1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-PSMA-GUL (99.0%), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-PSMA-GUL (99.3%), and 175LuCl3 (99.9%) were obtained from CellBion (Seoul, Korea). Esomeprazole, sodium acetate, and acetic acid were purchased from Sigma Aldrich Chemical Co. (Milwaukee, WI, USA). Hydrochloric acid was obtained from Samchun Chemical Co., Ltd (Seoul, Korea). High-performance liquid chromatography (HPLC) grade acetonitrile, methanol, and water were purchased from J.T. Baker Co. (Philipsburg, NJ, USA).
Preparation of 175Lu-DOTA-PSMA-GUL solution
The drug solution of 175Lu-DOTA-PSMA-GUL was prepared according to the method provided by Cellbion Co. (Seoul, Korea). Briefly, 175Lu-DOTA-PSMA-GUL was synthesized by mixing DOTA-PSMA-GUL solution and 175LuCl3 solution. DOTA-PSMA-GUL solution was prepared by dissolving 50 mg of DOTA-PSMA-GUL powder in 20 mL of 0.5 M sodium acetate buffer (pH 4.5). 175LuCl3 solution was prepared by dissolving 19 mg of 175LuCl3 in 10 mL of 0.04 M HCl. Then, 20 mL of DOTA-PSMA-GUL and 8.5 mL of 175LuCl3 were mixed for 20 min at 40 °C in a shaking water bath. The mixture was finally tested for the purity of 175Lu-DOTA-PSMA-GUL via HPLC–UV method using Waters 2695 separation module coupled with Waters 2487 dual wavelength absorbance detector (Waters, Milford, MA, USA). 175Lu-DOTA-PSMA-GUL was separated on an Agilent Zorbax 300SB-C18 (4.6 × 250 mm i.d., 5 μm, Agilent, Santa Clara, CA, USA) and detected at 244 nm.
LC–MS/MS analysis condition
Liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis was performed by an Agilent 6490 triple-quadrupole mass spectrometer coupled with an Agilent 1260 HPLC (Agilent Technologies, Santa Clara, CA, USA). 175Lu-DOTA-PSMA-GUL in the rat biometrics (plasma, urine, feces, and 12 different tissue samples) was separated on an Agilent Zorbax SB-Aq column (100 × 2.1 mm, i.d., 3.5 μm, Agilent). Chromatographic separations were performed by using a binary gradient mobile phase composed of mobile phase A (1% formic acid in distilled water) and mobile phase B (1% formic acid in methanol). The gradient elution profile and flow rate was set as: 0 min, A:B = 95:5 (v/v), 0.3 mL/min; 8 min, 0:100, 0.3 mL/min; 10 min, 0:100, 0.3 mL/min; 10.01 min, 95:5, 0.5 mL/min; 15 min, 100:0, 0.5 mL/min; 15.01 min, 95:5, 0.3 mL/min; 22 min, 95:5, 0.3 mL/min. The gradient profile was optimized to improve the peak response and achieve rapid wash-out interference and equilibrate the column with the initial mobile phase condition for the next injection. The total run time was 22 min, and the column oven temperature was 40 °C. The sample injection volume was 5 μL.
The electrospray ionization (ESI) source was operated in positive mode, and the mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. The observed MRM transitions and mass spectrometry settings are summarized in Supplementary Table 1.
Preparation of stock solutions, calibration standards, and quality control samples
The stock solutions of 175Lu-DOTA-PSMA-GUL were prepared by diluting 2.1 mg/mL synthesized solution in methanol to 400 μg/mL. The stock solutions of NOTA-PSMA-GUL (internal standard 1, IS1) and esomeprazole (internal standard 2, IS2) were prepared by separately dissolving 10 mg of each in 10 mL of methanol (1 mg/mL).
Calibration standards and quality control samples
For drug analysis in the plasma, calibration curves were constructed by spiking 50 μL of working stock solutions to blank plasma (50 μL each) to provide 175Lu-DOTA-PSMA-GUL concentrations at 20,000, 10,000, 5000, 1000, 500, 100, 50, and 20 ng/mL. The plasma was spiked with 50 μL of IS1 solution and 150 μL of methanol and mixed on a vortex mixer. The mixture was then centrifuged for 10 min at 4,000 rpm (3220 × g), and 100 μL of the supernatant was transferred to a plastic vial. After 100 μL of distilled water was added to the supernatant, the mixture was vortex-mixed for 10 min and 5 μL of the mixture was injected onto the LC–MS/MS. Quality control (QC) samples were prepared by spiking the working drug solutions to blank rat plasma to provide high concentration QC (16,000 ng/mL), middle concentration QC (8,000 ng/mL), low concentration QC (80 ng/mL) and lower limit of quantification (LLOQ) QC (20 ng/mL).
Similarly, calibration standards and QC samples were prepared for drug analysis in urine, feces, and twelve different tissues. Calibration ranges were 100–20,000 ng/mL for urine, 100–5000 ng/mL for feces and tissues. High, middle, and low QC sample concentrations were 16,000, 8000, and 400 ng/mL for urine, 4000, 1600, and 400 ng/mL for feces and tissue matrices.
