Use of bandage contact lenses for treatment of spontaneous chronic corneal epithelial defects in dogs.

OBJECTIVE: To determine whether polyxylon bandage contact lenses influence healing time and ocular comfort in the management of spontaneous chronic corneal epithelial defects in dogs. METHODS: Twenty dogs with spontaneous chronic corneal epithelial defects were included. All dogs were treated by debridement under topical anaesthesia at the first presentation. Ten dogs were assigned to the study group (application of a polyxylon bandage contact lens), and the remaining ten served as a control group. The healing time and ocular (dis)comfort were evaluated by assessment of the clinical findings and an owner-based questionnaire. All dogs received the same topical and systemic medication. RESULTS: Healing time for dogs in the study population was significantly shorter (mean 14±0 days) than for dogs in the control group (mean 36±17 days; P=0·005). The spontaneous chronic corneal epithelial defects had completely healed at the first recheck in all dogs with a polyxylon bandage contact lens. The duration of blepharospasm following debridement was significantly shorter in the study population (mean 4±4 days) than in the control group (mean 30±20 days; P=0·001). CLINICAL SIGNIFICANCE: The use of polyxylon bandage contact lenses is beneficial in the management of spontaneous chronic corneal epithelial defects.

Surveillance of aminoglycoside resistance. European data.

The susceptibility patterns of gram-negative aerobic organisms to aminoglycosides differ widely from one European health care center to another and depend upon local antibiotic prescribing policies. Reports of the susceptibility of Pseudomonas aeruginosa to gentamicin and tobramycin have ranged from as low as 49.8 percent and 77.7 percent, respectively, in Greece, to as high as 96.6 percent and 99.2 percent, respectively, in the United Kingdom. The susceptibility of P. aeruginosa to gentamicin, tobramycin, and amikacin decreased in our hospital from 73.1 percent, 94.8 percent, and 95.6 percent, respectively, in 1982, to 43.1 percent, 70.6 percent, and 74.3 percent, respectively, in 1984. A prospective surveillance study of the susceptibility of gram-negative aerobic bacilli to four aminoglycosides (gentamicin, tobramycin, amikacin, and netilmicin) was performed over a period of 17 months. Gentamicin and tobramycin were freely used, while the use of amikacin was restricted throughout the hospital during a four-month baseline period (May through August 1984). Gentamicin and tobramycin accounted for 94 percent of the aminoglycoside use. During the following 13 months (September 1984 through September 1985), amikacin was used as the first-line aminoglycoside and accounted for more than 97 percent of the aminoglycoside usage. A total of 1,866 organisms were analyzed during the baseline period; 5,429 were analyzed during the amikacin-usage period. The overall susceptibility to gentamicin, tobramycin, amikacin, and netilmicin increased from 86.9 percent, 90.4 percent, 94.2 percent, and 88.3 percent, respectively, to 92.3 percent, 94.0 percent, 97.3 percent, and 92.3 percent, respectively. P. aeruginosa isolates had the most striking changes, with the susceptibility to gentamicin, tobramycin, amikacin, and netilmicin increasing from 43.1 percent, 70.6 percent, 74.3 percent, and 50.6 percent, respectively, during the baseline period, to 64.5 percent, 81.6 percent, 90.8 percent, and 65.1 percent, respectively, during the amikacin-usage period. The use of amikacin as a first-line aminoglycoside, while use of the other aminoglycosides was restricted, seemed to have a favorable influence on the susceptibility pattern of gram-negative aerobic isolates in our hospital.

Technetium-99m-L,L-ethylenedicysteine: a renal imaging agent. I. Labeling and evaluation in animals.

