Towards developing a non-SnCl2 formulation for RP444, a new radiopharmaceutical for thrombus imaging.

There are many factors influencing the yield and radiochemical purity (RCP) of the radiopharmaceutical RP444. These include heating temperature, heating time, pH, the use of a buffering agent and a bulking agent, as well as the component (XV066, tricine, TPPTS, and Na99mTcO4) concentration. Through a series of radiolabeling experiments, we found that a formulation comprised of 20 microg of XV066, 6.5 mg of tricine, 40 mg of mannitol, 5 mg of TPPTS, and 0.1 mg of Pluronic acid dissolved in 1.0 mL of 250 mM succinate buffer (pH 5.0) gives the best RCP for RP444. The formulation can be lyophilized to form a stable crystal "cake". The radiolabeling is achieved by adding 1.5 mL generator eluant (33-133 mCi of Na99mTcO4) to a lyophilized vial and heating the reaction mixture at 100 degrees C for 10 min. Using this formulation, RP444 is prepared consistently in high yield with RCP > or = 90%. Formation of [99mTc]colloid is minimal (< 0.5%).

Synthesis of stable hydrazones of a hydrazinonicotinyl-modified peptide for the preparation of 99mTc-labeled radiopharmaceuticals.

Hydrazones of a 6-hydrazinonicotinyl-modified cyclic peptide IIb/IIIa receptor antagonist were prepared in order to protect the hydrazine moiety from reaction with trace aldehyde and ketone impurities encountered during the process of manufacturing and compounding lyophilized kits used in radiolabeling with (99m)Tc. Hydrazones were prepared by either a direct reaction of the 6-hydrazinonicotinyl-modified cyclic peptide with carbonyl compounds or by conjugation of the cyclic peptide with hydrazones of succinimidyl 6-hydrazinonicotinate. Stability of the hydrazones was evaluated by treatment with formaldehyde. Hydrazones derived from simple aliphatic aldehydes underwent an exchange reaction with formaldehyde, while hydrazones of aromatic aldehydes and ketones provided the greatest level of stability when challenged with formaldehyde. We have been successful in protecting 6-hydrazinonicotinyl-modified cyclic peptides from reacting with formaldehyde, while still allowing sufficient reactivity for radiolabeling with (99m)Tc. The hydrazones of succinimidyl 6-hydrazinonicotinate are convenient and general reagents for forming 6-hydrazinonicotinyl conjugates with amino-functionalized bioactive molecules.

99mTc-labeling of hydrazones of a hydrazinonicotinamide conjugated cyclic peptide.

Eight HYNICtide hydrazones (three with aliphatic substituents and five with aromatic groups) were studied for their potential use as the final intermediate for preparation of RP444, a new radiopharmaceutical under development for imaging thrombosis. The goal of this study is to screen various hydrazones through stability testing and radiolabeling and find those which are able to remain stable without significant degradation in the manufacturing process and at the same time are reactive to produce enough free hydrazine in situ for successful (99m)Tc-labeling. In an initial screening study, only hydrazones 6 and 8, which contain aliphatic substituents, gave satisfactory (>/=90%) yields of RP444 using 50 degrees C and 30 min of heating. However, their solution instability excludes them from being used as commercial reagents. Hydrazones 1 and 4 gave >/=90% yields when the reaction mixtures were heated at 80 degrees C for 30 min. Both hydrazone 1 and hydrazone 4 can be used as the final intermediate for preparation of RP444. The combination of 40 mg of tricine, 1-10 mg of TPPTS, 20-40 microg of hydrazone 1 or 4 for 50 mCi of [(99m)Tc]pertechnetate, 20-50 microg of stannous chloride, pH 4.5 +/- 0.5, and heating at 80 degrees C for 30 min gives the best yield for RP444. It is surprising that hydrazones 1 and 4 have both the solution stability with respect to decomposition and to reaction with aldehydes and ketones and yet are able to hydrolyze in situ to produce enough free HYNICtide for the (99m)Tc-labeling.

Labeling cyclic glycoprotein IIb/IIIa receptor antagonists with 99mTc by the preformed chelate approach: effects of chelators on properties of [99mTc]chelator-peptide conjugates.

