Boronic acid adducts of technetium dioxime (BATO) complexes derived from quinuclidine benzilate (QNB) boronic acid stereoisomers: syntheses and studies of their binding to the muscarinic acetylcholine receptor.

We have investigated the possibility of using BATO complexes derivatized with the muscarinic acetylcholine receptor (mAChR) antagonist, quinuclidinyl benzilate (QNB), for mAChR imaging. The BATO complexes, TcCl(DMG)3B-QNB, were prepared using QNB derivatives containing a 4'-boronic acid substituent on one of the benzilic benzene rings (QNB-boronic acid). The QNB-boronic acid molecule has two chiral centers, and all four QNB-BATO stereoisomers were made and evaluated. When studied using in vitro receptor binding assays based on tissue from rat brain caudate-putamen (which contains primarily M1 and M4 mAChR) and rat heart (M2 mAChR), the QNB-boronic acid stereoisomers had binding affinities (KA) in the range 2 x 10(5)-1 x 10(8), at least 10-fold lower than the KA for QNB (ca 2 x 10(9)). The stereochemistry of both centers had some influence on the affinity constant. When the TcCl(DMG)3B-QNB complexes were studied, none of the stereoisomeric complexes displayed measurable specific binding (KA < 10(6)), but all showed high non-specific binding. In vitro autoradiography with rat brain slices confirmed the absence of specific binding in these tracers. In vivo, the 99mTcCl(DMG)3B-QNB complexes displayed minimal brain uptake, and modest heart uptake; the latter was unlikely to be related to uptake by the mAChR. In light of these findings, we conclude that the interaction between the TcCl(DMG)3B-QNB complexes and biological membranes is dominated by the hydrophobicity of the BATO moiety. The TcCl(DMG)3B-QNB complexes, therefore, have little potential for mAChR imaging.

[99mTc] Teboroxime and [99mTc]Cl(DMG)3B2MP: binding characteristics andmetabolism of two [99mTc]BATOs in blood and tissues.

Studies were performed in vitro and in vivo to evaluate the binding properties and metabolism of [99mTc]Cl(CDO)3BMe (Teboroxime) and [99mTc]Cl(DMG)3B2MP in blood and target tissues of rats. Both radiopharmaceuticals displayed rapid binding (within 1-3 min) with high affinity to plasma proteins and blood cells. The amounts of radioactivity associated with blood components became progressively greater with time of exposure to either compound. There was a higher proportion of the radiopharmaceuticals associated with blood components during in vivo conditions, likely due, at least in part, to clearance of the free fraction from the plasma pool. Exposure of both compounds to blood results in axial ligand exchange of the chloro atom to a hydroxyl. The results suggest that it is the free species that is extracted primarily by tissues.

PET scanning with hydroxyephedrine: an approach to the localization of pheochromocytoma.

Pheochromocytomas are potentially curable causes of hypertension. These tumors are currently located by functional imaging with meta-iodobenzylguanidine (MIBG), usually labeled with 131I, or anatomic imaging (computed tomography, magnetic resonance). Hydroxyephedrine (HED) is a newly developed radiotracer that concentrates in adrenergic nerve terminals. When HED is labeled with 11C, its distribution can be mapped in vivo using PET. The purposes of this investigation were to characterize the uptake of 11C-HED in pheochromocytoma and to determine the feasibility and advantages of utilizing this compound as a tumor imaging agent. Ten patients with known or suspected pheochromocytoma were studied. Each patient underwent PET scanning with 11C-HED and conventional scintigraphy with MIBG. Pheochromocytomas were localized by PET scanning in 9 of the 10 patients. Image quality was excellent and superior to that obtained from planar and tomographic MIBG studies. The uptake of 11C-HED into pheochromocytomas was rapid; tumors were evident within 5 min following intravenous injection. All lesions within the field of view that were identified by MIBG scintigraphy were readily apparent. PET scanning with 11C-HED localizes pheochromocytoma using a specifically designed radiotracer and advanced imaging technology. The method has promise for locating the more elusive tumors.

