Yilong Cui

PET Imaging–Based Evaluation of Hepatobiliary Transport in Humans with (15R)-11C-TIC-Me

It is well accepted that drug transporters play a pivotal role in hepatobiliary excretion of anionic drugs, in which drug–drug interactions and genetic polymorphisms are known to cause variations. However, PET probes for in vivo functional characterization of these transporters have not been established yet. We used PET to investigate hepatic uptake and subsequent canalicular efflux of 11C-labeled (15R)-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin methyl ester [(15R)-11C-TIC-Me)] in healthy subjects. Methods: Serial PET scans of the abdominal region in healthy male subjects were obtained with or without the organic anion–transporting polypeptide (OATP) inhibitor rifampicin after intravenous injection of (15R)-11C-TIC-Me as a radiotracer. Venous blood samples and PET images were obtained at frequent intervals up to 30 min after administration of the PET tracer. Dynamic imaging data were evaluated by integration plots of data collected for 2–10 min and for 10–30 min after tracer administration for the determination of tissue uptake clearance and biliary efflux clearance, respectively. Results: After rapid hydrolysis in blood, the acid form—11C-labeled (15R)-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin [(15R)-11C-TIC]—accumulated in the liver (37% of the dose by 17 min), and the radioactivity was then excreted into the bile (6.2% by 30 min). Rifampicin (600 mg by mouth), a potent OATP inhibitor, significantly reduced the radioactivity excreted into the bile (by 44%) by inhibiting both uptake (by 45%) and subsequent canalicular efflux (by 62%). (15R)-11C-TIC is an in vitro substrate of OATP1B1 and OATP1B3, and clinically relevant concentrations of rifampicin inhibited uptake by OATP1B1 and OATP1B3. These results demonstrated that in humans, (15R)-11C-TIC–associated radioactivity is excreted into the bile by organic anion transport systems. Conclusion: We demonstrated that PET image analysis with (15R)-11C-TIC-Me is useful for investigating variations in OATP function in the human hepatobiliary transport system.

CNS-specific prostacyclin ligands as neuronal survival-promoting factors in the brain

Prostacyclin (PGI2) is a critical regulator of the cardiovascular system, via dilatation of vascular smooth muscle and inhibition of platelet aggregation (Moncada, S. 1982, Br. J. Pharmacol., 76, 3). Our previous studies demonstrated that a novel subtype of PGI2 receptor, which is clearly distinct from a peripheral subtype in terms of ligand specificity, is expressed in the rostral region of the brain, e.g. cerebral cortex, hippocampus, thalamus and striatum, and that (15r)‐16‐m‐17,18,19,20‐tetranorisocarbacyclin (15r‐TIC) and 15‐deoxy‐16‐m‐17,18,19,20‐tetranorisocarbacyclin (15‐deoxy‐TIC) specifically bind to the central nervous system (CNS)‐specific PGI2 receptor. Here, we report that these CNS‐specific PGI2 receptor ligands, including PGI2 itself, prevented the neuronal death. They prevented apoptotic cell death of hippocampal neurons induced by high (50%) oxygen atmosphere, xanthine + xanthine oxidase, and serum deprivation. IC50s for neuronal death were ∼ 30 and 300 nm for 15‐deoxy‐TIC and 15r‐TIC, respectively, which well correlated with the binding potency for the CNS‐specific PGI2 receptor. 6‐Keto‐PGF1α (a stable metabolite of PGI2), peripheral nervous system‐specific PGI2 ligands and other prostaglandins (PGs) than PGI2 did not show such neuroprotective effects. In vivo, 15r‐TIC protected CA1 pyramidal neurons against ischaemic damage in gerbils. These results indicate that CNS‐specific PGI2 ligands have neuronal survival‐promoting activity both in vitro and in vivo, and may represent a new type of therapeutic drug for neurodegeneration.

