In vitro and in vivo evaluation of novel glibenclamide derivatives as imaging agents for the non-invasive assessment of the pancreatic islet cell mass in animals and humans.

Pancreatic islet cell mass (PICM) is a major determinant of the insulin secretory capacity in humans. Currently, the only method for accurate assessment of the PICM is an autopsy study. Thus, development of a technique allowing the non-invasive quantification of PICM is of great interest. The aim of this study was to develop such a non-invasive technique featuring novel fluorine- and (99m)Tc-labelled glibenclamide derivatives. Despite the structural modifications necessary to introduce fluorine into the glibenclamide molecule, all derivatives retained insulin stimulating capacity as well as high affinity binding to human SUR1 when compared to the original glibenclamide. Contrastingly, the lipophilicity of the fluorine-labelled derivatives was altered depending on the particular modification. In the human PET-study a constant but weak radioactive signal could be detected in the pancreas using a fluorine-labelled glibenclamide derivative. However, a reliable assessment and visualisation of the PICM could not be obtained. It can be assumed that the high uptake of the fluorine-labelled tracer e.g. into the the liver and the high plasma protein binding leads to a relatively low signal-to-noise ratio. In case of the presented fluorine-labelled glibenclamide based compounds this could be the result of their invariably high lipophilicity. The development of a (99 m)Tc-labelled glibenclamide derivative with a lower lipophilicity and differing in vivo behaviour, glibenclamide based compounds for non-invasive imaging of the pancreatic islet cell mass may be possible.

Serial [18F]N-methylspiroperidol PET studies to measure changes in antipsychotic drug D-2 receptor occupancy in schizophrenic patients.

An indirect approach to the relationship among drug dose, plasma level, and the competition between a labeled neuroleptic drug [18F]N-methylspiroperidol (18F-NMS) for binding sites in striatal tissue in normal and schizophrenic subjects is described. The slope of the line plotting the ratio of activity in the striatum (As) to activity in the cerebellum (Ac) versus time up to 5 hr postinjection of 18F-NMS is taken as a marker of site occupancy. An inverse relation between labeled competitor uptake and drug plasma level has been demonstrated for the classes of antipsychotic drug studied. Striatal uptake studies showed a progressive increase in all subjects following drug withdrawal up to 156 hr postwithdrawal. Uptake and clearance of 18F-NMS in cerebellar tissue was not appreciably affected by antipsychotic medication or drug withdrawal.

Glucose metabolic rate kinetic model parameter determination in humans: the lumped constants and rate constants for [18F]fluorodeoxyglucose and [11C]deoxyglucose.

The rate constants and lumped constants (LCs) for [18F]fluorodeoxyglucose ([18F]FDG) and [11C]deoxyglucose ([11C]DG) were determined in humans for the glucose metabolic rate kinetic model used to measure local cerebral glucose consumption. The mean values (+/- SE) of the LCs for [18F]FDG and [11C]DG are 0.52 +/- 0.028 (n = 9) and 0.56 +/- 0.043 (n = 6), respectively. The mean values (+/- SE) of the rate constants k*1, k*2, k*3, and k*4 for [18F]FDG for gray matter are 0.095 +/- 0.005, 0.125 +/- 0.002, 0.069 +/- 0.002, and 0.0055 +/- 0.0003, respectively. The corresponding values for white matter are 0.065 +/- 0.005, 0.126 +/- 0.003, 0.066 +/- 0.002, and 0.0054 +/- 0.0006, respectively. Using these values and previously published values for the rate constants for [11C]DG, the average whole-brain metabolic rates for glucose in normal subjects measured with [18F]FDG and [11C]DG are 5.66 +/- 0.37 (n = 6) and 4.99 +/- 0.23 (n = 6) mg/100 g/min, respectively. These values are not significantly different (t = 1.56, p greater than 0.10) and agree well with reported values in the literature determined by means of the Kety-Schmidt technique.

Dopamine blockade and clinical response: evidence for two biological subgroups of schizophrenia.

