Glut-1 and hexokinase expression: relationship with 2-fluoro-2-deoxy-D-glucose uptake in A431 and T47D cells in culture.

Uptake of 2-[18F]-2-deoxy-D-glucose (FDG) has been used as a marker of increased glucose metabolism to visualize, stage, and monitor progression of human cancers with positron emission tomography. Many human tumors have been shown to overexpress the Glut-1 glucose transport protein. The aim of this study is to define whether a quantitative relationship exists between the amount of Glut-1 expressed by cells and their ability to accumulate FDG. We characterized the expression of the known facilitative and sodium-dependent glucose transporter isoforms in six different cancer cell lines used in our laboratory (A431, MDA-MB-231, T47D, CaCo II, MCF7, and HepG2). A431 and T47D cells express, respectively, the highest and lowest amount of Glut-1 mRNA by Northern blot of all of the cells analyzed, and no other glucose transporter isoforms were detectable by this method in both cell lines. Both total and plasma membrane-associated Glut-1 protein levels were higher in A431 than in T47D cells, consistent with the higher Glut-1 mRNA levels. However, T47D cells accumulate FDG more rapidly than do A431 cells. 3-O-Methylglucose transport is higher in A431 cells. Although hexokinase I and II mRNA levels are higher in A431 cells than in T47D cells, the ability of mitochondrial preparations to phosphorylate FDG is higher in T47D cells. Our data indicate that in these cultured cells, FDG uptake correlates better with FDG phosphorylating activity of mitochondrial preparations rather than the level of expression of the Glut-1 or hexokinase I and II genes.

[125I]17-alpha-iodovinyl 11-beta-methoxyestradiol interaction in vivo with estrogen receptors in hormone-independent MCF-7 human breast cancer transfected with the v-rasH oncogene.

[125I]17-alpha-Iodovinyl 11-beta-methoxyestradiol [( 125I]MIVE2) has been evaluated as a potential radiotracer for the diagnostic imaging of estrogen receptor (ER)-positive human breast cancer. In vivo distribution experiments with athymic ovariectomized nude mice bearing human breast tumors revealed an apparent correlation between uptake of 125I-labeled compound and estrogen receptor concentration in the tumors. At 4 h after i.v. injection of [125I]MIVE2, HS578T (ER negative), ZR75-B (intermediate ER), and MCF-7ras (high ER) tumors accumulated 0.320 +/- 0.186, 0.679 +/- 0.467, and 2.6163 +/- 1.0121% injected dose/g, respectively. With coinjection of unlabeled 17-beta-estradiol, levels of radioactivity in MCF-7ras tumors were decreased to 0.4859 +/- 0.1424% injected dose/g, indicating a receptor-mediated process. Peak activity of radioligand in MCF-7ras tumors and uteri was observed at 2 h and was retained for the 8-h time course. Blood and nontarget tissue, such as muscle, revealed a rapid clearance of 125I-labeled compound by 8 h. Eight hours after injection, uterus and tumor-to-blood ratios were calculated to be 225 and 21, respectively. Also, MCF-7ras tumors were shown to accumulate 6.5-fold more radioactivity than muscle. These data suggest that [125I]MIVE2 has the capability of interacting specifically and with high affinity with estrogen receptors in human breast tumors in nude mice and may possibly be used for imaging receptor-positive tumors in breast cancer patients with very low serum estrogen levels. Selective uptake of compound in MCF-7ras tumors emphasizes the usefulness of an estrogen receptor-positive tumor model which has a unique ability to grow in a host system without circulating estrogens.

Binding characteristics and biological activity of 17 alpha-[125I]iodovinyl-11 beta-methoxyestradiol, an estrogen receptor-binding radiopharmaceutical, in human breast cancer cells (MCF-7).

