Radiosynthesis and in vivo evaluation of 1-[18F]fluoroelacridar as a positron emission tomography tracer for P-glycoprotein and breast cancer resistance protein

Aim of this study was to label the potent dual P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) inhibitor elacridar (1) with (18)F to provide a positron emission tomography (PET) radiotracer to visualize Pgp and BCRP. A series of new 1- and 2-halogen- and nitro-substituted derivatives of 1 (4a-e) was synthesized as precursor molecules and reference compounds for radiolabelling and shown to display comparable in vitro potency to 1 in increasing rhodamine 123 accumulation in a cell line overexpressing human Pgp (MDCKII-MDR1). 1-[(18)F]fluoroelacridar ([(18)F]4b) was synthesized in a decay-corrected radiochemical yield of 1.7±0.9% by a 1-step no-carrier added nucleophilic aromatic (18)F-substitution of 1-nitro precursor 4c. Small-animal PET imaging of [(18)F]4b was performed in na?ve rats, before and after administration of unlabelled 1 (5 mg/kg, n=3), as well as in wild-type and Mdr1a/b((-/-))Bcrp1((-/-)) mice (n=3). In PET experiments in rats, administration of unlabelled 1 increased brain activity uptake by a factor of 9.5 (p=0.0002, 2-tailed Student's t-test), whereas blood activity levels remained unchanged. In Mdr1a/b((-/-))Bcrp1((-/-)) mice, the mean brain-to-blood ratio of activity at 60 min after tracer injection was 7.6 times higher as compared to wild-type animals (p=0.0002). HPLC analysis of rat brain tissue extracts collected at 40 min after injection of [(18)F]4b revealed that 93±7% of total radioactivity in brain was in the form of unchanged [(18)F]4b. In conclusion, the in vivo behavior of [(18)F]4b was found to be similar to previously described [(11)C]1 suggesting transport of [(18)F]4b by Pgp and/or BCRP at the rodent BBB. However, low radiochemical yields and a significant degree of in vivo defluorination will limit the utility of [(18)F]4b as a PET tracer.     

Formation of glutamine from [13n]ammonia, [13n]dinitrogen, and [14C]glutamate by heterocysts isolated from Anabaena cylindrica.

A method is described for the isolation of metabolically active heterocysts from Anabaena cylindrica. These isolated heterocysts accounted for up to 34% of the acetylene-reducing activity of whole filaments and had a specific activity of up to 1,560 nmol of C2H4 formed per mg of heterocyst chlorphyll per min. Activity of glutamine synthetase was coupled to activity of nitrogenase in isolated heterocysts as shown by acetylene-inhibitable formation of [13N]NH3 and of amidelabeled [13N]glutamine form [13N]N2. A method is also described for the production of 6-mCi amounts of [13N]NH3. Isolated heterocysts formed [13N]glutamine from [13N]NH3 and glutamate, and [14C]glutamine from NH3 and [14C]glutamate, in the presence of magnesium adenosine 5'-triphosphate. Methionine sulfoximine strongly inhibited these syntheses. Glutamate synthase is, after nitrogenase and glutamine synthetase, the third sequential enzyme involved in the assimilation of N2 by intact filaments. However, the kinetics of solubilization of the activity of glutamate synthase during cavitation of suspensions of A. cylindrica indicated that very little, if any, of the activity of that enzyme was located in heterocysts. Concordantly, isolated heterocysts failed to form substantial amounts of radioactive glutamate from either [13N]glutamine or alph-[14C]ketoglutarate in the presence of other substrates and cofactors of the glutamate synthase reaction. However, they formed [14C]glutamate rapidly from alpha-[14C]ketoglutarate by aminotransferase reactions, with various amino acids as the nitrogen donor. The implication of these findings with regard to the identities of the substances moving between heterocysts and vegetative cells are discussed.     