Direct analysis of whole blood by internal surface reversed-phase chromatography: an examination of the binding and metabolism of technetium dioxime complexes.

We have developed a method using internal surface reversed-phase (ISRP) packing for rapid on-line separation of small hydrophobic compounds from cellular whole blood components. This is achieved by the use of 75-microns ISRP chromatographic material packed into a small high-performance liquid chromatographic (HPLC) column, in conjunction with column switching. We have applied this analytical method to study the in vitro metabolism of 99mTc-BATO (boronic acid adducts of technetium dioxime) cerebral and myocardial perfusion tracers in whole blood. The results from the ISRP procedure were compared with a conventional centrifugation method of analysis. This novel HPLC methods provides a rapid, convenient and reliable method for the analysis of radioactive and non-radioactive lipophilic components in whole blood.

Myocardial extraction of teboroxime: effects of teboroxime interaction with blood.

The isolated perfused rat heart preparation was used to determine whether the interaction of blood with either 99mTc-teboroxime, 99mTc-sestamibi or 201TI affects the extraction of these myocardial perfusion agents. Hearts were retrogradely perfused at 72 cm H2O with Krebs-Henseleit buffer equilibrated with O2:CO2 (95:5). The hearts were paced at 5 Hz. Single-pass extraction of 99mTc-teboroxime (96% +/- 1%) was greater than that of 99mTc-sestamibi (15% +/- 1%) or 201TI (30% +/- 5%). Extraction of the hydroxide form of 99mTc-teboroxime was only 43% +/- 4%. When arterial blood obtained from rats administered 99mTc-teboroxime was injected into the perfused heart, extraction of 99mTc-teboroxime decreased progressively as its time in circulation was lengthened. Similar experiments using either 99mTc-sestamibi or 201TI showed that extraction of these agents was neither affected by the presence of blood nor residence in circulation. For 99mTc-teboroxime, extraction was 99.5% +/- 0.5%, 57% +/- 13%, 20% +/- 2% at 1, 5, and 60 min postinjection, respectively. In separate experiments, HPLC analysis of blood at 5, 15 and 60 min postinjection indicated that only 34% +/- 4%, 13% +/- 2%, and 2% +/- 1%, respectively, of the total 99mTc-teboroxime was free and was associated with extraction values of 44% +/- 7%, 28% +/- 5%, and 19% +/- 3%, respectively. The percentage of this free radioactivity that converted from the chloro to the hydroxide form was 9% +/- 2%, 6% +/- 2%, and 2% +/- 1%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of aspartame-derived phenylalanine on neutral amino acid uptake in human brain: a positron emission tomography study.

The possible effects of elevation of the plasma phenylalanine level secondary to the ingestion of aspartame on brain amino acid uptake in human subjects have been investigated by means of positron emission tomography (PET). 1-[11C]Aminocyclohexanecarboxylate [( 11C]ACHC) is a poorly metabolized synthetic amino acid that crosses the blood-brain barrier by the same carrier that transports naturally occurring large neutral amino acids. Quantitative test-retest PET studies were performed on 15 individuals. Seven received two identical baseline scans, whereas eight received a baseline scan followed by a scan performed approximately 40-45 min following ingestion of an orange-flavored beverage containing 34 mg/kg of body weight of the low-calorie sweetener aspartame, a dose equivalent to the amount in 5 L of diet soft drink consumed all at once by the study subjects, weighing an average of 76 kg. The 40-45-min interval was selected to maximize the detection of possible decreases in ACHC uptake resulting from increased competition for the carrier, because the plasma phenylalanine level is known to peak at this time. We observed an 11.5% decrease in the amino acid transport rate constant K1 and a smaller decrease in the tissue distribution volume of ACHC (6%). Under conditions of normal dietary use, aspartame is thus unlikely to cause changes in brain amino acid uptake that are measurable by PET.

