Analysis of ADAM12-Mediated Ephrin-A1 Cleavage and Its Biological Functions.

Accumulating evidence indicates that an elevated ephrin-A1 expression is positively correlated with a worse prognosis in some cancers such as colon and liver cancer. The detailed mechanism of an elevated ephrin-A1 expression in a worse prognosis still remains to be fully elucidated. We previously reported that ADAM12-cleaved ephrin-A1 enhanced lung vascular permeability and thereby induced lung metastasis. However, it is still unclear whether or not cleaved forms of ephrin-A1 are derived from primary tumors and have biological activities. We identified the ADAM12-mediated cleavage site of ephrin-A1 by a Matrix-assisted laser desorption ionization mass spectrometry and checked levels of ephrin-A1 in the serum and the urine derived from the primary tumors by using a mouse model. We found elevated levels of tumor-derived ephrin-A1 in the serum and the urine in the tumor-bearing mice. Moreover, inhibition of ADAM-mediated cleavage of ephrin-A1 or antagonization of the EphA receptors resulted in a significant reduction of lung metastasis. The results suggest that tumor-derived ephrin-A1 is not only a potential biomarker to predict lung metastasis from the primary tumor highly expressing ephrin-A1 but also a therapeutic target of lung metastasis.

Imaging of intracellular fatty acids by scanning X-ray fluorescence microscopy.

Fatty acids are taken up by cells and incorporated into complex lipids such as neutral lipids and glycerophospholipids. Glycerophospholipids are major constituents of cellular membranes. More than 1000 molecular species of glycerophospholipids differ in their polar head groups and fatty acid compositions. They are related to cellular functions and diseases and have been well analyzed by mass spectrometry. However, intracellular imaging of fatty acids and glycerophospholipids has not been successful due to insufficient resolution using conventional methods. Here, we developed a method for labeling fatty acids with bromine (Br) and applied scanning X-ray fluorescence microscopy (SXFM) to obtain intracellular Br mapping data with submicrometer resolution. Mass spectrometry showed that cells took up Br-labeled fatty acids and metabolized them mainly into glycerophospholipids in CHO cells. Most Br signals observed by SXFM were in the perinuclear region. Higher resolution revealed a spot-like distribution of Br in the cytoplasm. The current method enabled successful visualization of intracellular Br-labeled fatty acids. Single-element labeling combined with SXFM technology facilitates the intracellular imaging of fatty acids, which provides a new tool to determine dynamic changes in fatty acids and their derivatives at the single-cell level.-Shimura, M., Shindou, H., Szyrwiel, L., Tokuoka, S. M., Hamano, F., Matsuyama, S., Okamoto, M., Matsunaga, A., Kita, Y., Ishizaka, Y., Yamauchi, K., Kohmura, Y., Lobinski, R., Shimizu, I., Shimizu, T. Imaging of intracellular fatty acids by scanning X-ray fluorescence microscopy.

Synthesis of 24,24-Difluoro-1,3-cis-25-dihydroxy-19-norvitamin D3 Derivatives and Evaluation of Their Vitamin D Receptor-Binding Affinity.

Two vitamin D3 derivatives, namely 24,24-difluoro-1ß,3ß,25-dihydroxy-19-norvitamin D3 (6a) and 24,24-difluoro-1α,3α,25-dihydroxy-19-norvitamin D3 (6b), were synthesized via a convergent route employing Julia-Kocienski olefination as a key step. Compounds 6a and b bound to vitamin D receptor (VDR) with IC50 values of 64.8 and 57.6 nM, respectively, exhibiting about 400- and 30-fold greater binding affinity than the corresponding non-fluorinated derivatives 5a and b.

Synthesis of Diastereomers of 1,3-cis-25-Dihydroxy-19-norvitamin D3.