17 alpha-[125I]Iodovinyl-11 beta-methoxyestradiol ([125I]MIVE2), a gamma-emitting analogue of estradiol, previously shown to bind to rat uterine estradiol receptor, was studied to determine the binding characteristics and biological activity in human breast cancer cells. In vitro determination of receptor binding by dextran-coated charcoal assays indicates that [125I]-MIVE2 binds specifically and with a high affinity to cytosolic estrogen receptors in the human breast cancer cell line, MCF-7. [3H]Estradiol binds to the receptor with approximately four times the affinity of [125I]-MIVE2 (Kd = 2.55 X 10(-9) M for [125I]MIVE2; Kd = 6.4 X 10(-10) M for [3H]estradiol). Unlabeled MIVE2 produces estrogenic effects similar to those of estradiol such as progesterone receptor induction and increases in thymidine incorporation in MCF-7 cells in culture. Cytosolic progesterone receptor levels were elevated 2.8-fold over control levels by 6 X 10(-9) M MIVE2. Stimulation of thymidine incorporation (approximately 300% above control levels) was observed after exposure to 1 X 10(-9) M MIVE2. Preliminary data show receptor-mediated uptake by the uterus in biodistribution studies in athymic nude mice given injections of [125I] MIVE2 (32-34 microCi). At 4 h, uterus:blood ratios are 20.5 and target tissue:nontarget tissue ratios are 12.9. In light of the fact that this compound can be prepared with a high specific activity, [125I]MIVE2 may have potential as a radiotracer for imaging estrogen receptor-positive breast tumors or metastatic lesions in human breast cancer patients.

Biodistribution of 18F- and 125I-labeled anti-Tac disulfide-stabilized Fv fragments in nude mice with interleukin 2 alpha receptor-positive tumor xenografts.

We evaluated the biodistribution, pharmacokinetics, and generation of catabolites of an 18F- and 125I-labeled anti-Tac disulfide-stabilized Fv fragment (dsFv) in tumor-bearing nude mice. This dsFv is genetically engineered from a murine monoclonal antibody that recognizes the alpha subunit of the interleukin 2 (IL-2 alpha) receptor. Labeling was performed with 18F using N-succinimidyl 4-([18F]fluoromethyl)benzoate or with 125I using the Iodo-Gen method. The immunoreactivities of the radiolabeled anti-Tac dsFv were > 82%. The biodistribution was evaluated (at 15, 45, and 90 min and 6 h) in athymic nude mice (approximately five/group) bearing s.c. tumor xenografts. Cell line A431 served as the IL-2 receptor-negative control tumor, whereas the ATAC4 cell line served as our IL-2 receptor-positive tumor. Animals received injections of 18F-labeled anti-Tac dsFv (0.7-1.4 megabecquerels/1.5-3 micrograms) and 125I-labeled anti-Tac dsFv (0.1-0.4 megabecquerels/0.9-1 microgram). Blood clearance for both preparations was rapid, with < 10% retained in the blood by 15 min. Maximum accumulation in ATAC4 tumors occurred between 45 and 90 min and peaked at a mean of 4.2% injected dose/g (18F) and 5.6% of injected dose/g (125I). At 6 h, the ATAC4 tumors contained 11 times more 18F and 3 times more 125I than did the A431 tumors. The ATAC4 tumor:blood ratios for the 18F and 125I were > 12:1 and > 1.4:1 at 6 h, respectively, whereas the ratios for the antigen-negative A431 tumor were less than 1. The kidneys were the major route of elimination. Catabolites appeared quickly and were identified as [125I]iodide and predominantly N-epsilon-[18F]4-fluoromethylbenzoyl(alpha-N-acetyl) lysine. This is the first study to evaluate the biodistribution of an 18F-labeled Fv fragment in vitro and in vivo. In vivo, the dsFv was taken up rapidly by the kidneys, producing lysine-containing catabolites for 18F-labeled dsFv and [125I]iodide for 125I-labeled dsFv.

Muscarinic cholinergic receptor measurements with [18F]FP-TZTP: control and competition studies.