L,L-ethylenedicysteine (L,L-EC) can be labeled efficiently with 99mTc at pH 12 to obtain a highly pure and very stable tracer agent (99mTc-L,L-EC). The biological behavior of 99mTc-L,L-EC was studied in mice and a baboon. In mice, 99mTc-L,L-EC demonstrated a more rapid urinary excretion and less retention in the kidneys, the liver, the intestines, and the blood than did 99mTc-MAG3 at 10 and 60 min p.i. Urinary excretion decreased in probenecid pretreated mice, which indicates active tubular transport. In the baboon, the renograms for 99mTc-MAG3 and 99mTc-L,L-EC were comparable. Plasma-protein binding of 99mTc-L,L-EC was lower than that of 99mTc-MAG3 while its distribution volume and 1-hr plasma clearance were clearly higher. The promising results of the animal experiments suggest that 99mTc-L,L-ethylenedicysteine may be a useful alternative to 99mTc-MAG3 for renal function studies in humans.

Clinical evaluation of technetium-99m-L,L-ethylenedicysteine in patients with chronic renal failure.

UNLABELLED: Technetium-99m-L,L-ethylenedicysteine (99mTc-L,L-EC), a new renal radiopharmaceutical, has been shown to have similar excretion characteristics but a higher plasma clearance than 99mTc-mercaptoacetyltriglycine (99mTc-MAG3) in normal volunteers and patients with obstructive nephropathy. This study evaluated 99mTc-L,L-EC in patients with chronic renal failure. METHODS: The clearance of 99mTc-L,L-EC was compared with that of 125l-hippuran in 26 patients with varying degrees of chronic renal impairment (serum creatine 168-1163 mumol/liter). All 26 patients also were imaged with 99mTc-L,L-EC (70-80 MBq). Fifteen patients had further imaging with 99mTc-MAG3 (100 MBq) the following day. RESULTS: A subjective analysis of the 99mTc-L,L-EC images revealed that all were of acceptable quality regardless of creatinine level. In the 15 patients who were imaged with both 99mTc-L,L-EC and 99mTc-MAG3, general image quality and target-to-background ratios were similar. Time-activity curves and mean parenchymal transit times obtained with the two agents were almost identical. Plasma clearance values (mean +/- s.d.) of 99mTc-L,L-EC and 125l-hippuran were 81 +/- 68 ml/min and 114 +/- 104 ml/min, respectively. Mean 99mTc-L,L-EC clearance was 71% of the mean 125l-hippuran value. CONCLUSION: Technetium-99m-L,L-EC provides equally high-quality images to 99mTc-MAG3 in patients with chronic renal failure. Technetium-99m-L,L-EC clearance more closely resembles that of hippuran than does 99mTc-MAG3 clearance. These features together with its ease of preparation make 99mTc-L,L-EC an attractive alternative to 99mTc-MAG3 in patients with chronic renal failure.

Comparison of modified technetium-99m albumin and technetium-99m red blood cells for equilibrium ventriculography.

UNLABELLED: A newly developed modified form of 99mTc-labeled human serum albumin reconstituted from a kit (99mTc-dimercaptopropionyl-human serum albumin; 99mTc-DMP-HSA) was prospectively compared to 99mTc-labeled red blood cells (RBC) in patients referred for equilibrium radionuclide ventriculography at rest to evaluate its potential use as a blood-pool imaging agent. METHODS: A Paired comparison between 99mTc-DMP-HSA and either in vitro or in vivo 99mTc-labeled RBC was performed within 2 days in 20 patients'. For each study, two sets of images were acquired, starting at 15 min and 180 min postinjection, respectively. Each set consisted of a gated blood-pool cardiac study and a planar static image centered on the patient's thorax. All data were processed by two independent observers. Early and late postinjection parameters were calculated: ejection fraction (EF) value, activity within the main organs surrounding the left ventricle (LV), ratio of activity between the LV and these surrounding organs for each study separately, and temporal (late/early) evolution of the intraorgan activities and of the LV/organ ratios after decay correction. RESULTS: The images and the visual wall-motion analysis were of good quality with both agents in most patients, without significant image degradation at 180 min postinjection. Calculated EF values were highly comparable with the two tracers. Interobserver variability was 0.17% (RBC) and 1.08% (DMP-HSA) for the early EF value (EF1), and 0.62% (RBC) and 0.27% (DMP-HSA) for the late EF (EF2). Mean difference between EF2 and EF1 was 0.74% (Observer 1) and 0.28% (Observer 2) for 99mTc-RBC, and -2.88% (Observer 1) and -2.07% (Observer 2) for 99mTc-DMP-HSA. When comparing 99mTc-DMP-HSA to 99mTc-RBC the mean difference was 1.27% (Observer 1) and 0.36% (Observer 2) for EF1, and -2.35% (Observer 1) and -1.99% (Observer 2) for EF2. Also, the biodistribution and temporal evolution of the organ repartition of both compounds were stable and similar, with values of late/early activity ratios very close to one for all the studied organs [mean intraorgan ratio: 0.946 for 99mTc-RBC (range: 0.881-1.086) and 0.979 for 99mTc-DMP-HSA (range: 0.914-1.141); mean late/early LV/organ ratio: 0.964 for 99mTc-RBC (range: 0.919-1.016) and 0.967 for 99mTc-DMP-HSA (range: 0.912-1.035)]. CONCLUSION: Paired comparison of kit-prepared 99mTc-DMP-HSA to 99mTc-labeled RBC demonstrated that both agents were very closely related regarding as well the calculated EF value as the in vivo stability up to more than 3 hr postinjection. Technetium-99m-DMP-HSA may constitute a practical and useful replacement for 99mTc-labeled RBC.

