Preclinical evaluation of 99m Tc-labeled LHRH as GnRH receptor imaging

doi:10.21203/rs.3.rs-3813155/v1

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Preclinical evaluation of ^99m Tc-labeled LHRH as GnRH receptor imaging

https://doi.org/10.21203/rs.3.rs-3813155/v1

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Breast cancer stands as the principal cause of cancer-related mortality among women in the developed world. Notably, receptors of Luteinizing Hormone-Releasing Hormone (LHRH or GnRH) exhibit overexpression in this malignancy. This study aimed to develop a new molecular prove [^99mTc] Tc-HYNIC-GSG-LHRH(D-Lys6)/Tricine/Nicotinic Acid (NA) as a novel molecular imaging agent for breast cancer. HYNIC-GSG-LHRH(D-Lys6) was acquired and radiolabeled with [^99mTc] Tc. The radiochemical purity and stability in different conditions were evaluated by Instant thin-layer chromatography (ITLC) and High performance liquid chromatography (HPLC). Lipophilicity was performed by the distribution coefficient test. In vitro cell binding studies were performed in different human and mice breast cancer cell lines (MDA-MB-231, MDA-MB-435, MCF-7, BT474 and 4T1) as well as in normal murine fibroblasts (NIH-3T3) and CHO-K1 as negative control. Biodistribution studies were performed in normal Balb/c mice and 4T1 tumor-bearing Balb/c mice up to 6 h post-injection. SPECT/CT images were performed in 4T1 tumor-bearing Balb/c mice up to 5 h post injection (p.i). [^99mTc] Tc-HYNIC-GSG-LHRH(D-Lys6)/Tricine/NA complex was labeled with a high radiochemical purity (> 98%) and stable up to 4 hs. It presented a good hydrophilicity (Log P = − 2.82 ± 0.04). It also yields a relevant and specific binding in all breast cell lines evaluated. Biodistributions studies showed a high renal clearance and low unspecific binding (< 2% Act/g) in most organs, as well as appreciable tumor uptake (5.8 ± 0.5%ID/g 1 h p.i) and high tumor/muscle ratio (maximum of 30.5 ± 11.2 at 1 h p.i). SPECT/CT of 4T1-tumor bearing Balb/c mice images revealed similar results to biodistribution studies, with a Tumor/Non-Tumor ration of > 3.5 at all times evaluated. In vivo blockage studies showed specificity for the LHRH-R; demonstrating a substantial potential for in vivo visualization of LHRH-R expression in breast cancer.

Biological sciences/Cancer/Breast cancer

Biological sciences/Cancer/Cancer imaging

Cancer persists as a formidable global public health challenge, solidifying its position as a prominent cause of mortality. In 2021, it was reported to be either the first or second leading cause of death prior to the age of 70 in 121 out of 183 countries, predominantly in developed nations[1]. The escalating incidences and mortality rates of cancer worldwide are fueled by an interplay of factors, such as demographic shifts, aging, and changes in risk factor prevalence and distribution, often dovetailing with socio-economic trends [2, 3].

Breast cancer conspicuously emerges not only as the most commonly diagnosed but also as the principal cause of cancer-related deaths among women [1]. The disease's development is influenced by various risk factors including genetics, late menopause, benign hyperplasia, and the use of estrogens or oral contraceptives. It categorically bifurcates into hormone-dependent and hormone-independent types. Notably, tumors expressing estrogen (33%) or both estrogen and progesterone receptors (50–70%) have shown favorable responses to hormone therapy [4].

Timely detection remains pivotal, dramatically enhancing prospects for successful treatment and potential cure, underscoring the paramount importance of education, prevention, early diagnosis, and screening. Our expanding comprehension of tumor causes and developmental mechanisms, particularly at the molecular and genetic levels, coupled with an understanding of their heterogeneity, has catalyzed the advent of novel, efficacious imaging techniques. These methods transcend mere anatomical description, facilitating cellular and molecular-level tissue characterization [5].

It is well known that early detection greatly increases the chances of successful treatment and cure. This includes both education and prevention as well as early diagnosis and screening. We now have a better understanding of the cause and develop mechanism of the tumors, both at the molecular and genetic level, as well as its heterogeneity, which have led to the development of new, fully functional, imaging techniques, capable of detecting tissue characteristic at the cellular and molecular level and not just provide a simple anatomical description [5].