1ß,3ß,25-Dihydroxy-19-norvitamin D3 (4a) and 1α,3α,25-dihydroxy-19-norvitamin D3 (4b) were synthesized by employing a new A-ring synthon, (1R,3S)-3-((tert-butyldimethylsilyl)oxy)-5-oxocyclohexyl benzoate (19), which was derived from D-(-)-quinic acid in 12 steps. The A-ring was coupled with the circular dichroism (CD) ring by means of Julia-Kocienski olefination to construct the diene unit. The structures of the products were confirmed by (1)H-NMR and nuclear Overhauser effect (NOE) experiments.

Synthesis of dibarrelane, a dibicyclo[2.2.2]octane hydrocarbon.

The synthesis of a novel hydrocarbon, dibarrelane, has been accomplished in 11 steps via an intramolecular REDDA reaction of a masked o-benzoquinone, followed by Clemmensen reduction and Barton decarboxylation. The twisted structure of the tetracyclic dibarrelane skeleton was also clarified via X-ray crystallography. Finally, it was proposed that dibarrelane has C2 symmetry rather than C(2v) symmetry.

Lurasidone suppresses rapid eye movement sleep and improves sleep quality in rats.

Patients with psychiatric disorders, including schizophrenia, are reported to suffer from sleep disorders. In this study, we investigated the effects of lurasidone, an atypical antipsychotic, on sleep architecture in rats using sleep electroencephalography. The course of sleep in rats was classified into 3 stages: WAKE, non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep. Lurasidone shortened REM duration and prolonged the mean duration of one bout in WAKE and NREM. Quantitative frequency band analysis during NREM sleep revealed that lurasidone increases slow waves and decreases fast waves. These results suggest that lurasidone ameliorates sleep disorders associated with psychosis.

DSP-6745, a novel 5-hydroxytryptamine modulator with rapid antidepressant, anxiolytic, antipsychotic and procognitive effects.

BACKGROUND: Current treatment of major depressive disorder is facing challenges, including a low remission rate, late onset of efficacy, and worsening severity due to comorbid symptoms such as psychosis and cognitive dysfunction. Serotonin (5-HT) neurotransmission is involved in a wide variety of psychiatric diseases and its potential as a drug target continues to attract attention. OBJECTIVES: The present study elucidates the effects of a novel 5-HT modulator, DSP-6745, on depression and its comorbid symptoms. RESULTS: In vitro radioligand binding and functional assays showed that DSP-6745 is a potent inhibitor of 5-HT transporter and 5-HT2A, 5-HT2C, and 5-HT7 receptors. In vivo, DSP-6745 (6.4 and 19.1 mg/kg as free base, p.o.) increased the release of not only 5-HT, norepinephrine, and dopamine, but also glutamate in the medial prefrontal cortex. The results of in vivo mouse phenotypic screening by SmartCube® suggested that DSP-6745 has a behavioral signature combined with antidepressant-, anxiolytic-, and antipsychotic-like signals. A single oral dose of DSP-6745 (6.4 and 19.1 mg/kg) showed rapid antidepressant-like efficacy in the rat forced swim test, even at 24 h post-dosing, and anxiolytic activity in the rat social interaction test. Moreover, DSP-6745 (12.7 mg/kg, p.o.) led to an improvement in the apomorphine-induced prepulse inhibition deficit in rats. In the marmoset object retrieval with detour task, which is used to assess cognitive functions such as attention and behavioral inhibition, DSP-6745 (7.8 mg/kg, p.o.) enhanced cognition. CONCLUSIONS: These data suggest that DSP-6745 is a multimodal 5-HT receptor antagonist and a 5-HT transporter inhibitor and has the potential to be a rapid acting antidepressant with efficacies in mitigating the comorbid symptoms of depression.

Wheel-shaped icosanuclear homo- and heterometallic complexes of Ni(II), Co(II), and Cu(II) ions supported by unsymmetrical aminoalcohol ligands.

