High-affinity no-carrier-added 99mTc-labeled chemotactic peptides for studies of inflammation in vivo.

Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys, a chemotactic peptide that binds with high affinity to the chemoattractant receptor on granulocytes and monocytes, was labeled with 99mTc using the diaminedithiol (DADT) chelating system to coordinate the Tc. 99mTc labeling of the DADT-coupled peptide was accomplished in 84% overall yield (room temperature for 10 min) using [99mTc]glucoheptonate as the donor of prereduced Tc. HPLC analysis showed two major 99mTc-labeled peptide peaks, 99mTc-DADT-Pep-I and 99mTc-DADT-Pep-II, were obtained in a ratio of 1:0.85. Using an iodoacetamide-derivatized gel to remove unlabeled peptide from the 99mTc labeling mixtures, essentially no-carrier-added (nca) high-specific activity 99mTc-labeled chemotactic peptides were obtained. The 99Tc analogues of the peptides were synthesized (72% yield) in a similar fashion and correlated with 99mTc complexes I and II by HPLC. In vitro competitive receptor binding assays of the isolated 99Tc analogues were performed against the tritiated chemotactic peptide [3H]N-for-Met-Leu-Phe ([3H]fMLF) using isolated granulocytes. The 99Tc-derivatized peptides showed similar binding affinities to the chemoattractant receptor as the unlabeled Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys. The nca 99mTc-labeled peptides gave high contrast images of experimental inflammation in rabbits without causing neutropenia. Thus, it is feasible to attach the Tc-DADT chelate to low-molecular weight receptor binding chemotactic peptides and retain substantial binding to the receptor. Chemotactic peptides labeled with 99mTc via the DADT ligand system have the potential for imaging focal sites of inflammation without toxic effects, an important consideration in the successful utilization of chemotactic peptide agonists.

[125/123I]IPH: a radioiodinated analog of epibatidine for in vivo studies of nicotinic acetylcholine receptors.

Tomographic imaging of central nicotinic acetylcholine receptors (nAChRs) via single photon emission computed tomography (SPECT) has been hampered by the lack of a radioligand with suitable in vivo binding characteristics. Therefore, a novel analog of epibatidine, (+/-)-exo-2-(2-iodo-5-pyridyl)-7-azabicyclo[2.2.1]heptane (IPH), labeled with [(125)I] or [(123)I] was evaluated as an in vivo marker of central nicotinic acetylcholine receptors (nAChRs). [(125)I]IPH showed substantial brain penetration (4.2% of the injected dose at 30 min) and a cerebral biodistribution in mice consistent with the in vivo labeling of nAChRs (% injected dose/gram of thalamus, superior colliculi >> cerebellum). [(125)I]IPH binding sites were shown to be saturable with unlabeled IPH (ED50 approximately 1 microg/kg). The uptake of [(125)I]IPH was blocked significantly by the nicotinic agonists, cytisine, lobeline, and (-)-nicotine, but not by the noncompetitive nAChR antagonist, mecamylamine. Antagonists of muscarinic (scopolamine), serotonin (ketanserin), and opioid (naloxone) receptors had no significant effect on [(125)I]IPH binding. A preliminary SPECT imaging study with [(123)I]IPH in a baboon showed [(123)I]IPH to localize in nAChR-rich areas of brain (thalamus > frontal cortex > cerebellum). [(123)I]IPH binding in baboon brain was also displaced (35-45% displacement) by a challenge dose of cytisine showing that a well-characterized nicotinic agonist effectively competes for [(123)I]IPH binding sites. [(123)I]IPH seems well suited for imaging studies of nAChRs and, to our knowledge, is the first SPECT agent that has allowed for the visualization of nAChRs in primate brain.

Fluorine-18-FPH for PET imaging of nicotinic acetylcholine receptors.

