(99m)Tc-MAG3-aptamer for imaging human tumors associated with high level of matrix metalloprotease-9.

The human matrix metalloprotease 9 (hMMP-9) is involved in many physiological processes such as tissue remodeling. Its overexpression in tumors promotes the release of cancer cells thus contributing to tumor metastasis. It is a relevant marker of malignant tumors. We selected an RNA aptamer containing 2'-fluoro, pyrimidine ribonucleosides, that exhibits a strong affinity for hMMP-9 (K(d) = 20 nM) and that discriminates other human MMPs: no binding was detected to either hMMP-2 or -7. Investigating the binding properties of different MMP-9 aptamer variants by surface plasmon resonance allowed the determination of recognition elements. As a result, a truncated aptamer, 36 nucleotides long, was made fully resistant to nuclease following the substitution of every purine ribonucleoside residue by 2'-O-methyl analogues and was conjugated to S-acetylmercaptoacetyltriglycine for imaging purposes. The resulting modified aptamer retained the binding properties of the originally selected sequence. Following (99m)Tc labeling, this aptamer was used for ex vivo imaging slices of human brain tumors. We were able to specifically detect the presence of hMMP-9 in such tissues.

Preparation of 99m Tc-MAG3: the effect on radiochemical purity of using sodium chloride injection from plastic ampoules that have been exposed to light.

OBJECTIVE: To determine the circumstances under which sodium chloride injection (SCI) that has been exposed to fluorescent light then used to prepare 99m Tc-MAG3 causes low radiochemical purity (RCP). METHODS: Two brands of SCI in plastic ampoules (Braun and Steri-Amp) and one in glass vials (Drytec) were exposed to light for up to 7 days then used to prepare 99m Tc-MAG3. RCP was measured by liquid chromatography. To study the effect on the labelling reaction, the reconstituted MAG3 kit was analysed before and after boiling and the formation of the 99m Tc-tartrate intermediate was investigated. Exposed water from plastic ampoules was analysed by mass spectrometry. RESULTS: After no exposure, each brand resulted in high RCP 99m Tc-MAG3 (>94%). Drytec SCI produced high RCP throughout (96.7 +/- 0.3%, n=5, 7 days). The RCP produced by Steri-Amp and Braun fell to 85.2 +/- 5.2% and 93.5 +/- 1.6% after exposure for 2 and 4 days, respectively. The chromatogram before boiling contained peaks corresponding to 99m Tc-tartrate and 99m Tc-pertechnetate. After boiling with unexposed SCI, these were minimal and a 99m Tc-MAG3 peak dominated. After boiling with exposed SCI, 99m Tc-pertechnetate and 99m Tc-MAG3 peaks were present. Measurements on tartrate showed a high level of 99m Tc-tartrate before and after boiling with unexposed SCI but a high level of 99m Tc-pertechnetate after boiling with exposed SCI. Mass spectrometry showed that compounds leach into the solution upon exposure to light. CONCLUSION: Preparing 99m Tc-MAG3 using SCI from plastic ampoules that have been exposed to light causes reduced RCP. Exposure of plastic ampoules to light causes leaching of many compounds into the solution. An unknown leached compound destabilizes the 99m Tc-tartrate intermediate complex.

Validation of alternative methods of preparing 99mTc-MAG3.

Parameters in the preparation of 99mTc-mertiatide (99mTc-MAG3) were investigated to determine the importance of total activity, activity concentration, boiling time, and delay before boiling for the radiochemical purity (RCP) and stability of the product. Satisfactory RCP results (>90%) were obtained over a range of concentrations including a dilute preparation for paediatric use. RCP was not affected by the time between the addition of pertechnetate and boiling, but low RCP (<60%) resulted when the kit was boiled for less than 10 min.

Preparation of 99mTc-MAG3: radiochemical purity is affected by the residence time of sodium chloride injection in a three-part syringe.

