Sensitivity of [11C]phenylephrine kinetics to monoamine oxidase activity in normal human heart.

UNLABELLED: Phenylephrine labeled with 11C was developed as a radiotracer for imaging studies of cardiac sympathetic nerves with PET. A structural analog of norepinephrine, (-)-[11C]phenylephrine (PHEN) is transported into cardiac sympathetic nerve varicosities by the neuronal norepinephrine transporter and stored in vesicles. PHEN is also a substrate for monoamine oxidase (MAO). The goal of this study was to assess the importance of neuronal MAO activity on the kinetics of PHEN in the normal human heart. MAO metabolism of PHEN was inhibited at the tracer level by substituting deuterium atoms for the two hydrogen atoms at the alpha-carbon side chain position to yield the MAO-resistant analog D2-PHEN. METHODS: Paired PET studies of PHEN and D2-PHEN were performed in six normal volunteers. Hemodynamic and electrocardiographic responses were monitored. Blood levels of intact radiotracer and radiolabeled metabolites were measured in venous samples taken during the 60 min dynamic PET study. Myocardial retention of the tracers was regionally quantified as a retention index. Tracer efflux between 6 and 50 min after tracer injection was fit to a single exponential process to obtain a washout half-time for all left ventricular regions. RESULTS: Although initial heart uptake of the two tracers was similar, D2-PHEN cleared from the heart 2.6 times more slowly than PHEN (mean half-time 155+/-52 versus 55+/-10 min, respectively; P < 0.01). Correspondingly, heart retention of D2-PHEN at 40-60 min after tracer injection was higher than PHEN (mean retention indices 0.086+/-0.018 versus 0.066+/-0.011 mL blood/ min/mL tissue, respectively; P < 0.003). CONCLUSION: Efflux of radioactivity from normal human heart after uptake of PHEN is primarily due to metabolism of the tracer by neuronal MAO. Related mechanistic studies in the isolated rat heart indicate that vesicular storage of PHEN protects the tracer from rapid metabolism by neuronal MAO, suggesting that MAO metabolism of PHEN leaking from storage vesicles leads to the gradual loss of PHEN from the neurons. Thus, although MAO metabolism influences the rate of clearance of PHEN from the neurons, MAO metabolism is not the rate-determining step in the observed efflux rate under normal conditions. Rather, the rate at which PHEN leaks from storage vesicles is likely to be the rate-limiting step in the observed efflux rate.

Clinical evaluation of carbon-11-phenylephrine: MAO-sensitive marker of cardiac sympathetic neurons.

UNLABELLED: The sympathomimetic drug phenylephrine recently has been labeled with 11C for use in PET studies of cardiac sympathetic innervation. Previous reports using isolated perfused rat heart models indicate that phenylephrine is metabolized by intraneuronal monoamine oxidase (MAO). This report compares the imaging characteristics, neuronal selectivity and kinetics of (-)-[11C]phenylephrine (PHEN) to the structurally similar but MAO-resistant analog (-)-[11C]-meta-hydroxyephedrine (HED), an established heart neuronal marker. METHODS: Fourteen healthy volunteers were studied with PET and PHEN. Ten had paired studies with HED; four of the 10 were scanned a second time with each tracer after oral administration of desipramine, a selective neuronal transport blocker. Hemodynamic and electrocardiographic responses were monitored. Blood levels of intact radiotracer and radiolabeled metabolites were determined from venous blood samples taken during the PET study. Myocardial retention indices for both tracers were calculated. RESULTS: No hemodynamic or electrocardiographic effects were observed with either tracer. PHEN showed reduced myocardial retention at 50 min compared to HED; however, image quality and uniformity of distribution were comparable. PHEN cleared from myocardium with a mean half-time of 59 +/- 5 min, while myocardial levels of HED remained constant. PHEN metabolites appeared in the blood approximately three times faster than HED metabolites. Desipramine pretreatment markedly reduced (> 60%) myocardial retention of both PHEN and HED. CONCLUSION: PHEN provides PET images of human heart comparable in quality and uniformity to HED. Like HED, PHEN localizes in the sympathetic nerves of the heart. However, the more rapid efflux of PHEN, that is likely mediated by MAO, may provide information on the functional status of cardiac sympathetic neurons unobtainable with HED.

Synthesis and preliminary evaluation of [11C]-(-)-phenylephrine as a functional heart neuronal PET agent.

