PREPARATION OF THE RADIOPHARMACEUTICAL [177Lu]-PSMA-617 FOR TARGETED RADIONUCLIDE THERAPY OF PROSTATE CANCER

ABSTRACT

This work is devoted to the study of the conditions for labeling the peptide-ligand of prostate-specific membrane-active antigen (PSMA) with the radionuclide of lutetium-177 from substance "Lutetium chloride (¹⁷⁷LuCI₃) with ¹⁷⁷Lu, carrier free" with a specific activity of 92-96 Ci per mg/Lu  produced by the State Enterprise "Radiopreparat"

The radiopharmaceutical [¹⁷⁷Lu]-PSMA-617 was synthesized by introducing the substance ¹⁷⁷LuCI₃, carrier free in a 0,04 M HCI solution with activity from 3,7 GBq to 18,5 GBq into a solution of peptide-ligand of PSMA-617 in an ascorbate buffer solution with molar ratios of ¹⁷⁷Lu : PSMA from 1 nmol : 1 nmol to 1 nmol : 4,0 nmol respectively. The radiochemical yield of the labeling and the radiochemical purity were determined by thin layer and paper chromatography.

The results of the studies showed that during the synthesis of the radiopharmaceutical [¹⁷⁷Lu]-PSMA-617 with a peptide-ligand of PSMA-617 content of 4,8 nmol, the amount of added ¹⁷⁷Lu should not exceed 1,5 GBq while the radiochemical yield is ≥98,5±0,5%.

АННОТАЦИЯ

Настоящая работа посвящается исследованию условий мечения пептида-лиганда простат-специфического мембранного антигена (ПСМА) с радионуклидом лютеций-177 субстанции «Лютеция хлорид (¹⁷⁷LuCI₃) c ¹⁷⁷Lu, без носителя» с удельной активностью 92-96 Кюри/мг производства ГП «Радиопрепарат».

Радиофармацевтический препарат [¹⁷⁷Lu]-ПСМА-617 был синтезирован путем введения субстанции ¹⁷⁷LuCI₃ без носителя в 0,04 М растворе HCI активностью от 3,7 ГБк до 18,5 ГБк в раствор пептида-лиганда ПСМА-617 в аскорбатном буферном растворе с мольными соотношениями ¹⁷⁷Lu : PSMA от 1 nMol :1 nmol до 1 nMol : 4,0 nMol соответственно. Радиохимический выход мечения и радиохимическую чистоту определили методами тонкослойной и бумажной хроматографии.

Результаты исследований показали, что при синтезе радиофармацевтического препарата [¹⁷⁷Lu]-PSMA-617 с содержанием пептида-лиганда PSMA-617 4,8 nmol количество добавляемого ¹⁷⁷Lu не должно превышать 1,5 ГБк, что при этом радиохимический выход составляет ≥98,5±0,5%.

Keywords: lutetium-177, labeling, prostate-specific membrane antigen (PSMA-617), prostate cancer, radiopharmaceutical of [¹⁷¹Lu]-PSMA-617, tin layer chromatography, radiochemical yield, radiochemical purity.

Ключевые слова: Лютеций-177, мечение, простат-специфический мембранный антиген (ПСМА-617), рак предстательной железы, радиоактивный фармацевтический препарат [¹⁷¹Lu]-PSMA-617, тонкослойная хроматография, радиохимический выход, радиохимическая чистота.

Introduction

Currently, a promising direction in the development of modern oncology is radionuclide therapy, with the use of radiopharmaceutical aimed, i.e. targeted effect on tumor tissues. Prostate cancer is the second most commonly diagnosed cancer in men and the fifth leading cause of cancer death worldwide [1]. According to the GLOBOCAN 2020 estimate, more than 1.4 million patients were diagnosed with prostate cancer in 2020, resulting in 375 304 associated deaths worldwide [2]. Based on this, it can be concluded that in order to reduce the mortality rate of patients with prostate cancer, early screening and diagnosis is necessary, followed by treatment with an appropriate therapeutic regimen using radiopharmaceuticals specific to prostate tumor cells. These promising radiopharmaceuticals include [¹⁷⁷Lu]-PSMA-617, labeled with the lutetium-177 radionuclide, carrier free, which is currently a new radiopharmaceutical for theranostics of prostate cancer [3]. [¹⁷⁷Lu]-PSMA-617, which is associated with high specificity for prostate-specific membrane antigen (PSMA), allowing beta-particle therapy to target metastatic castration-resistant prostate cancer, as well as mild gamma irradiation, allowing observation of accumulation of [¹⁷⁷Lu]-PSMA-617 in humans. The successful use of the radiopharmaceutical [¹⁷⁷Lu]-PSMA-617 in the clinical treatment of prostate cancer with encouraging therapeutic efficacy is evidenced by the results [1, 4-8].