In recent decades, the identification of specific receptors for peptide hormones, which are overexpressed on neoplastic cells, has propelled advances in personalized therapy and treatment. The development of new cytotoxic and radiolabeled hormone analogs, capable of localizing tumors and delivering precisely targeted therapy, has demonstrated efficacy in preclinical trials [6, 7].

In the early 1970s, the research teams of Schally and Guillemin unraveled the structure of the Gonadotropin-Releasing Hormone (GnRH or LHRH), laying a foundational understanding of ovarian and testicular function in humans and subsequently being recognized with a Nobel Prize [8, 9]. This hormone decapeptide is known to stimulate the generation of Luteinizing Hormone (LH), Follicle Stimulating Hormone (FSH), estradiol, and testosterone in organisms under normal conditions [10], by binding to it receptor (GNRH-R or LHRH-R)

In mammals, there are two major forms of the receptor, each encoded by a separate gene; besides, there are different isoforms of the receptor, encoded by alternative splicing. These are part of the transmembrane G-coupled protein receptors, which activate the phosphoinositide pathway one bound to its ligand, leading to different outcomes in the cell once it´s bound to its specific ligand [11–15].

It has been proved that the exogenous injection of the decapeptide in patients with hypothalamic deficiencies stimulates the production of estradiol and testosterone ^10–16. This was the basis for the development of more potent LHRH analogs (Leuprolide, Decapeptyl and Buserlin) for pharmaceutical use, which in the long term led to the suppression of the axis –creating a new therapeutic target-. The use of antagonist has also been successfully reported as a therapeutic agent (Cetrorelix, Ganirelix), leading to chemical castration [11, 16–19].

The low toxicity of the compounds made them optimal for treatment of patients with endocrine-dependent tumors; and animal studies demonstrated the regression of hormone-dependent breast and prostate tumors using these agents; these results were later successfully translated to clinical trials [20–25].

Given that LHRH receptors and mRNA are overexpressed in several solid tumors — approximately 80% of human endometrial and ovarian cancers, 86% of prostate cancers, and about 50% of breast cancers, including triple-negative breast cancer, as well as in various other cancer types [18, 26–29] — the broad and successful use of LHRH analogs and agonists for treatment grounds the rationale for utilizing this decapeptide as an imaging agent [30, 31]. Previous research on the labeling of LHRH analogs with various agents, including [⁶⁸Ga]Ga, [¹²³I]I, [¹⁸F]F, [¹¹¹In]In, and [^99mTc]Tc, has provided invaluable insights, although none have yet transitioned into the clinical phase [32–39].

It has been recorded that the motifs pGlu1-His2-Trp3 and Arg8-Pro9-Gly10-NH2 of the peptide LHRH (pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) are essential for receptor binding. It is also documented that the change of Gly6 with a D-amino acid results in an increase in the binding union, a shorter blood retention time and better therapeutic outcomes [25].

The primary aim of this research entailed formulating a novel [^99mTc]Tc radiopharmaceutical for the molecular imaging of breast cancer. Our strategy honed in on employing a Luteinizing Hormone-Releasing Hormone (LHRH) analog scaffold, specifically, HYNIC-GSG-LHRH(D-Lys6) (C72H100N24O18), leveraging its potential to accurately detect LHRH receptor (LHRH-R) expression. To implement this, we meticulously introduced a D-Lysine substitution at the sixth position (D-Lys6) which not only preserved the integral receptor binding affinity but also provided a pivotal anchor point for a flexible linker (GSG) and our chosen chelator (HYNIC)

HYNIC-GSG-LHRH (D-Lys6) analog Synthesis

The molecular weight of HYNIC-GSG-LHRH(D-Lys6) (Supplementary Information, Figure S1) analog was confirmed by Mass Spectrometry (MS) and UV-HPLC performed by SIQUIMIA S.A, where the relation m/z of LHRH analog of (MH) ⁺ was of 1454.6 Da. This analog was conjugated with our chelator (HYNIC) by a GSG linker; after which new studies of MS and UV-HPLC were performed, finding a m/z relation of (MH)⁺ of 1589.71 Da (Supplementary Information, Figure S1 and S2).

