Analysis of 99mTc-pyridoxylidene-glutamate complex formation and its preparation with tin-adsorbed-resin: Sn-resin kit method

^99mTc-Pyridoxylidene-glutamate (Pc-PG) was introduced in 1975 by Baker et al. as a cholescintigraphic agent. Nevertheless its routine use has been limited due to the autoclaving process involved. To shorten the labeling procedure, an analysis of Tc-PG complex formation using the stannous chloride method was carried out. Sn-Resin (stannous ion adsorbed onto cation exchange resin) was used for labeling a stable Tc-PG with high efficiency. A preautoclaved complex of pyridoxal and glutamate was required but the labeling procedure took only 10–15 min after the elution of ^99m-TcO ₄ ^- from the generator.Formation of a complex other than a ^99mTc-complex of the Schiff-base ligand pyridoxylidene-glutamate is discussed.The new formulation of a Tc-PG kit (Sn-Resin) simplified the labeling method and reproducible data to that already reported by Baker was obtained in chromatographic studies. Its suitability in hospital departments has already been shown in clinical studies.     

The effect of formulation conditions on the distribution of 99mTc(NaBH4)-HEDP components separated by anion exchange HPLC

Reduction of ^99mTcO₄⁻ by NaBH₄ in the presence of HEDP leads to a mixture of ^99mTc-containing complexes which can be separated by anion exchange high performance liquid chromatography (HPLC). The distribution of complexes within this mixture can be varied by controlling pH, the concentration of HEDP, the concentration of technetium, and the presence of air. Suitable control of these formulation conditions can yield mixtures which consist of essentially (85%) one component. Arguments are presented to support the view that the components of ^99mTc(NaBH₄)-HEDP and ^99mTc(NaBH₄)-MDP mixtures are in fact oligomeric or polymeric complexes that can contain technetium centers in at least two different oxidation states.     

Can technetium 99m bisdiethylphosphinoethanebis-t butylisocyanide (99mTc-DEPIC) be used for routine radionuclide ventriculography?

Radionuclide ventriculography is a useful investigation in the evaluation of cardiac function. Generally, in vivo technetium 99m-labelled red blood cells (RBC) yield good quality images in ventriculography. However, it is widely believed that some drugs have an adverse effect on RBC labelling. Zanelli et al. (1987) developed a radiopharmaceutical (technetium 99m bisdiethylphosphinoethanebis-t-butylisocyanide,^99mTc-DEPIC) to obtain better results in patients using such drugs. We untertook a prospective study of 6 patients with cardiovascular and/or pulmonary disease using several kinds of drugs to evaluate imaging of the cardiac blood pool with^99mTc-DEPIC and in vivo labelled^99mTc-RBC. After injection, blood samples were taken, and gated equilibrium blood pool studies were performed. The radiochemical purity of the injected^99mTc-DEPIC varied from 76.4 to 93.6% (mean 86.4%, SD 5.7%). The protein (pre-albumin) binding was 100%. Biological half-life in blood varied from 3.3 to 4.7 h (mean 4.1 h, SD 0.5 h). For^99mTc-RBC no significant blood disappearance was seen for 8 h. The percentage of RBC-bound^99mTc varied from 96.9% to 98.3% (mean 97.0%, SD 0.5%) and was stable for at least 8 h. The heart-to-lung, heart-to-spleen, and heart-to-liver ratios were higher for^99mTc-RBC than for^99mTc-DEPIC. Furthermore,^99mTc-DEPIC showed a significant decline of the ejection fraction with time. Visually, the images with^99mTc-RBC were superior to those with^99mTc-DEPIC, especially a few hours after injection. According to our findings, in vivo labelling of^99mTc-RBC is still the method of choice for routine radionuclide ventriculography. The decline of the ejection fraction, the short blood half-life, and the intense liver uptake make^99mTc-DEPIC less suitable for this purpose.     