Thus, the need to study the conditions for labeling the radionuclide ¹⁷⁷Lu in the form of chloride with the prostate-specific membrane antigen (PSMA) and the stability of the resulting [¹⁷⁷Lu]-PSMA complex is very relevant. In this work, we studied the conditions for labeling PSMA-617 with the radionuclide lutetium-177, carrier free, and the stability of the obtained [¹⁷⁷Lu]-PSMA-617 both in physiological saline and in human blood serum.

Production of ¹⁷⁷Lu radionuclide in a nuclear reactor

The radionuclide of ¹⁷⁷Lu can be obtained in two methods:

In the first method, ¹⁷⁷Lu is obtained by irradiating the starting material with neutrons from a nuclear reactor, enriched in Lu-176 by the nuclear reaction ¹⁷⁶Lu (n, γ) ¹⁷⁷Lu. In this case, the value of the specific activity of the resulting ¹⁷⁷Lu is significantly lower than the theoretical specific activity, as well as the inevitability of the formation of a long-lived radionuclide ^177mLu (Т_1/2= 160 days), the share (according to activity) of which in ¹⁷⁷Lu can achieved 3%.

In the second method, ¹⁷⁷Lu is obtained by irradiating a target material containing Yb enriched in the stable isotope ¹⁷⁶Yb by the nuclear reaction ¹⁷⁶Yb (n, γ) ¹⁷⁷Yb (β-) ¹⁷⁷Lu with neutrons from a nuclear reactor. In this case, regardless of the intensity of the neutron flux, ¹⁷⁷Lu is formed with a specific activity close to the theoretical specific activity, and the absence of an admixture of a long-lived radionuclide of ^177mLu.

Experimental part

Materials and equipment

As the irradiation material for obtaining the lutetium-177 radionuclide was used ytterbium oxide enriched by the stable isotope of ytterbium-176 with a degree of enrichment of 99,5% converted into the form of nitrate ¹⁷⁶Yb(NO₃)₃. The samples were irradiated in the vertical channels of the WWR-SM research reactor (Institute of Nuclear Physics, Academy of Sciences of the Republic of Uzbekistan). The samples were irradiated for 120 effective hours. The irradiated sample was held for 24 hours to decay the activity of the short-lived radionuclide ¹⁷⁷Yb (T_1/2 = 1,911 hours).

Reagents

DOWEX 50x8 cation exchange resin, 200-400 mesh, (Sigma Aldrich), in the H⁺ form, which was converted to the NH₄⁺ form by treatment with a 0,5 M NH₄CI solution was used us the ion exchange resin.

Alpha-hydroxyisobutyric acid (α-HIBA) (Sigma Aldrich) was used as the complexing agent. Peptide-ligand of PSMA-617 was purchased from MedChemExpress (USA). All reagents and laboratory reagents used in the work were of the highest purity (unless otherwise indicated).

The chromatographic column used in the separation of ¹⁷⁷Lu, carrier free from ytterbium-175,176, had a size of d=16 mm and H=500 mm.

The process of ion-exchange chromatography in the gradient mode was carried out on a high-performance liquid chromatograph Gilson-305. Measurement of the solution medium was carried out on a Seven Easy pH meter.

Measurements of quantitative and qualitative activities of radionuclides were carried out on a gamma spectrometric device ASPECT SU-03P with a semiconductor Ge(Li) detector. Radionuclides were identified by their gamma lines.

Membrane filters with a pore size of 0,22 µm manufactured by Millipore Express (PES) were used to filter the solutions. The process of labeling PSMA-617 with lutetium-177 was carried out in a STEGLER WB-4 thermostatic water bath.

