Uptake and transport of superparamagnetic iron oxide nanoparticles through human brain capillary endothelial cells.

The blood-brain barrier (BBB) formed by brain capillary endothelial cells (BCECs) constitutes a firm physical, chemical, and immunological barrier, making the brain accessible to only a few percent of potential drugs intended for treatment inside the central nervous system. With the purpose of overcoming the restraints of the BBB by allowing the transport of drugs, siRNA, or DNA into the brain, a novel approach is to use superparamagnetic iron oxide nanoparticles (SPIONs) as drug carriers. The aim of this study was to investigate the ability of fluorescent SPIONs to pass through human brain microvascular endothelial cells facilitated by an external magnet. The ability of SPIONs to penetrate the barrier was shown to be significantly stronger in the presence of an external magnetic force in an in vitro BBB model. Hence, particles added to the luminal side of the in vitro BBB model were found in astrocytes cocultured at a remote distance on the abluminal side, indicating that particles were transported through the barrier and taken up by astrocytes. Addition of the SPIONs to the culture medium did not negatively affect the viability of the endothelial cells. The magnetic force-mediated dragging of SPIONs through BCECs may denote a novel mechanism for the delivery of drugs to the brain.

A straightforward synthetic approach for roxindole.

A concise synthetic approach to the dopamine autoreceptor agonist roxindole 1 has been devised. The key step in the novel route is the addition of succinic anhydride to 5-methoxyindolylmagnesium bromide, which circumvents the cumbersome construction of the indole moiety - via the Japp Klingemann- Fischer indole methodology - in the original route. This novel route will enable a quick, multi-gram synthesis of roxindole from cheap starting materials.

Imaging beta-adrenoceptors in the human brain with (S)-1'-[18F]fluorocarazolol.

UNLABELLED: We evaluated the suitability of fluorocarazolol for in vivo studies of cerebral beta-adrenoceptors because (S)-1'-[18F]fluorocarazolol has a higher affinity to beta-adrenoceptors than to serotonergic receptors (pKi beta 1 9.4, beta 2 10.0, 5HT1A 7.4, 5HT1B 8.1) and rapidly crosses the blood-brain barrier. METHODS: The (S)-[18F]fluorocarazolol (74 MBq, > 37 TBq/mmol) was intravenously administered to healthy volunteers on two separate occasions with an interval of at least 1 wk. The initial injection was without pretreatment, but before the second injection, the volunteers received the beta blocker (+/-)-pindolol (3 x 5 mg orally, during 18 hr). The brain was studied with a PET camera in dynamic mode. RESULTS: Uptake of radioactivity delineated gray matter and was particularly high in the posterior cingulate, precuneus and striatum. Low uptake occurred in the thalamus, whereas the lowest uptake was observed in the white matter of the corpus callosum. After pindolol pretreatment, uptake was reduced and its distribution became homogeneous throughout the brain. The ratio of total-to-nonspecific binding was about 2 at 60 min, increasing to 2.5-2.75 at longer intervals. CONCLUSION: Fluorocarazolol is the first radioligand that can visualize cerebral beta-adrenoceptors and may enable monitoring of these binding sites during disease.

Characterization of pulmonary and myocardial beta-adrenoceptors with S-1'-[fluorine-18]fluorocarazolol.

UNLABELLED: S-1'-[18F]fluorocarazolol was administered to healthy volunteers to assess its potential for noninvasive measurement of regional pulmonary and myocardial beta-adrenoceptor densities. METHODS: High-specific activity fluorocarazolol was intravenously injected on two separate occasions within a 1-wk interval. The initial injection was without pretreatment, but before the second injection, the volunteers either inhaled salbutamol (2 x 200 micrograms aerosol) or they ingested pindolol (3 x 5 mg during a 12-hr interval). Twenty-eight PET time frames of 31 planes were acquired over a period of 60 min after each injection. Blood samples were drawn and analyzed for the presence of fluorocarazolol and radioactive metabolites. RESULTS: Uptake of fluorocarazolol in the target tissues was hardly affected by salbutamol but was strongly depressed by pindolol. Pulmonary and myocardial tissue-to-plasma concentration ratios of fluorocarazolol reached plateau values of 11.6 +/- 0.6 (lungs) and 18.1 +/- 1.0 (heart) at 45-50 min postinjection. These values were reduced to 2.0 +/- 0.4 and 2.0 +/- 0.6 after treatment with pindolol. CONCLUSION: These data indicate that: 1. Pulmonary and myocardial uptake of radioactivity after intravenous administration of S-1'-[18F]fluorocarazolol represents radioligand binding to beta-adrenoceptors. 2. Pulmonary binding occurs mainly in alveoli rather than in airway smooth muscle under these conditions. 3. Binding kinetics do not preclude quantification of receptors with compartment models.