Because CNS neuroleptic concentration cannot be directly measured in patients, the relation between clinical response and extent of dopamine receptor blockade is unknown. This relationship is critical in ascertaining whether nonresponse to neuroleptics is the result merely of inadequate CNS drug levels or of more basic biological differences in pathophysiology. Using [18F]N-methylspiroperidol and positron emission tomography, the authors assessed dopamine receptor occupancy in 10 schizophrenic patients before and after treatment with haloperidol. Responders and nonresponders had virtually identical indices of [18F]N-methylspiroperidol uptake after treatment, indicating that failure to respond clinically was not a function of neuroleptic uptake or binding in the CNS.

Effects of chronic cocaine abuse on postsynaptic dopamine receptors.

To assess the effects of chronic cocaine intoxication on dopamine receptors in human subjects, the authors evaluated [18F]N-methylspiroperidol binding using positron emission tomography in 10 cocaine abusers and 10 normal control subjects. Cocaine abusers who had been detoxified for 1 week or less showed significantly lower values for uptake of [18F]N-methylspiroperidol in striatum than the normal subjects, whereas the cocaine abusers who had been detoxified for 1 month showed values comparable to those obtained from normal subjects. The authors conclude that postsynaptic dopamine receptor availability decreases with chronic cocaine abuse but may recover after a drug-free interval.

Synthesis and biological activity of some 8-substituted selenoguanosine cyclic 3',5'-phosphates and related compounds.

8-Bromoguanosine cyclic 3',5'-monophosphate, 8-bromoguanosine 5'-monophosphate, and 8-bromoguanosine served as intermediates for the chemical synthesis of a series of 8-substituted seleno cyclic nucleotides, nucleotides, and their nucleosides. Selenourea was found to be a useful reagent in synthesizing these seleno-substituted nucleoside, nucleotide, and cyclic nucleotide. A nucleic acid analyzer was used to study the hydrolysis of these cyclic nucleotides by phosphodiesterase. It was found that all of the 8-substituted selenoguanosine cyclic 3',5'-phosphates synthesized, except 8-MeSe-cGMP, were resistant to hydrolyze by phosphodiesterase. These 8-substituted seleno cyclic GMP derivatives showed some antitumor activities against murine leukemic cells (L5178Y) in vitro and in vivo.

Syntheses and specific activity determinations of no-carrier-added (NCA) F-18-labeled butyrophenone neuroleptics--benperidol, haloperidol, spiroperidol, and pipamperone.

A general method for the syntheses of no-carrier-added (NCA) 18F-labeled butyrophenone neuroleptics--benperidol, haloperidol, spiroperidol, and pipamperone is described. These 18F-labeled neuroleptic drugs are synthesized by a multistep synthesis in an overall radiochemical yield of 10-20% at end of bombardment (EOB) in a synthesis time of 90 min from EOB. The sequence involves the synthesis of NCA p-[18F]fluorobenzonitrile from NCA [18F]-fluoride and p-nitrobenzonitrile using the rapidly converted to gamma-chloro-p-[18F]fluorobutyrophenone which is alkylated with appropriate amines to give NCA 18F-labeled benperidol, haloperidol, spiroperidol, and pipamperone. The final product is purified by preparative high performance liquid chromatography (HPLC). The 18F solution used in the synthesis as determined by ion chromatography contains 15.3 +/- 9.0 nmol of stable fluoride. The specific activities of the resulting butyrophenone neuroleptics were determined to be 3 Ci/mumol (at EOB) (range 1-6 Ci/mumol) as determined by radioreceptor assay and HPLC assay.

No-carrier-added N-(3-[18F]fluoropropyl)spiroperidol: biodistribution in mice and tomographic studies in a baboon.

Two potential radioligands, no-carrier-added (NCA) N-(2-[18F]fluoroethyl)spiroperidol (3) and N-(3-[18F]fluoropropyl)spiroperidol (4) have been synthesized for PET imaging of dopamine receptors in humans. Compounds 3 and 4 were synthesized by N-alkylation of spiroperidol with NCA 1-bromo-2-[18F]-fluoroethane (2b), 1-[18F]fluoro-3-iodopropane (2c) and 1-bromo-3-[18F]fluoropropane (2d) respectively. The biodistribution of 4 in mice showed that the mouse brain uptake of radioactivity was similar to that of [18F]-N-methylspiroperidol (1.1% of the administered dose), but the activity in bone (femur) increased with time. The kinetic distribution of compound 4 in baboon brain was similar to that of [18F]-N-methylspiroperiodol, and the striatal accumulation of radioactivity was also blocked stereoselectively by butaclamol. The ratio of striatum to cerebellum radioactivities at 3 hr after injection was 5.9. Analysis of the metabolic stability of 4 in mouse brains for 1 hr indicated that, like [18F]-N-methylspiroperidol, it is relatively stable to metabolic transformation in the central nervous system. These results suggest that compound 4 may be a useful radioligand for PET studies of the dopamine receptor in humans.

