cGMP-Compatible preparative scale synthesis of near-infrared fluorophores

Abstract

Image-guided surgery using optical imaging requires the availability of large quantities of clinical-grade fluorophores. We describe the cGMP-compatible synthesis of the zwitterionic heptamethine indocyanine near-infrared fluorophore ZW800-1 at the 10 g scale (~1000 patient doses) using facile and efficient solvent purification, and without the need for column chromatography. ZW800-1 has >90% yield at the final step and >99% purity as measured by fluorescence and evaporative light scatter detection. We describe an analytical framework for qualifying impurities, as well as a detailed analysis of counterion identities. Finally, we report the unique in vivo properties of ZW800-1 in large animals approaching the size of humans, thus laying the foundation for rapid clinical translation of these methods.

Figure 1. cGMP-compatible synthesis of ZW800-1

a. Scheme. AcOH, acetic acid; EA, ethyl acetate; KOH, potassium hydroxide; iPrOH, 2-propanol; MeOH, methanol; DW, distilled water; ACN, acetonitrile; DMF, N,N-dimethylformamide; POCl₃, phosphorus oxychloride; Hex, n-hexane; MTBE, methyl tertiary butyl ether; AcONa, sodium acetate; EtOH, ethanol; NaOH, sodium hydroxide; DMSO, dimethyl sulfoxide. b. LC-MS analysis using a photodiode array (PDA) at 210 nm, PDA at 770 nm, fluorescence detector (FLD; 770 nm excitation/790 nm emission), evaporative light scatter detection (ELSD), and electrospray time-of-flight mass spectrometry (TOF-MS ES+). Mobile phases were water supplemented with 0.1% formic acid (FA) and ACN, and separation occurred using a gradient of 0% to 90% ACN over 20 min at a flow rate of 1 mL/min.

Figure 1. cGMP-compatible synthesis of ZW800-1

a. Scheme. AcOH, acetic acid; EA, ethyl acetate; KOH, potassium hydroxide; iPrOH, 2-propanol; MeOH, methanol; DW, distilled water; ACN, acetonitrile; DMF, N,N-dimethylformamide; POCl₃, phosphorus oxychloride; Hex, n-hexane; MTBE, methyl tertiary butyl ether; AcONa, sodium acetate; EtOH, ethanol; NaOH, sodium hydroxide; DMSO, dimethyl sulfoxide. b. LC-MS analysis using a photodiode array (PDA) at 210 nm, PDA at 770 nm, fluorescence detector (FLD; 770 nm excitation/790 nm emission), evaporative light scatter detection (ELSD), and electrospray time-of-flight mass spectrometry (TOF-MS ES+). Mobile phases were water supplemented with 0.1% formic acid (FA) and ACN, and separation occurred using a gradient of 0% to 90% ACN over 20 min at a flow rate of 1 mL/min.

Figure 2. Optical properties of ZW800-1 in aqueous buffers and organic solvents

a. Absorbance (Abs) and fluorescence (FL) spectra of 1 μM ZW800-1 after incubating in FBS supplemented with 50 mM HEPES (pH 7.4) at 37°C for 4 h. b. Optical properties of preparative scale ZW800-1 in various aqueous buffers and organic solvents. c. Effect of fluorophore concentration on total fluorescence yield (i.e., quenching) in PBS and FBS as measured using a 90° (left) or 0° (right) excitation/emission geometry.

Figure 3. Biodistribution and clearance of ZW800-1 in mice and pigs

a. Dose-dependent clearance of ZW800-1 in mice. ZW800-1 was injected intravenously into CD-1 mice at a dose of 30 nmol (1.35 mg/kg), 60 nmol (2.7 mg/kg), and 120 nmol (5.5 mg/kg), 4 h prior to imaging. Shown are representative images from N = 3 animals per dose. NIR fluorescence images for each condition have identical exposure times and normalizations. Scale bars = 1 cm. b. Blood clearance (%ID/g), blood half-life (mean ± 95% confidence intervals), and elimination of the injected dose into urine in 35 kg Yorkshire pigs. Each data point is the mean ± S.D. from N = 3 animals. c. Tissue/organ uptake in 35 kg Yorkshire pigs 1 h post-intravenous injection of 11.5 mg of ZW800-1 or ICG. Shown are color and 800 nm NIR fluorescence images of the skin and surgically exposed organs/tissues. NIR fluorescence images have identical exposure and normalizations. Abbreviations used are: Bl, bladder; BD, bile duct; Du, duodenum; He, heart; Ki, kidneys; Li, liver; Lu, lung; Pa, pancreas; Sk, skin; Sp, spleen; St, stomach; Ur, ureter. Scale bars = 1 cm. d. Gel-filtration chromatography (top) and ESI-TOF mass spectrometry (bottom) of ZW800-1 (m/z = 943) in PBS (left), urine from a mouse 4 h postinjection (middle), and urine from a pig 4 h postinjection (right). Inserts show absorbance and fluorescence spectra of the gel-filtration peak (top) and high-resolution masses (bottom). Data are representative of N = 3 independent experiments per condition.