Biodistribution and absorbed radiation dose estimates of 99mTc-labeled dimercaptopropionyl human serum albumin.

UNLABELLED: The use of 99mTc-labeled red blood cells (RBC) for the evaluation of left ventricular function using equilibrium-gated blood-pool imaging suffers from several problems and potential risks. In this study, we estimated the absorbed radiation dose of 99mTc-labeled dimercaptopropionyl human serum albumin (DMP-HSA) as a potential alternative to 99mTc-RBC. METHODS: After the administration of 99mTc-DMP-HSA, whole-body imaging was performed up to 48 h after injection in five volunteers. The heart contents, liver and remainder of the body were used as source organs. Multicompartment modeling of the biodistribution was performed and absorbed radiation dose estimates for 99mTc-DMP-HSA were obtained using the Medical Internal Radiation Dose (MIRD) calculation. RESULTS: Residence times of 0.62 and 0.43 h were obtained for the heart contents and liver, respectively. Radiation dose estimates yielded an effective dose of 0.0055 mSv/MBq. CONCLUSION: 99MTC-DMP-HSA yielded absorbed radiation doses comparable with those of 99mTc-RBC. Therefore, the radiation properties of 99mTc-DMP-HSA are such that it can be used for clinical diagnostic studies.

Ocular penetration of (125I)IVDU, a radiolabeled analogue of bromovinyldeoxyuridine.

Following topical application of (125)IVDU, the radiolabeled analogue of bromovinyldeoxyuridine ([E]-5-[2-bromovinyl]-2'-deoxyuridine), as 0.5% or 0.3% eyedrops, to rabbits, (125I)IVDU appeared in the anterior chamber fluid at drug levels well above the minimum concentration (0.01 microgram/mL) required for inhibition of herpes simplex virus type 1 replication. These findings are consistent with the efficacy of 0.5% bromovinyldeoxyuridine eyedrops in the topical treatment of herpes simplex uveitis.

Evaluation of 99mTc-MAMA-chrysamine G as an in vivo probe for amyloidosis.