Reactions of M(OAc)2·4H2O (M = Ni, Co) with 3-[benzyl(2-hydroxyethyl)amino]-1-propanol (H2L) in the presence of pyridine or triethylamine afforded novel homometallic icosanuclear wheel-shaped complexes [M20L4(HL)4(OAc)28] (M = Ni (1), Co (2)), which consist of a central M(II)12 single-stranded, nearly planar loop with four peripheral [M2(HL)(OAc)2] fragments attached in an S4 symmetrical fashion. The complexes can alternatively be recognized as saddle-shaped wheel structures, in which four tetranuclear units of [M4L(HL)(OAc)7](2-) are connected by four M(2+) ions (M5). The tetranuclear unit itself can be derived from an ideal C2 symmetrical [M4(HL)2(µ-η(2)-OAc)4(µ-η(1),η(1)-OAc)2(η(1),η(1)-OAc)](-) structure through deprotonation of the HL(-) ligand, and is composed of two plane-shared M3O4 incomplete cubanes in which the M2 and M3 atoms are involved in the central fused plane and the M1 and M4 atoms are disposed at the apex sites. Mixed-metal icosanuclear complexes [NixM20-xL4(HL)4(OAc)28] (3, M = Co, x = 9.5) and [Ni12M8L4(HL)4(OAc)28] (4, M = Cu) were also synthesized by using equimolar amounts of Ni(II) and M(II) ions, and were shown to have similar structures to 1 and 2. X-ray crystallographic and fluorescent analyses revealed that complex 3 contains nonstoichiometric amounts of Ni(2+) and Co(2+) ions in the ratio of 9.5:10.5 and that these are disordered at every metal site. In striking contrast, complex 4 has a stoichiometric formula of Ni12Cu8, which was confirmed by the Jahn-Teller elongation of Cu(2+) ions, and consequently, the M2 and M5 positions are occupied exclusively by the Cu(2+) ions. The temperature-dependent direct current (dc) magnetic susceptibility data showed the presence of ferromagnetic exchange interactions in the Ni homometallic (1) and NiCu bimetallic (4) complexes, while the Co homometallic (2) and NiCo bimetallic (3) complexes exhibited antiferromagnetic interactions due to spin-orbit coupling effects of the octahedral Co(II) ions. The present results demonstrate that the unsymmetrical aminoalcohol ligand H2L is quite effective in organizing the homo- and heterometallic icosanuclear wheel-shaped metal arrangements.

Light-at-night exposure affects brain development through pineal allopregnanolone-dependent mechanisms.

The molecular mechanisms by which environmental light conditions affect cerebellar development are incompletely understood. We showed that circadian disruption by light-at-night induced Purkinje cell death through pineal allopregnanolone (ALLO) activity during early life in chicks. Light-at-night caused the loss of diurnal variation of pineal ALLO synthesis during early life and led to cerebellar Purkinje cell death, which was suppressed by a daily injection of ALLO. The loss of diurnal variation of pineal ALLO synthesis induced not only reduction in pituitary adenylate cyclase-activating polypeptide (PACAP), a neuroprotective hormone, but also transcriptional repression of the cerebellar Adcyap1 gene that produces PACAP, with subsequent Purkinje cell death. Taken together, pineal ALLO mediated the effect of light on early cerebellar development in chicks.

DSP-6952, a novel 5-HT4 receptor partial agonist, inhibits visceral hypersensitivity and ameliorates gastrointestinal dysfunction in experimental animals.

The pharmacological profile of DSP-6952, a novel 5-HT4 receptor partial agonist, was investigated to evaluate the potential use for GI disorders, and to compare its effects in some GI dysfunction models with those of clinically efficacious prokinetic agents. DSP-6952 enhanced gastric motility and caused colonic giant migrating contractions (GMCs) associated with defecation in conscious dogs, having ED50 value for inducing GMCs of 1.56 mg/kg. DSP-6952 (3-10 mg/kg, i.g.) significantly enhanced colonic transit rate in guinea pigs; this enhancement was antagonized by SB-207266, a selective 5-HT4 receptor antagonist. DSP-6952 (1-10 mg/kg, p.o.) rapidly increased fecal wet weight without increasing fluid content in mice. Sennoside (30-100 mg/kg, p.o.) also increased fecal wet weight; however, it significantly increased fluid content with diarrhea. DSP-6952 dose-dependently improved clonidine- and morphine-induced delay in whole-gut transit in mice (ED50= 0.429 mg/kg and 0.310 mg/kg, respectively), which represented atonic and spastic constipation models, respectively. In viscerally hypersensitive rats treated with acetic acid, DSP-6952 (10 mg/kg, i.p., 30 mg/kg, p.o., 30 mg/kg, i.c.) and tegaserod (1 mg/kg, i.p.), but not prucalopride (10 mg/kg, i.p.), significantly inhibited the increase in colorectal distension-induced visceromotor response; these findings suggest that DSP-6952 and tegaserod inhibit visceral hypersensitivity in rats. It was concluded that DSP-6952, a novel and orally available 5-HT4 receptor agonist, induced colonic GMCs, enhanced colonic transit, increased defecation without inducing diarrhea, improved drug-induced delay in whole-gut transit, and inhibited visceral hypersensitivity in experimental animals. Therefore, DSP-6952 is expected to become a useful drug for treatment of IBS-C and chronic constipation.