The in vivo behavior of (-)-[11C]phenylephrine (PHEN) is compared with the structurally similar but monoamine oxidase (MAO)-resistant analog (-)-[11C]-m-hydroxyephedrine (HED), which is an established heart neuronal marker. The chiral synthesis of PHEN has been achieved by direct methylation of (-)-m-octopamine with either 11CH3I or CF3SO311CH3. These synthetic methods produced PHEN with a specific activity ranging from 500-1000 Ci/mmol, in a radiochemical yield of > 50% (EOS) and with an enantiomeric purity of 94-96%. Biodistribution studies indicate the initial uptake of PHEN in rat heart is approximately half that of HED. Following PHEN injection, radioactivity egresses from the rat heart rapidly, with 50% washout occurring from 5 to 60 min. HED washout over this interval was less than 20%. The heart neuronal selectivity determined by desipramine blockade of the amine neuronal transporter was 75-77% compared to 92-95% for HED. Ring-labeled (-)-[3H]phenylephrine gave tissue-to-blood concentration ratios and heart clearance times very similar to PHEN. Rats pretreated with the MAO A inhibitor clorgyline showed higher levels of activity in the heart at 15 and 60 min. Tandem PET studies with PHEN and HED in the closed-chest dog provided excellent heart images with both tracers.

Synthesis and in vivo evaluation of a 99m/99Tc-DADT-benzovesamicol: a potential marker for cholinergic neurons.

The diaminodithiol (DADT) ligand has been conjugated to the neuromuscular blocking agent benzovesamicol (BVM) in the 5-position. DADT-BVM 1 was synthesized by coupling of 5-aminomethylbenzovesamicol with a BCA thiolactone reagent. 99mTc radiolabeling of 1 with [99mTc]glucoheptonate gave a 4.7:1 mixture of two 99mTc complexes as determined by HPLC. Biodistribution data of the major [99mTc]-1 complex in CD-1 mice (n = 4-5) showed very little uptake and no regional selectivity in the mouse brain. At all time points examined, the lung and liver showed the highest uptake. For whole brain, the % injected dose values were 0.27, 0.12, 0.04 and 0.01% at t = 1, 5, 30 and 240 min. The major [99mTc]-1 product exhibited a log P = 3.13 +/- 0.06 (SD) with an IC50 = 140-280 nM for the corresponding [99Tc]-1 vs (-)-N-[3H]methyl-5-aminobenzovesamicol. The low brain uptake of [99mTc]-1 vs 5-iodobenzovesamicol is attributed to its higher molecular weight (752) and lower binding affinity.

Biotinylated iodo-polylysine for pretargeted radiation delivery.

Efforts to achieve rapid specific targeting of radioisotopes to disease processes using antibodies conjugated with avidin or streptavidin for pretargeting and radiobiotin derivatives for isotope delivery are attracting substantial interest. At present, these approaches appear to be limited by low delivery of radiotracer to the target. As an alternate radiobiotin tracer, biotinylated/iodinated polylysine (BIP) was prepared by conjugating poly-L-lysine (MW approximately 10,200) with biotin succinimide esters and the Bolton-Hunter reagent. This reagent was then radioiodinated with 125I via the lodogen method. BIP was characterized by radio-HPLC and its in vitro binding to streptavidin. The in vivo localization of BIP was evaluated in a rat model in which streptavidin agarose beads were physically localized to precapillary arterioles in the lungs. Biodistribution and blocking studies performed at 4 and 24 hr after BIP injection indicated specific binding and localization of the radiolabeled peptide to the lungs (lung-to-blood ratio approximately 8 at 4 hr postinjection). Comparative studies of BIP and 111In chelated to biotin showed BIP to have two-fold higher lung targeting and lower splenic and hepatic uptake than the 111In biotin derivative. Our study demonstrates: (1) the feasibility of using a small peptide as a biotin carrier for pretargeting (and for solubilizing organic tracers which may otherwise be difficult to administer in vivo) and (2) that BIP and BIP-like compounds may be suitable and simple alternatives to radiometal-labeled biotin for pretargeting and may offer improved targeting to prelocalized streptavidin.

Synthesis of a nonpeptide carbon-11 labeled substance P antagonist for PET studies.

CP 96,345 is a nonpeptide high affinity antagonist of the substance P (NK1) receptor. The radiosynthesis of [11C]CP 96,345 suitable for Positron Emission Tomography (PET) applications is described. [11C]CP 96,345 was prepared by O-methylation of a desmethyl precursor via in situ generation of its phenolate salt. The in vivo tissue distribution of [11C]CP 96,345 in guinea pigs (n = 2) at 5 and 30 min was determined. Uptake was low in brain (approximately 0.04% dose/g) and highest (approximately 1-2% dose/g) in the spleen and lungs. The present findings indicate that the use of [11C]CP 96,345 in PET might be more applicable to the study of substance P receptors in peripheral tissues involved with inflammatory disease and arthritis.