Positron Emission Tomography Studies Using (15R)-16-m-[11C]tolyl-17,18,19,20-tetranorisocarbacyclin Methyl Ester for the Evaluation of Hepatobiliary Transport

A quantitative positron emission tomography (PET) methodology was developed for in vivo kinetic analysis of hepatobiliary transport. Serial abdominal PET scans were performed on normal and multidrug resistance-associated protein 2 (Mrp2)-deficient rats after intravenous injection of (15R)-16-m-[11C]tolyl-17,18,19,20-tetranorisocarbacyclin methyl ester (15R-[11C] TIC-Me) as a radiotracer. 15R-[11C]TIC-Me was rapidly converted to its acid form in blood within 10 s. PET scans revealed that 15R-[11C]TIC was localized mainly in the liver within 5 min of injection. By 90 min, total radioactivity in bile of Mrp2-deficient rats was significantly reduced compared with controls. Metabolite analysis by thin-layer chromatography autoradiography showed that 15R-[11C]TIC is converted to at least three metabolites (M1, M2, and M3), and M2 and M3 are the major metabolites in plasma and bile, respectively. Hepatic uptake clearance of total radioactivity in normal rats was close to the hepatic blood flow rate and slightly higher than that in Mrp2-deficient rats. The intrinsic canalicular efflux clearance of M3 (CLint,bile,M3) in Mrp2-deficient rats was decreased to 12% of controls, whereas clearance of M2 was moderately decreased (54%). An in vitro transport assay detected ATP-dependent uptake of both M2 and M3 by rat Mrp2-expressing membrane vesicles. These results demonstrated that M3 is excreted primarily into the bile by Mrp2 in normal rats. We conclude that PET studies using 15R-[11C]TIC-Me could be useful for in vivo analyses of Mrp2-mediated hepatobiliary transport.

Evaluation of Oatp and Mrp2 Activities in Hepatobiliary Excretion Using Newly Developed Positron Emission Tomography Tracer [11C]Dehydropravastatin in Rats

We developed a pravastatin derivative, sodium (3R,5R)-3,5-dihydroxy-7-((1S,2S,6S,8S)-6-hydroxy-2-methyl-8-((1-[11C]-(E)-2-methyl-but-2-enoyl)oxy)-1,2,6,7,8,8a-hexahydronaphthalen-1-yl)heptanoate ([11C]DPV), as a positron emission tomography (PET) probe for noninvasive measurement of hepatobiliary transport, and conducted pharmacokinetic analysis in rats as a feasibility study for future clinical study. Transport activities of DPV in freshly isolated rat hepatocytes and rodent multidrug resistance–associated protein 2 (rMrp2; human, MRP2)-expressing membrane vesicles were similar to those of pravastatin. Rifampicin diminished the uptake of DPV and pravastatin by the hepatocytes, with similar inhibition potency. [11C]DPV underwent biotransformation to produce at least two metabolites in rat, but metabolism of [11C]DPV occurred negligibly in human hepatocytes during a 90-minute incubation. After intravenous injection, [11C]DPV was mainly distributed to the liver and kidneys, where the tissue uptake clearances (CLuptake,liver and CLuptake,kidney) were blood-flow–limited (73.6 ± 4.8 and 24.6 ± 0.6 ml/min per kilogram, respectively). Systemic elimination of [11C]DPV was delayed in rifampicin-treated rat and an Mrp2-deficient mutant rat, Eisai hyperbilirubinemic mutant rat (EHBR). Rifampicin treatment decreased both CLuptake,liver and CLuptake,kidney of [11C]DPV by 30% (P < 0.05), whereas these parameters were unchanged in EHBR. Meanwhile, the canalicular efflux clearance (CLint,bile) of [11C]DPV, which was 12.2 ± 1.5 ml/min per kilogram in the control rat, decreased by 60% and 89% in rifampicin-treated rat and EHBR (P < 0.05), respectively. These results indicate that [11C]DPV is taken up into the liver by organic anion-transporting polypeptides (rodent, Oatps; human, OATP) and excreted into bile by Mrp2 in rat, and that rifampicin may inhibit Mrp2 as well as Oatps, and consequently increase systemic exposure of [11C]DPV. PET using [11C]DPV is feasible for studies prior to the future clinical investigation of OATP and MRP2 functionality, especially for personalized medicine.