Lack of dopamine supersensitivity in rats after chronic administration of blonanserin: Comparison with haloperidol.

Long-term treatment with antipsychotic drugs in patients with schizophrenia can lead to dopamine supersensitivity psychosis. It is reported that repeated administration of haloperidol caused dopamine supersensitivity in rats. Blonanserin is an atypical antipsychotic drug with high affinity for dopamine D2, D3 and serotonin2A receptors. In this study, we investigated whether chronic administration of blonanserin leads to dopamine supersensitivity. Following oral treatment with blonanserin (0.78 mg/kg) or haloperidol (1.1 mg/kg) twice daily for 28 days, the dopamine D2 agonist quinpirole-induced hyperlocomotion test and a dopamine D2 receptor binding assay were conducted. We found that haloperidol significantly enhanced both quinpirole-induced hyperlocomotion and striatal dopamine D2 receptor density in rats. On the other hand, repeated administration of blonanserin had no effect on either locomotor activity or striatal dopamine D2 receptor density. Further, our results show that mRNA levels of dopamine D2 and D3 receptors in several brain regions were unaffected by repeated administration of both agents. In addition, we examined the effect of the dopamine D3 receptor antagonist PG-01037 on development of dopamine supersensitivity induced by chronic haloperidol treatment and showed that PG-01037 prevents the development of supersensitivity to quinpirole in chronic haloperidol-treated rats. Given the higher affinity of blonanserin at dopamine D3 receptors than haloperidol, antagonism of blonanserin at dopamine D3 receptors may play a role in lack of dopamine supersensitivity after chronic administration. The present findings suggest long-term treatment with antipsychotic dose of blonanserin may be unlikely to lead to dopamine supersensitivity.

The in vitro pharmacology and non-clinical cardiovascular safety studies of a novel 5-HT4 receptor agonist, DSP-6952.

The pharmacological activity of DSP-6952, a novel compound was investigated, compared to that of clinically efficacious gastrointestinal (GI) prokinetic 5-hydroxytryptamine4 (5-HT4) receptor agonists. DSP-6952 had a strong affinity of Ki = 51.9 nM for 5-HT4(b) receptor, and produced contraction in the isolated guinea pig colon with EC50 of 271.6 nM and low intrinsic activity of 57%, similar to tegaserod and mosapride. In the development of the 5-HT4 receptor agonists, cardiovascular risk was deliberately evaluated, because some related prokinetics were reported to cause with cardiovascular adverse events, such as ventricular arrhythmias or ischemia. DSP-6952 showed minimal effects up to 100 µM in human ether-a-go-go-related gene (hERG) channels or guinea pig cardiomyocytes. In telemetered conscious monkeys, DSP-6952 did not affect blood pressure or any electrocardiogram (ECG) up to 180 mg/kg, p.o.; however, DSP-6952 transiently increased heart rate, as well as in anesthetized dogs. The positive chronotropic effects of DSP-6952 were completely antagonized by a 5-HT4 receptor antagonist, and another 5-HT4 receptor agonist, TD-5108 also increased heart rate. These effects are considered a class effect seen in clinically developing and marketed 5-HT4 receptor agonists, and have not been regarded as a critical issue in clinical use. DSP-6952 did not induce contraction in the rabbit coronary artery up to 100 µM, which differed from tegaserod or sumatriptan. These results show that DSP-6952 does not have cardiac ischemic risk via coronary vasoconstriction. In conclusion, DSP-6952 is a promising GI prokinetic compound with partial 5-HT4 receptor agonistic activity as well as a favorable cardiovascular safety profile.