UNLABELLED: Visualization of central nicotinic acetylcholine receptors (nAChRs) with modern PET or SPECT imaging techniques has been hampered by the lack of a radioligand with suitable in vivo binding characteristics (i.e., high target-to-nontarget ratios and kinetics appropriate for the half-life of the tracer and imaging modality used). This paper describes in vivo binding, kinetics and pharmacology of a highly potent 18F-labeled analog of epibatidine, (+/-)-exo-2-(2-[18F]fluoro-5-pyridyl)-7-azabicyclo[2.2.1]heptane ([18F]FPH), in the mouse brain with the view towards application of this tracer for PET imaging of nAChR in human brain. METHODS: Fluorine-18-FPH was administered intravenously to mice, and time-activity curves were determined for several regions in the brain and other organs. Saturation and pharmacology of [18F]FPH binding was demonstrated in vivo by preinjecting unlabeled FPH or other drugs with known pharmacological action before [18F]FPH was injected. The effect of the drugs on [18F]FPH accumulation was evaluated. RESULTS: [18F]FPH was rapidly incorporated into the mouse brain; peak activity (2.4% of the injected dose) was measured at 5 min after intravenous administration, followed by washout to 1.1% injected dose (ID) at 60 min. Highest concentrations of 18F occurred at 15 min in areas known to contain high densities of nAChR ¿e.g., thalamus [9.7% of injected dose per gram tissue (ID/g¿] and superior colliculus (8.3% ID/g)]. Accumulation of the 18F tracer in hippocampus, striatum, hypothalamus and cortical areas was intermediate (5.0, 5.6, 4.2 and 5.6% ID/g, respectively) and low in the cerebellum (2.8% ID/g). The distribution of [18F]FPH in the mouse brain matched that of other in vivo nAChR probes such as 3H-labeled epibatidine or norchloroepibatidine, [3H](-)-nicotine and [3H]cytisine and that of nAChR densities determined in postmortem autoradiographic studies in rodents. Preinjection of blocking doses of unlabeled epibatidine, (-)-nicotine, lobeline and cytisine significantly inhibited [18F]FPH binding in thalamus and superior colliculus, but not in cerebellum, whereas drugs that interact with binding sites other than acetylcholine recognition sites of nAChR (e.g., mecamylamine, scopolamine, N-methylspiperone and ketanserin) had no effect on [18F]FPH accumulation in any of the brain regions examined. CONCLUSION: Fluorine-18-FPH labels nAChR in vivo in the mouse brain. Because of its high uptake into the brain and high ratios of specific-to-nonspecific binding, this radioligand appears to be ideally suited for PET imaging of nAChR in the mammalian brain.

Synthesis and in vivo studies of a selective ligand for the dopamine transporter: 3 beta-(4-[125I]iodophenyl) tropan-2 beta-carboxylic acid isopropyl ester ([125I]RTI-121).

A selective ligand for the dopamine transporter 3 beta-(4-iodophenyl)tropan-2 beta-carboxylic acid isopropyl ester (RTI-121) has been labeled with iodine-125 by electrophilic radioiododestannylation. The [125I]RTI-121 was obtained in good yield (86 +/- 7%, n = 3) with high radiochemical purity (> 99%) and specific radioactivity (1210-1950 mCi/mumol). After i.v. administration of [125I]RTI-121 to mice, the rank order of regional brain tissue radioactivity (striatum > olfactory tubercles > > cortex, hippocampus, thalamus, hypothalamus, cerebellum) was consistent with dopamine transporter labeling. Specific in vivo binding in striatum and olfactory tubercles was saturable, and was blocked by the dopamine transporter ligands GBR 12,909 and (+/-)-nomifensine. By contrast, binding was not reduced by paroxetine, a serotonin transporter inhibitor, or desipramine, a norepinephrine transporter inhibitor. A variety of additional drugs having high affinities for recognition sites other than the neuronal dopamine transporter also had no effect. The [125I]RTI-121 binding in striatum and olfactory tubercles was inhibited by d-amphetamine in dose-dependent fashion. Nonmetabolized radioligand represents 85% of the signal observed in extracts of whole mouse brain. Thus, [125I]RTI-121 is readily prepared, and is a useful tracer for dopamine transporter studies in vivo.

In vivo labeling of delta opioid receptors in mouse brain by [3H]benzylidenenaltrexone, a ligand selective for the delta 1 subtype.

(E)-7-Benzylidenenaltrexone (BNTX) is a selective ligand for the putative delta 1 (delta 1) opioid receptor. To explore the feasibility of labeling delta 1 sites in vivo; we determined the cerebral distribution of radioactivity after systemic administration of [3H]BNTX to CD1 mice. Uptake was highest in striatum and lowest in cerebellum throughout the 4 hr time course. Specific radioligand binding, approximated as the difference in radioactivity concentrations between striatum and cerebellum, peaked at 0.32 percent injected dose/g at 30 min and comprised a modest 23% of total striatal radioactivity. For seven brain regions, radioactivity concentrations correlated with delta site densities known from prior in vitro studies (rS = 0.79, p = 0.03), and also with the uptake of N1'-([11C]methyl)naltrindole in vivo (rS = 0.78, p = 0.04) in mice. Specific binding in striatum, olfactory tubercles and cortical regions was saturable by BNTX, and was inhibited stereoselectively by the optical isomers of naloxone. Naltrindole and naltriben (NTB), delta antagonists, blocked 65-99% of [3H]BNTX specific binding at a dosage of 5.0 mumol/kg. Similar doses of the mu antagonist cyprodime, or the kappa agonist U50,488H, did not inhibit binding. Adjusted for the four-fold greater brain penetration of NTB relative to BNTX, dose-response studies suggested that delta 1 selective BNTX (ED50 = 1.51 mumol/kg) was 50% more potent than delta 2 selective NTB (ED50 = 0.56 mumol/kg) in blocking specific [3H]BNTX binding in striatum. In CXBK mice, a strain with functional delta 1 but not delta 2 receptors in antinociceptive assays, radioligand uptake and distribution proved similar to that in CD1 mice. In sum, [3H]BNTX labels murine delta opioid receptors in vivo with a low extent of specific binding. The data is consistent with, but not conclusive for, selective labeling of the delta 1 subtype.