Stereoselective synthesis and biodistribution of potent [11C]-labeled antagonists for positron emission tomography imaging of muscarinic receptors in the airways.

Quantitation of muscarinic receptors in the lungs in vivo with positron emission tomography (PET) is of clinical interest. For that purpose we decided to develop [11C]-labeled ligands with a high affinity (KD < 0.1 nM). Three quaternary muscarinic antagonists, racemic N-methylpiperidin-4-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate methiodide 1a (pKB = 10.39), its (R)-isomer 1b (pKB = 11.08), and (R,R)-quinuclidin-3-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate methiodide 2 (pKB = 11.28), were labeled by reacting [11C]CH3I with their tertiary amine precursors. The enantiomerically pure tertiary amine precursors were prepared by stereoselective synthesis starting from (R)-(-)-mandelic acid. In vitro binding assay of 1b and 2 demonstrated that both ligands bind with very high affinity to the muscarinic receptor subtypes M1, M2, and M3. They are more potent than the muscarinic antagonist (R)-N-methylquinuclidinyl benzilate ((R)-MQNB). Distribution studies with 1a, 1b, and 2 in control and atropine-treated male Wistar rats demonstrated significant specific binding (90-99% of total issue uptake) in tissues containing cholinoceptors (heart, intestine, lung, pancreas, spleen, stomach, submandibular gland). Because the tissue/plasma concentration ratios of 1b are most favorable, this ligand was used for further evaluation. Analysis of plasma samples showed a very rapid clearance (t1/2 = 0.3 min) of the radioligand 1b and a relatively slow appearance of a hydrophilic metabolite. At 15 min postinjection of 1b, analysis of heart, lungs, and liver showed that respectively 99%, 88%, and 8% of the tissue radioactivity corresponded with the parent compound. Ligand 1b appears to be an excellent candidate for PET studies of mAChR receptors in heart and lungs.

Carbon-11-labeled daunorubicin and verapamil for probing P-glycoprotein in tumors with PET.

UNLABELLED: One of the mechanisms for multidrug resistance (MDR) of tumors is an overexpression of the P-glycoprotein (P-gp). The cytostatic agent daunorubicin and the modulator verapamil were labeled with 11C to probe P-gp with PET. METHODS: Carbon-11-daunorubicin was prepared from 11CCH2N2 with an aldehyde precursor, followed by hydrolysis. Carbon-11-verapamil was synthesized by 11C-methylation. Both tracers were evaluated by investigating pharmacokinetics in rats and in vitro cell kinetics using human ovarian carcinoma cells. RESULTS: Amounts of 111 MBq 11C-daunorubicin were prepared. Biodistribution studies of 11C-daunorubicin in male Wistar rats showed dose-dependent pharmacokinetics, whereas with 11C-verapamil the pharmacokinetics were dose independent. In in vitro experiments with cells, the ratio of accumulation of 11C-daunorubicin in drug sensitive/resistant cell lines was 16. Addition of verapamil resulted in increased accumulation of 11C-daunorubicin in the resistant cell line. The ratios of 11C-verapamil accumulation in drug-sensitive versus the MDR counterpart were 4-5. CONCLUSION: Carbon-11-daunorubicin and 11C-verapamil both have potential for in vivo probing of P-glycoprotein with PET.

Quantification of the beta-adrenoceptor ligand S-1'-[18F]fluorocarazolol in plasma of humans, rats and sheep.