For plasma samples, NOTA-PSMA-GUL (internal standard 1, IS1) solution 50 μL was added to 50 μL of the rat plasma. As a precipitation solvent, 200 μL of methanol was added, and the mixture was mixed on a vortex mixer for 10 min, followed by centrifugation for 10 min at 4000 rpm (3220 × g). After taking 100 μL of the supernatant, 100 μL of distilled water was added, vortex-mixed for 10 min. Finally, 5 μL of the prepared mixture was injected onto the LC–MS/MS. Since several plasma samples showed concentrations above the ULOQ, those samples were diluted 10- or 20-fold for analysis.
For urine and fecal homogenate samples, the working IS2 solution 50 μL was added to 50 μL of the homogenate samples. The samples were precipitated with methanol (900 μL) on a vortex mixer for 10 min, followed by centrifugation for 10 min at 4000 rpm (3220 × g). After taking 100 μL of the supernatant, 100 μL of distilled water was added, vortex-mixed for 10 min.
For tissue homogenate samples, the working IS2 solution 50 μL was added to 50 μL of tissue homogenate samples. The samples were precipitated with methanol (400 μL) on a vortex mixer for 10 min, followed by centrifugation for 10 min at 4,000 rpm (3220 × g). After taking 200 μL of the supernatant, 200 μL of distilled water was added to the supernatant and centrifuged for 10 min again. After the second centrifugation, 100 μL of supernatant and the same volume of distilled water was mixed for 10 min. Finally, 5 μL of the prepared mixture was injected onto the LC–MS/MS.
In vivo pharmacokinetic studies in rats
Male Sprague–Dawley rats (7 weeks, 190–210 g; DBL co., Eumsung, Korea) were kept in plastic cages with free access to a standard diet (Youngbio, Seong-nam, Korea) and water. All experiments were performed in accordance with the relevant guidelines and regulations. The animal study protocol was approved by the Institutional Animal Care and Use Committee of Sungkyunkwan University (SKKUIACUC2018-07–25-1). Studies involving animals are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).
Pharmacokinetics of 175Lu-DOTA-PSMA-GUL after I.V. bolus injection
Freshly prepared 175Lu-DOTA-PSMA-GUL solution (2.1 mg/mL) was administered by i.v. bolus injection via the penile vein (n = 5–7) at three doses of 1, 2, and 5 mg/kg. Approximately 0.3 mL of the jugular venous blood samples were collected at predetermined times after i.v. injection. Plasma samples were harvested by centrifugation of the blood samples at 3220 × g for 10 min. Urine and feces samples were collected at 4, 8, 12, and 24 h after i.v. injection. All samples were stored at -70 °C until analysis.
Tissue distribution of 175Lu-DOTA-PSMA-GUL after I.V. infusion
Tissue distribution of 175Lu-DOTA-PSMA-GUL was examined under two different steady-state conditions after i.v. infusion of 175Lu-DOTA-PSMA-GUL. Two target steady-state plasma concentrations (Css) were set as 3000 and 6000 ng/mL. Rats were surgically cannulated with polyethylene tubing (0.58 mm i.d. and 0.96 mm o.d.; Natume, Tokyo, Japan) in the left jugular vein for blood sampling and femoral vein for i.v. injection and infusion. After one day of recovery, 175Lu-DOTA-PSMA-GUL was administered by i.v. injection as a loading dose (LD) and i.v. infusion for 3 h to achieve the target Css. The i.v. bolus LD and i.v. infusion rates (K0) were calculated by LD = Css,target·Vss and K0 = Css,target·CL, respectively24. The volume of distribution (Vss , 0.20 L/kg) and clearance (CL, 607.55 mL/h/kg) of 175Lu-DOTA-PSMA-GUL were obtained from the i.v. injection study. The calculated LD was 0.60 mg/kg and 1.20 mg/kg for the target Css of 3,000 ng/mL and 6,000 ng/mL, respectively. The calculated K0 was 1.82 mg/h/kg and 3.65 mg/h/kg for the target Css of 3,000 ng/mL and 6,000 ng/mL, respectively.
Blood samples were collected at 1.5, 2, 2.5, and 3 h during i.v. infusion, and centrifuged at 3,200 × g for 10 min. At the end of the infusion, rats were sacrificed, and brain, lung, heart, spleen, small intestine, stomach, kidney, liver, prostate, fat, muscle, and testis were excised and immediately homogenized in normal saline. All samples were stored at -70 °C until analysis.
The plasma concentration–time data were analyzed by non-compartmental method using Phoenix® WinNonlin® (Pharsight, NC, USA). The fraction of 175Lu-DOTA-PSMA-GUL excreted into urine (Furine) and feces (Ffeces) were calculated by the ratio of the total amount of drug excreted in the urine and feces to the fraction of the dose, respectively. The tissue-to-plasma partition coefficients (KP) were calculated as the tissue-to-plasma concentration ratios.
Dose proportionality was tested for Cmax, AUCall, and AUCinf based on power model. Assuming the natural logarithm of the pharmacokinetic parameter is linearly related to the natural logarithm of dose as in the following equation: ln(PK parameter) = β0 + β1 × ln(dose), the slope coefficient (β1) and its two-sided 95% confidence intervals (CI) were estimated.
The data were statistically tested by the unpaired t-test to compare between two means and by one-way analysis of variance (ANOVA) followed by scheffe or games-howell post hoc test. The statistical significance level was set at p < 0.05. All the statistical analyses were performed by using IBM® SPSS® Statistics 26 (IBM, Armonk, NY, USA).