PET Imaging of the Gastrointestinal Absorption of Orally Administered Drugs in Conscious and Anesthetized Rats

This study assessed the process of gastrointestinal drug absorption in vivo using PET. Methods: 18F-FDG was used as a model probe and was orally administered to rats as a solution. PET scans were obtained of the whole body and abdominal region under conscious and anesthetized conditions. Blood samples were routinely taken from the femoral vein during scanning. The rate of gastric emptying and intestinal absorption of 18F-FDG was estimated from the time profiles of radioactivity in the stomach and small intestine. In addition, nonradiolabeled 2-fluoro-2-deoxy-d-glucose (2-FDG) was used in an intestinal closed-loop experiment to compare the intestinal permeability of 2-FDG with that of d-glucose. Results: In conscious rats, gastrointestinal absorption of 18F-FDG was rate-limited by the process of intestinal membrane permeation, because the permeability of 2-FDG through the intestinal membrane was low compared with that of d-glucose. In anesthetized rats, the gastric emptying rate of 18F-FDG decreased dramatically whereas the intestinal absorption rate constant was not significantly different from that in conscious rats. As a result, the rate-limiting step of gastrointestinal absorption of 18F-FDG was shifted to the gastric emptying process by anesthesia. Conclusion: PET technology is a powerful tool for in vivo analysis of the gastrointestinal absorption of orally administered drugs.

Neuroprotection by a central nervous system-type prostacyclin receptor ligand demonstrated in monkeys subjected to middle cerebral artery occlusion and reperfusion : A positron emission tomography study

Background and Purpose— Recently, we found that a novel subtype of prostacyclin (PGI2) receptor clearly distinct from the peripheral subtype in terms of ligand specificity is expressed in the central nervous system (CNS). (15R)-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin (15R-TIC) was synthesized and demonstrated to be a specific ligand for this CNS-type PGI2 receptor. Previously, we demonstrated 15R-TIC to be neuroprotective in vivo during transient forebrain ischemia in gerbils and permanent middle cerebral artery occlusion (MCAO) in rats. Furthermore, this compound was shown to exert an anti-apoptotic effect on primary cultured hippocampal neurons, indicating its neuroprotective effect against ischemic insults occurs via direct action on CNS-type PGI2 receptor. Methods— Local cerebral hemodynamics and oxygen metabolism were measured simultaneously by using positron emission tomography with the 15O steady-state method, before and up to 18 hours after 3-hour transient MCAO reperfusion in cynomolgus monkeys. Methyl ester of 15R-TIC (50 &mgr;g/kg, n=4) or its vehicle (10% Intralipos, n=4) was injected intravenously within 5 minutes after onset of MCAO and continuously infused for 5 hours (50 &mgr;g/kg per hour). Results— Neuropathology showed that 15R-TIC significantly reduced cortical damage after 3-hour MCAO. Positron emission tomography results showed 15R-TIC significantly reduced the volume of “infarct” region of interest and attenuated the decrease in cerebral metabolic rate of oxygen and oxygen extraction fraction, and these protective effects were not attributable to improvement of cerebral circulation. Conclusions— These results suggest that 15R-TIC has a potent neuroprotective effect against focal cerebral ischemia in a monkey MCAO via its direct action on CNS-type PGI2 receptors.

Role of cortical spreading depression in the pathophysiology of migraine

A migraine is a recurring neurological disorder characterized by unilateral, intense, and pulsatile headaches. In one-third of migraine patients, the attacks are preceded by a visual aura, such as a slowly-propagating scintillating scotoma. Migraine aura is thought to be a result of the neurovascular phenomenon of cortical spreading depression (SD), a self-propagating wave of depolarization that spreads across the cerebral cortex. Several animal experiments have demonstrated that cortical SD causes intracranial neurogenic inflammation around the meningeal blood vessels, such as plasma protein extravasation and pro-inflammatory peptide release. Cortical SD has also been reported to activate both peripheral and central trigeminal nociceptive pathways. Although several issues remain to be resolved, recent evidence suggests that cortical SD could be the initial trigger of intracranial neurogenic inflammation, which then contributes to migraine headaches via subsequent activation of trigeminal afferents.