Short-term metabolic fate of L-[13N]glutamate in the Walker 256 carcinosarcoma in vivo.

In vivo studies with L-[13N]glutamate in the Walker 256 carcinosarcoma implanted under the renal capsule of female Sprague-Dawley rats demonstrate that uptake of glutamate and the rate of incorporation of the nitrogen label from this amino acid into metabolites is slower in the tumor than in nontumorous kidney tissue. Glutamate dehydrogenase, glutaminase, and alanine aminotransferase activities are significantly lower within the tumor than within the adjoining kidney. However, the tumor expresses high levels of aspartate aminotransferase, attesting to the importance of this enzyme in the metabolism of glutamate. Indeed, high performance liquid chromatographic analysis showed that the principal metabolic fate of label derived from L-[13N]glutamate in the tumor is incorporation into aspartate. Measurement of specific activity ratios of glutamate to aspartate shows that the transfer of nitrogen from glutamate to aspartate is rapid and that equilibration of label among components of the aspartate aminotransferase reaction is attained within minutes after tumor uptake. Analyses of the nontumorous portion of the implanted kidney also showed that aspartate is the major recipient of glutamate nitrogen. However, high performance liquid chromatographic analyses of deproteinized tissue revealed that glutamine and ammonia are also significant 13N-labeled metabolites formed from L-[13N]glutamate within the kidney. Proportionately lower amounts of these labeled metabolites were found in the tumor.

Synthesis and preliminary evaluation of carbon-11-meta-hydroxyephedrine: a false transmitter agent for heart neuronal imaging.

Carbon-11-meta-hydroxyephedrine is a new radiotracer developed for mapping the sympathetic nerves of the heart. Carbon-11-meta-hydroxyephedrine is synthesized by direct N-methylation of metaraminol with [11C]methyl iodide in dimethyl formamide/dimethyl sulfoxide and purified by semi-preparative reversed-phase HPLC. Total synthesis time is 45 min from end-of-bombardment. Carbon-11-meta-hydroxyephedrine is produced in 40%-50% corrected radiochemical yield with a specific activity of 900 Ci/mmol. Routine radiochemical and chemical purity are 95% and 98%, respectively. Biodistribution studies in rats show high myocardial uptake. Pretreatment with desipramine, a drug known to selectively block neuronal uptake, results in a 92% decrease in tracer accumulation in the myocardium. Metabolic studies in guinea pigs show less than 5% metabolites in heart tissue 30 min after intravenous injection suggesting that [11C]meta-hydroxyephedrine is suitable for kinetic modeling. These preliminary results support this new tracer as a clinical agent for neuronal imaging of the heart.

Noninvasive quantification of regional blood flow in the human heart using N-13 ammonia and dynamic positron emission tomographic imaging.

Evaluation of regional myocardial blood flow by conventional scintigraphic techniques is limited to the qualitative assessment of regional tracer distribution. Dynamic imaging with positron emission tomography allows the quantitative delineation of myocardial tracer kinetics and, hence, the measurement of physiologic processes such as myocardial blood flow. To test this hypothesis, positron emission tomographic imaging in combination with N-13 ammonia was performed at rest and after pharmacologically induced vasodilation in seven healthy volunteers. Myocardial and blood time-activity curves derived from regions of interest over the heart and ventricular chamber were fitted using a three compartment model for N-13 ammonia, yielding rate constants for tracer uptake and retention. Myocardial blood flow (K1) averaged 88 +/- 17 ml/min per 100 g at rest and increased to 417 +/- 112 ml/min per 100 g after dipyridamole infusion (0.56 mg/kg) and handgrip exercise. The coronary reserve averaged 4.8 +/- 1.3 and was not significantly different in the septal, anterior and lateral walls of the left ventricle. Blood flow values showed only a minor dependence on the correction for blood metabolites of N-13 ammonia. These data demonstrate that quantification of regional myocardial blood flow is feasible by dynamic positron emission tomographic imaging. The observed coronary flow reserve after dipyridamole is in close agreement with the results obtained by invasive techniques, indicating accurate flow estimates over a wide range. Thus, positron emission tomography may provide accurate and noninvasive definition of the functional significance of coronary artery disease and may allow the improved selection of patients for revascularization.