Amine inversion in proteins. A 13C-NMR study of proton exchange and nitrogen inversion rates in N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine,N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine methyl ester, and reductively methylated concanavalin A

Exchange rates were calculated as a function of pH from line widths of methylamine resonances in 13C-NMR spectra of N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine (TML) and N epsilon,N epsilon,N alpha,N alpha-tetramethyllysine methyl ester (TMLME). The pH dependence of the dimethyl alpha-amine exchange rate could be adequately described by assuming base-catalyzed chemical exchange between two diastereotopic methyl populations related by nitrogen inversion. Deprotonation of the alpha-amine was assumed to occur by proton transfer to (1) OH-, (2) water, (3) a deprotonated amine or (4) RCO2-. Microscopic rate constants characterizing each of these transfer processes (k1, k2, k3 and k4, respectively) were determined by fitting the rates calculated from line width analysis to a steady-state kinetic model. Using this procedure it was determined that for both TML and TMLME k2 approximately equal to 1-10 M-1 s-1, k3 approximately equal to 10(6) M-1 s-1 and ki, the rate constant for nitrogen inversion was about 10(8)-10(9) s-1. Upper limits of 10(12) and 10(3) M-1 s-1 could be determined for k1 and k4, respectively. A similar kinetic analysis was used to explain pH-dependent line-broadening effects observed for the N-terminal dimethylalanyl resonance in 13C-NMR spectra of concanavalin A, reductively methylated using 90% [13C]formaldehyde. From exchange data below pH 4 it could be determined that amine inversion was limited by the proton transfer rate to the solvent, with a rate constant estimated at 20 M-1 s-1. Above pH 4, exchange was limited by proton transfer to other titrating groups in the protein structure. Based upon their proximity, the carboxylate side chains of Asp-2 and Asp-218 appear to be likely candidates. The apparent first-order microscopic rate constant characterizing proton transfer to these groups was estimated to be about 1 X 10(4) s-1. Rate constants characterizing nitrogen inversion (ki), proton transfer to OH- (k1) and proton transfer to the solvent (k2) were estimated to be of the same order of magnitude as those determined for the model compounds. On the basis of our results, it is proposed that chemical exchange processes associated with base-catalyzed nitrogen inversion may contribute to 15N or 13C spin-lattice relaxation times in reductively methylated peptides or proteins.     

Microbial synthesis of L-[15N]leucine L-[15N]isoleucine, and L-[3-13C]-and L-[3'-13C]isoleucines studied by nuclear magnetic resonance and gas chromatography-mass spectrometry

The preparation of leucine and isoleucine labeled with 15N and of site-specific 13C-labeled isoleucines is described. This method is based on the induction of the biosynthetic pathways specific for branched chain amino acids in glutamic acid producing bacteria, and controlled provision of stable isotope labeled precursors. Corynebacterium glutamicum (ATCC 13032), a glutamic acid overproducer, was incubated in leucine production medium which consisted of a basal medium supplemented with [15N]ammonium sulfate, glucose, and sodium alpha-ketoisocaproate. production of L-[15N]leucine reached 138 mumol/ml at an isotopic efficiency of 90%. It was purified and checked by proton NMR and GC-MS. The electron impact (EI) spectrum showed 95 atom% enrichment. The cultivation of C. glutamicum in a similar medium containing alpha-ketobutyrate yielded L-[15N]isoleucine at a concentration of 120 mumol/ml. The GC-MS EI and chemical ionization (CI) spectra confirmed enrichment of 96 atom% 15N as that of the labeled precursors. The biosynthesis of L-[13C]isoleucine was carried out by induced cells which were transferred to a similar medium in which [2-13C]- or [3-13C]pyruvic acid replaced glucose. 13C NMR of the product isoleucine revealed single-site enrichment at C-3 or at C-3' respective to the precursor [13C]pyruvate; i.e., C-3 was labeled from [2-13C]pyruvate and C-3' from [3-13C]pyruvate. Mass spectrometric analysis confirmed that all molecules were labeled only in one carbon. This site-specific incorporation of [13C]pyruvate is contrasted with the labeling pattern obtained when producing cells were supplied with [2-13C]acetate, instead of pyruvate, when most label was incorporated into carbons 3 and 3' of the same isoleucine molecule.     