BACKGROUND: Routine technetium-99m mercaptoacetyltriglycine (99mTc-MAG3) radiochemical purity measurements have revealed occasional unacceptably low values. Preliminary investigations suggested a causal link with the residence time of sodium chloride injection in the syringe used to reconstitute the MAG3 kit. OBJECTIVES: To investigate the cause of this phenomenon, determine how it can be avoided and establish whether it occurs with other 99mTc radiopharmaceuticals. METHODS: 99mTc-MAG3 was prepared by drawing sodium chloride injection into a lubricated, three-part, 10 ml Plastipak syringe and using it to reconstitute a MAG3 kit immediately or after a 15 min incubation period. The radiochemical purity was measured by high-performance liquid chromatography. The experiment was repeated using lubricant-free, two-part, Norm-Ject syringes and lubricated, two-part, Monoject syringes (15 min incubation only). To investigate the influence of Plastipak's rubber components on the radiochemical purity, samples were prepared using sodium chloride injection that had been incubated with lubricated or lubricant-free rubber plunger ends. Similar experiments were performed to determine the effect of Plastipak on 99mTc-exametazime, 99mTc-sestamibi and 99mTc-tetrofosmin. RESULTS: The radiochemical purities of 99mTc-MAG3 prepared with sodium chloride injection incubated for 0 and 15 min in Plastipak syringes were 96.4+/-0.5% and 89.4+/-5.5%, respectively. The difference was significant (P<0.05, n=10). With Norm-Ject syringes, the radiochemical purities were 96.5+/-0.5% and 96.6+/-0.5%, respectively. The difference was not significant (P>0.05, n=10). With Monoject syringes, the radiochemical purity was 96.6+/-0.4% (n=10). 99mTc-MAG3 prepared using sodium chloride injection treated with lubricated and unlubricated syringe rubber plunger ends had radiochemical purities of 85.3+/-6.6% and 82.1+/-6.5% (n=5), respectively. The radiochemical purities of other 99mTc radiopharmaceuticals prepared using sodium chloride injection incubated for 0 or 15 min in Plastipak syringes were as follows: 99mTc-exametazime, 95.3+/-0.6% and 94.5+/-1.8%; 99mTc-sestamibi, 98.0+/-0.6% and 97.7+/-0.6%; 99mTc-tetrofosmin, 96.5+/-0.2% and 97.0+/-0.4%. None of the differences was significant (P>0.05, n=5). CONCLUSIONS: A lipophilic impurity, originating from the rubber plunger of a three-part Plastipak syringe, is formed in 99mTc-MAG3 when the sodium chloride injection used to reconstitute the kit is in the syringe for a prolonged time. The effect is eliminated by using a two-part syringe or by injecting the sodium chloride injection into the kit immediately. The phenomenon does not occur with 99mTc-exametazime, 99mTc-sestamibi or 99mTc-tetrofosmin.

Investigation of the labelling characteristics of 99mTc-mercaptoacetyltriglycine.

S-Benzyl-, S-benzamidomethyl- and S-benzoylmercaptoacetyltriglycine were synthesized and compared in exchange labelling experiments for the preparation of 99mTc-MAG3. The rate of exchange from 99mTc-tartrate to 99mTc-MAG3 starting from the respective precursors was determined in different conditions. Labelling proceeded most rapidly starting from the S-benzoyl protected precursor but efficient labelling was also accomplished using the more stable S-benzamidomethyl- and S-benzylmercaptoacetyltriglycine. 99mTc-MAG3 was also prepared by direct labelling of unprotected mercaptoacetyltriglycine at alkaline pH. Radiochemical purity in these conditions is mainly dependent on the pH during labelling.

Anomalies in hepatobiliary excretion of technetium-99m-MAG3 preparations.

Technetium-99m-MAG3 is accepted as a renal tubular function agent. However, sporadic liver and gall bladder visualisation during its clinical use is clearly a disadvantage. HPLC-purified 99mTc-MAG3 samples exhibited appreciable hepatobiliary uptake (7%), and an elevated level of such uptake was observed in unpurified kit preparations, which was stated to be associated with the excretory property of the radiolabeled kit impurities. To verify this we attempted to quantitate the hepatobiliary uptake of the kit preparations with that of its radiolabeled components. The contribution of each component toward hepatobiliary uptake of the sample was calculated from their abundance in the chelate mixture and the individual biodistribution of the isolated components. However, the anticipated hepatobiliary uptake of different preparations of 99mTc-MAG3 calculated in this way was always lower than that of the experimental value determined directly. Further work is needed to explain the anomaly.

Synthesis, radiolabelling and biological characteristics of a bombesin peptide analog as a tumor imaging agent.

BACKGROUND: Several human cancers, including small cell lung, prostate, breast, gastric, colon and pancreatic cancers, express receptors for bombesin-like peptides. Bombesin (BN) peptides that bind specifically to these receptors are useful for detection of bombesin receptor-expressing cancers in vivo. A new 99mTc-labelled-BN peptide for targeting bombesin receptor-expressing cancers was prepared and characterized. MATERIALS AND METHODS: MAG3-coupled BN peptide (MAG3-BN) was prepared by solid-phase synthesis and radiolabelled with 99mTc by an exchange method. In vitro cell binding assays were conducted on human breast cancer cell lines, MDA-MB-231 and MCF-7. In vivo biodistribution studies were performed in normal and nude mice bearing bombesin receptor-positive tumors. RESULTS: Radiolabelling of MAG3-BN with 99mTc produces a single radioactive species (> 95%). In vitro cell-binding indicated the affinity and specificity of 99mTc-MAG3-BN towards bombesin receptors. In vivo biodistribution in mice demonstrated that 99mTc-MAG3-BN cleared rapidly from the blood and most non-targeted tissues and was excreted mainly via the kidneys. Uptake in bombesin receptor-positive tissues and in the tumor was low to moderate. CONCLUSION: 99mTc-MAG3-BN displays good radiolabelling together with certain favorable biological characteristics and might be a useful peptide radiopharmaceutical in the detection of bombesin receptor-expressing cancers in vivo.

Preparation of 99Tcm-MAG3: no confirmation that sodium chloride injections from plastic containers affect radiochemical purity.