The radiochemical purity (RCP) of the obtained ¹⁷⁷LuCl₃ and the radiochemical labeling yield (RY) of the peptide-ligand PSMA-617 with ¹⁷⁷LuCl₃ were evaluated by thin layer chromatography (TLC) and paper chromatography (PC). Methyl ethyl ketone (MEK) or 0,1 M sodium citrate solution pH=5,0-5,5 was used as the mobile phase in the case of TLC, and «DC-Alufolien Kieselgel 60», Merck was used as the stationary phase, and in the case of In Paper Chromatography, the solvent mixture NH₄OH:CH₃OH:H₂O, phase ratio 1:5:10, was used as the mobile phase, and Whatman brand 3MM chromatographic paper was used as the stationary phase.

Results and discussions

Producing of ¹⁷⁷LuCl₃

To obtain lutetium-177 chloride (¹⁷⁷LuCI₃), 100 mg of stable ytterbium enriched in ytterbium-176 (with an enrichment degree of 99.5) in the form of ¹⁷⁶Yb(NO₃)₃ nitrate was irradiated in a quartz ampoule in a thermal neutron flux for 120 hours. After irradiation, the sample was kept for 20 hours for the decomposition of short-lived ¹⁷⁷Yb (T_1/2= 1,911 hours) and dissolved in a 0,01 M nitric acid solution. The resulting solution was loaded into a chromatographic column, filled with cation exchange resin Dowex-50X8, with a particle size of 200-400 mesh, pre-treated with 0,1 M ammonium chloride solution. The column was washed with bidistilled water and then eluted with ¹⁷⁷Lu and ^175,176Yb with a 0,15 M solution of the ammonium salt of alpha-hydroxyisobutyric acid with an eluent concentration gradient from 0,05 M to 0,15 M over 300 minutes. The fractions of eluates with Lu-177 and ^175,176Yb were collected separately for 340 to 440 min. Fractions with a solution with ¹⁷⁷Lu were acidified with 6,0 M hydrochloric acid to pH=1,0, passed through a column filled with solid extractant HDEHP/FT-4, prepared by impregnation of liquid extractant cation exchanger (HDEHP) Di(2-ethylhexyl)orthophosphoric acid.

The column was washed with 0,1 M HCI and ¹⁷⁷Lu was eluted with 6,0 M HCI. Then the solution was evaporated to dryness on a rotary evaporator, and the dry residue was dissolved in the required amount of 0,04 M HCI solution, and passed through a membrane filter with a pore size of 0,22 μm. As a result, lutetium-177 chloride, (¹⁷⁷LuCI₃) carrier free was obtained in 0,04 M HCI solution with a radiochemical purity of 99,9 ± 0,05% and a specific activity of ≥92,0 Curie/mg.

Quality control of the substance "Lutetium chloride (¹⁷⁷LuCI₃) with ¹⁷⁷Lu, carrier free" in 0,04 M HCI solution.

To determine the RCP of the substance, the test stock solution of the substance was diluted to a volume activity of 740 MBq/ml and an aliquot was applied to chromatographic strips of 7,4 × 10⁵ Bq.

 A plate with a thin layer of silica gel (TLC) was cut into strips 15 × 140 and 10 × 100 mm in size, a sample of the test solution with a volume of 1,0-1,5 μl was applied to the start line (15 mm from the edge of the strip). The plates of the substance deposited on it were dried in air. The plates were then transferred to a TLC chamber and the start line end was immersed in the solvent (mobile phase) in a vertical position for ascending chromatography.

The results of studies for the determination of RCP substances are shown in Figures -1 and 2.