Simultaneous acquisition of [13C,15N]- and [15N,15N]-separated 4D gradient-enhanced NOESY spectra in proteins

Summary The simultaneous acquisition of a 4D gradient-enhanced and sensitivity-enhanced [¹³C,¹⁵N]/[¹⁵N,¹⁵N]-separated NOESY is presented for the 74-residue [¹³C,¹⁵N]-labeled N-terminal SH3 domain of mGrb2 complexed with a peptide gragment from mSOS-2 in 90% H₂O. The method readily accommodates different ¹³C and ¹⁵N spectral widths, but requires that the same number of increments be collected for both ¹³C and ¹⁵N in the simultaneous dimension (F₂). For purposes of display and analysis, the two 4D spectra can be deconvolved during the processing stage by the appropriate linear combination of separately stored FIDs. Compared to collecting each of these two 4D data sets separately, the presented method is a factor (2)^1/2 more efficient in sensitivity per unit acquisition time. The interleaved nature of this method may also lead to improved peak registration between the two 4D spectra.     

Effects of [Nphe1]nociceptin(1-13)NH2, [Phe1(CH2-NH)Gly2]nociceptin(1- 13)NH2, and nocistatin on nociceptin inhibited constrictions of guinea pig isolated bronchus

Electric field stimulation (EFS) causes excitatory non adrenergic-non cholinergic (eNANC) and cholinergic constrictions in the guinea pig isolated bronchus, the activation of eNANC and cholinergic nerves respectively. We investigated the effects of [Nphe1]nociceptin(1-13)NH2 ([Nphe1]NC(1-13)NH2), [Phe1(CH2-NH)Gly2]nociceptin(1- 13)NH2 ([F/G] NC(1-13)NH2), and nocistatin (NST) on nociceptin (NC) inhibited constrictions in isolated bronchus of guinea pig. The results show that NC (1 micromol/L) inhibited EFS-induced eNANC and cholinergic constrictions compared with the control, in which nociceptin was not applied. After pretreatment with [Nphe1]NC(1-13)NH2, [F/G]NC(1-13)NH2, or NST, the inhibitions of NC were antagonized by [Nphe1]NC(1-13)NH2 and [F/G]NC(1-13)NH2 but not NST. However, [Nphe1]NC(1-13)NH2, [F/G]NC(1-13)NH2, and NST did not affect the inhibitions induced by morphine. Furthermore, [Nphe1]NC(1-13)NH2, [F/G]NC(1-13)NH2 and NST did not cause any appreciable effects on EFS-induced eNANC and cholinergic constrictions in guinea pig bronchi. The results demonstrate that [Nphe1]NC(1-13)NH2 and [F/G]NC(1- 13)NH2 but not NST act as selective antagonists of the NC receptor and the effects of NC on EFS-induced constrictions of guinea pig isolated bronchus.     

Utilization of [11C]phosgene for radiosynthesis of N-(2-{3-[3,5-bis(trifluoromethyl)]phenyl[11C]ureido}ethyl)glycyrrhetinamide, an inhibitory agent for proteasome and kinase in tumors

N-(2-{3-[3,5-Bis(trifluoromethyl)]phenylureido}ethyl)glycyrrhetinamide (2), an ureido-substituted derivative of glycyrrhetinic acid (1), has been reported to display potent inhibitory activity for proteasome and kinase, which are overexpressed in tumors. In this study, we labeled this unsymmetrical urea 2 using [(11)C]phosgene ([(11)C]COCl(2)) as a labeling agent with the expectation that [(11)C]2 could become a positron emission tomography ligand for the imaging of proteasome and kinase in tumors. The strategy for the radiosynthesis of [(11)C]2 was to react hydrochloride of 3,5-bis(trifluoromethyl)aniline (4·HCl) with [(11)C]COCl(2) to possibly give isocyanate [(11)C]6, followed by the reaction of [(11)C]6 with N-(2-aminoethyl)glycyrrhetinamide (3).     