Synthesis and basic evaluation of 7α-(3-[18F]fluoropropyl)-testosterone and 7α-(3-[18F]fluoropropyl)-dihydrotestosterone.

OBJECTIVE: 7α-Substituted androgen derivatives may have the potential to visualize androgen receptors with positron emission tomography. In the present study, we synthesized fluoropropyl derivatives of 7α-(3-[18F]fluoropropyl)-testosterone ([18F]7) and 7α-(3-[18F]fluoropropyl)-dihydrotestosterone ([18F]15), and characterized their in vitro binding, in vivo biodistribution, and performed blocking studies in mature androgen deprived male rats. METHODS: We synthesized [18F]7 and [18F]15. In vitro binding to recombinant rat AR ligand binding domain protein was determined using a competitive radiometric ligand-binding assay with the high-affinity synthetic androgen [17α-methyl-3H]-methyltrienolone ([3H]R1881). In vivo biodistribution was performed in mature male rats treated with diethylstilbestrol (chemical castration). A blocking study was performed by co-administration of dihydrotestosterone (36 µg/animal). RESULTS: 7α-(3-Fluoropropyl)-testosterone (7) and 7α-(3-fluoropropyl)-dihydrotestosterone (15) showed competitive binding to recombinant rat AR ligand binding domain protein. The IC50 value of 15 (13.0 ± 3.3 nM) was higher than 7 (47.8 ± 10.0 nM). In contrast to the AR binding affinity, the ventral prostate uptake of [18F]7 and [18F]15 at 2 h post-injection was similar (0.07 % injected dose/g of tissue). A blocking study indicated that specific binding of [18F]15 is observed in the ventral prostate. [18F]7 and [18F]15 showed moderate levels of bone uptake, which indicates moderate metabolic de-fluorination in rodents. CONCLUSION: [18F]15 is better than [18F]7 in terms of radiochemical yield, in vitro binding affinity, prostate specific binding and stability against in vivo metabolic de-fluorination. However, the net uptake level of [18F]15 in prostate might be insufficient for in vivo visualization. Although [18F]7 and [18F]15 improved in vivo stability against de-fluorination, other basic characterization data in rodents were not superior to the current standard tracer 16ß-[18F]fluoro-5α-dihydrotestosterone. It is also revealed that the shorter side chain length of 7α-[18F]fluoromethyl-dihydrotestosterone is superior to the longer three carbon chain in [18F]15, in terms of net prostate uptake and in vivo metabolic stability.

(R)-[¹¹C]Emopamil as a novel tracer for imaging enhanced P-glycoprotein function.