Peptide radiolabeling and purification and Log P

HYNIC-GSG-LHRH(D-Lys6) was labeled with radiochemical purity of 99.8 ± 0.3% (RT-HPLC) and 99.5 ± 0.2 (ITLC-SG) when using Tricine/NA as co-ligand (mean ± SD, n = 4), with specific activities average between 1.85–5.55 MBq/µg, and a maximum of 18.5 MBq/µg (Fig. 1).

The hydrophilic character of the [^99mTc] Tc-HYNIC-GSG-LHRH(D-Lys6)/Tricine/NA complex was examined. The octanol-water partition coefficient values obtained was − 2.82 ± 0.04, showing a high hydrophilicity.

In vitro stability assays

The complex labeled using Tricine/NA as co-ligands exhibited stability > 95% radiochemical purity in PBS over a 4 h period, similar results were observed when studying the protein binding in human serum, which shown a low protein binding of 4.49 ± 0.12 up to 4 h and a radiochemical purity by HPLC-RT of > 98%.

Upon subjecting the [^99mTc] Tc-HYNIC-GSG-LHRH (D-Lys6)/Tricine/NA complex to various L-cysteine concentrations (0.1 mM, 1.0 mM, and 10.0 mM), the compound exhibited remarkable stability up to 4 hours, maintaining a radiochemical purity exceeding 95% across all evaluated concentrations.

Specific binding and internalization

In the control line (NIH-3T3), membrane-bound radiopeptide remained consistently below 0.5%, and internalization remained below 0.16% across all time points, mirroring the characteristics of the LHRH receptor-negative CHO-K1 cell line.

Conversely, all breast cancer cell lines examined displayed membrane-bound radiopeptide levels always exceeding 3%, with internalization levels surpassing 0.4%.

Notably, MCF-7, BT-474, and 4T1 cell lines exhibited the highest binding capacity for the [^99mTc] Tc-HYNIC-GSG-LHRH(D-Lys6)/Tricine/NA complex, with nearly 15% of the activity remaining membrane-bound up to 60 minutes and approximately 1.5% undergoing internalization, as depicted in Figs. 2 and 3. They were followed by MDA-MB 231 and, finally, MDA-MB 435.

Specific binding of labeled compounds was evaluated in all cell lines, choosing the time of the maximum peptide-bound for each cell line. We found all cell lines evaluated, except for negative control (NIH-3T3 and CHO-K1) presented an inhibition of over 40% (Fig. 4).

Biodistribution studies

The in vivo properties of [^99mTc] Tc-HYNIC-GSG-LHRH(D-Lys6)/Tricine/NA complex were evaluated both in normal female Balb/c and 4T1 tumor bearing mice up to 6 h p.i Supplementary Information, Table S1 and Fig. 5 shows results of the biodistribution studies in normal Balb/c mice, showing a clear renal clearance and low uptake in the rest of the organs (< 2%).

On the other hand, the evaluation on 4T1 bearing mice (Supplementary Information, Table S2, and Fig. 6), we observed the same biodistribution pattern (a clear renal clearance, followed by minimal accumulation-less than 5% ID/g uptake- in the rest of the organs), with a rapid tumor intake (5.8 ± 0.5%ID/g, 3.7 ± 0.4%ID/g and 3.1 ± 0.9%ID/g at 1,3 and 6 h pi respectively). Tumor-to-muscle uptake ratios of radiotracer were 30.5 ± 11.2, 14.5 ± 6.6, 13.7 ± 1.4 at 1,3 and 6 h p.i respectively, tumor-to-blood ration was also consistently over 2.

The blocking of receptors with predosing of unlabeled peptide significantly reduced tumor uptake at 3 h p.i (3,7 ± 0.4 vs 1.7 ± 0.9, P ≤ 0.05, Student t) by over 50% whereas the other normal organs affected was the kidney (55.1 ± 8.6 vs 33.4 ± 12.3, P ≤ 0.0001, Student t).