Metabolism of [13N]ammonia in rat lung

Bolus injection of [13N]ammonia into the femoral vein of pentobarbital-anesthetized rats was followed by rapid clearance from the blood and first-pass extraction of nearly 30% by the lungs. Of the label present in the lungs at 6 s after injection (about 27% of the dose), more than 20% was in metabolized form. Of the label present in the lungs at 2 min after injection (about 10% of the dose), 18-25% was in ammonia, about 75% was in glutamine (amide) and less than 1% was in glutamate and aspartate. Thus, despite the presence of significant amounts of glutamate dehydrogenase, the overwhelming route for metabolism of ammonia entering the rat lung in vivo was the glutamine synthetase reaction. Lung tissue that was removed 6 s after intravenous injection of [13N]ammonia and incubated in Krebs-Ringer glucose medium at 37 degrees C for 20 min, showed a significant increase (more than one-third), compared to unincubated lung tissue in the quantity of label in glutamine. Between 6s and 2 min after injection, during which time the total 13N content of the lungs decreased by more than 60%, the maintenance of a quasi-steady state in the concentration of labeled glutamine suggested a short-term balance between formation from extracted ammonia and loss of glutamine into the circulation. Our data support the concept that the lungs are a source of circulating glutamine in the rat. Despite the large fractional extraction of blood-borne [13N]ammonia by the lungs, only minute amounts of tracer (0.2-0.6 ppm of the injected dose) were detected in the expired air within the first 5 min after administration of [13N]ammonia to anesthetized rats, so that pulmonary excretion was not a significant pathway of ammonia elimination. The present findings emphasize the importance of the lungs in the maintenance of whole-body nitrogen homeostasis and suggest the use of [13N]ammonia and 13N-labeled amino acids as non-invasive probes in the study of normal and diseased lung metabolism.     

Biosynthetic preparation of L-[13C]- and [15N]glutamate by Brevibacterium flavum.

The biosynthesis of isotopically labeled L-glutamic acid by the microorganism Brevibacterium flavum was studied with a variety of carbon-13-enriched precursors. The purpose of this study was twofold: to develop techniques for the efficient preparation of labeled L-glutamate with a variety of useful labeling patterns which can be used for other metabolic studies, and to better understand the metabolic events leading to label scrambling in these strains. B. flavum, which is used commercially for the production of monosodium glutamate, has the capability of utilizing glucose or acetate as a sole carbon source, an important criterion from the standpoint of developing labeling strategies. Unfortunately, singly labeled glucose precursors lead to excessive isotopic dilution which reduces their usefulness. Studies with [3-13C]pyruvate indicate that this problem can in principle be overcome by using labeled three-carbon precursors; however, conditions could not be found which would lead to an acceptable yield of isotopically labeled L-glutamate. In contrast, [1-13C]- or [2-13C]acetate provides relatively inexpensive, readily available precursors for the production of selectively labeled, highly enriched L-glutamate. The preparation of L-[15N]glutamate from [15N]ammonium sulfate was carried out and is a very effective labeling strategy. Analysis of the isotopic distribution in labeled glutamate provides details about the metabolic pathways in these interesting organisms.     

Fixation of [13N]N2 and transfer of fixed nitrogen in the Anthoceros-Nostoc symbiotic association