Myocardial and pulmonary beta-adrenoceptors can be imaged with 2-(S)-(-)-(9H-carbazol-4-yl-oxy)-3-[1-(fluoromethyl)ethyl]amino-2- propanol (S-1'-[18F]fluorocarazolol, I). Quantification of unmodified fluorocarazolol in plasma is necessary for analysis of PET images in terms of receptor densities. We have determined I and its radioactive metabolites in rat, sheep and human plasma, using (1) solid-phase extraction (C18) followed by reversed-phase HPLC and (2) direct injection of untreated plasma samples on an internal-surface reversed-phase (ISRP) column. The two methods were in good agreement. Unmodified I decreased from over 99% initially to less than 5%, 5-10% and 20% at 60 min post-injection in rats, sheep and human volunteers, respectively. Protein binding in sheep and human plasma was determined by ultrafiltration. The fraction of total plasma radioactivity bound to protein and the fraction representing unmodified radioligand were linearly correlated, suggesting that fluorocarazolol was more than 70% protein-bound, whereas its metabolites showed negligible protein binding. Direct injection of plasma on an ISRP column seems a convenient method for quantification of lipophilic radioligands such as fluorocarazolol.

In vitro evaluation of N-(fluoro)isopropyl norephedrine as potential cardiac imaging agents for PET.

N-Isopropylnorephedrine (INE) and N-fluoroisopropylnorephedrine (FINE) were found to have a poor affinity for either beta-adrenoceptors and the norepinephrine carrier protein. The low affinity of both compounds for Uptake-1 is probably due to the introduction of a bulky substituent on the nitrogen atom. It is concluded that INE and FINE cannot be used for cardiac imaging with PET.

(S,S)- and (S,R)-1'-[18F]fluorocarazolol, ligands for the visualization of pulmonary beta-adrenergic receptors with PET.

The beta-adrenoceptor antagonist carazolol has been labelled with fluorine-18 in the isopropyl group via a reductive alkylation by [18F]-fluoroacetone of the corresponding (S)-desisopropyl compound according to a known procedure. The introduction of fluorine in the isopropyl group creates a new stereogenic centre resulting in the formation of (S,S)- and (S,R)-1'-[18F]fluorocarazolol, which were separated by HPLC. Tissue distribution studies were performed in male Wistar rats. Both the (S,S)- and (S,R)-diastereomers (S.A. 500-2000 Ci/mmol; 18.5-74 TBq/mmol) showed high uptake in lung and heart, which could be blocked by pretreatment of the animals with (+/-)-propranolol. No significant differences were observed between the biodistribution of the two diastereomers. Metabolite analysis showed a rapid appearance of polar metabolites in plasma, while at 60 min postinjection 92% and 82% of the total radioactivity in lung and heart was unmetabolized 1'-[18F]fluorocarazolol. In a PET-study with male Wistar rats, the lungs were clearly visualized and the pulmonary uptake was decreased after pretreatment of the animals with (+/-)-propranolol. The heart could not be visualized. Similar results were obtained in PET-studies with lambs.

Brain tumors: L-[1-C-11]tyrosine PET for visualization and quantification of protein synthesis rate.

PURPOSE: Positron emission tomography (PET) with the amino acid tracer L-[1-C-11]-tyrosine was evaluated in 27 patients with primary and recurrent brain tumors. MATERIALS AND METHODS: Patients underwent either static (n = 14) or dynamic PET (n = 13), with quantification of protein synthesis rate (PSR) and tumor-to-background ratio. Findings were compared with histologic findings. RESULTS: Primary brain tumor was proved in 22 patients histologically, as well as metastatic cancer of unknown origin, primary non-Hodgkin lymphoma, meningioma, atypical infarction, and vasculitis in one patient each. At PET, 20 of 22 primary tumors, the metastasis, and non-Hodgkin lymphoma were correctly depicted. A false-positive finding was obtained with the infarction, and the meningioma and vasculitis were not depicted. The calculated sensitivity was 92%; specificity, 67%; and accuracy, 89%. There were no statistically significant relationships between histologic findings, PSR, and tumor-to-background ratio. CONCLUSION: L-[1-C-11]-tyrosine is a valid tracer for diagnosis of brain tumors and allowed quantification of PSR.

Synthesis and organ distribution of [18F]fluoro-Org 6141 in the rat: a potential glucocorticoid receptor ligand for positron emission tomography.