SPECT/CT imaging

SPECT/CT images of [^99mTc] Tc-HYNIC-GSG-LHRH(D-Lys6)/Tricine/NA complex was conducted in Balb/c female mice models bearing intramammary 4T1 breast cancer cell line xenografts. The 4T1 xenografted tumors were detectable in the SPECT images. The tumor-to-non-tumor ratios (T/NT) exceeded 3 at all-time points evaluated (3.81 ± 0.06; 8.0 ± 1.8 and 12.7 ± 3.9, at 1,3 and 5 h respectively, calculated manually by volume of interest (VOIs) (Fig. 7).

Blocking receptor experiments with the pre-dosing of unlabeled peptide (100 µg/100 µL, administered 30 min prior to injection) at the 1, 3, and 5-hour time points reveled discernible differences at the 3-hour between non blocked and blocked tumor respectively, and T/NT ratios show a significant difference at the 5-hour time frames (12.7 ± 3.9 vs. 6.1 ± 0.8, Student's t-test, P ≤ 0.05), calculated manually by volume of interest (VOIs).

The decapeptide analog HYNIC-GSG-LHRH (D-Lys6) (C₇₂H₁₀₀N₂₄O_18,) was synthetized with a purity exceeding 95% and a molecular weight of of 1589.71 g/mol (Supplementary Information, Figure S1 and S2). It was successfully radiolabeled with [^99mTc] Tc using Tricine and Nicotinic acid (NA) as co-ligands, with a high radiochemical purity (> 98%, Fig. 1) and high specific activity (maximum 18.5 MBq/µg).

It is of vital importance for a tumor-targeting peptide to display proteolytic and radiolytic stability over time, especially in human plasma, in order to release the maximal action over the tumor. [^99mTc] Tc-HYNIC-GSG-LHRH (D-Lys6)/Tricine/NA complex showed remarkable stability up to 4 h in all conditions evaluated, maintaining a radiochemical purity exceeding 90%. These results are similar to the ones achieved by Masteri Farahani A et al [38] when radiolabeling a similar LHRH analog ([^99mTc] Tc -HYNIC-Ahx-DLys6GnRH) with [^99mTc] Tc via HYNIC, although this study used Tricine and EDDA as co-ligands.

[^99mTc] Tc-HYNIC-GSG-LHRH(D-Lys6) with Tricine and NA as co-ligands exhibited specific binding to the LHRH receptors in all breast cancer cell lines evaluated. These results were statistically significant when compared to the negative control CHO-K1 cell line (Figs. 2 and 3) [35]. Previous studies with similar molecules, such as Okarvi SM et al [39] with [¹⁷⁷Lu]Lu/[⁶⁸Ga]Ga radiolabeled leuprolide also found considerable binding to the cell lines evaluated, in the order of nM ratio, although we could not replicate the high internalization of this biosimilar. Blocking studies also demonstrated the specificity of binding to the LHRH receptor across all evaluated breast cancer cell lines, with a significant decrease (Student t) compared to the non-blocked counterpart (Fig. 4). It is worth noting that blocking excess was of around 400X of radiolabeled compound, newer studies recommend using 500-1000x; better results could be obtained this way [38]. Given that the percentage of internalization was notably low (below 2% in all instances), we opted not to perform an efflux assay, deeming it to be without substantive purpose under these conditions.

We demonstrate that in vivo, the peptide exhibits a clear renal clearance (highest activity observed in kidneys, bladder, and urine) while the rest of the organs show no discernible radiotracer uptake (%ID/g less than 2%) (Fig. 5).

As we expected by the in vitro studies, the peptide exhibited a rapid tumor intake (5.8 ± 0.5%ID/g maximum at 1 h p.i) (Fig. 6). The tumor-to-muscle uptake ratios of radiotracer were also high, with 30.5 ± 11.2 maximum at 1 h pi.; tumor-to-blood ration was also considerably high. These results are similar to the ones previously reported by Zoghi et al [34] with [¹⁷⁷Lu] Lu-DOTA‐GnRH‐I agonist in 4T1 xenografts.