[18F]Fluoropropyl-TZTP (FP-TZTP) is a subtype-selective muscarinic cholinergic ligand with potential suitability for studying Alzheimer's disease. Positron emission tomography studies in isofluorane-anesthetized rhesus monkeys were performed to assess the in vivo behavior of this radiotracer. First, control studies (n = 11) were performed to characterize the tracer kinetics and to choose an appropriate model using a metabolite-corrected arterial input function. Second, preblocking studies (n = 4) with unlabeled FP-TZTP were used to measure nonspecific binding. Third, the sensitivity of [18F]FP-TZTP binding to changes in brain acetylcholine (ACh) was assessed by administering physostigmine, an acetylcholinesterase (AChE) inhibitor, by intravenous infusion (100 to 200 microg x kg(-1) x h(-1)) beginning 30 minutes before tracer injection (n = 7). Tracer uptake in the brain was rapid with K1 values of 0.4 to 0.6 mL x min(-1) x mL(-1) in gray matter. A model with one tissue compartment was chosen because reliable parameter estimates could not be obtained with a more complex model. Volume of distribution (V) values, determined from functional images created by pixel-by-pixel fitting, were very similar in cortical regions, basal ganglia, and thalamus, but significantly lower (P < 0.01) in the cerebellum, consistent with the distribution of M2 cholinergic receptors. Preblocking studies with unlabeled FP-TZTP reduced V by 60% to 70% in cortical and subcortical regions. Physostigmine produced a 35% reduction in cortical specific binding (P < 0.05), consistent with increased ACh competition. The reduction in basal ganglia (12%) was significantly smaller (P < 0.05), consistent with its markedly higher AChE activity. These studies indicate that [18F]FP-TZTP should be useful for the in vivo measurement of muscarinic receptors with positron emission tomography.

Kinetic analysis of the 5-HT2A ligand [11C]MDL 100,907.

The goal of this study was to develop a suitable kinetic analysis method for quantification of 5-HT2A receptor parameters with [11C]MDL 100,907. Twelve control studies and four preblocking studies (400 nmol/kg unlabeled MDL 100,907) were performed in isoflurane-anesthetized rhesus monkeys. The plasma input function was determined from arterial blood samples with metabolite measurements by extraction in ethyl acetate. The preblocking studies showed that a two-tissue compartment model was necessary to fit the time activity curves of all brain regions including the cerebellum--in other words, the need for two compartments is not proof of specific binding. Therefore, a three-tissue compartment model was used to analyze the control studies, with three parameters fixed based on the preblocking data. Reliable fits of control data could be obtained only if no more than three parameters were allowed to vary. For routine use of [11C]MDL 100,907, several simplified methods were evaluated. A two-tissue (2T') compartment with one fixed parameter was the most reliable compartmental approach; a one-compartment model failed to fit the data adequately. The Logan graphical approach was also tested and produced comparable results to the 2T' model. However, a simulation study showed that Logan analysis produced a larger bias at higher noise levels. Thus, the 2T' model is the best choice for analysis of [11C]MDL 100,907 studies.

An affinity column method for determination of the immunoreactivity of 131I-chimeric L6 monoclonal antibody and comparison to in vivo tumor localization.

An affinity column method was developed to determine the immunoreactivity of 131I-ChiL6 (chimeric L6 monoclonal antibody), a candidate for radioimmunotherapy. This method involved assessing the binding of the radiolabeled antibody to antigen containing membranes bound to a Reacti-gel agarose matrix. The immunoreactivity determined by the affinity column method correlated to other in vitro binding assays including the Lindmo infinite antigen excess method. In tumor-bearing mice which had been injected with 131I-ChiL6, which possessed high immunoreactivities (90-82%), a high tumor uptake (13.5-10.5% ID/g) was observed. A decrease in tumor uptake (5.2-4.8% ID/g) was observed with 131I-ChiL6 samples of low immunoreactivity (42% and 31%, respectively). While a moderate loss of immunoreactivity (4-18%) of the 131I-ChiL6 samples could be detected by the affinity column method, the loss of tumor uptake in vivo observed was not as significant. This method was found to be an efficient and sensitive method for detecting damage to the antibody during radiolabeling and applicable as a quality control method for clinical trials. This rapid method, compared to the other in vitro binding assays (including the Lindmo infinite antigen excess method) has distinct advantages as a quality control method since it requires less manipulation and can be semi-automated.

Regional brain uptake of the muscarinic ligand, [18F]FP-TZTP, is greatly decreased in M2 receptor knockout mice but not in M1, M3 and M4 receptor knockout mice.