For the synthesis of [18F]Fluoro-Org 6141 via a nucleophilic substitution reaction with 18F-, the tosyl group was chosen as the leaving group because of its stability and excellent leaving group ability. The biodistribution of the high affinity and moderate lipophilicity (log P = 2.66, calculated value) ligand [18F]Fluoro-Org 6141 (specific activity 8.2 to 37 TBq/mmol, yield 10% at EOB) was examined in sham adrenalectomized (sADX) and adrenalectomized (ADX) male Wistar rats. Two days after ADX or sADX, the animals were anesthetized and 0.37 to 1.85 MBq of [18F]Fluoro-Org 6141 was administered intravenously. Kinetics of 18F activity uptake were monitored for 3 h using a stationary double-headed positron emission tomography (PET) camera, and the biodistribution was assessed by ex vivo determination of radioactivity in several tissues and different brain areas. One hour after injection of the radioligand, the bladder, kidney, liver, trachea, and bone of sADX animals contained more concentration on a wet weight basis than blood. Three hours post injection, radioactivity was retained in bladder, trachea, and bone. The accumulation of radioactivity in brain corresponded to the concentration of activity in the blood within the first hours after injection. ADX animals showed a higher uptake of 18F activity in spleen, testes, and brain areas (hippocampus and brainstem) but a lower uptake in bone than sADX rats. PET scans suggested that 18F activity uptake in the brain had not yet reached a maximum at this interval. Although [18F]Fluoro-Org 6141 is not useful for PET studies of glucocorticoid receptors (GRs), the results obtained with this compound indicate a synthetic strategy suitable for the synthesis of high-affinity radioligands for GRs.

Feasibility of planar fluorine-18-FDG imaging after recent myocardial infarction to assess myocardial viability.

UNLABELLED: The aim of this study was to define the clinical feasibility of planar myocardial 18F-fluorodeoxyglucose (FDG) imaging and to assess the relation between 201Tl, FDG and left ventricular function early after myocardial infarction. METHODS: Fifty-one patients were studied 5 +/- 2 days after infarction. Scintigraphic images were visually and quantitatively analyzed using a circumferential profiles technique. FDG uptake was normalized to the area with maximal 201Tl uptake. Scintigraphic data were compared with left ventricular wall motion as assessed by ventriculography in 22 patients. Relative regional 201Tl uptake was categorized as normal (> or = 75% of peak activity), moderately reduced (50%-75%) or severely reduced (< 50%). These tracer defects were considered viable if FDG uptake exceeded 201Tl uptake by > or = 20% and/or if FDG uptake was normal (> or = 75%). All regions with FDG uptake 20% less than 201Tl uptake were considered nonviable. RESULTS: Four hundred forty-one myocardial regions were analyzed; 200 showed normal 201Tl uptake, 241 had reduced uptake, 191 had moderately reduced 201Tl uptake and 50 regions had severely reduced uptake. Viability for moderately and severely reduced regions was observed in 62% and 48%, respectively. A concordance between flow and metabolism was observed in 38% and 52%, respectively. CONCLUSION: Myocardial FDG imaging is feasible with standard gamma camera systems and enables the identification of regions with preserved glucose metabolism in patients shortly after infarction.

N-[18F]-fluoroalkylation of noraporphines: microwave versus thermal treatment.

A comparable study of microwave versus thermal heating is described for the N-[18F]-fluoroalkylation of noraporphines. As compared to thermal treatment, different products were obtained during microwave treatment. Thermal treatment resulted in the loss of the protection of the catechol functionality of the noraporphines (O-deacylation), whereas during microwave treatment N-[18F]-fluoroalkylation was observed. The results described in this report might suggest that the influence of microwaves on chemical transformations is not exclusively thermal.

Myocardial and pulmonary uptake of S-1'-[18F]fluorocarazolol in intact rats reflects radioligand binding to beta-adrenoceptors.