The initial product of fixation of [¹³N]N₂ by pure cultures of the reconstituted symbiotic association between Anthoceros punctatus L. and Nostoc sp. strain ac 7801 was ammonium; it accounted for 75% of the total radioactivity recovered in methanolic extracts after 0.5 min and 14% after 10 min of incubation. Glutamine and glutamate were the primary organic products synthesized from [¹³N]N₂ after incubation times of 0.5–10 min. The kinetics of labeling of these two amino acids were characteristic of a precursor (glutamine) and product (glutamate) relationship. Results of inhibition experiments with methionine sulfoximine (MSX) and diazo-oxonorleucine were also consistent with the assimilation of N₂-derived NH ₄ ⁺ by Anthoceros-Nostoc through the sequential activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1), with little or no assimilation by glutamate dehydrogenase (EC 1.3.1.3). Isolated symbiotic Nostoc assimilated exogenous ¹³NH ₄ ⁺ into glutamine and glutamate and their formation was inhibited by MSX, indicating operation of the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway: However, relative to free-living cultures, isolated symbiotic Nostoc assimilated 80% less exogenous ammonium into glutamine and glutamate, implying that symbiotic Nostoc could assimilate only a fraction of N₂-derived NH ₄ ⁺ . This implication was tested by using Anthoceros associations reconstituted with wild-type or MSX-resistant strains of Nostoc incubated with [¹³N]N₂ in the presence of MSX. The results of these experiments indicated that, in situ, symbiotic Nostoc assimilated about 10% of the N₂-derived NH ₄ ⁺ and that NH ₄ ⁺ was made available to Anthoceros tissue where it was apparently assimilated by the GS-GOGAT pathway. Since less than 1% of the fixed N₂ was lost to the suspension medium, it appears that transfer of NH ₄ ⁺ from symbiont to host tissue was very efficient in this extracellular symbiotic association.Abbreviations DON 6-diazo-5-oxo-l-norleucine - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSX l-methionine-dl-sulfoximine     

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.     

Radiosyntheses of two positron emission tomography probes: [11C]Oseltamivir and its active metabolite [11C]Ro 64-0802

Oseltamivir phosphate (Tamiflu, 1.H(3)PO(4) is an orally active anti-influenza drug, which is hydrolyzed by esterase to its carboxylate metabolite Ro 64-0802 (2) with potent activity inhibiting neuraminidase. In this study, for the first time, we synthesized carbon-11-labeled oseltamivir ([(11)C]1) and Ro 64-0802 ([(11)C]2) as two novel positron emission tomography probes and demonstrated that [(11)C]1 had twofold higher radioactivity concentration in the mouse brains than [(11)C]2.     

Radiosynthesis of three [11C]ureido-substituted benzenesulfonamides as PET probes for carbonic anhydrase IX in tumors

Three ureido-substituted benzenesulfonamides 1a-c have been developed as potent inhibitors for carbonic anhydrase IX, which is overexpressed in hypoxic tumors. In this study, we labeled these unsymmetrical ureas 1a-c using [(11)C]phosgene ([(11)C]COCl(2)) as a labeling agent with the expectation that [(11)C]1a-c could become promising positron tomography probes for imaging carbonic anhydrase IX in tumors. The strategy for radiosynthesis of [(11)C]1a-c was to react hydrochloride of anilines 2a-c with [(11)C]COCl(2) to give isocyanate [(11)C]4a-c, followed by a reaction with 4-aminobenzenesulfonamide (3).     

N,N-bis(2-mercaptoethyl)methylamine: a new coligand for Tc-99m labeling of hydrazinonicotinamide peptides