A muscarinic receptor radioligand, 3-(3-(3-fluoropropyl)thio) -1,2,5,thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (fP-TZTP) radiolabeled with the positron emitting radionuclide (18)F ([(18)F]FP-TZTP) displayed regional brain distribution consistent with M2 receptor densities in rat brain. The purpose of the present study is to further elucidate the subtype selectivity of [(18)F]FP-TZTP using genetically engineered mice which lacked functional M1, M2, M3, or M4 muscarinic receptors. Using ex vivo autoradiography, the regional brain localization of [(18)F]FP-TZTP in M2 knockout (M2 KO) was significantly decreased (51.3 to 61.4%; P<0.01) when compared to the wild-type (WT) mice in amygdala, brain stem, caudate putamen, cerebellum, cortex, hippocampus, hypothalamus, superior colliculus, and thalamus. In similar studies with M1KO, M3KO and M4KO compared to their WT mice, [(18)F]FP-TZTP uptakes in the same brain regions were not significantly decreased at P<0.01. However, in amygdala and hippocampus small decreases of 19.5% and 22.7%, respectively, were observed for M1KO vs WT mice at P<0.05. Given the fact that large decreases in [(18)F]FP-TZTP brain uptakes were seen only in M2 KO vs. WT mice, we conclude that [(18)F]FP-TZTP preferentially labels M2 receptors in vivo.

Synthesis and evaluation of (17 alpha,20E)-21-[125I]iodo-19-norpregna-1,3,5(10),20-tetraene-3,17 -diol and (17 alpha,20E)-21-[125I]iodo-11 beta-methoxy-19-norpregna-1,3,5(10),20-tetraene-3,17-diol (17 alpha-(iodovinyl)estradiol derivatives) as high specific activity potential radiopharmaceuticals.

Two 17 alpha-[125I]iodovinyl estradiol derivatives 4b,d possessing high specific activity have been prepared and tested as potential radiopharmaceuticals. The use of the 3-acetyl derivatives 2c,e and the replacement of iodine monochloride with sodium iodide and Chloramine-T in THF/phosphate buffer (pH 7.0) permitted us to synthesize no-carrier-added (17 alpha,20E)-21-[125I]iodo-19-norpregna-1,3,5(10),20-tetraene-3,17-d iol (4b) and (17 alpha,20E)-21-[125I]iodo-11 beta-methoxy-19-norpregna-1,3,5(10),20-tetraene-3,17-diol (4d) with 50% radiochemical yield and high purity. Although the specific activity represents only half of the theoretical value in some cases, this modified approach is a substantial improvement over the previously published method. Our preliminary distribution studies indicate that although both 4b and 4d localize in the tissues known to have a large concentration of estrogen receptors, 4d accumulates in higher amounts in target tissues and provides a high target to nontarget ratio.

Analogues of 3-quinuclidinyl benzilate.

A number of analogues of 3-quinuclidinyl benzilate (QNB) have been synthesized and their affinities to muscarinic receptor from rat or dog ventricular muscle measured. We have determined that the muscarinic receptor can to a different degree accommodate either a halogen in the ortho, meta, or para position of one phenyl ring or the replacement of one phenyl ring with an alkyl group. Our in vitro competition studies show that the affinities lie within a 270-fold range, from the highest affinity compound, 3-quinuclidinyl alpha-hydroxy-alpha-cyclopentylphenylacetate (2), to the lowest affinity compound, 3-quinuclidinyl alpha-hydroxy-alpha-2-propargylphenylacetate (11).

Cardioselectivity of beta-adrenoceptor blocking agents. 2. Role of the amino group substituent.

A series of 1-(aralkylamino)-3-(aryloxy)propan-2-ols were synthesized, and their apparent dissociation constants (Kapp) were determined by using rat ventricular muscle (RVM) and rat lung membrane (RLM) preparations. Analysis of the binding studies suggests the existence of different modes of binding dependent on the presence or absence of the 4-substituent in the aryloxy ring and the nature of that ring. Without 4-substitution only one compound (4), bearing the 2-(2-methoxyphenoxy)ethyl substituent on the amino group, shows high cardioselectivity. Introduction of the 4-acylamido substituent into the phenoxy ring renders all compounds cardioselective. The cardioselective influence of 4-substitution is diminished or eliminated when the phenoxy ring is replaced by naphth-1-yloxy.

Development of fluorine-18-labeled 5-HT1A antagonists.