Figure 1. Chromatogram of the substance "Lutetium chloride (¹⁷⁷LuCI₃) with ¹⁷⁷Lu, carrier free" in 0.04 M HCI solution. A) Mobile phase MEK (Methyl ethyl ketone), strip length 10 cm. RCP 99,5%, n=3

Figure 2. Chromatogram of the substance "Lutetium chloride (¹⁷⁷LuCI₃) with ¹⁷⁷Lu, carrier free" in 0,04 M HCI solution. B) Mobile phase 0,1 M Cit, pH 5.0, strip length 12 cm, RCP 99,2%, n=3

Preparation of the radiopharmaceutical [¹⁷⁷Lu]-PSMA-617

The radiopharmaceutical [¹⁷⁷Lu-PSMA-617] was obtained by mixing a solution of the peptide-ligand PSMA-617 in ascorbate buffer solution (ABS) with a solution of the substance «Lutetium chloride (¹⁷⁷LuCI₃) with ¹⁷⁷Lu, carrier free» and incubation in a water bath at a temperature of 90 -95 ⁰C for 20 minutes, periodically stirring the solution of the reaction mixture. To do this, a certain amount of the peptide-ligand PSMA-617 was dissolved in water for injection and dispensed in Eppendorf plastic tubes into several portions and left for storage in a Kelvinator at a temperature of -50 ⁰C. A portion/package of the frozen peptide/ligand PSMA-617 solution was taken, thawed and ascorbic buffer solution (ABS) was added and mixed well. The content of ABS ranged from 50 to 150 mg. After that, a solution of the substance «Lutetium chloride (¹⁷⁷LuCI₃) with ¹⁷⁷Lu, carrier free» was added to the solution of the peptide/ligand PSMA-617 and incubated in a thermostatic water bath at a temperature of 90-95 ⁰C for 20 minutes. The amount of ¹⁷⁷Lu activity ranged from 0,37 to 1,85 GBq. After the completion of the incubation process, the test tube with the solution was removed from the thermostat bath and cooled to room temperature, and the volume of the reaction mixture was brought to 3,0 ml with a sodium chloride solution at the rate of 8,0-10,0 mg/ml of NaCI, transferred into a drug vial. The radiochemical yield of the labeling was evaluated by determining the radiochemical purity of each reaction mixture by the quality control procedure described above.

Radiochemical yield of the peptide-ligand PSMA-617 labeling with ¹⁷⁷LuCI₃

The determination of the radiochemical yield of the process of labeling the peptide-ligand PSMA-617 with the ¹⁷⁷Lu radionuclide was carried out by determining the RCP of the radiopharmaceutical [¹⁷⁷Lu]-PSMA-617. The determination of RCP of the radiopharmaceutical [¹⁷⁷Lu]-PSMA-617 was carried out by TLC and PC.

The radioactive concentration of the tested initial solutions of radiopharmaceuticals was adjusted to a radioactive concentration of 740 MBq/ml and an aliquot was applied to chromatographic strips of 7,4 × 10⁵ Bq.

In the case of PC, a sheet of Whatman 3MM chromatographic paper was used, strips of 15x100 mm in size were cut, and in the case of TLC, strips of 15x160 mm in size were cut from the plate with a thin layer of silica gel, and a sample of the test solution with a volume of 1,0-1,5 µl was applied to the start line (15 mm from the edge of the strip). The [¹⁷⁷Lu]-PSMA-617, deposited on the strips were dried in air. The strips were then transferred to a chromatography chamber and the start line end was immersed in the solvent (mobile phase) in a vertical position for ascending chromatography. As a mobile phase in the case of TLC, a 0,1 M solution of sodium citrate with pH = 5,0-5,5 was used, and in the case of PC, was used as a mobile phase with a mixture of solvents NH₄OH : CH₃OH : H₂O with a phase ratio of 1:5:10.

The results of studies on the determination of radiochemical yield of the radiopharmaceutical [¹⁷⁷Lu]-PSMA-617 are shown in Figures 3-4 and 5-6.

The results of studies of the RY of labeling the peptide-ligand PSMA-617 with the radionuclide lutetium-177 by determining the RCP of the radiopharmaceutical [¹⁷⁷Lu]-PSMA-617 with different activity of the ¹⁷⁷LuCI₃ added to the reaction mixture of the peptide-ligand PSMA-617 during the synthesis of the radiopharmaceutical [¹⁷⁷Lu]-PSMA-617 are given in figure 3.