Hydrazinonicotinamide (HYNIC) forms stable coordination complexes with Tc-99m when reacted with Tc(V)oxo species such as Tc-mannitol or other Tc-polyhydric complexes. However, radio-HPLC of [Tc-For-MLFK-HYNIC] labeled via Tc-polyhydric ligands demonstrated multiple radiochemical species each with unique biodistribution patterns. This is likely due to the fact that Tc can bind to the hydrazino moiety, as well as polyhydric ligands, in a variety of coordination geometries. Tridentate ligands, such as bis(mercaptoethyl)methylamine (NS2), may constrain the possible coordination geometries and improve overall stability. To investigate this, we synthesized NS2, converted the [Tc-mannitol-For-MLFK-HYNIC] to the corresponding NS2-containing complex [Tc-NS2-For-MLFK-HYNIC], and compared its infection imaging and biodistribution properties with [Tc-mannitol-For-MLFK-HYNIC]. Conversion to the NS2 complex was confirmed by HPLC which showed a single unique hydrophobic species with retention time greater than the [Tc-mannitol-For-MLFK-HYNIC] complex. Imaging experiments with both preparations were performed in rabbits with E. coli infections in the left thigh. Tissue radioactivity measurements demonstrated that compared to Tc-mannitol-peptide, accumulation of Tc-NS2-peptide was lower in blood, heart, and normal muscle and higher in spleen, infected muscle, and pus (p < 0.01). These results indicate that the Tc-NS2-peptide complex is chemically more homogeneous and exhibits improved infection localization and biodistribution properties. In an effort to model the interactions of the metal-HYNIC core with NS2 and related ligand types, the reactions of [ReCl3(NNC5H4NH)(NHNC5H4N)] and [99TcCl3(NNC5H4NH)(NHNC5H4N)], effective structural analogues for the [M(NNC5H4NH(x))2] core, with NS2, C5H3N-2,6-(CH2SH)2, O(CH2CH2SH)2, and S(CH2CH2SH)2 were investigated and the compounds [M[CH3N(CH2CH2S)2](NNC5H4N)(NHNC5H4N] (M = 99Tc (5a), Re (5b)), [Re[C5H3N-2,6-(CH2S)2](NNC5H4N)(NHNC5H4N)].CH2Cl2.0.5MeOH (7), [Re[SCH2CH2)2O] (NNC5H4N)(NHNC5H4N)] (8), and [Re[(SCH2CH2)2S](NNC5H4NH)(NHNC5H4N)]Cl (9) were isolated. Similarly, the reaction of [ReCl3(NNC5H4NH)(NHNC5H4N)] with the bidentate ligands pyridine-2-methanethiol and 3-(trimethlysilyl)pyridine-2-thiol led to the isolation of [ReCl(C5H4N-2-CH2S) (NNC5H4N)(NHNC5H4N)] (10) and [Re(2-SC5H3N-3-SiMe3)2 (NNC5H4N)(NHNC5H4N)] (11), respectively, while reaction with N-methylimidazole-2-thiol yielded the binuclear complex [Re(OH)Cl(SC3H2N2CH3)2(NNC5H4N)2 (NHNC5H4N)2] (12). The analogous metal-(HYNIC-OH) precursor, [ReCl3[NNC5H3NH(CO2R)] [NHNC5H3N(CO2R)]] (R = H, 13a; R = CH3, 13b) has been prepared and coupled to lysine to provide [RCl3[NNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)] [NHNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)]].2HCl (14.2HCl), while the reaction of the methyl ester 13b with 2-mercaptopyridine yields [Re(2-SC5H4N)2[NNC5H3N(CO2Me)][NHNC5H3N(CO2Me)]] (15). While the chemical studies confirm the robustness of the M-HYNIC core (M = Tc, Re) and its persistence in ligand substitution reactions at adjacent coordination sites of the metal, the isolation of oligomeric structures and the insolubility of the peptide conjugates of 13, 14, and 15 underscore the difficulty of characterizing these materials on the macroscopic scale, an observation relevant to the persistent concerns with reagent purity and identity on the tracer level.     

Radiosynthesis and biodistribution of cyclic RGD peptides conjugated with novel [18F]fluorinated aldehyde-containing prosthetic groups

Achieving high-yielding, robust, and reproducible chemistry is a prerequisite for the (18)F-labeling of peptides for quantitative receptor imaging using positron emission tomography (PET). In this study, we extend the toolbox of oxime chemistry to include the novel prosthetic groups [(18)F]-(2-{2-[2-(2-fluoroethoxy)ethoxy]ethoxy}ethoxy)acetaldehyde, [(18)F]5, and [(18)F]-4-(3-fluoropropoxy)benzaldehyde, [(18)F]9, in addition to the widely used 4-[(18)F]fluorobenzaldehyde, [(18)F]12. The three (18)F-aldehydes were conjugated to the same aminooxy-bearing RGD peptide and the effect of the prosthetic group on biodistribution and tumor uptake studied in mice. The peptide conjugate [(18)F]7 was found to possess superior in vivo pharmacokinetics with higher tumor to blood, tumor to liver, tumor to muscle, and tumor to lung ratios than either [(18)F]10 or [(18)F]13. The radioactivity from the [(18)F]7 conjugate excreted more extensively through the kidney route with 79%id passing through the urine and bladder at the 2 h time point compared to around 55%id for the more hydrophobic conjugates [(18)F]10 and [(18)F]13. The chemical nature of a prosthetic group can be employed to tailor the overall biodistribution profile of the radiotracer. In this example, the hydrophilic nature of the ethylene glycol containing prosthetic group [(18)F]5 clearly influences the overall excretion pattern for the RGD peptide conjugate.     