Rapid blood clearance was seen, and low activity was retained in blood at 6 h p.i (0.7 ± 0.1%ID/g). Comparisons between normal Balb/c and 4T1 tumor bearing mice (Student-t) only demonstrate a significant difference between kidney at 1 hs and 6 hs p.i (38.4 ± 3.5 vs 72.8 ± 15.8 and 30.53 ± 0.04 vs 57.5 ± 5.4 %ID/g, P ≤ 0.0001, Student t), stomach at 6 h p.i (,3 ± 0.2 vs 4.1 ± 1.3, P ≤ 0.01,Student t) and urine plus bladder and gastro intestinal tract (GIT) at 1 h pi (47.3 ± 0.5 vs 10.4 ± 1.3 and 1.6 ± 0.6 vs 3.7 ± 0.6 %ID, P ≤ 0.0001, Student t) while the rest of the organs did not show significant differences (P > 0.05, Student t). Blocking studies with previous injection of cold peptide confirmed that the tumor uptake of the radiolabeled peptide was LHRH-R specific. These findings once again affirm the specificity of our radiotracer for in vivo LHRH-R receptor argeting.

The initial assessment of the tumor imaging potential of the [^99mTc] Tc-HYNIC-GSG-LHRH(D-Lys6)/Tricine/NA complex by SPECT/CT in 4T1 xenografted tumors showed a detectable tumor, despite observing a high concentration of radioactivity in the kidneys and urinary bladder, consistent with biodistribution study findings. Note that some hot spots are also observable in some of the tumors, though their inconsistency makes it hard to analyze them as a group (Fig. 7). Notably, other non-target organs exhibited low levels of radioactivity uptake. The tumor-to-non-tumor ratios (T/NT) were also high, with a maximum 12.7 ± 3.9 at 5 h p.i

To further confirm the specificity of our radiotracer for LHRH-R receptors in vivo, we conducted blocking receptor experiments with the pre-dosing of unlabeled peptide. The SPECT images revealed a significant blockage of the tumor at the 5-hour time frames (12.7 ± 3.9 vs. 6.1 ± 0.8, Student's t-test, P ≤ 0.05), calculated by volume of interest (VOIs). These findings once again affirm the specificity of our radiotracer for in vivo LHRH-R receptor targeting.

In this study, the successful optimization of labeling the LHRH analog, HYNIC-GSG-LHRH(D-Lys6), with [^99mTc] Tc was efficiently and swiftly achieved. Contrasting with prior direct and indirect radiolabeling of LHRH peptides [32–39], the utilization of the D-Lys6 analog and the GSG-HYNIC motif enabled the [^99mTc] Tc-HYNIC-GSG-LHRH/Tricine/NA complex to achieve optimal radiochemical purity (≥ 98%), along with high stability in PBS, human serum, and during the competition assay, establishing it as a potent radiopharmaceutical peptide for diagnostic purposes.

Moreover, the receptor specificity of [^99mTc] Tc-HYNIC-GSG-LHRH(D-Lys6)/Tricine/NA was retained during evaluations across diverse breast cancer cell lines, exhibiting a substantial membrane-bound activity that, in all instances, was blocked (≥ 40%) when contested with the free agent. The biodistribution profiles observed were analogous to those found in preceding studies [32–35, 37, 39], demonstrating explicit renal clearance and bladder uptake, albeit with seemingly lower stomach absorption using this compound. The minimal absorption by the thyroid, indicative of effective labeling technique and stability of the imaging agent, is also noteworthy. SPECT/CT imaging disclosed significant and specific tumor uptake compared to non-targeted organs, accentuating only the compound's elimination route (kidney, bladder), thereby validating its efficacy as an imaging agent. Our group is presently developing xenografted models of human breast cancer cell lines (MCF-7 and BT-474) with the aim of evaluating our new radiotracer in these future models.

In summary, the LHRH peptide and its analogs demonstrate potential utility as breast carcinoma imaging agents. Nonetheless, additional preclinical investigations are required to fully unravel the extensive potential of these molecules.

