclide therapy of metastatic breast cancer.

Author contributions

A. M. performed radiolabeling of H(htbh) with [¹⁸⁶Re]ReO₄, physicochemical and biological quality control of [¹⁸⁶Re]Re-(htbh)₂, synthesis and characterization of the cold [¹⁸⁵Re]Re complex, and made the first draft of the manuscript. A. C. conducted the in-vitro and in-vivo studies of [¹⁸⁶Re]Re-(htbh)₂ and wrote the related manuscript sections. T. U. carried out SPECT/CT imaging of tumour-xenografted SCID mice. S. S. standardized and validated radio-HPLC methods. M. T. performed culture work of MCF7 and other cell lines. S. C., R. C., and K. V. V. produced ¹⁸⁶Re from enriched ¹⁸⁵Re targets, processed and purified it to [¹⁸⁶Re]ReO₄⁻. S. K. conducted and recorded ¹H-NMR spectra of [¹⁸⁵Re]Re-(htbh)₂. D. S. and S. G. (Snigdha Gangopadhyay) carried out the chemical synthesis of the ligand, S. G. (Sanchita Goswami) provided the resources and arranged for funding from CSIR for carrying out the studies related to synthesis and characterization of the cold ligand. S. G. (Sanchita Goswami), S. G. (Snigdha Gangopadhyay) and P. K. G. jointly conceived the work. S. B. conceptualized the project and the overall radioactive studies, led the project, ensured isotope availability, provided resources and overall administrative oversight, and reviewed and edited the final manuscript.

Conflicts of interest

There are no conflicts of interest to declare.

Data availability

All data supporting the findings of this study are included within the article. Raw datasets underlying the analyses are available from the corresponding author upon reasonable request. Source data are provided with this manuscript to facilitate transparency and reproducibility.

Acknowledgements

The authors sincerely thank the staff of the Hospital Radiopharmacy Section, Scintigraphy Section and the Animal House Facility of the Radiation Medicine Centre, Bhabha Atomic Research Centre (BARC), for providing the necessary facilities to carry out this work. The assistance of Professor D. Maiti, Department of Chemistry, IIT Bombay, in acquiring HRMS data is gratefully acknowledged. We also thank Bio-Organic Division, BARC, for providing the FT-IR data. The authors also acknowledge the support and encouragement of the Director, Medical Group, BARC. One of the authors, Sanchita Goswami acknowledges the support from CSIR [Sanction No.: 01(2869)/17/EMR-II] and is thankful to DST-FIST, DST-PURSE, and CAS(V) of the Department of Chemistry, University of Calcutta, for access to instrumental facilities, required for synthesis of the non-radioactive ligand.

Notes and references

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Footnote

† Equal contribution first authors.

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