Rate of binding of various inhibitors at the dopamine transporter in vivo.

The rate of entry of drugs into brain is thought to be a factor in their abuse liability. In this investigation, we have examined the rate of entry and binding at dopamine transporters in mouse striatum for a variety of dopamine transporter inhibitors. The method utilized was based on measuring the displacement of 3H-WIN 35,428 from striatal dopamine transporter sites in vivo at different times. Eleven cocaine analogs (RTI-31, RTI-32, RTI-51, RTI-55, RTI-113, RTI-114, RTI-117, RTI-120, RTI-121, WIN 35,065-2, and WIN 35,428) as well as other dopamine uptake site blockers (bupropion, nomifensine, and methylphenidate) were compared with (-) cocaine for their rates of displacement of 3H-WIN 35,428 binding in vivo. The drugs that displayed the fastest occupancy rates were bupropion, (-) cocaine, nomifensine, and methylphenidate. RTI-51, RTI-121, RTI-114, RTI-117, RTI-120, RTI-32, RTI-55, and RTI-113, showed intermediate rates, whereas RTI-31, WIN 35,065-2, and WIN 35,428 exhibited the slowest rates of displacement. While many of the cocaine analogs have proven to be behaviorally and pharmacologically more potent than (-) cocaine, their rates of entry and binding site occupancy were slower than that for (-) cocaine. Earliest times of transporter occupancy by the different drugs were correlated (although weakly) with their degree of lipophilicity (r = 0.59; P < 0.02). Kinetic effects and metabolism of the compounds could complicate the interpretations of these data.(ABSTRACT TRUNCATED AT 250 WORDS)

[3H]A-69024: a non-benzazepine ligand for in vitro and in vivo studies of dopamine D1 receptors.

[3H]A-69024 has been prepared as a radioligand for studying the dopamine D1 receptor. [3H]A-69024 binds to rat striatal membranes with a KD = 14.3 +/- 3.2 nM (mean +/- SEM; n = 3) and Bmax = 63.5 +/- 12.8 fmol/mg wet tissue (1.8 +/- 0.3 pmol/mg protein). This ligand binds to only one site with a Hill coefficient close to unity. The in vivo biodistribution of [3H]A-69024 showed a high uptake in the striatum (5.9% ID/g) at 5 min followed by clearance. As a measure of specificity, the striatum/cerebellar ratio reached a maximum of 6.7 at 30 min post-injection. Pre-treatment with the D1 antagonist R(+)SCH 23390 (1 mg/kg) reduced this ratio to unity. The dopamine antagonist (+)butaclamol and unlabeled A-69024 inhibited striatal uptake by 70 and 51%, respectively. Spiperone (D2/5-HT2A) and ketanserin (5-HT2A/5-HT2C) at doses of 1 mg/kg had no inhibitory effect on [3H]A-69024 uptake in the striatum; however, increased uptake of [3H]A-69024 by > 30% in the whole brain was observed. The selectivity and affinity of [3H]A-69024 suggests that this non-benzazepine radioligand may be useful for in vitro and in vivo studies of the dopamine D1 receptor.

(+)-[C-11]-cis-N-benzyl-normetazocine: a selective ligand for sigma receptors in vivo.