To date, systemic amyloidosis is diagnosed histologically using Congo red staining or in vivo using iodine-123 labelled serum amyloid P component (123I-SAP) scintigraphy. We developed 99mTc-MAMA-CG, a 99mTc-labelled derivative of the lipophilic Congo red analogue chrysamine G (CG), as a possible alternative to 123I-SAP. In vivo 99mTc-MAMA-CG scintigraphy, performed in chickens with spontaneous joint amyloidosis, resulted as soon as 10 min after injection in scintigraphic images showing uptake of activity in amyloid-loaded organs (liver, joints). One of these chickens was studied also with 123I-SAP resulting in scintigraphic images revealing 123I-SAP binding to amyloid deposits in the liver. However, up to 11 h after injection no radioactivity was visible in the amyloid positive joints. In vitro autoradiography, performed on sections of chicken joints with Enterococcus faecalis induced amyloid arthropathy (chjAA), demonstrated the failure of 99mTc-MAMA-CG to bind significantly to amyloid deposits in the presence of 10 microM Congo red The specificity of 99mTc-MAMA-CG localisation was also established by the absence of 99mTc-MAMA-CG binding in non-amyloidotic organs in vitro and in vivo. 99mTc-MAMA-CG did not show any sign of acute toxicity. These findings establish the usefulness of 99mTc-MAMA-CG as a non-invasive in vivo diagnostic probe in chickens with amyloid arthropathy and suggest that it may also be applicable to human amyloidosis.

Synthesis and biological evaluation of the four isomers of technetium-99m labeled ethylenecysteamine cysteine ((99m)Tc-ECC), the mono-acid derivative of (99m)tc-L,L-ethylenedicysteine.

A few years ago (99m)Tc-ethylenedicysteine ((99m)Tc-L,L-EC) had been proposed as an interesting substitute for technetium-99m labeled mercaptoacetyltriglycine (MAG3) as renal function tracer agent. It possesses in its structure two carboxylate functions and is in this respect different from other renal tracers such as (99m)Tc-N, N'-bis-(mercaptoacetyl)-2,3-diaminopropionate ((99m)Tc-CO(2)DADS), (99m)Tc-MAG3, and Hippuran, which have only one carboxylic group. To study whether both carboxylic acid groups of (99m)Tc-L,L-EC contribute to the efficient renal handling of this compound we synthesized and biologically evaluated the technetium-99m labeled isomers of L- and D-ethylenecysteamine cysteine (ECC), the mono-acid derivative of (99m)Tc-L,L-EC. Labeling of L-ECC or D-ECC with (99m)Tc using a direct or exchange labeling method yields for each of them two diastereomeric (99m)Tc complexes (A and B, in the order of elution during reversed phase high performance liquid chromatography) in relative amounts depending on the pH during labeling. In mice, all four isomers of (99m)Tc-ECC (LA, LB, DA, and DB) are cleared rapidly from the blood, mainly by the renal system. The isomers LB and DB show the most efficient renal handling, but none of the mono-acid derivatives has a urinary excretion rate as high as that of (99m)Tc-L,L-EC. The renal handling of the isomers of (99m)Tc-ECC is partly due to tubular secretion because the urinary excretion of these compounds is significantly lower in mice pretreated with probenecid. In the baboon, isomers DA and DB show a plasma clearance comparable to that of (99m)Tc-L,L-EC. The plasma clearance of isomers LA and LB is lower but still comparable to or higher than that of (99m)Tc-MAG3. In a human volunteer, isomer DB shows a plasma clearance rate only slightly lower than that of (99m)Tc-L,L-EC. Thus, it appears that the presence of one carboxylate in (99m)Tc-EC-like compounds can be sufficient for efficient renal handling. However, it is also evident that the configuration at the chiral carbon atom and the orientation of the oxotechnetium core determine in a significant way the biological characteristics.

Importance of the two ester functions for the brain retention of 99mTc-labelled ethylene dicysteine diethyl ester (99mTc-ECD).