Technetium 99m mercaptoacetyltriglycine gamma camera clearance calculations: methodological problems

Major sources of errors in the gamma-camera methods for the calculation of renal clearance are the accuracy of background correction for obtaining the true renal time-activity curve and the validity of the externally recorded pre-cordial activity as an estimate of the plasmatic time-activity curve. With technetium 99m mercaptoacetyltriglycine (^99mTc-MAG3), because of its high protein plasma binding, one could expect minimal extravascular diffusion and hence a more accurate externally detected plasmatic curve. The high extraction rate should reduce the influence of the background, but, on the other hand, the effect of hepatobiliary excretion on the calculation of renal clearance might be significant. Our results suggest that the hepatobiliary excretion of^99mTc-MAG3 does not influence the gamma-camera renal clearance determination, even in patients with low renal function. However, the pre-cordial curve does not reflect accurately the plasmatic disappearance curve; its calibration with a single plasma sample taken at the 20th min is responsible for significant errors, probably because of an unfavourable ratio between the intravascular and extravascular activities at the 20th min. Offprint requests to: M. TondeurParts of this work have been presented at the 17th Annual Meeting of the British Nuclear Medicine Society, London, in April 1989.     

99mTc-Diethyl-HIDA

Sephadex column chromatography has shown that ^99mTc-diethyl-HIDA consists of two clearly separable components. One of them seems to be an intermediate compound changing into the other, almost completely within two hours after reconstitution of the diethyl-HIDA (Solco HIDA) with pertechnetate. The two components could be the mono- and biscomplex of N-(2,6-diethyl-acetanilid)-imino diacetic acid with one atom of technetium. Animal experiments show that there is no important difference in the biological distribution in mice between the two components.     

Preparation and evaluation of 99mTc-labeled cyclic arginine–glycine–aspartate (RGD) peptide for integrin targeting

Technetium coordination chemistry has been a subject of interest in the development of radiopharmaceuticals, especially imaging radiotracers. Due to the extensive work done on developing chelates for ^99mTc, various chelators have been investigated and applied to radiopharmceuticals. Previous studies on the coordination chemistry of the [^99mTc=O] core have established peptide-derived sequences as effective chelating ligands. These observations led to the design of tetradentate ligands derived from amino acid sequences. Such amino acid sequences provide a tetradentate coordination site for chelation to the radionuclide and an effective functional group for conjugation to biomolecules using conventional solid-phase synthetic routes. A derivative of a novel tripeptide chelating sequence, Pro–Gly–Cys (PGC) has been developed where it is possible to form stable technetium complexes with the [^99mTc=O] via N₃S₁ tetradentate coordination core that serves this function and can be readily incorporated into biomolecules using solid-phase synthesis techniques. As a model system, the RGD peptide was selected which has been well known to target the integrin receptor for angiogenesis and tumor imaging agents. The results of in vivo studies with these novel radiolabeled compounds in tumor xenografts demonstrated a distribution in tumor targeting and other organs, such as kidney, liver and intestines.     

99mTc-gentamicin: Chemical and biological evaluation

^99mTc-Gentamicin complex, a new agent for renal studies, is prepared by the reduction of pertechnetate by stannous chloride. Organ distribution in rats and paper chromatography with Whatman 3MM paper [developed in n-butanol, acetic acid, and water (4:1:1)] were favored as analytical tools for determination of labeling parameters of ^99mTc-Gentamicin. A higher concentration of stannous ion is responsible for the formation of insoluble technetium species which concentrate in the liver. The mean kidney: liver ratio of 10.4:1 was obtained 1 h after injection. This confirms that scintigraphic delineation of kidneys is highly satisfactory.     

Transport mechanisms of hepatic uptake and bile excretion in clinical hepatobiliary scintigraphy with 99mTc-N-pyridoxyl-5-methyltryptophan