Patterns of [13N]ammonium uptake and assimilation by Frankia HFPArl3

Nitrogen-starved cells of Frankia strain HFPArl3 incorporated [¹³N]-labeled ammonium into glutamine serine (glutamate, alanine, aspartate), after five-minute radioisotope exposures. High initial endogenous pools of glutamate were reduced, while total glutamine increased, during short term NH _inf4 ^sup+ incubation. Preincubation of cells in methionine sulfoximine (MSX) resulted in [¹³N]glutamine reduced by more than 80%, while [¹³N]glutamate and [¹³N]alanine levels increased. The results suggest that glutamine synthetase is the primary enzyme of ammonium assimilation, and that glutamate dehydrogenase and alanine dehydrogenase may also function in ammonium assimilation at low levels. Efflux of [¹³N]serine and lesser amounts of [¹³N]glutamine was detected from the Frankia cells. The identity of both Ser and Gln in the extracellular compartment was confirmed with gas chromatography/mass spectrometry. Serine efflux may be of significance in nitrogen transfer in Frankia.Abbreviations Pthr phosphothreonine - Aad -amino-adipate - MSX methionine sulfoximine     

Receptor binding and cell-mediated metabolism of [125I]monoiodoglucagon by isolated canine hepatocytes

We have developed a reverse-phase HPLC method to purify 125I-labeled products resulting from the chloramine-T-based iodination of glucagon and have used the products [(125I)iodoTyr13]glucagon, [(125I)iodoTyr10,13]glucagon, and [(125I)iodoTyr10]glucagon) to study the receptor binding of glucagon and the cell-mediated metabolism of the hormone by isolated canine hepatocytes. The extent of binding of the three labeled glucagons to cell receptors differed at steady state (8.5, 11.9, and 12.6% of the three peptides, respectively, becoming cell-associated), but each of the labeled glucagons approached steady state binding at the same rate. Further, unlabeled glucagon competed for the binding of each of the labeled peptides in parallel under steady state conditions, and each of the peptides showed potent activity in inhibiting [14C]fructose incorporation into glycogen. Gel filtration of the acetic acid-extracted, cell-associated products of radiolabeled glucagon binding revealed 10-20% of the material as a shoulder on the descending limb of the peak of hormone for each of the three labeled peptides. Trypsin digestion of the lower molecular weight peptide derived from [(125I)iodoTyr13]glucagon resulted in a fragment containing residues 13 to 17 as the only detectable radiolabeled product. On the other hand, trypsin digestion of the analogous peptide derived from [(125I)iodoTyr10]glucagon revealed, in addition to the radiolabeled fragment containing residues 1 to 12, a major fragment identified by radiosequence analysis to contain residues 4 to 12 and a minor fragment identified to contain residues 7 to 12. We conclude that (a) notwithstanding apparent differences in affinities exhibited by [(125I)iodoTyr13]glucagon, [(125I)iodoTyr10,13]glucagon, and [(125I)iodoTyr10]glucagon for binding to canine hepatocytes, the interactions of all three peptides with the glucagon receptor are functionally equivalent, and (b) the cell-mediated metabolism of receptor-bound glucagon involves the formation of hormone-derived peptides in which the biologically important NH2-terminal region of the hormone has been modified by limited proteolytic cleavage.     

Asymmetric induction in the [4+2]cycloaddition of cyclopentadiene and furan to chiral derivatives of fumaric acid.

[4+2]Cycloaddition reactions of cyclopentadiene (1a) and furan (1b) to N,N'-fumaroyldi[(2R)-bornane-10,2-sultam] (2) and to N,N'-fumaroyldi[(2R)-bornane-10,2-(2'-phenyl-pyrazol-3'-one)] (3) are presented. A correlation between the solvent polarity and the logarithm of the diastereoisomer ratio (dr) was found for the uncatalyzed [4+2]cycloaddition of 1a to 3.     

