酶促萘普生酯的选择性水解拆分

柱状假丝酵母脂肪酶可以选择性催化S－萘普生甲酯、乙酯和乙氧基乙基酯发生水解,从而对化学合成的混旋萘普生进行拆分,制备具有高光学纯度的S－对映体。利用中等极性大孔吸附树脂HZ－806作为固定化载体,可在酶分子周围营造一个有利于反应的微环境,有效地提高酶的催化活力及解决酶的回收。在中等极性大孔吸附树脂固定化酶填充床反应器中,混旋乙氧基乙基萘普生酯可被连续水解拆分,当流量为72mL/h时,酯的水解率为17％,光学纯度为89．1％。     

A Novel Program Trains Community‐Academic Teams to Build Research and Partnership Capacity

The Community‐Engaged Research Team Support (CERTS) program was developed and tested to build research and partnership capacity for community‐engaged research (CEnR) teams. Led by the Northwestern University Clinical and Translational Sciences Institute (NUCATS), the goals of CERTS were: (1) to help community‐academic teams build capacity for conducting rigorous CEnR and (2) to support teams as they prepare federal grant proposal drafts. The program was guided by an advisory committee of community and clinical partners, and representatives from Chicago''s Clinical and Translational Science Institutes. Monthly workshops guided teams to write elements of NIH‐style research proposals. Draft reviewing fostered a collaborative learning environment and helped teams develop equal partnerships. The program culminated in a mock‐proposal review. All teams clarified their research and acquired new knowledge about the preparation of NIH‐style proposals. Trust, partnership collaboration, and a structured writing strategy were assets of the CERTS approach. CERTS also uncovered gaps in resources and preparedness for teams to be competitive for federally funded grants. Areas of need include experience as principal investigators, publications on study results, mentoring, institutional infrastructure, and dedicated time for research.     

[18F]DPA‐C5yne,a novel fluorine‐18‐labelled analogue of DPA‐714: radiosynthesis and preliminary evaluation as a radiotracer for imaging neuroinflammation with PET

DPA‐C5yne, the lead compound of a novel series of DPA‐714 derivatives in which the fluoroethoxy chain linked to the phenylpyrazolopyrimidine scaffold has been replaced by a fluoroalkyn‐1‐yl moiety, is a high affinity (K_i: 0.35 nM) and selective ligand targeting the translocator protein 18 kDa. In the present work, DPA‐C5yne was labelled with no‐carrier‐added [¹⁸F]fluoride based on a one‐step tosyloxy‐for‐fluorine nucleophilic substitution reaction, purified by cartridge and HPLC, and formulated as an i.v. injectable solution using a TRACERLab FX N Pro synthesizer. Typically, 4.3–5.2 GBq of [¹⁸F]DPA‐C5yne, ready‐to‐use, chemically and radiochemically pure (> 95%), was obtained with specific radioactivities ranging from 55 to 110 GBq/µmol within 50–60 min, starting from a 30 GBq [¹⁸F]fluoride batch (14–17%). LogP and LogD of [¹⁸F]DPA‐C5yne were measured using the shake‐flask method and values of 2.39 and 2.51 were found, respectively. Autoradiography studies performed on slices of ((R,S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolopropionique (AMPA)‐lesioned rat brains showed a high target‐to‐background ratio (1.9 ± 0.3). Selectivity and specificity of the binding for the translocator protein was demonstrated using DPA‐C5yne (unlabelled), PK11195 and Flumazenil (central benzodiazepine receptor ligand) as competitors. Furthermore, DPA‐C5yne proved to be stable in plasma at 37°C for at least 90 min.     

Determination of [11C]PBR28 binding potential in vivo: a first human TSPO blocking study

Positron emission tomography (PET) targeting the 18 kDa translocator protein (TSPO) is used to quantify neuroinflammation. Translocator protein is expressed throughout the brain, and therefore a classical reference region approach cannot be used to estimate binding potential (BP_ND). Here, we used blockade of the TSPO radioligand [¹¹C]PBR28 with the TSPO ligand XBD173, to determine the non-displaceable volume of distribution (V_ND), and hence estimate the BP_ND. A total of 26 healthy volunteers, 16 high-affinity binders (HABs) and 10 mixed affinity binders (MABs) underwent a [¹¹C]PBR28 PET scan with arterial sampling. Six of the HABs received oral XBD173 (10 to 90 mg), 2 hours before a repeat scan. In XBD173-dosed subjects, V_ND was estimated via the occupancy plot. Values of BP_ND for all subjects were calculated using this V_ND estimate. Total volume of distribution (V_T) of MABs (2.94±0.31) was lower than V_T of HABs (4.33±0.29) (P<0.005). There was dose-dependent occupancy of TSPO by XBD173 (ED50=0.34±0.13 mg/kg). The occupancy plot provided a V_ND estimate of 1.98 (1.69, 2.26). Based on these V_ND estimates, BP_ND for HABs is approximately twice that of MABs, consistent with predictions from in vitro data. Our estimates of [¹¹C]PBR28 V_ND and hence BP_ND in the healthy human brain are consistent with in vitro predictions. XBD173 blockade provides a practical means of estimating V_ND for TSPO targeting radioligands.     

