This study investigated the mechanism of myocardial retention of technetium-99m-sestamibi. 99mTc-sestamibi was injected intravenously into guinea pigs, and the myocardium was homogenized and fractionated by differential centrifugation. More than 90% of myocardial 99mTc-sestamibi was localized within the mitochondrial fraction. Calcium was found to release 99mTc-sestamibi from the mitochondrial fraction, with an IC50 of 2.54 +/- 0.98 mM. This effect was potentiated by NaCl, and inhibited by the mitochondrial calcium channel blocker ruthenium red. In vitro uptake of 99mTc-sestamibi was found to increase from 10.5% +/- 3.0% to 61.2% +/- 0.2% with the addition of 10 mM succinate, indicating that respiration is involved. Since irreversible ischemia results in cellular and mitochondrial calcium "overload" and loss of mitochondrial metabolic function, 99mTc-sestamibi should not be retained in necrotic or irreversibly ischemic myocardium, and could potentially act as a sensitive indicator of myocardial cell viability.

Ternary ligand technetium complexes of a hydrazinonicotinamide-conjugated cyclic peptide (HYNICtide: cyclo(D-Val-NMeArg-Gly-Asp-Mamb(5-(6-(6-hydrazinonicotinamido)hexanamide)))) and 2-hydrazinopyridine (HYPY) were prepared and characterized by various spectroscopic methods. The HPLC concordance experiments for (99m)Tc and (99)Tc analogues show clearly that the same complexes are prepared on the no-carrier-added ((99m)Tc) and the carrier-added ((99)Tc) levels. Using a chirality experiment, it was demonstrated that the presence of two radiometric peaks in the HPLC chromatograms of RP444, RP445, and RP446 is due to the resolution of diastereomers, which result from the presence of chiral cyclic peptide and the formation of two enantiomers of the technetium chelate. In a ligand challenge experiment, we found that the high solution stability of these ternary ligand [(99m)Tc]HYNICtide complexes is due to their kinetic inertness. The 1:1:1:1 composition for Tc:HYNICtide:L:tricine (L = TPPTS, TPPDS, and TPPMS) in these ternary ligand [(99)Tc]HYNICtide complexes is confirmed by (1)H NMR and FAB mass spectral data and is completely consistent with that determined on the tracer ((99m)Tc) level. In addition, the IC(50) values of RP444, RP445, and RP446 and the two isomeric forms of RP444 were determined using a platelet IIb/IIIa binding assay. Both isomeric forms of RP444 were found to have the same binding affinity (IC(50) = 13 +/- 2 nM). Complexes [(99)Tc(HYPY)(PPh(3))(2)Cl(2)] and [(99)Tc(HYPY)(PPh(3))(tricine)] were isolated from the reaction of HYPY with [n-Bu(4)N][TcOCl(4)(-)] in the presence of excess tricine and triphenylphosphine. [(99)Tc(HYPY)(PPh(3))(tricine)] serves as a model for ternary ligand [(99m)Tc]HYNICtide complexes. Both complexes have been characterized by HPLC, spectroscopic (IR, NMR, and FAB-MS) methods, and elemental analysis. The HPLC concordance for complexes [(99m)Tc(HYPY)(PPh(3))(tricine)] and [(99)Tc(HYPY)(PPh(3))(tricine)] shows that the two complexes are identical. The NMR ((1)H and (13)C) data suggests that the complex [(99)Tc(HYPY)(PPh(3))(tricine)] have an octahedral coordination geometry with a monodentate diazenido HYPY, a tetradentate tricine, and a monodentate triphenylphosphine coligand.

A HYNIC-conjugated chemotactic peptide (fMLFK-HYNIC) was labeled with (99m)Tc using tricine and TPPTS as coligands. The combination of fMLFK-HYNIC, tricine, and TPPTS with (99m)Tc produced a ternary ligand complex [(99m)Tc(fMLFK-HYNIC)(tricine)(TPPTS)] (RP463). RP463 was synthesized either in two steps, in which the binary ligand complex [(99m)Tc(fMLFK-HYNIC)(tricine)(2)] (RP469) was formed first and then reacted with TPPTS, or in one step by direct reduction of [(99m)Tc]pertechnetate with stannous chloride in the presence of fMLFK-HYNIC, tricine, and TPPTS. The radiolabeling yield for RP463 was usually >/=90% using 10 microg of fMLFK-HYNIC and 100 mCi of [(99m)Tc]pertechnetate. Unlike RP469, which decomposed rapidly in the absence of excess tricine coligand, RP463 was stable in solution for at least 6 h. [(99)Tc]RP463 was prepared and characterized by HPLC and electrospray mass spectrometry. In an in vitro assay, [(99)Tc]RP463 showed an IC(50) of 2 nM against binding of [(3)H]fMLF to receptors on PMNs. [(99)Tc]RP463 also induces effectively the superoxide release of polymorphonuclear leukocytes (PMNs) with an EC(50) value of 0.2 +/- 0.2 nM. The localization of RP463 in the infection foci was assessed in a rabbit infection model. RP463 was cleared from the blood faster than RP469 and was excreted mainly through the renal system. As a result of rapid blood clearance and increased uptake, the target-to-background ratios continuously increased from 1.5 +/- 0.2 at 15 min postinjection to 7.5 +/- 0.4 at 4 h postinjection. Visualization of the infected area could be as early as 2 h. A transient decrease in white blood cell count of 35% was observed during the first 30 min after injection of the HPLC-purified RP463 in the infected rabbit. This suggests that future research in this area should focus on developing highly potent antagonists for chemotactic peptide receptor or other receptors on PMNs and monocytes.

A series of 99mTc-labeled cyclic glycoprotein IIb/IIIa receptor antagonists, [99mTcO(L1-III)]-, [99mTcO-(L6-III)]-, [99mTcO(L1-V)]-, and [99mTcO(L6-V)]-, were evaluated in a canine arteriovenous (AV) shunt model for their potential use as thrombus imaging agents. The thrombus formed consists of a platelet-rich head and a fibrin-rich tail. All four agents were incorporated into the growing thrombus under both arterial (platelet-rich) and venous (platelet-poor) conditions. The rank order for uptake was [99mTcO(L1-V)]- > [99mTcO(L6-V)]- > [99mTcO(L6-III)]- > [99mTcO(L1-III)]- (arterial range, 5.8-0.47% id/g; venous range, 0.58-0.04% id/g). The uptakes of both [99mTcO(L6-III)]- and [99mTcO-(L1-III)]- under both arterial and venous conditions were not significantly greater than that of [99mTc]-albumin and [125I]fibrinogen. In contrast, the uptakes of both [99mTcO(L1-V)]- and [99mTcO(L6-V)]- were significantly greater than those of [99mTc]albumin and [125I]fibrinogen and comparable to that of [111In]platelets under both arterial and venous conditions. All four [99mTc]chelator-peptide conjugates are cleared faster than the controls with the clearance of the conjugates of peptide III faster than that of the conjugates of peptide V. The differences in incorporation are attributable to the effect of both the cyclic peptide and the chelator. The conjugate [99mTcO(L1-V)]- was also studied using a canine DVT (deep vein thrombosis) model. [99mTcO(L1-V)]- was actively incorporated into the growing thrombus with images clearly detectable within 15 min postinjection. At 2 h postinjection, thrombus/blood and thrombus/muscle ratios [region of interest (ROI)/background] were approximately 7/1 and 10/1, respectively. This clearly demonstrated that the conjugate [99mTcO(L1-V)]- has the potential for rapid diagnosis of thrombolic events occurring under both arterial and venous conditions.