Neuronal mapping of the heart with 6-[18F]fluorometaraminol.

The false neurotransmitter metaraminol labeled with fluorine-18 has been used to noninvasively assess regional adrenergic nerve density in the canine heart. Intravenous administration of 6-[18F]fluorometaraminol (FMR) results in high, selective accumulation of radioactivity in the heart; drug blocking studies with desipramine and reserpine confirm the neuronal locus of FMR. Iodine-125 labeled metaraminol, however, shows no selective accumulation in the canine heart. Positron emission tomography (PET) analyses with FMR of closed-chest dogs bearing left ventricular neuronal defects clearly delineate the region of neuronal impairment; blood perfusion in the left ventricle wall was homogeneous as determined by [13N]NH3 tomograms. The accumulation of FMR in regionally denervated dog heart correlates closely (r = 0.88) with endogenous norepinephrine concentrations. PET-generated 18F time-activity curves demonstrate marked kinetic differences between normal and denervated myocardium. FMR/PET analysis could be used to assess the heterogeneity of sympathetic innervation in human heart disease contingent on the development of FMR with sufficiently high specific activity to clearly avoid pressor activity.

Metabolic fate of [13N]ammonia in human and canine blood.

Nitrogen-13- ([13N]) ammonia is a widely used tracer for PET myocardial blood flow studies. Quantification of blood flow using tracer kinetic principles requires accurate determination of [13N]ammonia activity in blood. Since [13N] ammonia is rapidly metabolized, the arterial input function may be contaminated by labeled metabolites. We, therefore, characterized the 13N-labeled metabolites in blood after intravenous (i.v.) injection of 20 mCi [13N]ammonia in nine healthy volunteers. Utilizing a series of ion exchange resins, 13N-labeled compounds were separated into four groups: ammonia, neutral amino acids, acidic amino acids, and urea. Analysis of the metabolic fate of [13N]ammonia indicates that over 90% of the blood activity within the first two minutes after injection is present as [13N]ammonia. However, there is considerable contamination of the blood activity at 3-5 min by [13N]glutamine (amide) and urea, which collectively represent 18%-50% of the blood activity. Thus, correction of the arterial input function for 13N-metabolites is required to accurately quantify the arterial input function of [13N]ammonia in myocardial blood flow studies.

Methods for the enzymatic synthesis of tyrosine and phenylalanine labeled with nitrogen-13.

L-[13N]Tyrosine and L-[13N]phenylalanine were synthesized using immobilized enzymes by two methods. In method 1, [13N]ammonia is converted to L-[13N]glutamate; transamination with p-hydroxyphenylpyruvate yields L-[13N]tyrosine. [13N]Tyrosine is separated from other labeled intermediates on a Poropak Q column. In method 2, phenylalanine dehydrogenase catalyzes the reversible reductive [13N]amination of either phenylpyruvate or p-hydroxyphenylpyruvate to form L-[13N]phenylalanine or L-[13N]tyrosine, respectively. The feasibility of labeling DOPA and tryptophan with 13N was also demonstrated.

Metabolic fate of the heart agent [18F]6-fluorometaraminol.

Studies were performed to determine whether [18F]6-fluorometaraminol (18F-FMR), a new neuronal heart radiopharmaceutical, is metabolized in vivo and if the metabolites are taken up in heart. Rat, dog, baboon and guinea pig were injected with 18F-FMR and tissue samples were analyzed for metabolites by HPLC. Liver contained the most metabolites of the tissues studied with 25-90% of the radioactivity present as metabolites at 1 h in all the species studied. While metabolites of 18F-FMR are found in blood, no significant accumulation of these metabolites is found in heart (less than or equal to 0.3%) 1 h after i.v. administration in any species except rat. These studies suggest that 18F-FMR is a suitable agent for quantitative imaging of the heart by positron emission tomography.