We have synthesized five fluorinated derivatives of WAY 100635, N-{2-[4-(2-methoxyphenyl)piperazino]ethyl}-N-(2-pyridyl)cyclohe xaneca rboxamide (4a), using various acids in place of the cyclohexanecarboxylic acid (CHCA, 2a) in the reaction scheme. The five acids are 4-fluorobenzoic acid (FB, 2b), 4-fluoro-3-methylbenzoic acid (MeFB, 2c), trans-4-fluorocyclohexanecarboxylic acid (FC, 2d), 4-(fluoromethyl)benzoic acid (FMeB, 2e), and 3-nitro-4-(fluoromethyl)benzoic acid (NFMeB, 2f) (see Scheme 1). These compounds were radiolabeled with fluorine-18, and their biological properties were evaluated in rats and compared with those of [11C]carbonyl WAY 100635 ([carbonyl-11C]4a). [Carbonyl-11C]4a cleared the brain with a biological half-life averaging 41 min. The metabolite-corrected blood radioactivity had a half-life of 29 min. [18F]FCWAY ([18F]4d) gave half-lives and intercepts comparable to [carbonyl-11C]4a in the brain, but the blood clearance was faster. [18F]FBWAY ([18F]4b) showed an early rapid net efflux from the whole brain, clearing with a biological half-life of 35 min. The metabolite-corrected blood half-life was 41 min. The comparable whole brain and blood half-lives for Me[18F]FBWAY ([18F]4c) were 16 and 18 min, respectively. For each compound, the corresponding carboxylic acid was identified as a major metabolite in blood. Fluoride was also found after injection of [18F]4d. However, for all compounds there was a good correlation (R > 0.97) between the differential uptake ratio (DUR, (%ID/g) x body weight (g)/100) in individual rat brain regions at 30 min after injection and the concentration of receptors as determined by in vitro quantitative autoradiography in rat. Specific binding ratios [region of interest (ROI)/cerebellum-1] in control studies for cortex (Ctx) and hippocampus (H) were higher for [carbonyl-11C]4a and [18F]4d compared to [18F]4b and [18F]4c. [18F]4d has similar pharmacokinetic properties and comparable specific binding ratios to [carbonyl-11C]4a. Fifty nanomoles of 4a blocked only 30% of the specific binding of [18F]4d, while complete blockade was obtained from co-injection of 200 nmol of 4a (H/Cb-1 from 17.2 to 0.6). [18F]4b and [18F]4c showed lower specific binding ratios than [carbonyl-11C]4a and [18F]4d. [18F]4c was superior to [18F]4b since its specific binding was more readily blocked by 4a. These studies suggest that [18F]4c should be a useful compound to assess dynamic changes in serotonin levels while [18F]4d, with its high contrast and F-18 label, should provide better statistics and quantification for static measurement of 5-HT1A receptor distribution.

Synthesis and evaluation of radioiodinated derivatives of 1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(4-iodophenyl)-alpha- phenylacetate as potential radiopharmaceuticals.

Two derivatives of (RS)-1-azabicyclo[2.2.2]oct-3-yl (RS)-alpha-hydroxy-alpha-(4-iodophenyl)-alpha-phenylacetate (1a) and three partially resolved (R)- or (S)-1-azabicyclo[2.2.2]oct-3-yl (RS)-alpha-hydroxy-alpha-(4-iodophenyl)-alpha-phenylacetates labeled with no carrier added iodine-125 (1b, 18, and 19) and iodine-123 (1c and 18a) were synthesized by the Wallach triazene approach. We have found that this approach is necessary to obtain no carrier added labeling and gives far better results than the direct electrophilic iodination. The obtained yields were 7 to 18% when using iodine-123 (yield dependent on the source of iodide) and up to 17% for iodine-123 (yield dependent on the source of iodide) and up to 17% for iodine-125 labeled compounds. Our preliminary distribution studies indicate that 1b localizes in the organs known to have a large concentration of muscarinic receptors and that this localization is due to binding to those receptors.

Evaluation of human transferrin radiolabeled with N-succinimidyl 4-[fluorine-18](fluoromethyl) benzoate.