IntroductionIn clinical hepatobiliary scintigraphy, ^99mTc-N-pyridoxyl-5-methyltryptophan (^99mTc-PMT) is an effective radiotracer among the ^99mTc-pyridoxylaminates. However, the mechanisms of human hepatic uptake and bile excretion transport of ^99mTc-PMT have not been determined. We thus investigated the transport mechanisms of human hepatic uptake and bile excretion in hepatobiliary scintigraphy with ^99mTc-PMT.MethodsFour solute carrier (SLC) transporters involved in hepatic uptake were evaluated using human embryonic kidney (HEK) and HeLa cells with high expression of SLC transporters (organic anion transporting polypeptide (OATP)1B1, OATP1B3, OATP2B1, organic anion transporters (OAT)2 and organic cation transporters (OCT)1) after 5 min of ^99mTc-PMT incubation. Metabolic analysis of ^99mTc-PMT was performed using pooled human liver S9. Adenosine triphosphate (ATP)-binding cassette (ABC) transporters for bile excretion were examined using hepatic ABC transporter vesicles human expressing multiple drug resistance 1 (MDR1), multidrug resistance-associated protein 2 (MRP2), breast cancer resistance protein or bile salt export pump. ^99mTc-PMT was incubated for 1, 3 and 5 min with ATP or adenosine monophosphate and these vesicles. SPECT scans were performed in normal and Eisai hyperbilirubinemic (EHBR) model rats, deficient in Mrp2 transporters, without and with verapamil (rat Mdr1 and human MDR1 inhibitor) after intravenous injection of ^99mTc-PMT.ResultsUptake of ^99mTc-PMT in HEK293/OATP1B1 and HeLa/OATP1B3 was significantly higher than that in HEK293- and HeLa-mock cells. ^99mTc-PMT was not metabolized in the human liver S9. In vesicles with high expression of ABC transporters, uptake of MDR1 or MRP2 was significantly higher at all incubation times. Bile excretion of ^99mTc-PMT was also identified by comparison between normal and EHBR rats with and without verapamil on in-vivo imaging.ConclusionsHuman hepatic uptake of ^99mTc-PMT was transferred by OATP1B1 and OATP1B3, and excretion into bile canaliculi via MDR1 and MRP2. ^99mTc-PMT hepatobiliary scintigraphy may be a useful ligand as a noninvasive method of visualizing and quantifying hepatobiliary transporter functionality, which could predict drug pharmacokinetics.     

Quantitative analysis by digital computer of 99mTc-N-pyridoxyl-5-methyltryptophan (99mTc-PMT) hepatogram in diffuse parenchymal liver diseases

^99mTc-N-pyridoxyl-5-methyltryptophan (^99mTc-PMT) hepatograms were analyzed to provide information about the liver and bile duct. Calculations were based on a four compartmental model and included corrections for blood, tissue, hepatic parenchymal and bile backgrounds. The time activity curves for ^99mTc-PMT in the cardiac region were described as the sum of two exponential functions, while curves for the hepatic regions were described as the sum of three exponential components. The measured hepatograms were compared with simulations and good agreement between the two curves showed that the compartmental model adequately described the blood and bile activities in vivo. Hepatic excretion rate constants were 0.179±0.026 in three normal subjects, 0.102±0.012 in four patients with chronic hepatitis and 0.082±0.034 in six patients with liver cirrhosis. In the cases of diffuse parenchymal liver disease, there were lower rate constants for the excretion from the liver to the bile ducts than in normals, and the relative distribution volumes were also larger than normal. Prior to the development of this compartmental model, no useful kinetic model had been found which could satisfactorily explain the time activity curves. Experience in human studies proves this method to be accurate in determining the rate constants for the hepatobiliary transport of ^99mTc-PMT     

The behavior of99mTc-hexamethylpropyleneamineoxime (99mTc-HMPAO) in blood and brain

^99mTc-hexamethylpropyleneamineoxime (^99mTc-HMPAO) is a reagent for scanning cerebral blood flow. We investigated how^99mTc-HMPAO changed in the blood and brain. The^99mTc-HMPAO, which was prepared by adding of^99mTcO _- ⁴ to HMPAO and Sn(II), consisted of primary and secondary complexes, reduced hydrolyzed^99mTc, and^99mTc0pertechnetate. The percentage of the primary complex in^99mTc-HMPAO decreased with time after preparation. The primary complex converted to the secondary one very rapidly in the presence of plasma. When^99mTc-HMPAO was injected into patients,^99mTc activity was immediately partitioned in the plasma fraction, with approximately 60% in whole blood. In plasma,^99mTc was found to be associated with proteins such as albumin and globulin.^99mTc trapped in red cells was not washed out with either plasma or saline. Biodistribution studies showed that the less lipophilic compounds of^99mTc-HMPAO could not pass through the blood brain barrier (BBB), and therefore did not accumulate in the brain. The results of gel chromatography and equilibrium dialysis indicated that no specific^99mTc binding protein was present in the brain. Considering the instability of^99mTc-HMPAO in vivo, we proposed that the speed at which the primary complex converted to the less lipophilic compounds was important in allowing^99mTc-HMPAO to pass through the BBB and to be fixed in the brain.     