Fluorine-18-N-methylspiroperidol: radiolytic decomposition as a consequence of high specific activity and high dose levels.

High specific activity [18F]N-methylspiroperidol(8-[4-(4-[18F]fluorophenyl)-4-oxobutyl]-3-me thyl l-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one, 5-10 mCi/ml, 4-8 Ci/mumol at EOB) in saline solution undergoes significant radiolytic decomposition resulting in a decrease in radiochemical purity of 10-25% during the first hour. The rate of decomposition is affected by the specific activity, total dose to and chemical composition of the solution. That radiolysis is responsible for the observed decomposition was verified by the observation that unlabeled N-methylspiroperidol is decomposed in the presence of [18F]fluoride.

Transient toxicity of 2-deoxy-2-[18F]fluoro-D-glucose in mammalian cells: concise communication.

The kinetics of uptake and toxicity of the positron emitter F-18 have been examined in a cultured cell line. 2-Deoxy-2[18F]fluoro-D-glucose (18FDG) concentrated rapidly within Chinese hamster V79 cells, and the uptake was linear with the extracellular radioactive concentrations. Whereas 18FDG synthesized 2 hr before the incubation did not appear to be toxic, that synthesized 5 hr previously was highly toxic. Toxicity was transient and independent of both the extracellular/intracellular radioactive concentration and the energy released from the decay of fluorine-18. Similarly synthesized nonradioactive FDG and Na 18F were not toxic under comparable experimental conditions. We conclude that this transient toxicity is due to an unidentified chemical species that is cytocidal following intracellular localization. These toxic levels are not likely to be achieved in the clinical use of 18FDG due to dilution factors that are orders of magnitude greater than those used in these in vitro studies.

No-carrier-added fluorine-18-labeled N-methylspiroperidol: synthesis and biodistribution in mice.

No-carrier-added fluorine-18- (18F) labeled N-methylspiroperidol (4) was synthesized from four different substrates: p-nitrobenzonitrile (1), cyclopropyl p-nitrophenyl ketone (2A), p-cyclopropanoyl-N,N,N-trimethylanilinium iodide (2B) and p-cyclopropanoyl-N,N,N-trimethylanilinium perchlorate (2C) using the nucleophilic aromatic substitution reaction. Radiochemical yield, synthesis time, experimental simplicity, and specific activity were compared. In addition, factors which influence the yield of the nucleophilic aromatic substitution were studied. Based on these studies, the synthesis of 4 from 2A maximizes product specific activity and experimental simplicity and provides 4 in 10-15% radiochemical yield [based on [18F-] with a mass of less than 2 nmol and a specific activity of greater than 10 Ci/mumol (EOB)]. The synthesis of 4 from 8-[4-(4-nitrophenyl)-4-oxobutyl]-3-methyl-1-phenyl-1,3,8-triazaspiro+ ++ [4.5]decan-4-one (5) and Cs[18F] using the nucleophilic aromatic substitution reaction gave unacceptably low and erratic yields. The biodistribution of 4 in mice showed a maximum brain uptake of 1.1% of the administered dose at 5 min and declined to approximately 0.6% at 120 min.

Improved delineation of human dopamine receptors using [18F]-N-methylspiroperidol and PET.

The brain uptake of [18F]-N-methylspiroperidol, a butyrophenone neuroleptic with high selectivity for the dopamine receptor, has been measured in three normal human volunteers using positron emission tomography for times up to 12 hr postinjection. These studies demonstrated two unique findings concerning the in vivo distribution of this neuroleptic: (a) it is tightly bound to dopamine D-2 receptors in the caudate-putamen brain regions, and (b) these regions are the only large brain structures which exhibit appreciable long-term retention. In addition, radioactivity clears rapidly from plasma, and the percentage of unchanged [18F]-N-methylspiroperidol in plasma declines rapidly. These results suggest that this compound binds irreversibly to dopamine D-2 receptors, and that there are few if any dopamine D-2 receptors in the human frontal cortex. These studies emphasize not only the importance of characterizing neurotransmitter receptors in living human brain using a ligand labeled with a positron emitting nuclide of sufficiently long half-life to allow monitoring of brain radioactivity distribution for several hours after the injection of radioligand, but also of accurately determining the amount of unchanged tracer in plasma for tracer kinetic modeling.