Streptavidin-biotinylated IgG conjugates: a simple procedure for reducing polymer formation.

Disulfide links of the IgG2ak anti-ovarian carcinoma antibody, 5G6.4, were site-specifically biotinylated [approximately 2 biotins/IgG2a] using a novel crosslinking procedure using the biotin derivatized ETAC (equilibrium transfer alkylation crosslink reagent) 1a. Complexation of ETAC 1a biotinylated 5G6.4 on a column of immobilized protein A at high dilution, followed by passage of [125I]streptavidin, washing and pH change leads to elution of a streptavidin-free product with a molecular mass in the 200-300 kDa range. By contrast, direct mixing with [125I]streptavidin rapidly gave larger oligomers of much greater than 669 and approximately 440-669 kDa molecular mass, respectively. The biodistribution of the 200-300 kDa complex showed significantly diminished liver, kidney and spleen uptake as well as higher blood activity than the 440-669 kDa complex. The methodology represent the first application of ETAC chemistry to disulfide-bond directed biotinylation of antibodies and the synthesis of streptavidin antibody conjugates which minimizes their polymerization.

Disulfide bond-targeted radiolabeling: tumor specificity of a streptavidin-biotinylated monoclonal antibody complex.

A site-specific labeling method was developed in which sulfhydryl groups of a murine IgG2a anti-ovarian monoclonal antibody, 5G6.4, were biotinylated with N-iodoacetyl-N'-biotinylhexylenediamine (Compound 1) following partial reduction of disulfide bonds with dithiothreitol. Reaction of 1-alkylated 5G6.4 with 125I-streptavidin gave immunoreactive streptavidin-1-biotinylated complexes. Radio-fast protein liquid chromatography data were consistent with the formation of a stable monovalent streptavidin-half-antibody complex as the major species. In vivo specific localization of these radioantibody conjugates to human tumor xenografts of ovarian carcinoma was confirmed by a comparative biodistribution study in nude mice using as a control the nonspecific 125I-streptavidin-1-alkylated UPC-10 (an irrelevant IgG2a monoclonal antibody) complex prepared analogously as described above. Tumor uptake for radiolabeled 5G6.4 [0.279 +/- 0.041% (SE) kg injection dose/g) was significantly greater [P less than 0.025] than for UPC-10 [0.165 +/- 0.027% kg injection dose/g]. The tumor:blood ratio (7.38 +/- 1.285) for 5G6.4 was approximately 3 times that for UPC-10 (2.48 +/- 0.708, P less than 0.01). This sulfhydryl site-directed approach demonstrated that reduced disulfides of monoclonal antibodies are viable sites for attaching labels without significant loss of in vitro and in vivo immunoreactivity.

Sulfhydryl site-specific cross-linking and labeling of monoclonal antibodies by a fluorescent equilibrium transfer alkylation cross-link reagent.

The site-specific intramolecular cross-linking of sulfhydryls of monoclonal antibodies via a new class of "equilibrium transfer alkylation cross-link (ETAC) reagents" is described. Following complete or partial reduction of interchain disulfides with dithiothreitol (DTT), two murine IgG2a monoclonal antibodies, 225.28S and 5G6.4, were reacted with alpha,alpha-bis[(p-tolylsulfonyl)methyl]-m-aminoacetophenone (ETAC 1a) and a fluorescent conjugated derivative, sulforhodamine B m-(alpha,alpha-bis(p-tolysulfonylmethyl)acetyl)anilide derivative (ETAC 1b). Reducing SDS-polyacrylamide gel electrophoresis analysis of the products from 1b indicated the formation of S-ETAC-S interchain heavy and light chain cross-links (approximately 23-34% overall yield by video-camera densitometry) which do not undergo disulfide-thiol exchange with DTT at 100 degrees C. In contrast, no interchain cross-links were observed upon reaction of unreduced or reduced antibody wherein the thiols have been previously alkylated with iodoacetamide. These results indicated site-specific cross-linking of interchain sulfhydryls and places their distance within 3-4 A. Flow cytometry of the ETAC 1b 5G6.4 cross-linked product using 77 IP3 human ovarian carcinoma target cells showed positive binding and retention of immunoreactivity. The in vivo biodistributions of 131I-labeled intact 5G6.4 and 125I-labeled reduced 5G6.4 + ETAC 1a product in rats were essentially identical over a period of 24 h. The present study illustrates the potential applications of labelable ETAC reagents as thiol-specific probes for a wide variety of immunological studies.