The effect of DMSO treatment on the composition of 99mTc(Sn)EHDP

To find an explanation for the reported positive effect of a dimethyl sulfoxide (DMSO) treatment on the performance of the bone scan agent technetium-(tin)-ethane-1-hydroxy-1, 1-diphosphonate, we compared the composition of the agent, prepared with and without treatment with DMSO by using high performance ion-pair chromatography (IPC).The preparation obtained with the DMSO treatment appeared to contain a larger fraction of large and highly charged polynuclear complexes than the preparation without the DMSO treatment. According to experiments by other investigators the smaller ^99mTc(Sn)EHDP complexes (early eluting components in IPC) give lower bone/blood ratios than the larger ones. The results presented in this paper show that the explanation for the effect of the DMSO treatment may be that the Sn-EHDP complexes which are removed by extraction with DMSO, give relatively small ^99mTc(Sn)EHDP complexes. Without these small complexes a superior bone scan agent is obtained.From experiments in which an excess of ⁹⁹TcO₄ over Sn(II) was used, it was concluded that at least one of the technetium complexes contains Sn(IV). On the other hand, the absence of Sn from a late eluting technetium complex was proven.     

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. Received 1 May and in revised form 18 July 1997     

Biodistribution of Tc-99m methoxy-isobutyl-isonitrile (MIBI) in humans

Hexakis (methoxyisobutilisonitrile) technetium(I), ^99mTc-MIBI, has been proposed for myocardial perfusion studies. We have evaluated the biodistribution of this new agent in normal volunteers at rest and after stress. The biodistribution of ^99mTc-MIBI is characterized by rapid blood clearance and a consequently early myocardial uptake. The initial intense hepatic activity is cleared into the gallbladder at 1 h after injection, and the best target to non target ratio is observed at 60–90 min after injection. Absorbed radiation dose calculations show that the thyroid is the critical target organ (230 mRad/mCi at rest), presumably because of ^99mTc-pertechnetate generated in vivo. Our results indicate that ^99mTc-MIBI is a promising tracer for myocardial perfusion imaging.     

Importance of precise determination of technetium-labeled DISIDA concentrations for in vitro hepatocellular transport studies

Technetium 99m-labeled diisopropyliminodiacetic (acid [^99mTc]DISIDA) has been used extensively in the evaluation of hepatobiliary diseases. As a result of investigation into transport mechanisms of [^99mTc]DISIDA, we undertook to determine precise concentrations of this compound in the standard clinical formulation. Calculations based on published formulas were used. These take into account column efficiency, the time since the previous elution of ^99mTc from the ⁹⁹Mo column, and the total quantity of ^99mTc eluted. Primary cultures of rat hepatocytes were used to evaluate the transport of Na^99mTcO₄ and the ^99mTc-labeled DISIDA monomer. Pertechnetate was not taken up by the hepatocytes, the amount of [^99mTc]DISIDa taken up was not dependent on the concentration of DISIDA but rather on the concentration of technetium, when the DISIDA/^99mTc ratio was varied in the culture medium. We conclude that calculation of the total technetium concentration is necessary to determine the amount of compound taken up by the liver, and to interpret kinetic studies of hepatocellular transport mechanisms.     

Search for 99mTc labeled DTS bifunctional radiopharmaceutical: Role of functional groups in myocardial accumulation

Development of ^99mTc-bifunctional radiopharmaceutical (BR) is attracting the interest of various research groups. In the present paper, various molecules containing a neutral ^99mTc-dithiosemicarbazone (DTS) structure as the technetium chelating site, along with various functional groups (amino, carboxyl or isobutyl group with diverse charge) are tested for their chemical or biological functions. The study on the effect of those functional groups is carried out in vitro and in vivo. The validity of introducing an amino group along with the technetium chelating site DTS for myocardial accumulation is discussed.     

Click chemistry for [99mTc(CO)3] labeling of Lys3-bombesin

^99mTc-HYNIC labeled Lys³-bombesin has shown specific binding to gastrin-releasing peptide receptors (GRP-r) over-expressed in cancer cells. Click chemistry offers an innovative functionalization strategy for biomolecules such as bombesin. The aim of this research was to apply a click chemistry approach for [^99mTc(CO)₃] labeling of Lys³-bombesin and to compare the in vitro MCF7 breast cancer cell uptake and biodistribution profile in mice with that of ^99mTc-EDDA/HYNIC-Lys³-bombesin. The results suggest a higher lipophilicity for ^99mTc(CO)₃-triazole-Lys³-bombesin which explains its higher in vivo hepatobiliary elimination. Pancreas-to-blood ratio for ^99mTc(CO)₃-triazole-Lys³-bombesin was 4.46 at 3 h and both bombesin radiopharmaceuticals showed specific recognition for GRP receptors in MCF7 cancer cells. Click chemistry is a reliable approach for [^99mTc(CO)₃] labeling of Lys³-bombesin.     