99mTc-ethylene dicysteine diethyl ester (99mTc-L,L-ECD) is a neutral lipophilic tracer agent that crosses the blood-brain barrier and is retained in the brain of primates following enzymatic hydrolysis of one of the ester functions to the ionized mono-ester, mono-acid metabolite. Up to now, it is not clear whether the second ethylcarboxylate group is essential for brain uptake and retention. Therefore, we have synthesized and studied two derivatives of 99mTc-L,L-ECD that contain only one ethylcarboxylate function, namely 99mTc-labelled L- and D-ethylene cysteamine cysteine ethyl ester (99mTc-ECCE). Direct labelling of L- or D-ECCE at neutral pH and room temperature resulted for each of them in the formation of two probably diastereomeric 99mTc-complexes in a 1:1 ratio. This means that four different isomers could be isolated. The 99mTc-labelled complexes formed after labelling ECCE (A and B, in order of elution during HPLC) are slightly less lipophilic than 99mTc-L,L-ECD. In mice, all four isomers show a low brain activity at 10 min post injection (p.i.), approximately 0.1% to 0.3% of the injected dose versus 0.9% for 99mTc-L,L-ECD. The clearance from the blood is comparable with (isomers LA and LB) or slower (isomers DA and DB) than that of 99mTc-L,L-ECD. Isomers LA and DA show high liver uptake and rapid excretion to the intestines. Both 99mTc-ECCE-LB and 99mTc-ECCE-DB, the most lipophilic isomers, are cleared from the blood mainly by the kidneys and excreted more efficiently to the urine. 99mTc-ECCE-LB is characterized by a surprisingly high heart uptake (about 1.5% of i.d.) at 10 min p.i. versus 1.0% for 99mTc-methoxyisobutylisonitrile (99mTc-MIBI) and 0.2-0.3% for the other isomers, but also lung uptake is relatively high. In the baboon, brain and heart uptake of isomers LA and LB of 99mTc-ECCE were negligible. Activity concentrated mostly in the hepatobiliary system for isomer LA and in the renal system for isomer LB. The results indicate a clear difference in biological behaviour between 99mTc-L,L-ECD and the four isomers of 99mTc-ECCE. This shows that the presence of both ester functions in 99mTc-L,L-ECD is an essential structural requirement for its brain uptake and retention in primates.

Influence of a 99mTcN core on the biological and physicochemical behavior of 99mTc complexes of L,L-EC and L,L-ECD.

99mTc-nitrido complexes of L,L-ethylene dicysteine (99mTcN-L,L-EC) and 99mTcN-L,L-ethylene dicysteine diethylester (99mTcN-L,L-ECD) were prepared and their characteristics compared to those of the respective 99mTc-oxo complexes. 99mTcN-L,L-EC and 99mTcO-L,L-EC migrate to similar extents during electrophoresis at pH 12, but, at pH6, 99mTcN-L,L-EC migrates further than 99mTcO-L,L-EC. Renal excretion of 99mTcN-L,L-EC is inferior to that of 99mTcO-L,L-EC, indicating that the TcN-glycine sequence has lower affinity for the renal tubular system. Both 99mTcO-L,L-ECD and 99mTcN-L,L-ECD are neutral, but 99mTcN-L,L-ECD is hydrophilic and shows minimal brain uptake in both mice and the baboon.

Complexes of technetium-99m with tetrapeptides, a new class of 99mTc-labelled agents.

Tetrapeptides are a class of N4-tetraligands that can efficiently bind 99mTc. In fact, tetrapeptides can be considered as derivatives of mercaptoacetyltriglycine (MAG3) in which the mercaptoacetyl moiety is replaced by a more stable and easier to handle aminoacyl group. Direct labelling of tetrapeptides with 99mTc in alkaline medium (pH > or = 11) in the presence of stannous ions gave a high yield (> 95%) of one or two (probably isomeric) radiochemical species. Exchange labelling at different pH values in the presence of stannous tartrate resulted in lower yields of the same 99mTc-labelled products as those formed during direct labelling. In addition, other radiochemical species were formed of which one was characterized as an oxotechnetium-complex with the cyclisized tetrapeptide. Tetrapeptides with a chiral centre in the first amino acid yield upon labelling with 99mTc two radiochemical species, probably the two diastereomers with an oxotechnetium core respectively syn and anti with respect to the substituent on the amino acid. Only one diastereomer was observed when the chiral carbon atom is located in the second or third amino acid. Electrophoresis indicated that these new 99mTc-labelled complexes are neutral in acidic medium and negatively charged in neutral and alkaline conditions. This correlates with a complex in which an oxotechnetium(V) group is bound to the ligand through three deprotonated nitrogen atoms of the amide functions and the free electron pair of the amine nitrogen atom. Biodistribution in mice showed for all studied 99mTc-labelled tetrapeptides a rapid clearance from the blood mainly by the renal system. The presence of a methyl substituent in the tetrapeptide increased the urinary excretion. 99mTc-labelled L-glycylalanylglycylglycine showed in mice a urinary excretion comparable to that of 99mTc-MAG3. Further rise of lipophilicity by introduction of a dimethyl, isopropyl or isobutyryl group leads to increased hepatobiliary handling. It is concluded that tetrapeptides are an interesting group of technetium complexing agents which can easily be labelled with 99mTc at room temperature in alkaline medium. This class offers the possibility of a wide variety of derivatives, just by substituting one or more amino acids. This group of ligands thus opens a new research field of 99mTc-complexes with potential usefulness in several areas.