Mapping of regional brain activation in response to fatigue-load and recovery in rats with c-Fos immunohistochemistry

Fatigue is known to be accompanied by a feeling of extreme physical or mental tiredness, resulting from severe stress and hard physical or mental work. To investigate the functional localization of neural activity related to fatigue and recovery, we examined brain c-Fos expression patterns in a rat in a state of fatigue in which rats kept in a cage filled with water to a height of 2.2cm for 1-5 days. A significant increase in the number of c-Fos-immunopositive cells was observed in the retrosplenial granular b cortex during the fatigue-loading and in the dentate gyrus of the ventral hippocampus after a 24-h recovery. In addition, variable increases in the number of c-Fos-immunopositive cells were observed in the cingulate cortex area 2, ventral part of the lateral septum nucleus, median preoptic nucleus, anterior part of the paraventricular thalamic nucleus, medial parvicellular part of the paraventricular hypothalamic nucleus, and lateral and ventrolateral periaqueductal gray during the fatigue-load period. These results indicate that such regional brain activity would be involved in fatigue or in subsequent recovery and might provide a foothold for further research into the nature of fatigue.

Intrapancreatic axonal hyperbranching of dorsal root ganglia neurons in chronic pancreatitis model rats and its relation to pancreatic pain.

Objectives: Increase in number of intrapancreatic nerve bundles has been implicated in the generation of persistent pain in chronic pancreatitis. To examine the origin of these nerve fibers and the mechanisms linking neural morphological change to pain generation, we used neuronal tracing techniques in combination with immunohistochemistry in spontaneous chronic pancreatitis in the Wistar Bonn/Kobori (WBN/Kob) rats. Methods: For retrograde tracing, horseradish peroxidase was injected into the pancreas, and labeled neurons in the sensory ganglia were counted. For anterograde tracing, biotinylated dextran amine was injected into the dorsal root ganglia (DRGs), and labeled intrapancreatic sensory fibers were histochemically assessed. For assessment of pain generation, we evaluated c-Fos-positive neurons in the spinal dorsal horn and behavioral changes of the animals. Results: In WBN/Kob rats, the numbers of horseradish peroxidase-labeled neurons were decreased in the DRGs, and the numbers of biotinylated dextran amine-labeled intrapancreatic nerve fibers and terminals were increased. Biotinylated dextran amine-labeled nerve fibers contained growth-associated protein 43. The number of c-Fos-positive neurons in the dorsal horn was also increased and was correlated with intrapancreatic growth-associated protein 43 immunoreactivity. Grooming behavior was reduced in WBN/Kob rats, and this reduction was facilitated by exocrine stimulation. Conclusions: Axonal branching in DRG neurons innervating the pancreas increases in WBN/Kob rats, and these morphological changes are likely involved in pain generation in chronic pancreatitis.

Chronic Degeneration of Dorsal Raphe Serotonergic Neurons Modulates Cortical Spreading Depression: A Possible Pathophysiology of Migraine

The vascular serotonergic system in the brain has been implicated in the pathophysiology of migraine, however, involvement of the serotonergic nervous system of the brain parenchyma in the pathophysiology remains unclear. To investigate whether the brain parenchymal serotonergic nervous system is involved in the etiology of migraine, we prepared an experimental model of migraine by generation of cortical spreading depression (SD), characterized by spreading of neuronal/glial membrane depolarization accompanied by temporal elevation of the cerebral blood flow (CBF) throughout the cerebral cortical hemisphere in rats, which underwent pharmacological treatment for degeneration of serotonergic neurons in the dorsal raphe nucleus. We show here that 1) significant degeneration of serotonergic neurons in the dorsal raphe nucleus and serotonergic fibers in the cerebral cortex was observed in treated rats, 2) spreading velocity of the CBF changes was significantly increased in these rats, and 3) calculated width of the depolarization wave was significantly extended in these rats. These results indicate that the dorsal raphe serotonergic neurons modulate cortical spreading depression and might be involved in migraine pathology via a similar mechanism.