Radioiodinated benzodiazepines: agents for mapping glial tumors.

Two isomeric iodinated analogues of the peripheral benzodiazepine binding site (PBS) ligand Ro5-4864 have been synthesized and labeled in high specific activity with iodine-125. Competitive binding assays conducted with the unlabeled analogues indicate high affinity for PBS. Tissue biodistribution studies in rats with these 125I-labeled ligands indicate high uptake of radioactivity in the adrenals, heart, and kidney--tissues known to have high concentrations of PBS. Preadministration of the potent PBS antagonist PK 11195 blocked in vivo uptake in adrenal tissue by over 75%, but to a lesser degree in other normal tissues. In vivo binding autoradiography in brain conducted in C6 glioma bearing rats showed dense, PBS-mediated accumulation of radioactivity in the tumor. Ligand 6 labeled with 123I may have potential for scintigraphic localization of intracranial glioma.

Short-term metabolic fate of 13N-labeled glutamate, alanine, and glutamine(amide) in rat liver.

Tracer quantities (in 0.2 ml) of 13N-labeled glutamate, alanine, or glutamine(amide) were administered rapidly (less than or equal to 2 s) via the portal vein of anesthetized adult male rats. Liver content of tracer at 5 s was 57 +/- 6 (n = 6), 24 +/- 1 (n = 3), and 69 +/- 7 (n = 3)% of the injected dose, respectively. Portal-hepatic vein differences for the corresponding amino acids were 17 +/- 6, 26 +/- 8, and 19 +/- 9% (n = 4), respectively, suggesting some export of glutamate and glutamine, but not of alanine, to the hepatic vein. Following L-[13N]glutamate administration, label rapidly appeared in liver alanine and aspartate (within seconds). The data emphasize the rapidity of nitrogen exchange via linked transaminases. By 30 s following administration of either L-[13N]glutamate or L-[13N]alanine, label in liver glutamate was comparable; yet, by 1 min greater than or equal to 9 times as much label was present in liver glutamine(amine) following L-[13N]glutamate administration than following L-[13N]alanine administration. Conversely, label in liver urea at 1 min was more pronounced in the latter case despite: (a) comparable total pool sizes of glutamate and alanine in liver; and (b) label incorporation from alanine into urea must occur via prior transfer of alanine nitrogen to glutamate. The data provide evidence for zonal differences in uptake of alanine and glutamate from the portal vein in vivo. The rate of turnover of L-[amide-13N]glutamine was considerably slower than that of L-[13N]alanine or of L-[13N]glutamate, presumably due in part to the higher concentration of glutamine in that organ. Nevertheless, it was possible to show that despite occasional suggestions to the contrary, glutamine(amide) is a source of urea nitrogen in vivo. The present findings continue to emphasize the rapidity of nitrogen exchange reactions in vivo.

High-performance liquid chromatographic on-line flow-through radioactivity detector system for analyzing amino acids and metabolites labeled with nitrogen-13.

A flow-through radioactivity detector was used for the high-performance liquid chromatographic determination of amino acids and other nitrogenous substances labeled with 13N, a short-lived (t1/2 9.96 min) positron-emitting radionuclide. 13N-Labeled compounds were analyzed using cation, anion and amino columns, or as the o-phthaldialdehyde derivative on an ODS column. Use of column-switching valves and a high-performance liquid chromatographic system with a quaternary eluting capability permits two to three 20-min analyses of labeled samples from a single 13N experiment to be carried out on different columns using a binary or a single mobile phase. Radioactivity in liver metabolites was quantified using an on-line flow-through monitor with data processing capability for integrating peaks and correcting for radioactivity decay. As an example, 1 min following an L-[13N]glutamate injection via the hepatic portal vein, 77% of the label in the liver was in a metabolized form; at least ten labeled products were formed.