3'-99mTc-labeling and biodistribution of a CAPL antisense oligodeoxynucleotide.

CAPL is a cancer-related gene shown to be overexpressed during tumor metastasis formation. A CAPL antisense oligodeoxynucleotide (ODN), GX-1, and a random control ODN (CTRL1) were 3'-conjugated to MAG3, labeled with technetium-99m, purified, and the biodistribution of the radiolabeled conjugates in normal mice was studied. A 99mTc-MAG3-GX-1 complex of >97% radiochemical purity was obtained and the product was stable for >6 h as determined by reversed phase high performance liquid chromatography (HPLC). Biodistribution studies of the 99mTc-MAG3-ODNs in groups of four normal mice, sacrificed 5 min and 60 min after injection, demonstrated that the radiolabeled ODNs were distributed in an unspecific manner. The excretion route was mainly urinary. At 60 min, 55.2% of the injected dose of 99mTc-MAG3-GX-1 and 72.4% of 99mTc-MAG3-CTRL1 was found in the urine. This finding is clearly different from previously reported data on tritiated 20-mer phosphodiester ODNs, as well as the unconjugated 99mTc-MAG3 chelate, suggesting that 99mTc-MAG3 coupled to the 3'-end of ODNs has an influence on the biodistribution of the oligo, and possibly has a protective function to enzymatic degradation in vivo.

Development and evaluation of a kit formulation for the preparation of 99mTc-DMP-HSA, a new tracer agent for radionuclide ventriculography.

This study presents the development of a kit formulation for the preparation of 99mTc-DMP-HSA, followed by a comparison of such kit-prepared 99mTc-DMP-HSA to 99mTc-RBCs in a volunteer. Reconstitution of the labeling kits with up to 5.55 GBq 99mTc afforded 99mTc-DMP-HSA preparations with a > 95% radiochemical purity for up to 8 h. Only minor differences were observed in the global distribution of both tracer agents, whereas the calculated ejection fractions were almost identical. The effective dose equivalent of 99mTc-DMP-HSA is 8.68 microSv/MBq.

Infections caused by Kingella kingae: report of four cases and review.

Kingella kingae, formerly known as Moraxella kingae, is a fastidious, non-motile, coccobacillary, fermentative Gram-negative rod that has been chiefly associated with two types of infections in man: bone and joint infections, and endocarditis. We describe four patients with K. kingae infections, one with septicaemia, two with endocarditis, and one with osteoarthritis. The current literature on infections with K. kingae is reviewed.

Development of a lyophilized kit formulation for labeling of DNA probes with 99mTc.