INTRODUCTION: 2-Isopropyl-5-[methyl-(2-phenylethyl)amino]-2-phenylpentanenitrile (emopamil; EMP) is a calcium channel blocker of the phenylalkylamine class, with weak substrate properties for P-glycoprotein (P-gp). A weak substrate for P-gp would be suitable for measuring enhanced P-gp function. This study was performed to synthesise (R)- and (S)-[(11)C]EMP and characterise their properties as P-gp tracers. METHODS: We synthesised (R)- and (S)-[(11)C]EMP and compared their biodistribution, peripheral metabolism, and effects of the P-gp inhibitor cyclosporine A (CsA, 50 mg/kg). We compared the brain pharmacokinetics of (R)-[(11)C]EMP and (R)-[(11)C]verapamil [(R)-[(11)C]VER] at baseline and CsA pretreatment with small animal positron emission tomography (PET). RESULTS: (R)- and (S)-[(11)C]EMP were synthesised from (R)- and (S)-noremopamil, respectively, by methylation with [(11)C]methyl triflate in the presence of NaOH at room temperature. (R)- and (S)-[(11)C]EMP yields were ~30%, with specific activity>74 GBq/µmol and radiochemical purity>99%. (R)-[(11)C]EMP showed significantly greater uptake in the mouse brain than (S)-[(11)C]EMP. Both showed homogeneous non-stereoselective regional brain distributions. (R)- and (S)-[(11)C]EMP were rapidly metabolised to hydrophilic metabolites. Unchanged plasma (S)-[(11)C]EMP level was significantly lower than that of (R)-[(11)C]EMP 15 minutes post-injection, whilst>88% of radioactivity in the brain was intact at 15 minutes post-injection and was non-stereoselective. CsA pretreatment increased brain activity ~3-fold in mice, but was non-stereoselective. The baseline area-under-the-curve (AUC) of brain radioactivity (0-60 minutes) of (R)-[(11)C]EMP was 2-fold higher than that of (R)-[(11)C]VER, but their AUCs after CsA pretreatment were comparable. CONCLUSIONS: (R)-[(11)C]EMP is a novel tracer for imaging P-gp function with higher baseline uptake than (R)-[(11)C]VER.

Optimization of the alkyl side chain length of fluorine-18-labeled 7α-alkyl-fluoroestradiol.

INTRODUCTION: Several lines of evidence suggest that 7α-substituted estradiol derivatives bind to the estrogen receptor (ER). In line with this hypothesis, we designed and synthesized (18)F-labeled 7α-fluoroalkylestradiol (Cn-7α-[(18)F]FES) derivatives as molecular probes for visualizing ERs. Previously, we successfully synthesized 7α-(3-[(18)F]fluoropropyl)estradiol (C3-7α-[(18)F]FES) and showed promising results for quantification of ER density in vivo, although extensive metabolism was observed in rodents. Therefore, optimization of the alkyl side chain length is needed to obtain suitable radioligands based on Cn-7α-substituted estradiol pharmacophores. METHODS: We synthesized fluoromethyl (23; C1-7α-[(18)F]FES) to fluorohexyl (26; C6-7α-[(18)F]FES) derivatives, except fluoropropyl (C3-7α-[(18)F]FES) and fluoropentyl derivatives (C5-7α-[(18)F]FES), which have been previously synthesized. In vitro binding to the α-subtype (ERα) isoform of ERs and in vivo biodistribution studies in mature female mice were carried out. RESULTS: The in vitro IC50 value of Cn-7α-FES tended to gradually decrease depending on the alkyl side chain length. C1-7α-[(18)F]FES (23) showed the highest uptake in ER-rich tissues such as the uterus. Uterus uptake also gradually decreased depending on the alkyl side chain length. As a result, in vivo uterus uptake reflected the in vitro ERα affinity of each compound. Bone uptake, which indicates de-fluorination, was marked in 7α-(2-[(18)F]fluoroethyl)estradiol (C2-7α-[(18)F]FES) (24) and 7α-(4-[(18)F]fluorobutyl)estradiol (C4-7α-[(18)F]FES) (25) derivatives. However, C1-7α-[(18)F]FES (23) and C6-7α-[(18)F]FES (26) showed limited uptake in bone. As a result, in vivo bone uptake (de-fluorination) showed a bell-shaped pattern, depending on the alkyl side chain length. C1-7α-[(18)F]FES (23) showed the same levels of uptake in uterus and bone compared with those of 16α-[(18)F]fluoro-17ß-estradiol. CONCLUSIONS: The optimal alkyl side chain length of (18)F-labeled 7α-fluoroalkylestradiol was the shortest: C1-7α-[(18)F]FES. Our results indicate that shorter chain lengths within the 4-Å ligand binding cavities of ERα are suitable for 7α-fluoroalkylestradiol derivatives.

Heat-evoked activation of the ion channel, TRPV4.