The biodistribution of S-(-)-4-(2-hydroxy-3-(1'-[18F]fluoroisopropyl)- aminopropoxy)carbazole ([18F]S-fluorocarazolol, a non-selective beta-adrenoceptor antagonist) was studied in rats (60 min after 18F injection when specific binding in peripheral organs was maximal). 18F uptake in brain, erythrocytes, heart and lung appeared to be linked to beta-adrenoceptors. CGP-20712A and ICI-89,406 inhibited 18F uptake in heart (predominantly beta 1-adrenoceptors) more potently than in lungs (predominantly beta 2-adrenoceptors). In contrast, ICI-118,551 and procaterol were more potent in the lungs than in the heart. ICI-118,551 inhibited 18F uptake in cerebellum (predominantly beta 2-adrenoceptors) more potently than in cerebral cortex (predominantly beta 1-adrenoceptors). Stereoselectivity of the in vivo binding was demonstrated since S-(-)-propranolol inhibited uptake in target tissues more effectively than R-(+)-propranolol. Myocardial and cerebral imaging may be hampered by poor heart-to-lung contrast and low signal-to-noise ratios, but [18F]S-fluorocarazolol seems suitable for positron emission tomography (PET) of pulmonary beta-adrenoceptors.

The presence of progesterone receptors in arachnoid granulations and in the lining of arachnoid cysts: its relevance to expression of progesterone receptors in meningiomas.

Progesterone receptors (PR) were identified with an enzyme immunoassay in cytosols from human arachnoid granulations and arachnoid cysts. Meningiomas presumably originate from subdural endothelium which is abundantly present in these structures. In the three cases studied, oestrogen receptors were absent. The presence of PR in subdural endothelium may provide further ground for the expression of PR in meningiomas.

Quantification of an 11C-labelled beta-adrenoceptor ligand, S-(-)CGP 12177, in plasma of humans and rats.

beta-Adrenoceptors in human lungs and heart can be imaged with the radioligand 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3- dihydro-2H-benzimidazol-2-11C-one (CGP 12177, [11C]I). For quantification of receptor density with compartment models by adjustment of rate constants, an 'input function' is required which consists of the integral of the concentration of unmodified ligand in arterial plasma over time. A discrepancy in the literature regarding metabolic stability of [11C]I prompted us to study metabolism in rats by reversed-phase HPLC (RP-HPLC) of trichloroacetic acid extracts of arterial plasma after i.v. injection of [11C]I (> 11.1 TBq/mmol, 11 MBq/kg). Some plasma samples were also directly applied to an internal-surface reversed-phase (ISRP) column. In parallel experiments, tritiated [11C]I was employed and methanol extracts of arterial plasma were analyzed by straight-phase TLC. The three methods were in excellent agreement. Unmodified [11C]I decreased from > 98.5% (3H) or > 99.9% (11C) initially to 57 +/- 7% at 80 min post injection due to formation of two polar metabolites. Using the RP-HPLC method, no metabolism was detectable in humans up to 30 min after injection of [11C]I (1851 MBq). Deproteinization of plasma with acetonitrile resulted in the formation of a radioactive species (artifact) which eluted immediately after the void volume in RP-HPLC and which could be mistakenly interpreted as a metabolite. Plasma protein binding was low (ca. 30%) in both humans and rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of a [18F]fluorine labeled progestin (21-[18F]fluoro-16 alpha-ethyl-19-norprogesterone) in humans: a clue for future investigations.

Assessment of estrogen receptors and progesterone receptors (PR) with PET may allow the determination of the hormone responsiveness of tumors without the need for multiple biopsies, and the monitoring of the effect of hormonal therapy. In spite of the favourable characteristics of 21-[18F]fluoro-16 alpha-ethyl-19-norprogesterone ([18F]FENP) found in preclinical studies, the compound failed to reveal the presence of PR in breast carcinomas and meningiomas. In view of the clinical significance of the PR assay in human breast cancer, it is worthwhile to explore mechanisms that are potentially involved in the inadequacy of [18F]FENP to image PR with PET. Our present study on the in vivo metabolism of [18F]FENP in humans demonstrates a rapid clearance and biotransformation of the compound. Results of incubation experiments suggest that the metabolic conversion of [18F]FENP is not restricted to the liver, but also occurs in blood cells (presumably the erythrocytes) and tumors (breast carcinomas and meningiomas). The predominant metabolite of [18F]FENP in plasma during the rapid distribution phase and in tumors is identified as 20-dihydro-[18F]FENP. The conversion of [18F]FENP to its 20 alpha- or 20 beta-hydroxy metabolite has a deleterious effect on the binding affinity for PR. Our findings do not justify a conclusion as to the extent of in vivo extrahepatic biotransformation of [18F]FENP, or its significance in the ineffectiveness of [18F]FENP as an imaging agent for PR. On the other hand, the ability of breast carcinomas and meningiomas to metabolize [18F]FENP avidly appears to preclude selective imaging of PR in these tumors during the time of a PET examination. It is imperative to evaluate the metabolic stability of a [18F]fluorine labeled progestin in an early stage of future screening procedures.