Several cyclic GPIIb/IIIa receptor antagonists were labeled with 99mTc by the preformed chelate approach using chelators such as H4L1 [4,5-bis(mercaptoacetamido)pentanoic acid], H4L2 [3,4-bis-(mercaptoacetamido)benzoic acid], H3L3 [2-(mercapto)ethylaminoacetyl-L-cysteine], H4L4 [N-(mercaptoacetyl)glycylglycylglycine], H4L5 [N-[2-(mercapto)propionyl]glycylglycylglycine], and H4L6 [N-[2-(mercapto)propionyl]glycylglycyl-gamma-aminobutyric acid]. In this approach, the [99mTc]chelator complexes are formed first, followed by the activation of the carboxylic group on the complex by formation of its tetrafluorophenol (TFP) ester and the conjugation of the TFP ester with an amino group of a cyclic GPIIb/IIIa receptor antagonist. The 99mTc-labeled cyclic GPIIb/IIIa receptor antagonists were characterized by radio-HPLC (high-performance liquid chromatography); differences in lipophilicity of the [99mTc]chelator-peptide conjugate are attributable to the effects of both the cyclic peptide and the chelator.

Labeling a hydrazino nicotinamide-modified cyclic IIb/IIIa receptor antagonist with 99mTc using aminocarboxylates as coligands.

A series of 99mTc complexes containing a hydrazinonicotinamide-conjugated cyclic IIb/IIIa receptor antagonist, cyclo(D-Val-NMeArg-Gly-Asp-Mamb-(hydrazinonicotinyl-5- (6-aminocaproic acid))), were synthesized in high yield using tricine or other aminocarboxylates as coligands. These 99mTc complexes have the potential to be used as thrombus imaging agents. The radiolabeling of the HYNIC-conjugated cyclic IIb/IIIa peptide (HYNICtide) was carried out by reaction with pertechnetate in the presence of excess tricine and stannous chloride at pH 4-5. The reaction time and temperature depend on the amount of the HYNICtide and pertechnetate used for the radiolabeling. Very high specific activity (> or = 20,000 mCi/mumol) can be achieved for the complex [99mTc(HYNICtide)(tricine)2] without postlabeling purification. The complex [99mTc(HYNICtide)(tricine)2] was found by two reversed phase HPLC methods to exist as multiple species, some of which interconvert, depending on the temperature, reaction time, and pH of the reaction mixture. The presence of these multiple species is most likely due to different bonding modalities of either the hydrazine moiety of the HYNICtide or the two tricine coligands. The complex [99mTc(HYNICtide)(EDDA)] (EDDA = ethylenediamine-N,N'-diacetic acid) was prepared either by reacting the cyclic IIb/IIIa HYNICtide with pertechnetate, excess EDDA, and stannous chloride at pH 4-5 and 75 degrees C for 30 min or by reacting excess EDDA with [99mTc(HYNICtide)(tricine)2]. The complex [99mTc(HYNICtide)(EDDA)] was found to be stable for at least 12 h in the reaction mixture. Three major species were detected in the radio-HPLC chromatograms, presumably due to the more limited number of possible coordination isomers. Similar results were obtained using other polydentate aminocarboxylates (such as HEDTA, N-(2-hydroxyethyl)ethylenediaminetriacetic acid) as coligands. It is clear that the replacement of tricine by other polydentate aminocarboxylates produces 99mTc-HYNICtide complexes with higher stability and fewer coordination isomers.

Potential 99mTc radiopharmaceuticals for renal imaging: tris(N-substituted-3-hydroxy-2-methyl-4-pyridinonato)technetium(IV) cations.

A series of monocationic complexes of N-substituted-3-hydroxy-2-methyl-4-pyridinones labeled with technetium(IV)-99m have been evaluated in vivo as potential radiopharmaceuticals. The pyridinones have different substituents at the ring nitrogen atom: ethyl, i-propyl, i-butyl, benzyl, phenyl, p-methoxyphenyl, 3-butoxypropyl and cyclohexyl. Biodistribution studies of the 99mTc complexes have been carried out in rabbits and mice. High kidney uptake and retention of the radionuclide has been shown in rabbits and mice with the cationic complexes of 3-hydroxy-1-(p-methoxyphenyl)-2-methyl-4-pyridinone and 1-(cyclohexyl)-3-hydroxy-2-methyl-4-pyridinone. These 99mTcL3+ compounds appear to be morphologic renal agents.