The mammalian nervous system constantly evaluates internal and environmental temperatures to maintain homeostasis and to avoid thermal extremes. Several members of the transient receptor potential (TRP) family of ion channels have been implicated as transducers of thermal stimuli, including TRPV1 and TRPV2, which are activated by heat, and TRPM8, which is activated by cold. Here we demonstrate that another member of the TRP family, TRPV4, previously described as a hypo-osmolarity-activated ion channel, also can be activated by heat. In response to warm temperatures, TRPV4 mediates large inward currents in Xenopus oocytes and both inward currents and calcium influx into human embryonic kidney 293 cells. In both cases these responses are observed at temperatures lower than those required to activate TRPV1 and can be inhibited reversibly by ruthenium red. Heat-evoked TRPV4-mediated responses are greater in hypo-osmotic solutions and reduced in hyperosmotic solutions. Consistent with these functional properties, we observed TRPV4 immunoreactivity in anterior hypothalamic structures involved in temperature sensation and the integration of thermal and osmotic information. Together, these data implicate TRPV4 as a possible transducer of warm stimuli within the hypothalamus.

Heat hyperalgesia after incision requires TRPV1 and is distinct from pure inflammatory pain.

Postoperative pain significantly impacts patient recovery. However, postoperative pain management remains suboptimal, perhaps because treatment strategies are based mainly on studies using inflammatory pain models. We used a recently developed mouse model of incisional pain to investigate peripheral and spinal mechanisms contributing to heat hyperalgesia after incision. Behavioral experiments involving TRPV1 KO mice demonstrate that, as previously observed in inflammatory models, TRPV1 is necessary for heat (but not mechanical) hyperalgesia after incision. However, in WT mice, neither the proportion of TRPV1 immunoreactive neurons in the DRG nor the intensity of TRPV1 staining in the sciatic nerve was different from that in controls up to 4 days after incision. This result was corroborated by immunoblot analysis of sciatic nerve in rats subjected to an incision, and is distinct from that following inflammation of the rat hind paw, a situation in which TRPV1 expression levels in sciatic nerve increases. In the absence of heat exposure, spinal c-Fos staining was similar between incised TRPV1 KO and WT mice. However, differences in c-Fos staining between heat exposed TRPV1 KO and WT mice after incision suggest that the incision-mediated enhancement of heat-evoked signaling to the spinal cord involves a TRPV1-dependent mechanism. Finally, heat hyperalgesia after incision was reversed by antagonism of spinal non-NMDA receptors, unlike inflammatory hyperalgesia, which is mediated via NMDA receptors . Thus, TRPV1 is important for the generation of thermal hyperalgesia after incision. Our observations suggest that all experimental pain models may not be equally appropriate to guide the development of postoperative pain therapies.

Enhanced thermal avoidance in mice lacking the ATP receptor P2X3.

P2X3 is an ATP-gated cation channel subtype expressed by a subpopulation of primary sensory neurons. In vivo spinal cord recordings in mice lacking P2X3 (P2X3-/-) have suggested that this protein may be important for the coding of peripheral warm stimuli. To explore this possibility more thoroughly, we examined behavioral and electrophysiological responses to thermal stimuli in P2X3-/- mice. As previously reported, recording from the spinal cord dorsal horn of anesthetized P2X3-/- mice revealed a blunted response of wide dynamic range neurons to hind paw heating. When placed in a thermal gradient, however, P2X3-/- mice exhibited an unexpectedly enhanced avoidance of both hot and cold temperatures, relative to controls. In the tail immersion test, mutant mice exhibited shorter withdrawal latencies at temperatures above and below thermoneutrality. Consistent with these changes, P2X3-/- mice exhibited enhanced induction of spinal cord c-FOS following hind paw heating to 45 degrees C. Thus, gain- and loss-of-function thermosensory phenotypes coexist in P2X3-/- mice. No changes in thermal preference were observed in wild-type mice injected subcutaneously with the P2X3 antagonist, A317491 or intrathecally with the P2X3 and P2X1 antagonist TNP-ATP. The reason for this apparent discrepancy is unclear, but we cannot exclude the possibility that compensatory events contribute, at least in part, to the P2X3-/- phenotype. Regardless, this study illustrates the utility of thermal preference assays as part of a comprehensive approach to the analysis of mouse thermosensation.