Synthesis of 6 alpha-[18F]fluoroprogesterone: a first step towards a potential receptor-ligand for PET.

6 alpha-[18F]Fluoroprogesterone 3 was prepared by the BF3.Et2O-catalyzed reaction of progest-5 alpha, 6 alpha-epoxy-3,20-bisketal 1 and [18F]fluoride as a possible route for the in vivo visualization of progesterone receptors by PET. The radiochemical yield of 3 was 25% (EOB) and the sp. act. was 5 MBq/mumol (100 Ci/mol, EOS).

Synthesis and preliminary evaluation of (R,S)-1-[2-((carbamoyl-4-hydroxy)phenoxy)-ethylamino]-3-[4-(1-[11C]-met hyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol ([11C]CGP 20712A) as a selective beta 1-adrenoceptor ligand for PET.

The most selective beta 1-adrenoceptor ligand known at this moment is (S)-1-[2-((carbamoyl-4-hydroxy) phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl) phenoxy]-2-propanol (CGP 26505), the S-isomer of CGP 20712A. We prepared the racemic 11C analogue by methylation with [11C]CH3I of the corresponding desmethyl compound using a microwave oven to accelerate the reaction. Several radioactive by-products (about 70% of the non-volatile radioactive products) were formed. After HPLC purification [11C]CGP 20712A with a specific activity of 35 TBq/mmol was dissolved in a propylene glycol-ethanol-saline mixture to prepare it for injection. The total preparation time was 35 min. The radiochemical yield was 5% (calculated from [11C]CH3I, not corrected for decay). The identity of [11C]CGP 20712A was proved by liquid chromatography-mass spectrometry (LC-MS). Tissue distribution studies in male Wistar rats have been performed. At 20 min after injection of the radioligand (0.1 nmol) the DAR [differential absorption ratio = (counts per minute recovered/g tissue)/(counts per min injected/g body weight)] in heart tissue decreased significantly (P < 0.005) from 1.84 +/- 0.11 to 1.21 +/- 0.12 after blocking of beta-adrenoceptors with 500 micrograms (R,S)-propranolol. A preliminary PET study in a Wistar rat showed maximal uptake in the time frame 10-20 min after injection. The ratio of specific/non-specific binding at this interval was 2.6.

Rodent biodistribution and metabolism of tritiated 4-DAMP, a M3 subtype-selective cholinoceptor ligand.

The biodistribution of [3H]4-DAMP (a M3-selective cholinoceptor antagonist) was studied in rats which had received either saline or saline containing atropine (to block cholinoceptors). Specific binding of the radioligand was observed in the urinary bladder, ileum, pancreas, stomach, submandibular gland and trachea. Maximal ratios of total-to-non-specific uptake reached values of 1.8 (trachea), 3.2 (bladder), 4.0 (stomach), 4.8 (ileum), 6.6 (pancreas) and 6.9 (submandibular gland) at 5-10 min post-injection; this rank order reflects the tissue densities of M3 cholinoceptors, 4-DAMP did not bind to blood cells and it was rapidly cleared from the circulation (> 90% with a half-life of 0.2 min, the remainder with a half-life of 9.4 min). Labelled metabolites appeared within 5 min in plasma, but metabolite uptake by the target organs was low (< 15% of total radioactivity 40 min post-injection). Although 4-DAMP binds to M3-cholinoceptors in vivo, its potential use as a radiopharmaceutical appears limited since the compound does not cross the blood-brain barrier and it does not show measurable specific binding in airways.