UNLABELLED: Iron metabolism plays a key role in cell proliferation and survival in rapidly growing cancer cells. Uptake is mediated by the carrier protein transferrin. The increased need for iron has been used as a method to target tumors and there is well-documented evidence that certain tumors can be imaged with tracers such as 67Ga, that mimic transferrin-mediated iron uptake. To obtain a tracer that would be better able to quantitate transferrin kinetics and indirectly evaluate iron metabolism, we have labeled human transferrin with the positron emitter, 18F, with a one-step high-specific activity method developed in our laboratory. METHODS: We measured the binding affinities of [18F]diferric (holo-) and iron-free (apo-) transferrin on two human cell lines. We also compared cellular uptake of [18F]holo-transferrin and [67Ga]citrate in various conditions, and washout of label incorporated into cells. RESULTS: The binding affinity of [18F]holo-transferrin was found to be the same as that reported for [125I]holo-transferrin. In our hands there was no significant difference in binding affinity between diferric holo-transferrin and iron-free apo-transferrin. [18F]holo-transferrin uptake rapidly reaches a steady-state equilibrium between the intracellular and extracellular environment, while gallium accumulation linearly increases with time. [18F]holo-transferrin is rapidly recycled out of the cell with similar kinetics to those reported for [125I]holo-transferrin. CONCLUSION: [18F]holo-transferrin displays the properties of native transferrin and appears suitable for quantitative evaluation of transferrin kinetics in vivo.

[I-125] 17 alpha-Iodovinyl 11 beta-methoxyestradiol: in vivo and in vitro properties of a high-affinity estrogen-receptor radiopharmaceutical.

17 alpha-[125I[Iodovinyl 11 beta-methoxyestradiol ([I-125]MIVE2) has been prepared with high specific activity (1500-2000 Ci/mmol) and a high affinity for the estrogen receptor (KA = 6.8 x 10(9) M/Ml). In vivo distribution studies using immature rats result in high levels of activity in the uterus (20-30% dose/g) with uterus-to-plasma ratios on the order of 68 to 100. Peak activity in the uterus is obtained between 2 and 4 hr, and by 6 hr 50% of the activity has washed out. The [I-125]MIVE2 exhibits a slower rate of washout relative to the washout of H-3 estradiol. By in vivo competition studies with nonradioactive estradiol, we found that 95% of the [I-125]MIVE2 bound in the uterus is specifically bound to estrogen receptors. The radioactive labeling of MIVE2 is sufficiently rapid so that [I-123]MIVE2 has been synthesized and is currently in clinical trials. These results suggest that MIVE2 would be an excellent agent for the study of estrogen receptors in vivo and in vitro.

The characteristics of I-125 4-IQNB and H-3 QNB in vivo and in vitro.

The accumulation of (R)-(H-3)-3-quinuclidinyl benzilate (H-3 QNB) and (R,S)-1-azabicyclo(2.2.2)oct-3-yl (R,S)-alpha-hydroxy-(4-[I-125]iodophenyl) benzeneacetate (I-125 4- IQNB ) in heart, caudate/putamen, and cerebellum of rats was determined at intervals from 15 min to 4 hr after injection. The behavior of the two radiotracers in the heart is consistent with in vitro results with respect to affinities and specificities. In the brain, however, the compounds differ in tissue selectivity. At high specific activity, neither compound provides localization that is consistent with the concentration of receptor in the tissues. The results of this study do not indicate quantification of receptor concentration by means of single external images.

Differences in affinities of muscarinic acetylcholine receptor antagonists for brain and heart receptors.

The affinities of atropine, scopolamine, 3-quinuclidinyl benzilate and twelve analogues of 3-quinuclidinyl benzilate were determined for the muscarinic acetylcholine receptor (m-AChR) using membrane preparations from caudate/putamen. The affinity constants thus obtained were compared with affinities previously reported for the m-AChR obtained from ventricular muscle. The affinities differed significantly for six of the compounds, the largest difference being 16-fold. Neither solubilization nor variation of physiologically significant salts led to a significant change in the affinity of that compound. These results are interpreted as supporting the subclassification of the muscarinic acetylcholine receptor.

Biologically stable [(18)F]-labeled benzylfluoride derivatives.

