DX600

Purpose: For the tumor-specific ACE2 expression, this research aimed to establish and verify ACE2-targeted PET imaging in differentiating tumors with distinct ACE2 expression. Methods: 68Ga-cyc-DX600 was synthesized as tracer of ACE2 PET. NOD-SCID mice were used to prepare the subcutaneous tumor models with HEK-293 or HEK-293T/hACE2 cells to verify ACE2 specificity, with other kinds of tumor cells to evaluate the diagnostic efficiency for ACE2 expression, additionally, immunohistochemical analysis and western blot were used to certify the findings on ACE2 PET, which was then performed on four cancer patients and compared with FDG PET. Results: The metabolic clearance of 68Ga-cyc-DX600 was initially completed in 60 min, realizing an ACE2-dependent and organ-specific background of ACE2 PET; meanwhile, tracer uptake of subcutaneous tumor models was of a definite dependence on ACE2 expression (r = 0.903, p < 0.05), and the latter served as the primary factor when ACE2 PET was used for the differential diagnosis of ACE2-related tumors. In pre-clinical practice, a comparable tumor-to-background ratio was acquired in ACE2 PET of a lung cancer patient at 50 and 80 min post injection; the quantitative values of ACE2 PET and FDG PET were negatively correlated (r = - 0.971 for SUVmax, p = 0.006; r = - 0.994 for SUVmean, p = 0.001) in an esophageal cancer patient, no matter the primary lesion or metastasis.

Angiotensin-converting enzymes, ACE and ACE2, are key members of renin angiotensin system. Activation of ACE2/Ang-(1-7) pathway enhances cardiovascular protective functions of bone marrow-derived stem/progenitor cells. The current study evaluated the selectivity of ACE2 inhibitors, MLN-4760 and DX-600, and ACE and ACE2 activities in human (hu) and murine (mu) bone marrow cells. Assays were carried out in hu and mu mononuclear cells (MNCs) and huCD34(+) cells or mu-lineage-depleted (muLin(-)) cells, human-recombinant (rh) enzymes, and mu-heart with enzyme-specific substrates. ACE or ACE2 inhibition by racemic MLN-4760, its isomers MLN-4760-A and MLN-4760-B, DX600 and captopril were characterized. MLN-4760-B is relatively less efficacious and less-selective than the racemate or MLN-4760-A at hu-rhACE2, and all three of them inhibited 43% rhACE. In huMNCs, MLN-4760-B detected 63% ACE2 with 28-fold selectivity over ACE. In huCD34(+) cells, MLN-4760-B detected 38% of ACE2 activity with 63-fold selectivity. In mu-heart and muMNCs, isomer B was 100- and 228-fold selective for ACE2, respectively. In muLin(-) cells, MLN-4760-B detected 25% ACE2 activity with a pIC50 of 6.3. The racemic mixture and MLN-4760-A showed lower efficacy and poor selectivity for ACE2 in MNCs and mu-heart. ACE activity detected by captopril was 32% and 19%, respectively, in huCD34(+) and muLin(-) cells. DX600 was less efficacious, and more selective for ACE2 compared to MLN-4760-B in all samples tested. These results suggest that MLN-4760-B is a better antagonist of ACE2 than DX600 at 10 µm concentration in human and murine bone marrow cells, and that these cells express more functional ACE2 than ACE.

Angiotensin-converting enzyme 2 (ACE2) is a component of the renin-angiotensin system, and its expression and activity have been shown to be reduced in cardiovascular diseases. Enzymatic activity of ACE2 is commonly measured by hydrolysis of quenched fluorescent substrates in the absence or presence of an ACE2-specific inhibitor, such as the commercially available inhibitor DX600. Whereas recombinant human ACE2 is readily detected in mouse tissues using 1 μM DX600 at pH 7.5, the endogenous ACE2 activity in mouse tissues is barely detectable. We compared human, mouse, and rat ACE2 overexpressed in cell lines for their sensitivity to inhibition by DX600. ACE2 from all three species could be inhibited by DX600, but the half maximal inhibitory concentration (IC(50)) for human ACE2 was much lower (78-fold) than for rodent ACE2. Following optimization of pH, substrate concentration, and antagonist concentration, rat and mouse ACE2 expressed in a cell line could be accurately quantified with 10 μM DX600 (>95% inhibition) but not with 1 μM DX600 (<75% inhibition). Validation that the optimized method robustly quantifies ACE2 in mouse tissues (kidney, brain, heart, and plasma) was performed using wild-type and ACE2 knockout mice. This study provides a reliable method for measuring human, as well as endogenous ACE2 activity in rodents. Our data underscore the importance of validating the effect of DX600 on ACE2 from each particular species at the experimental conditions employed.