Reagents

All chemicals, unless otherwise stated were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Water was purified and deionized (18 MΩ/cm2) via a Milli-Q water filtration system (Millipore Corp., Milford, MA). [⁹⁹Mo]Mo-[^99mTc]Tc generators were purchased from TecnoNuclear (Argentina). Radioactivity was counted in a CRC7 Capintec dose calibrator and in a solid scintillation counter detector with 3"x3" NaI(Tl) crystal associated with a single channel analyzer (ORTEC, Oak Ridge, TN). High performance liquid chromatography (HPLC) was performed on an Agilent 1200 Series Infinity Star equipped with GABI detector, a UV detector and a C18 column (Restek, Ultra C18, 250 mm x 4.6 x 10 microns). Radio-TLC detection was accomplished using instant thin-layer chromatography (ITLC) on silica gel strips (Pall Corporation, Port Washington, NY).

HYNIC-GSG-LHRH(D-Lys6) analog Synthesis

The decapeptide analog HYNIC-GSG-LHRH (D-Lys6) (C₇₂H₁₀₀N₂₄O_18, as shown in Supplementary Information, Figure S1) was purchased SIQUIMIA S.A (Uruguay) and pre-derivatized with the bifunctional agent Succinimidyl-hydrazinonicotinamide (HYNIC), with a purity exceeding 95% and a molecular weight of of 1454.6 g/mol (Supplementary Information, Figure S2) HYNIC was synthesized in our laboratory (as previously described in García et al [40]). It is important to note that the peptide, prior to the addition of HYNIC, exhibited a molecular weight of 1454.6 g/mol (as illustrated in Supplementary Information, Fig. 2). The HYNIC-GSG-LHRH (D-Lys6) peptide was provided in a lyophilized form.

Peptide radiolabeling and purification.

We used 150 µL of Tricine solution (100 mg/mL in H₂O, 83.7µmol) and 100 µL of nicotinic acid solution (20 mg/mL in H₂O, 16.2 µmol) to 20µg of HYNIC-GSG-LHRH(D-Lys6) (1µg/µL), followed by 15 µL of a fresh solution of SnCl₂ (1 mg/mL in 0.1 M HCl, 66.5 nmol) as a reducing agent and 37–111 MBq of [^99mTc]TcO₄⁻(up to 500 µL). The mixture (pH 4.5) was incubated for 30 min at 50°C.

The radiolabeled peptides were purified by HPLC on a C18 reverse phase column with a 20 min gradient consisting of the mobile phases: A) water/0.1% Trifluoroacetic Acid (TFA) and (B) acetonitrile/0.1% (TFA) (0–5 min 0–45% B, 5–10 min 45–65% B, 10–20 min 100% B). Radiochemical purity was determined by HPLC and instant thin layer chromatography (ITLC) on silica gel (ITLC-SG) as previously described. ITLC mobile phases were Acetone to determine free [^99mTc] TcO₄⁻ (Rf = 1) and methanol/1 M ammonium acetate (1/1) to determine ^99mTc-colloid (Rf = 0). Subsequently, the purified fraction by HPLC was diluted with approximately 500 µL of 0.1 M phosphate buffer saline (PBS) pH 7.4 and the acetonitrile was reduced by applying a N₂ flow. Finally, 0.1 M PBS pH 7.4 was added as necessary to achieve a neutral pH.

Calculation of Log P

Tubes containing 500 µL of octanol and 500 µL of the radiolabeled peptide (74 MBq) dissolved in 0.1 M PBS pH 7.4 were vigorously agitated for 1 min and centrifuged at 14,000 RPM for 10 min. Three fractions of 100 µL were collected from both phases, and the radioactivity counted in a NaI well counter. The partition coefficient (P) was calculated as follows: P = Counts Per Minute (cpm) in octanol / cpm in PBS and expressed as Log P (mean ± SD, n = 3). cpm = counts per minute

In vitro stability assays and protein binding studies.

In vitro stability was evaluated in PBS and in human serum (Sigma Aldrich) at 37 ºC up to 4 h post-labeling.

An aliquot containing 86 MBq (100 µL) of each purified complex was incubated in 500 µL of 0.1 M PBS pH 7.4 at 37 ºC for 1, 2, and 4 h. Each experiment was performed in triplicate and monitored by HPLC. For human serum stability study, we incubated aliquots of each purified complexes (37–86 mBq, 100 µL) in 400 µL of human serum at 37 ºC for 1, 2, and 4 h. Proteins were precipitated with 200 µL of EtOH (70%) and centrifuged (10000 g, 10 min, room temperature). Supernatant was removed, and the radioactivity in the supernatant was compared with that in the pellet, measured in a solid scintillation counter detector with 3"x3" NaI (Tl). Once supernatant was removed and measured, it was filtered by 0.22 um filter and analyzed by HPLC with the method previously described.