Reports have suggested that when sodium chloride injections from a plastic ampoule are used during the preparation of 99Tcm-mercaptoacetyltriglycine (99Tcm-MAG3), the radiochemical purity of the final product might be reduced. A study was therefore undertaken to examine the effect of sodium chloride injections from five manufacturers on the radiochemical purity and stability of 99Tcm-MAG3. One sodium chloride injection was supplied in a glass vial, three in plastic ampoules and one in a plastic infusion bag. Three batches of sodium chloride injections from each manufacturer were tested. The radiopharmaceutical was prepared at a radioactive concentration of 1.1 GBq in 10 ml according to the instructions of the manufacturer of TechneScan MAG3. Analysis of radiochemical purity was performed by high-performance liquid chromatography immediately after preparation and 6 h later. Using 95% as the minimum acceptable radiochemical purity, all the products were satisfactory over the 6 h test period. No manufacturer's sodium chloride injection was found to have a statistically significant effect on the radiochemical purity. Based on the 15 batches of sodium chloride injection tested, this study cannot confirm that sodium chloride injections from a plastic container affect the radiochemical purity of 99Tcm-MAG3. However, in view of the known sensitivity of some 99Tcm radiopharmaceuticals to external influences, it is probably good practice to test radiochemical purity when new batches of ancillary materials, such as sodium chloride injections, are introduced.

Testing different storage conditions for (99m)Tc-MAG3 kit: can hot fractioning reduce the cost per unit dose?

Since its release for routine clinical use, (99m)Tc-mercaptoacetyltriglycine (MAG3) has become an important alternative to (131)I-labeled orthoiodohippuran. The cold kit for MAG3 is expensive, especially in developing countries. Therefore, unique storage conditions should be provided for cost reduction. Cold fractioning is a well-known procedure but has special requirements, such as a nitrogen tank and a laminar flow hood. The aim of this study was to prolong the shelf life of (99m)Tc-labeled MAG3 by a hot fractioning method, which separates the patient doses after (99m)Tc labeling. The radiochemical purity of the (99m)Tc-labeled MAG3 kit was tested under different storage conditions. Hot fractioning of the (99m)Tc-labeled MAG3 kit was found to be a possible alternative to cold fractioning for routine clinical studies.

The influence of temperature and alkaline pH on the labeling of free and conjugated MAG3 with technetium-99m.

Benzoyl-protected mercaptoacetyltriglycine (S-benzoyl MAG3) is radiolabeled by tartrate transchelation at elevated temperatures or basic pH. The object of this investigation was to establish whether the same 99mTc labeled species are formed when S-acetyl (S-acetyl MAG3)-conjugated compounds are radiolabeled by tartrate transchelation at ambient temperature and neutral pH in contrast to labeling at 95 degrees C and pH 11. S-acetyl MAG3 was conjugated to biocytin and to the amine-derivitized oligomers DNA and PNA. Along with free S-acetyl MAG3, these were radiolabeled under the different conditions. Although labeling efficiencies were always lower when labeled at ambient temperature and neutral pH relative to labeling at 95 degrees C or pH 11 (free S-acetyl MAG3 could not be labeled at all), size exclusion and reverse phase HPLC showed no difference with labeling conditions in the radiochemical profiles for labeled DNA and biocytin. In the case of DNA, a cysteine challenge also failed to demonstrate a difference. However, in the case of PNA, some important differences were observed in the size exclusion HPLC radiochromatograms. In addition, PNA labeled at ambient temperature and neutral pH was less stable to transchelation to cysteine. In conclusion, S-acetyl MAG3 conjugated compounds may be radiolabeled at ambient temperature and neutral pH. In most cases, the radiochemical species produced appear to be identical to those formed when labeling is accomplished at 95 degrees C or pH 11.

Characteristics and biological behaviour of 99mTc-labelled hydroxyacetyltriglycine, a potential alternative to 99mTc-MAG3.

Substitution of the oxidation-sensitive thiol function of mercaptoacetyltriglycine (MAG3) by a hydroxyl group yields a tetraligand (hydroxyacetyltriglycine or HAG3) which is almost insensitive to oxidation and has the advantage over MAG3 that it can be stored safely without protection of the alcohol function. We found that deprotected HAG3 could be directly labelled at alkaline pH (pH>/=11.5) and room temperature in high yield (>95%). Results of electrophoresis experiments suggested a comparable structure for 99mTc-HAG3 and 99mTc-MAG3, namely binding of an oxotechnetium(V)core via three deprotonated amides and a deprotonated hydroxyl group. Biodistribution studies in mice at 10 min and 30 min p.i. showed a slightly higher urinary excretion, a faster renal transit and a significantly lower hepatobiliary handling for 99mTc-HAG3 than for 99mTc-MAG3. In a baboon, the 1-h plasma clearance of 99mTc-HAG3 was clearly higher than that of 99mTc-MAG3. Its plasma protein binding was in the same order as that of Hippuran and much lower than that of 99mTc-MAG3. Evaluation in a human volunteer confirmed the favourable biological characteristics of 99mTc-HAG3, namely a rapid renal excretion, a high 1-h plasma clearance and a negligible hepatobiliary handling. The results indicate that 99mTc-HAG3 may be an easy-to-prepare and practical substitute for 99mTc-MAG3 with improved renal excretion characteristics.