Synthesis and evaluation of 99mTc-DOTA-ARA-290 as potential SPECT tracer for targeting cardiac ischemic region

Abstract

Objectives: Myocardial infarction caused by ischemia of heart tissue is the main reason for death worldwide; therefore, early detection can reduce mortality and treatment costs. Erythropoietin (EPO) has protection effects on ischemic tissue due to nonhematopoietic peptide (pHBSP; ARA-290) which is derived from the B-subunit of EPO.

Materials and methods: We designed and synthesized a modified DOTA-(Lys-Dabcyl⁶, Phe⁷)-ARA-290 using Fmoc solid-phase peptide synthesis strategies. To improve serum stability, Fmoc-Lys-(Dabcyl)-OH as lipophilic amino acid was synthesized along with Fmoc-Phe-OH which then were substituted with Arg⁶ and Ala⁷, respectively; they were then investigated for the ability to detect ischemic cardiac imaging. DOTA-(Lys-Dabcyl⁶,Phe⁷)-ARA-290 was labeled with technetium 99m, and its radiochemical purity (RCP), stability in the presence of human serum and, specific bind to hypoxic H9c2 cells were evaluated. In vivo studies for biodistribution and SPECT scintigraphy were checked in a normal and cardiac ischemia rat model.

Results: Radiolabeling purity was obtained more than 96% by ITLC, and in vitro stability of the radiopeptide up to 6 hr was 85%. The binding of ^99mTc-ARA-290 to hypoxic cells was remarkably higher than normoxic cells (3 times higher than normoxic cells at 1 hr). Biodistribution and SPECT imaging on the cardiac ischemic model showed that radiopeptide considerably accumulated in the ischemic region (cardiac ischemic-to-lung rate = 3.65 ID/g % at 0.5 hr).

Conclusion: The results of studies, in vitro and in vivo, indicated that 99mTc-DOTA-(Lys-Dabcyl6,Phe7)-ARA-290 could be an appropriate candidate for early diagnosis of cardiac ischemia.

Keywords: ARA-290; Erythropoietin (EPO); Ischemia; Molecular imaging; Technetium-99m.

Scheme 1

Synthesis of 4-(4-hidroxyphenylazo)-benzoic acid (I). Reagent and condition: a) NaOH, NaNO_2, HCl , 25 min , 0-4 °C; b) Dimethylaniline, NaOH, 2 °C, 1 hr, 69%

Scheme 2

Synthesis of Dabcyl-OSu (IIa). Reagent and condition: a) THF, DCC, 1 hr, 0-2 °C; HOSu, 25 °C, 24 hr, 84%

Scheme 3

Synthesis of Lys-Cu-Lys. Reagent and condition: a) CuCO₃.Cu (OH)2.H₂O, 1 hr, 100 °C; H2O, NaHCO, 25 °C

Scheme 4

Synthesis of Dabcyl-Lys-Cu-Lys-Dabcyl. Reagent and condition: a) CuCO₃.Cu (OH)2.H₂O, 1 hr, 100 °C; H₂O, NaHCO, 25 °C

Scheme 5

Synthesis of Fmoc-Lys (Dabcyl)-OH (III). Reagent and condition: a) EDTA (1×2), 24 hr, 25 °C. b) CH3CN/DMF/H₂O (6:2: 1), 0-2 °C; Fmoc-SOu, 1 hr, 0-2 °C; HCl, Ether, 25 °C

Figure 1.

(A) The skeletal structure of DOTA-(Lys-Dabcyl⁶,Phe⁷)-ARA-290 and (B), Electrospray ionization-mass spectroscopy of DOTA-(Lys-Dabcyl⁶,Phe⁷)-ARA-290

Figure 2

Effect of SnCl₂ on labeling purity of ^99mTc-DOTA-(Lys-Dabcyl⁶,Phe⁷)-ARA-290 (mean ± SD, n=3)

Figure 3

Effect of reaction pH on ^99mTc-DOTA-(Lys-Dabcyl⁶,Phe⁷)-ARA-290 (mean ± SD, n=3)

Figure 4

Possible complex formed between technetium-99m and DOTA

Figure 5

Normal saline and human serum stability of ^99mTc-DOTA-(Lys-Dabcyl⁶,Phe⁷)-ARA-290(mean±SD, n=3)

Figure 6

Hypoxic test of ^99mTc-DOTA-(Lys-Dabcyl⁶,Phe⁷)-ARA-29 for different times (1, 2, 3, and 4 hr)

Figure 7

Demonstrative confirm ischemia created by surgery. Cardiac ischemic biomarkers in blood plasma in different groups of rats at 12 hr after LAD ligature: (A) CK, (B) cTnI, (C) CK-MB. Data are means±SD. (n=4, **P<0.01 for ischemic model contrasting to the sham group)

Figure 8

Rat myocardial ischemic scintigraphy images: 30 (A) and 90 (B) min after injecting into the tail vein of ^99mTc-DOTA-(Lys-Dabcyl⁶,Phe⁷)-ARA-290. Arrows indicate the high cardiac ischemic accumulation position. (C) Demonstrative normal rat images at 30 min post inection