A cysteine challenge assay was conducted to assess the in vitro stability of the best performing complexes at 37°C for 4 hours. Fresh solutions of L-cysteine (Sigma-Aldrich, USA) at concentrations of 0.1, 1.0, and 10 mM in 0.9% NaCl at pH 7.0 were meticulously prepared. Subsequently, 500 µL of the L-cysteine solutions were introduced to 100 µL of the radiotracer (37–86 MBq) and incubated at 37 ºC. After the incubation period, the reaction mixture was subjected to HPLC analysis, as previously described, at various time points ranging from 1 to 3 hours.

Cell culture

Human breast cell lines (MDA-MB-231, MDA-MB-435, MCF-7 and BT-474) were acquired by American Type Culture Collection (ATCC, Manassas, Va). They were grown on RPMI-1640 medium with stable L-glutamine (2mM) (Capricorn Scientific) supplemented with 10% Fetal Bovine Serum (FBS, Capricorn Scientific) (at 10%, 100 U/mL penicillin and 100 ug/mL Streptomycin (Capricorn Scientific). Mouse breast cell line 4T1 was acquired by ATCC and grown on DMEM high glucose medium (Capricorn Scientific) supplemented with FBS at 10% final concentration, 100 U/mL penicillin,100 ug/mL streptomycin. As a negative control, normal mice fibroblast cell line NIH-3T3 and CHO-K1 were also acquired by ATCC, growth on DMEM high glucose medium (Capricorn Scientific) supplemented with FBS (Capricorn Scientific) at 10% final concentration, 100 U/mL penicillin,100 ug/mL streptomycin and 100 ug/mL HEPES (Capricorn Scientific). All cultures were incubated at 37 ºC with 5% CO₂, and a 95% humidity atmosphere.

Specific binding and cellular internalization

Cell binding and internalization studies were performed in MDA-MB-231, MDA-MB-435, MCF-7, BT-474, and 4T1 breast cancer cell lines and as negative control using NIH-3T3 normal mice fibroblast cell line.

For each cell line, cells (0.5 x 10⁶) were seeded with culture medium in 24 well plates and allowed to grow overnight. After washing once with culture medium without FBS, the cells were incubated at 25 ° C for 15, 30, 45 and 60 min with ~ 100 nM (100000–300000 cpm) of [^99mTc] Tc-HYNIC-GSG-LHRH(D-Lys6)/Tricine/NA in 300 µL of culture medium. After incubation period, the reaction medium was aspirated and the cells were washed twice with 500 µL cold PBS-0.2% Bovine Serum Albumin (BSA, Sigma Aldrich), pH 7.4. Radiopeptide internalization was determined by quantifying the membrane bound and internalized activity. Cells were washed with an acid buffer solution (40 mM sodium acetate, pH 4.5) to remove membrane-bound radiopeptide. The remaining internalized activity in the cells was obtained by lysis with 1 M NaOH for 5 min. Membrane-bound and internalized activities were measured in a gamma detector. Nonspecific binding was determined by co-incubation with 20 µg of cold LHRH(D-Lys6) for 2 h at 25°C in culture medium, before the binding assay. For each cell line, the time with the highest binding was chosen. The cell binding ability was reported as the percentage of total added radioactivity that was bound or internalized by the cells and expressed as mean ± SD (cpm bound or internalized * 100/total cpm, n = 4). The results are expressed as mean ± SD.

Biodistribution studies

All animal procedures and protocols were approved by Institutional Animal Care and Use Committees of Facultad de Ciencias, Universidad del a República (protocol number #353, act 240011-001862-16).

Female Balb/c mice (6–10 weeks old, weighing 18–24 g) and female xenografted 4T1 Balb/c mice (10 weeks old) were used in this study. The xenografted mice were established by injecting 250,000 4T1 cells/100 µL PBS intramammary and allowing for a tumor growth period of 10–14 days.