Figure 3. Radiochemical Yield in % of the peptide-ligand PSMA-617 Labeling (4,8 nmol) with ¹⁷⁷Lu₃⁺ in the ratio ¹⁷⁷Lu: PSMA-617 from 1:1.6 to 1:4,1

As can be seen from Figure 3, when 4,8 nmol the peptide-ligand PSMA-617 is labeled with lutetium-177 radionuclide in the PSMA-617:¹⁷⁷Lu ratio from 4,8:1 to 2:1, the radiochemical labeling yield decreases from 99,7% to 98,2%, where the amount of added activity of the radionuclide lutetium-177 was 0,37; 0,55; 0,74; 0,94; 1,0; 1,11; 1,3; 1,48 GBq respectively. With an increase in the amount of added of activity ¹⁷⁷Lu of more than 1,5 GBq per 4,8 nmol the peptide-ligand PSMA-617, where the ratio of PSMA-617:¹⁷⁷Lu was less than 1:2, then the radiochemical yield of PSMA-617 labeling with the lutetium-177 radionuclide was below 96%.

To determine the optimal radioactive concentration added to the reaction mixture of lutetium chloride with lutetium-177, we studied the radiochemical yield of the peptide-ligand PSMA-617 labeling with the ¹⁷⁷Lu radionuclide. The ratio of PSMA-617:¹⁷⁷Lu was 2:1 and the total volume of the reaction mixture was 1,0 ml.

The results of studies of the synthesis of the radiopharmaceutical [¹⁷⁷Lu]-PSMA-617 with a ratio of PSMA-617 : ¹⁷⁷Lu 2:1, with different radioactive concentration are shown in Figure 4.

Figure 4. Radiochemical yield of the peptide-ligand PSMA-617 labeling with ¹⁷⁷LuCI₃ with various radioactive concentration

As can be seen from Fig.-4, when the radionuclide ¹⁷⁷Lu is introduced in the form of ¹⁷⁷LuCI₃ in a 0,04 M HCI solution to the reaction mixture peptide-ligand of PSMA-617 with a radioactive concentration of lutetium chloride less than 1000 mCi/ml, the radiochemical purity of the resulting radiopharmaceutical decreases on average to values below 98,0±0,3%.

This is apparently due to the fact that with an increase in the volume of ¹⁷⁷LuCI₃ in a 0,04 M solution of HCI acid introduced to a solution of the peptide-ligand  PSMA-617 reaction mixture, it negatively affects the reaction medium and leads to a decrease in the radiochemical yield of the complex formation reaction, which leads to a deterioration in the quality of the final product i.e. decrease the radiochemical purity.

To reliably determine the radiochemical yield of the peptide-ligand of PSMA-617 labeling with the lutetium-177 radionuclide, the RCP of the obtained radiopharmaceutical «[¹⁷⁷Lu]-PSMA-617» was determined in two systems.

Figure 5. Histogram of radiopharmaceutical 177Lu-PSMA-617, Whatman 3MM stationary phase, NH₄OH:CH₃OH:H₂O mobile phase, phase ratio 1:5:10, n=6, RCP=99,4±0,3%

Figure 6. Histogram of the radiopharmaceutical ¹⁷⁷Lu-PSMA-617, stationary phase "DC-Alufolien Kieselgel 60", mobile phase 0,1 M CitNa₃, pH=5,0-5,5, n=6, RCP=99,2±0,2%

In the system consisting of stationary phase Whatman 3MM and mobile phase NH₄OH :CH₃OH:H₂O, in a phase ratio 1:5:10, the free ¹⁷⁷Lu³⁺ ion remains at the sample application site with (Rf = 0,0), and the [¹⁷⁷Lu] labeled with PSMA-617 moves forward with mobile phase with retention factor Rf=0,75. At that time, in the second system, both the free ¹⁷⁷Lu³⁺ ion moves with the mobile phase to the finish line (Rf=0,95), and the labeled [¹⁷⁷Lu] with peptide-ligand of PSMA-617 moves with the mobile phase with Rf=0,55, and on the start line ( Rf=0,2) remains the colloidal form of ¹⁷⁷Lu.

Conclusion

Thus, it follows from the research results that in the synthesis of the radiopharmaceutical of ¹⁷⁷Lu-PSMA-617, the most optimal ratio of the ¹⁷⁷LuCI₃ substance in a 0,04 M solution of HCI acid with the peptide-ligand of PSMA-617 is 1:2, namely, when labeling 4,8 nmol PSMA-617 to 1,5 GBq ¹⁷⁷LuCI₃, the radiochemical purity of the obtained radiopharmaceutical is more than ≥98,5±0,5%. And this indicates a high radiochemical yield of labeling.