DNA fragments such as oligodeoxynucleotides (ODNs) are under investigation for a possible utilization in nuclear medicine. Until now, experiments on 99mTc-labeled ODNs in vitro or in vivo have required the application of time-consuming procedures to obtain and control the purity of the radiolabeled compound. A lyophilized labeling kit would ease and improve the reproducibility in further investigations with this class of promising biomolecules; therefore a study was initiated to evaluate the suitability of conjugates of ODNs and a bifunctional chelating agent to be part of lyophilized kit formulations. We report here the development of the first kit for one-step labeling of oligonucleotides with 99mTc. The formulation comprises 250-500 pmol S-benzoyl-mercaptoacetyldiglycine (MAG2)-ODN phosphorothioate conjugate, 5 mg potassium sodium tartrate tetrahydrate and 100 microg stannous chloride dihydrate in a lyophilized kit. Labeling yields above 90% were reproducibly achieved after addition of 0.1-1 GBq pertechnetate and subsequent heating in a boiling water bath. Once formed, the 99mTc-MAG2-ODN complexes were stable for at least 24 h. The shelf life of the kits is at least 10 weeks when stored protected from light at room temperature, but even kits stored at 40 degrees C gave labeling yields above 90% after 10 weeks.

RP-HPLC separation of the diastereomers of technetium-99m labelled tropanes and identity confirmation using radio-LC-MS.

99mTc-TRODAT-1 (technetium(V)-oxo-2-[[2-[[[3-(4-chlorophenyl)-8-methyl-8-azabicyclo[3.2.1]oct-2-yl]methyl](2-mercaptoethyl)amino]ethyl]]amino]-ethanethiolato(3-)) and 99mTc-TRODAT-M, the 4-methylphenyl derivative of 99mTc-TRODAT-1, are at this moment being evaluated in clinical trials as imaging agents for the central nervous dopamine transporter system. Both compounds are formed as a mixture of two major diastereomers. As the tracer concentration in preparations for clinical investigations is very low (30-150 pmol/ml), identification of these 99mTc-complexes was, up to now, carried out indirectly using X-ray diffraction analysis of the corresponding rhenium complexes which can be synthesized in gram amounts. In this study, we developed a convenient and practical reversed phase HPLC-method for purification and isolation of the respective diastereomers of 99mTc-TRODAT-1 and three of its derivatives using mixtures of solvents which are compatible with biological studies, i.e. aqueous buffers and ethanol. Furthermore, direct identity confirmation of the 99mTc-complexes using radio-LC-MS was successfully elaborated.

An efficient HPLC method for the analysis of isomeric purity of technetium-99m-exametazime and identity confirmation using LC-MS.

99mTc-exametazime (99mTc-d,l-HMPAO, 99mTc-d,l-hexamethylpropyleneamine oxime) is a neutral rather unstable complex of short-lived 99mTc (t(1/2)=6 h) with the d,l-isomer (mixture of D,D- and L,L-isomers) of a bis-amine bis-oxime tetraligand. It is widely used for measurement of regional cerebral perfusion in nuclear medicine. The meso-isomer (D,L-form) should not be present in a preparation as it is not retained in brain and thus does not provide clinically useful information. Meso-HMPAO is removed from the ligand during the synthesis procedure by repeated recrystallization, but can still be present as impurity in d,l-isomer. Due to the lack of a suitable chromatographic method for analysis of the isomeric purity of 99mTc-exametazime preparations, United States Pharmacopoeia 25 (USP 25) prescribes a biological test in rats for quality control purpose. In this study, we developed a suitable high-performance liquid chromatography (HPLC) method which allows to demonstrate the relative amounts of d,l- and meso-isomer in 99mTc-exametazime and so obviates the need for a biodistribution test in animals as part of the quality control. Due to the low concentrations in which 99mTc-d,l-HMPAO is obtained (typically 2-6 ng/ml), confirmation of the identity of 99mTc-d,l-HMPAO in the monograph of the European Pharmacopoeia is now performed only indirectly by TLC and assessment of its retention time on RP-HPLC. To investigate the potential of radio-LC-MS for assessment of the identity of 99mTc-exametazime, 99mTc-d,l-HMPAO and 99mTc-meso-HMPAO prepared using a Tc-rich eluate were analyzed using a radio-LC-MS system equipped with a time-of-flight mass spectrometer with electrospray ionization. The main peak in the radiometric channel coincided with the molecular ion mass of 99mTc-d,l-HMPAO in the mass spectrometer channel and the measured accurate mass differed only by 0.26 ppm from the theoretical mass. The identity of 99mTc-meso-HMPAO was also confirmed. Thus, radio-LC-MS allowed to obtain strong evidence for the structure of 99mTc-d,l-HMPAO and 99mTc-meso-HMPAO at nanomolar concentration. It is concluded that radio-LC-MS can become a sensitive aid in quality control of "no carrier added" radiopharmaceutical preparations.