All mice were administered 0.5-1 MBq (0.05–0.15 µg) of purified [^99mTc] Tc-HYNIC-GSG-LHRH(D-Lys6)/Tricine/NA intravenously via the tail vein. To assess non-specific uptake, a separate group of three mice received 100 µg of unlabeled peptide (block group) 30 min prior to the administration of the radiopeptide.

The animals were euthanized at predetermined time points, specifically at 1-, 3-, and 6-hours post-injection, with a sample size of n = 3–4 for each time point.

After each time point, they were sacrificed by cervical dislocation and dissected to study their radioactivity. Heart liver, lungs, thyroid, muscle, bone, kidneys, stomach, spleen, gastrointestinal tract, and bladder were extracted, cleaned, weighted, and measured for radioactivity in NaI(TI) detector. Blood and urine were also collected and measured. The radioactivity in tissues was expressed as percentage of injected dose per gram of tissue (% ID/g) or percent of injected dose (% ID). The results are expressed as mean ± SD.

All studies are reported in accordance to ARRIVE guidelines.

SPECT/CT images

Single-photon emission computed tomography/computed tomography (SPECT/CT) images were carried out on a nanoScan SPECT/CT (Mediso). After 10–15 days following 4T1 inoculation (250.000 cells/100 µL, intramammary) in female Balb/c mice, a mixture of 2-2.5% isoflurane and oxygen was used for anesthesia, followed by intravenous tail injection of 20 mg of arginine in 100 µl of gelofusine (Sigma-Aldrich) and after 5 minutes, injection of [^99mTc]Tc-HYNIC-GSG-LHRH(D-Lys6)/Tricine/NA ( 74–111 MBq/mouse). To perform the blocking study, 30 min prior to inoculation of radiolabeled LHRH analog, intravenous injection of unlabeled LHRH(D-Lys6) (100 µg) was performed. After 1, 2, and 5 h SPECT volumes were reconstructed using Tera Tomo 3D normal dynamic range method (medium filters, 128 resolution, 48 iterations, and 3 subsets). The reconstructed images were analyzed using PMOD software version 3.8 (PMOD Technologies, Ltd., Zurich, Switzerland). Ellipsoidal 3-dimensional Volumes of Interest (VOI) were manually drawn around the tumor and muscle by visual inspection.

Statistical analysis

The calculation of mean values and standard deviation were performed using Excel software. All graphs and Statistical analysis were done on GraphPad Prism 9 software (San Diego, CA) and performed by ANOVA or Student-t test as stated, with a significant value of P ≤ 0, 05 (*), ≤ 0.01 (**), ≤ 0.001 (***), < 0.0001 (****) and > 0.05 (ns or not stated for not significant).

Competing interests

The authors declare no competing interests.

Funding

This work was supported by Agencia Nacional de Investigación e Innovacion (ANII, POS_NAC_2016_1_129986, POS_NAC2018_1_151180) and Comisión Sectorial de Investigación (CSIC, 240600-000148-18) for funding.

Author Contribution

L.A., X.C., M.C., M.T., M.F.G., M.F.J.P.G., and P.C. participated in the peptide radiolabeling and in vitro in vivo studies. E.S., A.L.R., A.P., participated in the SPECT-CT imaging. L.A. drafted the manuscript. All authors participated in the editing and review of the manuscript.

Acknowledgements

This work is part of the Doctorate Program in Chemistry (PEDECIBA) from the Facultad de Química at the Universidad de la República, Uruguay, where the Bach. Lucía Alfaya is pursuing his doctorate in Chemistry.

Data availability

All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).

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Preclinical evaluation of ^99m Tc-labeled LHRH as GnRH receptor imaging

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Abstract

Figures

Introduction

Results

HYNIC-GSG-LHRH (D-Lys6) analog Synthesis

Peptide radiolabeling and purification and Log P

Specific binding and internalization

Biodistribution studies

SPECT/CT imaging

Discussion

Conclusions

Materials and Methods

Reagents

HYNIC-GSG-LHRH(D-Lys6) analog Synthesis

Calculation of Log P

Cell culture

Specific binding and cellular internalization

Biodistribution studies

SPECT/CT images

Statistical analysis

Declarations

Competing interests

Funding

Author Contribution

Acknowledgements

Data availability

References

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Supplementary Files

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