The optimal radioactive concentration of radionuclide lutetium-177 in a 0,04M solution of hidochloric acide added to the reaction mixture during the synthesis of the radiopharmaceutical [¹⁷⁷Lu]-PSMA-617 was determined, which, with a radioactive concentration of less than 1000 mCi/ml, the radiochemical purity of the resulting radiopharmaceutical decreases on average to values below 98,0 ± 0,3%.

Reproducible methods have been developed for determining the radiochemical purity of the radiopharmaceutical [¹⁷⁷Lu]-PSMA-617, where in the histograms the minimum distance between the peaks of the colloidal form ¹⁷⁷Lu; ions of ¹⁷⁷Lu₃⁺ and labeled [¹⁷⁷Lu]-PSMA-617 is more than 2,0 cm, so the mutual influence of the peaks is excluded.

Thus, from the obtained results of the work, we can conclude that during the synthesis of the radiopharmaceutical [¹⁷⁷Lu]-PSMA-617 with an peptide-ligand PSMA-617 content of 48 nmol, the amount of the added substance "Lutetium chloride (¹⁷⁷LuCI₃) with ¹⁷⁷Lu, carrier free" with a specific activity of more than 90 Curie /mg should not exceed 15 GBq with a radioactive concentration of at least 37 GBq/ml.

References:

1. Michael S Hofman, John V., Rodney J. H., Justin F., Sue P.T., Tim A. at all // [¹⁷⁷Lu]-psma-617 radionuclide treatment in patients with metastatic castration-resistant prostate cancer (lupsma trial): a single-centre, single-arm, phase 2 study// Lancet Oncology 2018; 19; рр. 825–833;
2. Hyuna Sung, Jacques F., Rebecca L., Mathieu L., Isabelle Soerjomataram., Ahmedin Jemal, Freddie Bray, //“Global cancer statistics 2020: GLOBOCAN estimates of incidence and Mortality Worldwide for 36 cancers in 185 countries,” A Cancer Journal for Clinicians 2020; vol. 71, Issue 3, pp. 209–249;
3. Kristell L.S.C., Sandra H., Mark K., Janneke D.M., Gerben M. F., Marian C., Margret S. at all //Towards Personalized Treatment of Prostate Cancer: PSMA I&T, a Promising Prostate-Specific Membrane Antigen-Targeted, Theranostic Agent, Theranostics 2016; Vol. 6, Issue 6, рр.849-861;
4. Yasemin S., Duygu Has S., Oner S., Rathan M. S., Ayse T. K. // ¹⁷⁷Lu-PSMA Therapy in Metastatic Castration-Resistant Prostate Cancer, Biomedecines, 2021; 9, 430
5. L. van Kalmthout, A. Braat, M. Lam et all. // “First experience with ¹⁷⁷Lu-PSMA-617 therapy for advanced prostate cancer in the Netherlands,” Clinical Nuclear Medicine, 2019, vol. 44, no. 6, pp. 446–451;
6. F. E. von Eyben, G. Roviello, T. Kiljunen et all. // “Third-line treatment and ¹⁷⁷Lu-PSMA radioligand therapy of metastatic castration-resistant prostate cancer: a systematic review,” European Journal of Nuclear Medicine and Molecular Imaging, 2018, vol. 45, no. 3, pp. 496–508;
7. Louise E., Kathy W., John V., Jane S, Ashley B., Jonathan Lee // Lutetium 177 PSMA radionuclide therapy for men with prostate cancer: a review of the current literature and discussion of practical aspects of therapy, Journal of Medical Radiation Sciences, 2017, (64), pp.52-60;
8. H. Ahmadzadehfar, S. Wegen, A. Yordanova et all. // “Overall survival and response pattern of castration-resistant meta-static prostate cancer to multiple cycles of radioligand ther-apy using [¹⁷⁷Lu]Lu-PSMA-617,” European Journal of Nuclear Medicine and Molecular Imaging, vol. 44, no. 9, pp. 1448–1454, 2017.