Use of the [(18)F]-fluoromethyl phenyl group is an attractive alternative to direct fluorination of phenyl groups because the fluorination of the methyl group takes place under milder reaction conditions. However, we have found that 4-FMeBWAY showed femur uptake equal to that of fluoride up to 30 min in rat whereas 4-FMeQNB had a significantly lower percent injected dose per gram in femur up to 120 min. For these and other benzylfluoride derivatives, there was no clear in vivo structure-defluorination relationship. Because benzylchlorides (BzCls) are known alkylating agents, benzylfluorides may be alkylating agents as well, which may be the mechanism of defluorination. On this basis, the effects of substitution on chemical stability were evaluated by the 4-(4-nitro-benzyl)-pyridine (NBP) test, which is used to estimate alkylating activity with NBP. The effect of substitution on the alkylating activity was evaluated for nine BzCl derivatives: BzCl; 3- or 4-methoxy (electron donation) substituted BzCl; 2-, 3-, or 4-nitro (electron withdrawing) substituted BzCl; and 2-, 3-, or 4-chloro (electron withdrawing) substituted BzCl. Taken together, the alkylating reactivity of 3-chloro-BzCl was the weakest. This result was then applied to [(18)F]-benzylfluoride derivatives and in vivo and in vitro stability were evaluated. Consequently, 3-chloro-[(18)F]-benzylfluoride showed a 70-80% decrease of defluorination in both experiments in comparison with [(18)F]-benzylfluoride, as expected. Moreover, a good linear relationship between in vivo femur uptake and in vitro hepatocyte metabolism was observed with seven (18)F-labeled radiopharmaceuticals, which were benzylfluorides, alkylfluorides, and arylfluorides. Apparently, the [(18)F]-fluoride ion is released by metabolism in the liver in vivo. In conclusion, 3-chloro substituted BzCls are the most stable, which suggests that 3-chloro benzylfluorides will be the most chemically stable compound. This result should be important in future design of radioligands labeled with a benzylfluoride moiety.

Experiment assessment of mass effects in the rat: implications for small animal PET imaging.

In vivo imaging using positron emission tomography (PET) is important in the development of new radiopharmaceuticals in rodent animal models for use as biochemical probes, diagnostic agents, or in drug development. We have shown mathematically that, if small animal imaging studies in rodents are to have the same "quality" as human PET studies, the same number of coincidence events must be detected from a typical rodent imaging "voxel" as from the human imaging voxel. To achieve this using the same specific activity preparation, we show that roughly the same total amount of radiopharmaceutical must be given to a rodent as to a human subject. At high specific activities, the mass associated with human doses, when administered to a rodent, may not decrease the uptake of radioactivity at non saturable sites or sites where an enzyme has a high capacity for a substrate. However, in the case of binding sites of low density such as receptors, the increased mass injected could saturate the receptor and lead to physiologic effects and non-linear kinetics. Because of the importance of the mass injected for small animal PET imaging, we experimentally compared high and low mass preparations using ex vivo biodistribution and phosphorimaging of three compounds: 2-fluoro-2-deoxyglucose (FDG), 6-fluoro-L-metatyrosine (FMT) and one receptor-directed compound, the serotonin 5HT1A receptor ligand, trans-4-fluoro-N-[2-[4-(2-methoxylphenyl) piperazino]ethyl]-N-(2-pyridyl) cyclohexane- carboxamide (FCWAY). Changes in the mass injected per rat did not affect the distribution of FDG, FMT, and FCWAY in the range of 0.6-1.9 nmol per rat. Changes in the target to nontarget ratio were observed for injected masses of FCWAY in the range of approximately 5-50 nmol per rat. If the specific activity of such compounds and/or the sensitivity of small animal scanners are not increased relative to human studies, small animal PET imaging will not correctly portray the "true" tracer distribution. These difficulties will only be exacerbated in animals smaller than the rat, e.g., mice.

Fluoro analogs of WAY-100635 with varying pharmacokinetics properties.

Radiolabeled derivatives of WAY-100635 have been shown to be important for imaging in vivo because of their antagonist properties and their specificity for the 5-hydroxytryptamine(1A) (5-HT(1A)) receptor. Our goal is to prepare a series of radiofluorinated derivatives of WAY-100635 that, in the rat, range in pharmacokinetic properties from nearly irreversible to reversible in their behavior. It appears that derivatives containing a cyclohexanecarboxylic acid (e.g., FCWAY) with its high affinity and high target to nontarget contrast, has properties suited to measure receptor concentration. Derivatives based on phenylcarboxamide (e.g., FBWAY and MeFBAWAY) have properties more suited to the measurement of changes in endogenous serotonin. The compound containing the pyrimidine moiety in place of the pyridine moeity in FBWAY (FBWAY 1,3N) appears to have intermediate properties.