Noninvasive detection of tumor hypoxia using the 2-nitroimidazole [18F]EF1.

UNLABELLED: The noninvasive assessment of tumor hypoxia in vivo is under active investigation because hypoxia has been shown to be an important prognostic factor for therapy resistance. Various nuclear medicine imaging modalities are being used, including PET imaging of 18F-containing compounds. In this study, we report the development of 18F-labeled EF1 for noninvasive imaging of hypoxia. EF1 is a 3-monofluoro analog of the well-characterized hypoxia marker EF5, 2(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetami de, which has been used to detect hypoxia in tumor and nontumor systems using immunohistochemical methods. METHODS: We have studied 2 rat tumor types: the hypoxic Morris 7777 (Q7) hepatoma and the oxic 9LF glioma tumor, each grown in subcutaneous sites. PET studies were performed using a pharmacological dose of nonradioactive carrier in addition to [18F]EF1 to optimize and assess drug biodistribution. After PET imaging of the tumor-bearing rats, tissues were obtained for gamma-counting of the 18F in various tissues and immunohistochemical detection of intracellular drug adducts in tumors. In one pair of tumors, Eppendorf needle electrode studies were performed. RESULTS: [18F]EF1 was excreted dominantly through the urinary tract. The tumor-to-muscle (T/M) ratio of [18F]EF1 in the Q7 tumors was 2.7 and 2.4 based on PET studies and 2.1, 2.5, and 3.0 based on gamma-counting of the tissues (n = 3). In contrast, the T/M ratio of [18F]EF1 in the 9LF glioma tumor was 0.8 and 0.5 based on PET studies and 1.0, 1.2, and 1.4 based on gamma-counting of the tissues (n = 3). Immunohistochemical analysis of drug adducts for the two tumor types agreed with the radioactivity analysis. In the Q7 tumor, substantial heterogeneous binding was observed throughout the tumor, whereas in the 9LF tumor minimal binding was found. CONCLUSION: [18F]EF1 is an excellent radiotracer for noninvasive imaging of tumor hypoxia.

A fluorinated glucose analog, 2-fluoro-2-deoxy-D-glucose (F-18): nontoxic tracer for rapid tumor detection.

Rapid uptake of F-18 FDG was observed in a variety of transplanted and spontaneous tumors in animals. The tumor uptake reached a peak by 30 min and remained relatively constant up to 60 min, with a very slow wash-out of F-18 activity from the tumor thereafter. Tumor-to-normal tissue and tumor-to-blood ratios ranged from 2.10-9.15 and 2.61-17.82, respectively, depending on the type of tumor. A scintiscan of a seminoma in a dog showed very high uptake in the viable part and lack of uptake in the necrotic mass. Toxicological studies in mice using 1000 times human tracer dose (HTD) per wk for 3 wk and in dogs using 50 times HTD per wk for 3 wk did not show any evidence of acute or chronic toxicity.

Considerations in setting up a positron emission tomography center.

Clinically oriented imaging with position emission tomography (PET) has come of age. Given an adequate referral base and physician interest, a compelling argument can be made at all levels of the review process for setting up a PET program in a clinical setting. PET is expensive. It is obvious that the cost of running a PET service depends heavily on an institution's ability to obtain reasonable financing. Educational institutions have the opportunity to acquire special funding through a variety of sources. On the other hand, money can be expensive for private entrepreneurs. It appears that in the near future PET centers will probably remain at educational institutions or large well-financed community hospitals able to raise money at reasonable rates until reimbursement issues are better resolved. Finally, the future of clinical PET may hinge significantly on the ability of commercial radiopharmaceutical suppliers to provide regional fluorodeoxyglucose distribution. As an institutional program development, PET offers opportunities by providing unique clinical data aiding the referral pattern. PET may serve as a magnet for recruitment in many areas and may promote interdisciplinary cooperation. A clinical PET center serves both as a model for future and more widespread use of PET and as a training ground for medical personnel. Finally, the unique capabilities of PET may facilitate grant opportunities.