Attenuated fever response in mice lacking TRPV1.

TRPV1, the capsaicin receptor, is expressed not only in nociceptive neurons, but also in other locations, including the hypothalamus. Studies involving systemic or intrahypothalamic capsaicin administration have suggested a role for TRPV1 in body temperature control. To explore this possibility, we examined thermoregulatory responses in TRPV1-/- mice. These mutant animals exhibited no obvious changes in circadian body temperature fluctuation, tolerance to increased (35 degrees C) or decreased (4 degrees C) ambient temperature or ethanol-induced hypothermia. In contrast, fever production in response to the bacterial pyrogen, lipopolysaccharide (LPS) was significantly attenuated in TRPV1-/- mice. Despite this finding, we detected no significant differences between TRPV1-/- and control mice in the extent of LPS-induced c-Fos expression in numerous fever-related brain subregions. These results suggest that TRPV1 participates in the generation of polyphasic fever, perhaps at sites outside the brain.

Synthesis and evaluation of 7α-(3-[(18)F]fluoropropyl) estradiol.

INTRODUCTION: Several lines of evidence suggest that C-7α-substituted estradiol derivatives are well tolerated by estrogen receptor (ER). In line with this hypothesis, we are interested in the design and synthesis of C-7α-substituted estrogens as molecular probes to visualize ER function. METHODS: We have synthesized 7α-(3-[(18)F]fluoropropyl) estradiol (C3-7α-[(18)F]FES) as a potential radiopharmaceutical for ER imaging by positron emission tomography (PET). In vitro receptor binding and in vivo biodistribution and blocking studies in mature female mice, and in vivo metabolite analysis were carried out. Furthermore, in vivo ER-selective uptake was confirmed using ER-positive T-47D and ER-negative MDA-MB-231 tumor-bearing mice. We also compared the in vivo biodistribution of C3-7α-[(18)F]FES with 16α-[(18)F]FES. RESULTS: C3-7α-[(18)F]FES was produced in moderate yields (30.7%±15.1%, decay corrected) with specific activity of 32.0±18.1GBq/µmol (EOS). The in vitro binding affinity of C3-7α-FES to the ERα isoform was sufficient and equivalent to that of estradiol. C3-7α-[(18)F]FES showed selective uptake in ER-rich tissues, such as the uterus (4.7%ID/g±1.2%ID/g at 15minutes) and ovary (4.0%ID/g±1.0%ID/g at 5minutes). The tissue time activity curves of these organs showed reversible kinetics, indicating suitability for quantitative analysis. The highest contrast was obtained at 120minutes after injection of C3-7α-[(18)F]FES in the uterus (uterus/blood=18, uterus/muscle=17.3) and ovary (ovary/blood=6.3, ovary/muscle=6.0). However, the level of selective uptake of C3-7α-[(18)F]FES was significantly lower than that of 16α-[(18)F]FES. Most radioactivity in the uterus was detected in unchanged form, although peripherally C3-7α-[(18)F]FES was rapidly degraded to hydrophilic metabolites. In accordance with this peripheral metabolism, gradual increases in bone radioactivity were observed, indicating defluorination. Coinjection with estradiol dose-dependently inhibited C3-7α-[(18)F]FES uptake in the uterus and ovary. The in vivo IC50 values of estradiol in the uterus and ovary were 34.4 and 38.5nmol/kg, respectively. Furthermore, in vivo tumor uptake of C3-7α-[(18)F]FES was significantly higher (unpaired t test with Welch's correction; p=0.015) in ER-positive T-47D tumors (2.3%ID/g±0.4%ID/g) than ER-negative MDA-MB-231 tumors (0.9%ID/g±0.1%ID/g). CONCLUSIONS: Although extensive metabolism was observed in rodents, C3-7α-[(18)F]FES showed promising results for quantitative analysis of ER density in vivo. However, the selective uptake of C3-7α-[(18)F]FES was lower than that of 16α-[(18)F]FES. Further optimizations and structure-activity relationship studies of the C-7α-substituted estradiol are needed.