[13N]Ammonia and L-[amide-13N]glutamine metabolism in glutaminase-sensitive and glutaminase-resistant murine tumors.

The short-term metabolic fate of labeled nitrogen derived from [13N]ammonia or from L-[amide-13N]glutamine was determined in murine tumors known to be resistant (Ridgeway Osteogenic Sarcoma (ROS] or sensitive (Sarcoma-180 (S-180)) to glutaminase therapy. At 5 min after intraperitoneal injection of [13N]ammonia or of L-[amide-13N]glutamine, only about 0.7% of the label recovered in both tumors was in protein and nucleic acid. After [13N]ammonia administration, most of the label (over 80%) was in a metabolized form; a large portion of this metabolized label (50-57%) was in the urea fraction with a smaller amount in glutamine (37-42%). The major short-term fate of label derived from L-[amide-13N]glutamine was incorporation into components of the urea cycle with smaller amounts in the acidic metabolites and in acidic amino acids. No labeled urea was found during in vitro studies in which S-180 tumor slices were incubated with [13N]ammonia, suggesting that the [13N]urea formed in the tumor in the in vivo experiments was not due to de novo synthesis through carbamyl phosphate in the tumor. Both tumors exhibited very low glutamine synthetase activity. Following glutaminase treatment, glutamine synthetase and gamma-glutamyltransferase activities, while remaining low, increased in the resistant tumor but not in the sensitive tumor; this increase may be related to the insensitivity of the ROS tumor toward glutaminase treatment.

Enzymatic syntheses of carbamyl phosphate, L-citrulline, and N-carbamyl L-aspartate labeled with either 13N or 11C.

[13N]- and [11C]carbamyl phosphate, L-[omega-13N]citrulline, L-[ureido-11C]citrulline, [carbamyl-13N]- and [carbamyl-11C]carbamyl-L-aspartate were synthesized using carbamyl phosphate synthetase co-immobilized with either aspartate transcarbamylase or ornithine transcarbamylase. Carbamyl L-[13N]aspartate was enzymatically prepared from carbamyl phosphate and L-[13N]aspartate. The tissue distribution of radioactivity in mice after injection of radiolabeled ammonia, carbamyl phosphate or citrulline was studied. The tissue distribution of isotope derived from [13N]carbamyl phosphate and [13N]ammonia were similar, with the exception of liver, brain and pancreas, in which 13NH3 uptake was higher after retroorbital injection. The distribution of label derived from L-[omega-13N]- and L-[ureido-11C]citrulline was similar. Substantial tumor (Sarcoma-180) uptake of label from L-citrulline was observed.

Investigation of the metabolic acitivity of bone in rheumatoid arthritis.

Uptake of 99mTc pyrophosphate was measured in the midthird of the radius, ulna femur and metacarpal bones of 22 men with rheumatoid arthritis and 18 control subjects. It was significantly elevated in the rheumatoid population. In a parallel study, the retention of 99mTc methylene diphosphonate was significantly increased in 12 of these rheumatoid patients compared to 6 of the control subjects. These results suggest that there is increased bone turnover occurring in "nonjoint" bone in patients with RA and that the use of such bone as a reference standard for joint imaging is likely to prove misleading.

Comparison of four methods of analysis of 99mTc pyrophosphate uptake in rheumatoid arthritic joints.

Knees, hands, forearms and thighs in 22 patients with classical rheumatoid arthritis and 18 controls were imaged with 99mTc pyrophosphate. The uptake of 99Tc pyrophosphate in the joints of the hand and knee was quantitated using a gamma camera interfaced to a computer. Quantitative measurements of joint activity were assessed by analyzing resultant data in a number of ways. Counts per unit area normalized for body weight and dose of radiopharmaceutical was found to be the most satisfactory method of quantitating differences in disease activity. However, the sensitivity of the method was not great enough to clearly distinguish normal and rheumatoid joints.