Synthesis and radiopharmacological characterization of 2beta-carbo-2'-[18F]fluoroethoxy-3beta-(4-bromo-phenyl)tropane ([18F]MCL-322) as a PET radiotracer for imaging the dopamine transporter (DAT)

The fluoroalkyl-containing tropane derivative 2beta-carbo-2'-fluoroethoxy-3beta-(4-bromo-phenyl)tropane (MCL-322) is a highly potent and moderately selective ligand for the dopamine transporter (DAT). The compound was labeled with the short-lived positron emitter (18)F in a single step by nucleophilic displacement of the corresponding tosylate precursor MCL-323 with no-carrier-added [(18)F]fluoride. The positron emission tomography (PET) radiotracer 2beta-carbo-2'-[(18)F]fluoroethoxy-3beta-(4-bromo-phenyl)tropane [(18)F]MCL-322 was obtained in decay-corrected radiochemical yields of 30-40% at a specific radioactivity of 1.6-2.4Ci/mumol (60-90GBq/mumol) at the end-of-synthesis (EOS). Small animal PET, ex vivo and in vivo biodistribution experiments in rats demonstrated a high uptake in the striatum (3.2% ID/g) 5min after injection, which increased to 4.2% ID/g after 60min. The uptake in the cerebellum was 1.8% ID/g and 0.6% ID/g after 5min and 60min post-injection, respectively. Specific binding to DAT of [(18)F]MCL-322 was confirmed by blocking experiments using the high affinity DAT ligand GBR 12909. The radiopharmacological characterization was completed with metabolite and autoradiographic studies confirming the selective uptake of [(18)F]MCL-322 in the striatum. It is concluded that the simple single-step radiosynthesis of [(18)F]MCL-322 and the promising radiopharmacological data make [(18)F]MCL-322 an attractive candidate for the further development of a PET radiotracer potentially suitable for clinical DAT imaging in the human brain.     

Recovery of (13)CO2 during rest and exercise after [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO3 infusions

For estimating the oxidation rates (Rox) of glucose and other substrates by use of (13)C-labeled tracers, we obtained correction factors to account for label dilution in endogenous bicarbonate pools and TCA cycle exchange reactions. Fractional recoveries of (13)C label in respiratory gases were determined during 225 min of rest and 90 min of leg cycle ergometry at 45 and 65% peak oxygen uptake (VO(2 peak)) after continuous infusions of [1-(13)C]acetate, [2-(13)C]acetate, or NaH(13)CO(3). In parallel trials, [6,6-(2)H]glucose and [1-(13)C]glucose were given. Experiments were conducted after an overnight fast with exercise commencing 12 h after the last meal. During the transition from rest to exercise, CO(2) production increased (P < 0.05) in an intensity-dependent manner. Significant differences were observed in the fractional recoveries of (13)C label as (13)CO(2) at rest (NaH(13)CO(3), 77.5 +/- 2.8%; [1-(13)C]acetate, 49.8 +/- 2.4%; [2-(13)C]acetate, 26.1 +/- 1.4%). During exercise, fractional recoveries of (13)C label from [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO(3) were increased compared with rest. Magnitudes of label recoveries during both exercise intensities were tracer specific (NaH(13)CO(3), 93%; [1-(13)C]acetate, 80%; [2-(13)C]acetate, 65%). Use of an acetate-derived correction factor for estimating glucose oxidation resulted in Rox values in excess (P < 0.05) of glucose rate of disappearance during hard exercise. We conclude that, after an overnight fast: 1) recovery of (13)C label as (13)CO(2) from [(13)C]acetate is decreased compared with bicarbonate; 2) the position of (13)C acetate label affects carbon dilution estimations; 3) recovery of (13)C label increases in the transition from rest to exercise in an isotope-dependent manner; and 4) application of an acetate correction factor in glucose oxidation measurements results in oxidation rates in excess of glucose disappearance during exercise at 65% of VO(2 peak). Therefore, bicarbonate, not acetate, correction factors are advocated for estimating glucose oxidation from carbon tracers in exercising men.