The in vivo biodistribution profile of the novel sigma (sigma) receptor ligand (+)-[C-11]-cis-N-benzyl-normetazocine ([C-11]-(+)-NBnNM) in mouse brain was examined. This radioligand displayed high brain uptake and a distribution consistent with the density of sigma receptors. Brain radioactivity levels peaked at 15 min postinjection and were largely maintained (ca. 80% of maximal values) up to 90 min postinjection. Pretreatment with several different sigma ligands (haloperidol, (+)-pentazocine, DuP 734, ifenprodil) effectively inhibited [C-11]-(+)-NBnNM binding in a dose-dependent manner in all brain regions. [C-11]-(+)-NBnNM binding sites were shown to be saturable with unlabeled (+)-NBnNM (ED50 = 0.02 mg/kg) and enantioselectively inhibited by the optical isomers of pentazocine. A blocking dose of the dopamine D2 antagonist spiperone (1 mg/kg) did not significantly inhibit [C-11]-(+)-NBnNM binding. Pretreatment with the phencyclidine (PCP) blocker 1-[1-(2-thienyl)cyclohexyl] piperidine (TCP) did not significantly alter total brain tissue radioactivity. Thus, [C-11]-(+)-NBnNM binds with high specificity and selectivity to sigma receptors in vivo and offers excellent potential to study sigma receptors in living human brain via positron emission tomography.

Repeated administration of MDMA causes transient down-regulation of serotonin 5-HT2 receptors.

The present study examined short- and long-term effects of MDMA (3,4-methylene-dioxymethamphetamine) on serotonin (5-HT2 and 5-HT1c) receptors in the brain of the rat. N1-Methyl-2-[125I]lysergic acid diethylamide ([125I]MIL) was used to label these receptors in vitro and in vivo. The usefulness of [125I]MIL for in vivo detection of changes in 5-HT2 receptors was confirmed in preliminary experiments in which rats were treated chronically with mianserin (5 mg/kg, once daily for 10 days). Decreases in specific in vivo binding of [125I]MIL, after treatment with mianserin were found to be of the same magnitude as those determined by others, using in vitro methods. The MDMA (8 doses; 5-20 mg/kg each) was administered to rats over a period of 4 days. At various times after administration of the last dose of MDMA, the binding of [125I]MIL was measured. Acutely, treatment with MDMA (20 mg/kg) reduced specific in vivo binding of [125I]MIL in all regions of brain studied. For example, in the frontal cortex, specific binding of [125I]MIL was decreased by 80% at 6 hr and by 62% at 24 hr, after cessation of treatment with MDMA. Twenty-one days after administration of MDMA however, the number of binding sites for [125I]MIL was back to control levels. Reductions in in vivo binding of [125I]MIL in frontal cortex were dependent on the dose of MDMA injected and were associated with decreases in the number of binding sites for [125I]MIL (Bmax values) in tissue homogenates of the same area. Autoradiographic studies of MDMA-treated rats confirmed the decreased density of 5-HT2 receptors and also suggested that the 5-HT1c receptor of the choroid plexus was not affected. These results indicate that repeated administration of MDMA caused transient down-regulation of 5-HT2 receptors in the brain of the rat. Further, they demonstrated that [125I]MIL is a suitable radioligand for labeling 5-HT2 receptors, both in vitro and in vivo. Once labeled with an appropriate radionuclide for SPECT (single photon emission computed tomography) or PET (positron emission tomography), MIL should prove useful for monitoring changes in the density of serotonin receptors in the living mammalian brain.

In vivo labeling of cocaine binding sites on dopamine transporters with [3H]WIN 35,428.

After in vivo administration, [3H]WIN 35,428 accumulated in mouse brain regions containing dopaminergic nerve terminals. The highest accumulation was in the striatum and it peaked between 30 and 60 min. The accumulation was saturable with increasing doses of WIN 35,428, and was blocked by compounds that bind to the dopamine transporter. Paroxetine, a drug that blocks serotonin transporters, had no effect. Moreover, the in vivo potency of cocaine analogs correlated with their in vitro potency. Thus, [3H]WIN 35,428 is a suitable ligand for labeling cocaine receptors associated with dopamine transporters in vivo.

In vivo biodistribution of two [18F]-labelled muscarinic cholinergic receptor ligands: 2-[18F]- and 4-[18F]-fluorodexetimide.

Two [18F]-labelled analogues of the potent muscarinic cholinergic receptor (m-AChR) antagonist, dexetimide, were evaluated as potential ligands for imaging m-AChR by positron emission tomography (PET). Intravenous administration of both 2-[18F]- or 4-[18F]-fluorodexetimide resulted in high brain uptake of radioactivity in mice. High binding levels were observed in m-AChR rich areas, such as cortex and striatum, with low levels in the receptor-poor cerebellum. Uptake of radioactivity was saturable and could be blocked by pre-administration of dexetimide or atropine. Drugs with different sites of action were ineffective at blocking receptor binding. The results indicate that both radiotracers are promising candidates for use in PET studies.

Radioiodinated D-(+)-N1-ethyl-2-iodolysergic acid diethylamide: a ligand for in vitro and in vivo studies of serotonin receptors.