Oxidation states of 99mTc in technetium chelates studied by the ICES method

The chemical shifts of ^99mTc core-electron binding energies were measured in solid samples by means of the internal conversion electron spectroscopy (ICES) method. Technetium chelates with citric acid, DTPA, EDTA and ethylenediamine-N,N′-tetraacetohydroxamic acid (EDTAHA) as ligands were prepared in solution at (2–4) × 10⁻⁷ M Tc concentration (no-carrier-added). The samples for ICES measurements were made by evaporation of the solution in vacuum to dryness. The following chemical shifts ΔE_B [K^99mTcO₄-^99mTc(chelate)] were found in the systems investigated: 1.9 and 4.1 eV for ^99mTc(Sn)citr; 2.1, 3.2 and 4.3 eV for ^99mTc(Sn)DTPA; 1.9 and 3.6 eV for ^99mTc(Sn)EDTA; 1.8 and 3.3 eV for ^99mTc(Sn)EDTAHA. Standard deviations of the shifts are 0.2 eV. These shifts were compared with those of inorganic technetium oxocompounds and correlated with oxidation states via a potential model. It was concluded, that these shifts refer to technetium oxidation states in chelates as follows: 1.8–2.1 eV to ^99mTc(V), 3.2–3.6 eV to ^99mTc(IV) and 4.1–4.3 eV to ^99mTc(III).     

Tc99m-hepatobiliary iminodiacetic acid (HIDA) scintigraphy in clinical practice

There have been evolutionary changes in the management of pathological conditions of the hepatobiliary system over recent years, particularly with an increasing emphasis on modern hepatobiliary surgical techniques. Concurrent advances have occurred in imaging technology and availability, leading to a greater use of ultrasound, multidetector computed tomography (CT), and magnetic resonance imaging (MRI) in the primary evaluation of hepatobiliary disease. Radionuclide imaging using technetium(99m) (Tc(99m)) hepatobiliary iminodiacetic acid (HIDA) derivatives is an established technique that complements morphological imaging, providing valuable functional information in both pre- and postoperative evaluation of patients with suspected or known hepatobiliary disease. This review discusses the current clinical indications for Tc(99m) HIDA scintigraphy using clinical cases to demonstrate how this technique continues to play a valuable diagnostic role in the assessment of the functional integrity of the hepatobiliary system.     

The binding of 99mTc(Sn)-MDP complexes to human serum albumin and other blood proteins determined with gel chromatography and ultrafiltration

The binding of ^99mTc(Sn)-MDP to human serum albumin and other blood proteins was investigated by gel chromatography and ultrafiltration.During gel chromatography dissociation of the ^99mTc(Sn)-MDP-protein complex occurs: thus, it is not a suitable technique for the determination of protein binding.The values found with ultrafiltration have to be corrected for non-ultrafiltrable TcO₂·nH₂O. From the corrected values it can be concluded that binding of ^99mTc(Sn)-MDP to blood proteins does not play a role in the biodistribution.     

Technetium Tc 99m plasmin in the diagnosis of inflammatory disease

The localization characteristics of technetium Tc 99m plasmin were studied in experimental animals to investigate the use of^99mTc-plasmin for imaging inflammatory processes. At various times after abscess induction using turpentine in rats, the in vivo distribution properties of^99mTc-plasmin, gallium citrate Ga 67,¹²⁵I-fibrinogen, and^99mTc-human serum albumin (HSA) were studied by gamma-camera imaging. The in vivo binding of each radiopharmaceutical was also tested in rat and human plasma clots. Region-of-interest analyses of gamma-camera images showed relatively poor^99mTc-plasmin localization at sites of abscess formation. The ratio of abscess-to-control activity of this radiopharmaceutical did not exceed that of⁶⁷Ga,¹²⁵I-fibrinogen, or^99mTc-HSA. In vitro assays of each of the radiopharmaceuticals in plasma clots showed^99mTc-phasmin and¹²⁵I-fibrinogen to have the best localization characteristics.