Mitochondrial ligand inhibits store-operated calcium influx and COX-2 production in human microglia

We used calcium sensitive fluorescence microscopy to investigate the actions of PK11195, a ligand for the mitochondrial peripheral benzodiazepine receptor (PBR), to modulate Ca2+ influx through store-operated channels (SOC) in human microglia. PK11195 effectively blocked SOC-mediated Ca2+ influx induced by platelet-activating factor (PAF) in a dose-dependent manner (IC50 of 9 microM). A prolonged SOC-mediated Ca2+ entry was also induced using the sarcoplasmic endoreticulum Ca2+-ATPase (SERCA) inhibitor cyclopiazonic acid (CPA) to deplete intracellular endoplasmic reticulum (ER) stores; a single concentration of PK11195 (at 20 microM) reduced SOC-mediated Ca2+ influx by 78%. RT-PCR and immunocytochemical analysis results showed PK11195 also inhibited the expression and production of cyclooxygenase-2 (COX-2) triggered by PAF stimulation. These results suggest that activation of the PBR in mitochondria is linked to reduced entry of Ca2+ through plasmalemmal SOC and subsequent modulation of cellular functions in human microglia.     

A genetic polymorphism for translocator protein 18?kDa affects both in vitro and in vivo radioligand binding in human brain to this putative biomarker of neuroinflammation

Second-generation radioligands for translocator protein (TSPO), an inflammation marker, are confounded by the codominant rs6971 polymorphism that affects binding affinity. The resulting three groups are homozygous for high-affinity state (HH), homozygous for low-affinity state (LL), or heterozygous (HL). We tested if in vitro binding to leukocytes distinguished TSPO genotypes and if genotype could affect clinical studies using the TSPO radioligand [¹¹C]PBR28. In vitro binding to leukocytes and [¹¹C]PBR28 brain imaging were performed in 27 human subjects with known TSPO genotype. Specific [³H]PBR28 binding was measured in prefrontal cortex of 45 schizophrenia patients and 47 controls. Leukocyte binding to PBR28 predicted genotype in all subjects. Brain uptake was ∼40% higher in HH than HL subjects. Specific [³H]PBR28 binding in LL controls was negligible, while HH controls had ∼80% higher binding than HL controls. After excluding LL subjects, specific binding was 16% greater in schizophrenia patients than controls. This difference was insignificant by itself (P=0.085), but was significant after correcting for TSPO genotype (P=0.011). Our results show that TSPO genotype influences PBR28 binding in vitro and in vivo. Correcting for this genotype increased statistical power in our postmortem study and is recommended for in vivo positron emission tomography studies.     

Radiosynthesis of [18F]PBR111, a selective radioligand for imaging the translocator protein (18 kDa) with PET

PBR111 (2‐(6‐chloro‐2‐(4‐(3‐fluoropropoxy)phenyl)imidazo[1,2‐a]pyridin‐3‐yl)‐N,N‐diethylacetamide) is a novel, reported, high‐affinity and selective ligand for the translocator protein (18 kDa). PBR111 has been labelled with fluorine‐18 (half‐life: 109.8 min) using our Zymate‐XP robotic system. The process involves (A) a simple one‐step tosyloxy‐for‐fluorine nucleophilic aliphatic substitution (performed at 165°C for 5 min in DMSO using K[¹⁸F]F‐Kryptofix^®222 and 6.8–7.6 µmol of the corresponding tosylate as precursor for labelling) followed by (B) C‐18 PrepSep cartridge pre‐purification and (C) semi‐preparative HPLC purification on a Waters Symmetry^® C‐18. Up to 4.8 GBq (130 mCi) of [¹⁸F]PBR111 could be obtained with specific radioactivities ranging from 74 to 148 GBq/µmol (2–4 Ci/µmol) in 75–80 min (HPLC purification and SepPak^®‐based formulation included), starting from a 37.0 GBq (1.0 Ci) [¹⁸F]fluoride batch. Overall non‐decay‐corrected isolated yields were 8–13% (13–21% decay‐corrected). Copyright © 2008 John Wiley & Sons, Ltd.     

The Peripheral Benzodiazepine Receptors: A Review

Peripheral benzodiazepine receptors (PBRs) have been identified in various peripheral tissues as well as in glial cells in the brain. This review describes the tissue and subcellular distribution of the PBR in mammalian tissues and analyzes its many putative endogenous ligands. It deals with the pharmacological, structural and molecular characterization of the PBR, the proteins associated with the receptor (VDAC, ANC, PRAX-1) and their roles in cell growth and differentiation, cancer, steroid biosynthesis, and other physiological roles.     

The high affinity peripheral benzodiazepine receptor ligand DAA1106 binds specifically to microglia in a rat model of traumatic brain injury: implications for PET imaging

Traumatic brain injury (TBI) is a significant cause of mortality, morbidity, and disability. Microglial activation is commonly observed in response to neuronal injury which, when prolonged, is thought to be detrimental to neuronal survival. Activated microglia can be labeled using PK11195, a ligand that binds the peripheral benzodiazepine receptor (PBR), receptors which are increased in activated microglia and sparse in the resting brain. We compared the binding properties of two PBR ligands PK11195 and DAA1106 in rats using the controlled cortical impact (CCI) model of experimental TBI. While both ligands showed relative increases with specific binding in the cortex ipsilateral to injury compared to the contralateral side, [(3)H]DAA1106 showed higher binding affinity compared with [(3)H](R)-PK11195. Combined immunohistochemistry and autoradiography in brain tissues near the injury site showed that [(3)H]DAA1106 binding co-registered with activated microglia more than astrocytes. Further, increased [(3)H]DAA1106-specific binding positively correlated with the degree of microglial activation, and to a lesser degree with reactive astrocytosis. Finally, in vivo administration of each ligand in rats with TBI showed greater retention of [(11)C]DAA1106 compared to [(11)C](R)-PK11195 at the site of the contusion as assessed by ex vivo autoradiography. These results in a rat model of TBI indicate that [(11)C]DAA1106 binds with higher affinity to microglia when compared with PK11195, suggesting that [(11)C]DAA1106 may represent a better ligand than [(11)C](R)-PK11195 for in vivo PET imaging of activated microglia in TBI.