Radioiodinated D-(+)-N1-ethyl-2-iodolysergic acid diethylamide ([125I]-EIL) has been evaluated as a ligand for in vitro and in vivo studies of cerebral serotonin 5-HT2 receptors. [125I]-EIL exhibited high affinity (KD = 209 pM) for 5-HT2 receptors with a high degree of specific binding (80-95%) in membranes from rat prefrontal cortex. The regional distribution of [125I]-EIL binding in vivo to seven areas of mouse brain correlated significantly (Rs = 0.93) with known densities of 5-HT2 receptors. In vivo specificity, defined by tissue to cerebellum radioactivity ratios, reached a maximum for frontal cortex at 6 hr (21.2) and persisted through 16 hr (8.8). Ketanserin, a 5-HT2 receptor antagonist, fully inhibited binding in a dose dependent fashion in all brain regions except cerebellum. By contrast, blockers for dopamine D2, alpha- or beta-adrenergic receptors did not significantly inhibit radioligand binding in any region. [125I]-EIL selectively labels 5-HT2 receptors in vivo with the highest specificity of any serotonergic ligand reported to date, indicating that [123I]-EIL should prove applicable to single photon emission computed tomography studies in living brain.

In vivo labeling of the dopamine D2 receptor with N-11C-methyl-benperidol.

A new dopamine D2 receptor radiotracer, N-11C-methyl-benperidol (11C-NMB), was prepared and its in vivo biologic behavior in mice and a baboon was studied. Carbon-11-NMB was determined to bind to specific sites characterized as dopamine D2 receptors. The binding was saturable, reversible, and stereospecific. Kinetic studies in the dopamine D2 receptor-rich striatum showed that 11C-NMB was retained five times longer than in receptor-devoid regions, resulting in a high maximum striatal-to-cerebellar ratio of 11:1 at 60 min after injection. From frontal cortex and cortex, on the other hand, the tracer washed out as rapidly as it did from cerebellum, resulting in tissue-to-cerebellar ratios close to one in these regions at any time after injection. Blocking studies confirmed the specificity and selectivity of the 11C-NMB binding to the dopamine D2 receptor. A PET study with 11C-NMB of the baboon brain revealed highly selective labeling of dopamine D2 receptor sites which was blocked by preinjection of raclopride.

In vitro and in vivo binding of (E)- and (Z)-N-(iodoallyl)spiperone to dopamine D2 and serotonin 5-HT2 neuroreceptors.

Apparent affinities (Ki) of (E)- and (Z)-N-(iodoallyl)spiperone [E)- and (Z)-NIASP) for dopamine D2 and serotonin 5-HT2 receptors were determined in competition binding assays. (Z)-NIASP (Ki 0.35 nM, D2; Ki 1.75 nM, 5-HT2) proved slightly more potent and selective for D2 sites in vitro than (E)-NIASP (Ki 0.72 nM, D2; Ki 1.14 nM, 5-HT2). In vivo, radioiodinated (E)- and (Z)-[125I]-NIASP showed regional distributions in mouse brain which are consonant with prolonged binding to dopamine D2 receptors accompanied by a minor serotonergic component of shorter duration. Stereoselective, dose-dependent blockade of (E)-[125I]-NIASP uptake was found for drugs binding to dopamine D2 sites, while drugs selective for serotonin 5-HT2, alpha 1-adrenergic and dopamine D1 receptors did not inhibit radioligand binding 2 hr postinjection. Specific binding in striatal tissue was essentially irreversible over the time course of the study, and (E)-[125I]-NIASP gave a striatal to cerebellar tissue radioactivity concentration of 16.9 to 1 at 6 hr postinjection. Thus, (E)-[125I]-NIASP binds with high selectivity and specificity to dopamine D2 sites in vivo.

Diagnosis of deep venous thrombosis using a Doppler ultrasonic technique.

A consecutive series of 118 patients was studied postoperatively by Doppler ultrasonic techniques and by either venography or radioiodinated fibrinogen. When using the latter diagnostic measures, 22 patients were shown to have deep venous thrombosis, an incidence of 18.6 per cent. The Doppler ultrasonic technique showed that 21 patients had deep venous thrombosis, an incidence of 17.7 per cent. When the patients diagnosed as having deep venous thrombosis by two separate methods were compared, it was shown that the Doppler technique gave two false-positive results and three false-negative results. It is concluded that this technique is accurate, and because of its convenience, lack of complications and ability to be repeated frequently, it should be the preferred screening technique for the diagnosis of postoperative deep venous thrombosis.