18F-5-Fluorouridine, a new probe for measuring the proliferation of tissue in vivo.

(1) Increased metabolic trapping of labeled fluorouridine reflects the interaction of three parameters in rapidly proliferating tissues: increased rates of intracellular phosphorylation, increased rates of transport, and increased rates of synthesis of RNA. (2) We have taken advantage of these metabolic phenomena, demonstrating in this paper that the uptake of 18F-5-fluorouridine, a positron-emitting radiopharmaceutical, can provide a very practical means for measuring changes in proliferative states of tissues in vivo. (3) Two major changes in proliferative states have been examined: one involves changes in growth of normal mouse tissues induced by pharmacological agents; the other involves tumor growth and neoplastic infiltration in mice and rabbits. (4) We describe tracer experiments with 18F-5-fluorouridylate, prepared by enzymatic means, and with 18F-5-fluorouridine, prepared by both enzymatic means and direct radiochemical procedures. (5) Uptakes of 18F after a pulse of 18F-5-fluorouridine were increased in mouse spleen following phenylhydrazine treatment to induce increased splenic erythropoiesis. (6) Uptakes of 18F in various mouse tissues were decreased following pretreatment with actinomycin D. This finding is consistent with the known inhibitory action of actinomycin on RNA synthesis. (7) Intracerebral Zimmerman ependymoblastoma tumors showed extraordinarily high uptakes of fluorine-18 in mice injected intravenously with 18F-5-fluorouridylate or with 18F-5-fluorouridine in contrast to very low uptakes by normal brain tissue. (8) After intracerebral injection of mice with suspensions of L1210 leukemia cells, distant organs such as lung, liver, and spleen became involved. These tissues showed significant increases of radioactivity after pulse labeling with 18F-5-fluorouridylate consistent with histological evidence for infiltration of these tissues by neoplastic cells. (9) Intramuscular VX2 carcinoma tumors in rabbits showed localized uptakes of 18F significantly higher than surrounding normal muscle tissue. (10) The most important clinical implication of the present work is the promise that 18F-5-fluorouridine uptakes can be followed in humans by positron emission tomography. This would provide a direct means of measuring different rates of in vivo proliferation in neoplasms, hematologic tissues and other organs undergoing rapid growth changes.

Comparative PET studies of the distribution of (-)-3,4-methylenedioxy-N-[11C]methamphetamine and (-)-[11C]methamphetamine in a monkey brain.

Carbon-11 labeled (-)-methamphetamine and (-)-3,4-methylenedioxy-N-methamphetamine were synthesized by methylation of the corresponding desmethyl precursors with [11C]H3I in 40-60% yield in a synthesis time of 30 min from EOB with a specific activity of 0.5-1.2 Ci/microM. PET studies in a Rhesus monkey revealed that the uptakes of both compounds in different brain regions were similar, and the retention of radioactivity in these brain regions remained constant throughout the study for the former while it was washed out slowly for the latter. The half-life of (-)-3,4-methylenedioxy-N-methamphetamine in monkey brain was approximately 70 min. Analyses of arterial plasma by HPLC revealed that 50% of radioactivity in the plasma remained as (-)-methamphetamine while only 3% remained as (-)-3,4-methylenedioxy-N-methamphetamine at 60 min post-injection. These results suggest that the uptakes of both compounds in monkey brain are probably not receptor mediated. Rather, blood flow, lipophilicity of the compounds or other transport mechanisms may play a role in their uptakes.

PET study of the distribution of [11C]fluoxetine in a monkey brain.

No-carrier-added [11C]fluoxetine (2) was synthesized by methylation of norfluoxetine (1) with [11C]H3I in 20% radiochemical yield in a synthesis time of 40 min from EOB with a specific activity of 0.48 Ci/microM (EOB). In vivo study in mouse indicated that the uptake of 2 in mouse tissues was high and the radioactivity remained constant throughout the study. The uptake of 2 in mouse brain was 4%/g. PET study in a Rhesus monkey also showed that the uptakes of 2 in different brain regions were similar and the retention of radioactivity in these regions remained constant throughout the study (80 min). Analysis of arterial plasma by HPLC showed that only 20% of radioactivity in the plasma remained as 2 at 30 min post-injection. These results suggest that the uptake of fluoxetine in monkey brain is probably not receptor mediated. Rather, blood flow, lipophilicity or other transport mechanisms may play a role in its uptake.