In vitro affinity of 99Tcm-labelled N2S2 conjugates of chrysamine G for amyloid deposits of systemic amyloidosis.

To date, systemic amyloidosis is diagnosed histologically in vitro using Congo red staining or in vivo using iodine-123 serum amyloid P component (123I-SAP) scintigraphy. 99Tcm-labelled derivatives of chrysamine G (CG), a lipophilic analogue of Congo red, were synthesized as potential tracer agents for direct and quantitative scintigraphic evaluation of amyloid deposits. To determine the affinity of 99Tcm-MAMA-CG, 99Tcm-Me4MAMA-CG and 99Tcm-MAMA-CG diethyl ester for amyloid, in vitro autoradiography was performed on sections of human kidney biopsy cylinders from kidneys with amyloid deposits (types AA, Alambda and Akappa) or control kidney tissue after incubation with the respective tracer agents. The binding of 99Tcm-MAMA-CG and its tetramethyl derivative was higher to kidney biopsy material with amyloid deposits of the AA, Alambda or Akappa type compared with control kidney tissue. This higher binding was prevented by the presence of 10 microM Congo red in the incubation medium. The diethyl ester of 9Tcm-MAMA-CG did not demonstrate increased binding to Congo red-positive kidney tissue. In conclusion, 99Tcm-MAMA-CG and 99Tcm-Me4MAMA-CG localize specifically to amyloid deposits in human kidney tissue, suggesting that these tracer agents may be applicable as specific targeting agents for diagnostic purposes in clinical amyloidosis.

Hybridization of a 99Tcm-labelled oligodeoxynucleotide to CAPL RNA.

Oligodeoxynucleotides (ODNs) labelled with an appropriate radionuclide could provide a means to identify serious diseases early on and thereby help initiate treatment at a very early phase. Regardless of important issues like in-vivo stability and membrane passage, the key issue for the oligonucleotide approach is the ability of the radiolabelled ODN to hybridize to the target mRNA. The secondary structure of mRNA does not permit all complementary ODNs to hybridize and a careful selection of the probe with consecutive testing is therefore necessary. This study was initiated to demonstrate hybridization of a 99Tcm-labelled 20-mer ODN to RNA of CAPL (S100A4), a gene reported to be overexpressed in metastatic cancers like breast carcinoma and osteosarcoma. The phosphodiester ODN GX-1 (antisense) and two control sequences (scrambled and random) were conjugated to the bifunctional chelating agent S-benzoyl-mercaptoacetyltriglycine (S-benzoyl-MAG3) and labelled with 99Tcm. The radiolabelled ODNs were purified on a C18 mini-column and characterized on a reverse-phase HPLC system. The radio-chemical purity was > 90% and the product was stable for > 6 h in aqueous medium. The hydrization properties of unlabelled, 32P-labelled and 99Tcm-labelled ODNs to transcribed RNA were studied using polyacrylamide gel electrophoresis (PAGE). Direct hybridization of GX-1 to transcribed RNA was demonstrated. A 50-fold excess of unlabelled ODN over transcribed RNA caused a near to complete consumption of RNA by RNase H activation. In 1:1 proportions of radiolabelled (32P and 99Tcm) ODNs to RNA, only radiolabelled GX-1 was found to hybridize to RNA in a PAGE system. The radiolabelled control ODNs did not show signs of hybridization. This study demonstrates that 3'-99Tcm-labelling of ODNs does not interfere with the hybridization properties of the ODNs in solution, making 99Tcm-labelling an attractive procedure for the future development of antisense technology in imaging.