Figure 3. Biodistribution and clearance of ZW800-1 in mice and pigs

a. Dose-dependent clearance of ZW800-1 in mice. ZW800-1 was injected intravenously into CD-1 mice at a dose of 30 nmol (1.35 mg/kg), 60 nmol (2.7 mg/kg), and 120 nmol (5.5 mg/kg), 4 h prior to imaging. Shown are representative images from N = 3 animals per dose. NIR fluorescence images for each condition have identical exposure times and normalizations. Scale bars = 1 cm. b. Blood clearance (%ID/g), blood half-life (mean ± 95% confidence intervals), and elimination of the injected dose into urine in 35 kg Yorkshire pigs. Each data point is the mean ± S.D. from N = 3 animals. c. Tissue/organ uptake in 35 kg Yorkshire pigs 1 h post-intravenous injection of 11.5 mg of ZW800-1 or ICG. Shown are color and 800 nm NIR fluorescence images of the skin and surgically exposed organs/tissues. NIR fluorescence images have identical exposure and normalizations. Abbreviations used are: Bl, bladder; BD, bile duct; Du, duodenum; He, heart; Ki, kidneys; Li, liver; Lu, lung; Pa, pancreas; Sk, skin; Sp, spleen; St, stomach; Ur, ureter. Scale bars = 1 cm. d. Gel-filtration chromatography (top) and ESI-TOF mass spectrometry (bottom) of ZW800-1 (m/z = 943) in PBS (left), urine from a mouse 4 h postinjection (middle), and urine from a pig 4 h postinjection (right). Inserts show absorbance and fluorescence spectra of the gel-filtration peak (top) and high-resolution masses (bottom). Data are representative of N = 3 independent experiments per condition.

Figure 3. Biodistribution and clearance of ZW800-1 in mice and pigs

a. Dose-dependent clearance of ZW800-1 in mice. ZW800-1 was injected intravenously into CD-1 mice at a dose of 30 nmol (1.35 mg/kg), 60 nmol (2.7 mg/kg), and 120 nmol (5.5 mg/kg), 4 h prior to imaging. Shown are representative images from N = 3 animals per dose. NIR fluorescence images for each condition have identical exposure times and normalizations. Scale bars = 1 cm. b. Blood clearance (%ID/g), blood half-life (mean ± 95% confidence intervals), and elimination of the injected dose into urine in 35 kg Yorkshire pigs. Each data point is the mean ± S.D. from N = 3 animals. c. Tissue/organ uptake in 35 kg Yorkshire pigs 1 h post-intravenous injection of 11.5 mg of ZW800-1 or ICG. Shown are color and 800 nm NIR fluorescence images of the skin and surgically exposed organs/tissues. NIR fluorescence images have identical exposure and normalizations. Abbreviations used are: Bl, bladder; BD, bile duct; Du, duodenum; He, heart; Ki, kidneys; Li, liver; Lu, lung; Pa, pancreas; Sk, skin; Sp, spleen; St, stomach; Ur, ureter. Scale bars = 1 cm. d. Gel-filtration chromatography (top) and ESI-TOF mass spectrometry (bottom) of ZW800-1 (m/z = 943) in PBS (left), urine from a mouse 4 h postinjection (middle), and urine from a pig 4 h postinjection (right). Inserts show absorbance and fluorescence spectra of the gel-filtration peak (top) and high-resolution masses (bottom). Data are representative of N = 3 independent experiments per condition.

Figure 3. Biodistribution and clearance of ZW800-1 in mice and pigs

a. Dose-dependent clearance of ZW800-1 in mice. ZW800-1 was injected intravenously into CD-1 mice at a dose of 30 nmol (1.35 mg/kg), 60 nmol (2.7 mg/kg), and 120 nmol (5.5 mg/kg), 4 h prior to imaging. Shown are representative images from N = 3 animals per dose. NIR fluorescence images for each condition have identical exposure times and normalizations. Scale bars = 1 cm. b. Blood clearance (%ID/g), blood half-life (mean ± 95% confidence intervals), and elimination of the injected dose into urine in 35 kg Yorkshire pigs. Each data point is the mean ± S.D. from N = 3 animals. c. Tissue/organ uptake in 35 kg Yorkshire pigs 1 h post-intravenous injection of 11.5 mg of ZW800-1 or ICG. Shown are color and 800 nm NIR fluorescence images of the skin and surgically exposed organs/tissues. NIR fluorescence images have identical exposure and normalizations. Abbreviations used are: Bl, bladder; BD, bile duct; Du, duodenum; He, heart; Ki, kidneys; Li, liver; Lu, lung; Pa, pancreas; Sk, skin; Sp, spleen; St, stomach; Ur, ureter. Scale bars = 1 cm. d. Gel-filtration chromatography (top) and ESI-TOF mass spectrometry (bottom) of ZW800-1 (m/z = 943) in PBS (left), urine from a mouse 4 h postinjection (middle), and urine from a pig 4 h postinjection (right). Inserts show absorbance and fluorescence spectra of the gel-filtration peak (top) and high-resolution masses (bottom). Data are representative of N = 3 independent experiments per condition.