Radiosynthesis and biodistribution of the S-[18F]fluoroethyl analog of McN5652.

[11C]McN5652 has been reported to exhibit favorable properties as a PET radiotracer for studying serotonin uptake sites. However, the use of this radiotracer may be limited by the short half-life of11C. To obtain a tracer with longer physical half-life, we have synthesized the S-[18F]fluoroethyl analog of McN5652 (trans-1,2,3,5,6,10b-hexahydro-6-[4-([18F]fluoroethylthio)-phenyl] pyrrolo-[2,1-a]-isoquinoline) ([18F]FEMcN) and evaluated as a PET radiotracer for imaging serotonin uptake sites. The radiosynthesis was performed via a one-pot, two-step procedure. In the first step, 1-bromo-2-[18F]fluoroethane was prepared from 2-bromoethyl triflate and K18F/Kryptofix 2.2.2. in THF at room temperature. The second step, the S-fluoroalkylation of the normethyl McN5652, a thiol, was carried out, without isolating the 1-bromo-2-[18F]fluoroethane, by adding the normethyl McN5652 to the reaction vial, which was warmed at 45 degrees C for 1 min. The fluoroalkylation reaction proceeded quickly, giving [18F]FEMcN in an average overall radio-chemical yield of 13 +/- 7%. The specific activity was 1593 +/- 625 mCi/mumol. Ex vivo autoradiographic studies revealed that [18F]FEMcN accumulated into regions with high densities of 5-HT uptake sites such as hypothalamus, substantia nigra, and raphe nuclei. With blockade by nitroquipazine, a selective and highly potent 5-HT uptake blocker, the activity level in these regions was close to that in regions low in 5-HT uptake sites such as cerebellum, suggesting that this radiotracer binds specifically to 5-HT uptake sites. The regional distribution of [18F]FEMcN at 60 min postinjection correlated with the distribution of [11C]McN5652 reported in the literature. The specific binding of this radiotracer determined as the difference in radioactivity accumulation with and without blocking by the 5-HT uptake blocker agreed with the distribution of the number of 5-HT uptake sites measured in vitro. Thus, 5-HT uptake sites were visualized in vivo with [18F]FEMcN. However, comparison with the in vivo behavior of [11C]McN5652 indicated less favorable properties of [18F]FEMcN as a PET radiotracer for imaging 5-HT uptake sites, including lower blood-brain barrier penetration and lower target-to-nontarget ratios.

Synthesis of (+/-)-[18F]BMY 14802, its enantiomers and their anatomical distributions in rodents.

A potential antipsychotic drug, BMY 14802 was labeled with 18F and its distribution in rodents was studied. No-carrier-added (NCA) (+/-)-[18F]BMY 14802 (5) was synthesized by two methods in 5-10% radiochemical yield in a synthesis time of 130-140 min from EOB with a specific activity of 0.5-1.5 Ci/microM. (+)- and (-)-[18F]BMY 14802 was synthesized by the chiral reduction of alpha-(4-[18F]fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazine-b utanone (4) with chiral reducing agent, (+)- and (-)-beta-chlorodiisopinocampheylborane [(+)- and (-)-DIP chloride] in 6-10% radiochemical yield in a synthesis time of 150 min from EOB. Animal studies in mouse and in rat revealed that the distribution of 5 in each tissue was high at 5 min, the radioactivity then declined rapidly in all tissues studied except in the liver and in the small intestine. The radioactivity in the femur did not increase with time indicating in vivo defluorination may not occur. The uptakes of (+/-)-[18F]BMY 14802 and its enantiomers, (+)- and (-)-[18F]BMY 14802 in rat cerebellum, brain stem, hippocampus and spinal cord were similar and were significantly reduced by prior treatment of rat with haldol. This suggests that (+/-)-[18F]BMY 14802 and its enantiomers bind to sigma-receptors in a similar fashion.