Synthesis of (S)-N-[methyl-11C]nicotine and its regional distribution in the mouse brain: a potential tracer for visualization of brain nicotinic receptors by positron emission tomography.

A nicotine agonist, 11C-labeled (S)-nicotine, was synthesized by N-methylation of (S)-nornicotine with [11C]-methyl iodide in dimethylformamide-dimethylsulfoxide in order to study nicotinic receptors in the human brain by positron emission tomography. The radiochemical yield of this N-methylation reaction was more than 90% within 5 min. After purification by high performance liquid chromatography the radiochemical purity of the product was more than 99% and the specific radioactivity was 7.4-11.1 GBq/mumol. The regional distribution of (S)-[11C]nicotine in the mouse brain after intravenous injection was compared with that of (R)-[11C]nicotine. After injection of (S)-[11C]nicotine, the regional uptake of radioactivity was in the following order: cortex greater than thalamu approximately hippocampus greater than striatum greater than hypothalamus greater than cerebellum. Moreover, (S)-[11C]nicotine was displaced from the brain by unlabeled (S)-nicotine, but unlabeled (R)-nicotine caused no change in uptake. In contrast, (R)-[11C]nicotine showed a lower brain uptake and lesser regional differences in radioactivity.     

Kinetic distribution of paeoniflorin in cortex of normal and cerebral ischemia-reperfusion rats after intravenous administration of Paeoniae Radix extract

The time course of paeoniflorin in the cortex of normal and cerebral ischemia-reperfusion rats, following intravenous administration of Paeoniae Radix extract at a dose of 60 mg/kg of paeoniflorin, was determined using high-performance liquid chromatographic (HPLC) assay. The results showed that paeoniflorin could penetrate through the blood-brain barrier to reach the cortex, and that the injuries of ischemia-reperfusion could play an important role in pharmacokinetic process of paeoniflorin in the cortex after intravenous administration of Paeoniae Radix extract. The cortex concentrations of paeoniflorin in cerebral ischemia-reperfusion rats were lower 5 min after dosing and declined more slowly than that in normal control.     

Synthesis of C-labeled imipramine and its biodistribution in mice: a potential tracer for positron emission tomography

A tricyclic antidepressant, C-labeled imipramine was synthesized by N-methylation of desipramine with 11CH3I to assist in the imaging of the human imipramine receptor by positron emission tomography. The radiochemical yield after purification of 11C-imipramine by high performance liquid chromatography was 28-63% at a specific activity of 26-53 Ci/mmol. The time required for synthesis, including purification was 30 min from the end of 11CH3I trapping. The organ distribution of 11C-imipramine was investigated in mice at various times after i.v. injection. The main accumulation of radioactivity was in the kidney, followed by the lung and the heart. In the brain, the radioactivity levels in the hypothalamus and striatum were the highest and remained constant, differentiating them from other portions of the brain. Furthermore, the result of a binding assay with 3H-labeled imipramine suggested that the regional distribution of 11C-imipramine in the same mouse brain correlated to that of the high affinity imipramine binding site.     

Automated method to estimate catecholamine and indoleamine content and turnover rates

A double-label isotopic method for estimation of the rate of formation of serotonin (5-HT) and dopamine (DA) in mouse striatum, hippocampus and cortex was standardized. Mice received an intravenous pulse injection of [3H]tryptophan (TRP) and [3H]tyrosine (TYR) at 2.5, 5, 10 or 20 min before sacrifice by microwave irradiation. Compounds of interest were separated by automated high-performance liquid chromatography and their contents were determined by electrochemical detection. Programmed collection of the TYR, DA, 5-HT and TRP peaks allowed determination of their radioactivity by liquid scintillation. Conversion of [3H]TYR to [3H]DA was nearly ten times greater in striatum than cortex, whereas the formation of [3H]5-HT from [3H]TRP was similar in striatum, cortex and hippocampus.     

High-performance liquid chromatography-fluorescent assay of catechol-O-methyltransferase activity in rat brain

A method to measure catechol- O-methyltransferase (COMT) activity using high performance liquid chromatography-fluorescence detection with norepinephrine (NE) as a natural substrate was optimized for both soluble (S-) and membrane-bound (MB-) COMT activities in rat brain areas, cerebral cortex, cerebellum, hippocampus, brain stem, hypophysis, and hypothalamus. The highest S-COMT activity in Sprague-Dawley rat brain was found in hippocampus. MB-COMT activities in all brain areas were about 3-8 times lower than S-COMT activities. However, considering Vmax/ Km values, specificity constants for NE to S- and MB-COMT contributes mainly to the metabolism of NE in cerebral cortex and cerebellum.     

Systemic Brain Delivery of Antisense Oligonucleotides across the Blood–Brain Barrier with a Glucose-Coated Polymeric Nanocarrier

Current antisense oligonucleotide (ASO) therapies for the treatment of central nervous system (CNS) disorders are performed through invasive administration, thereby placing a major burden on patients. To alleviate this burden, we herein report systemic ASO delivery to the brain by crossing the blood–brain barrier using glycemic control as an external trigger. Glucose-coated polymeric nanocarriers, which can be bound by glucose transporter-1 expressed on the brain capillary endothelial cells, are designed for stable encapsulation of ASOs, with a particle size of about 45 nm and an adequate glucose-ligand density. The optimized nanocarrier efficiently accumulates in the brain tissue 1 h after intravenous administration and exhibits significant knockdown of a target long non-coding RNA in various brain regions, including the cerebral cortex and hippocampus. These results demonstrate that the glucose-modified polymeric nanocarriers enable noninvasive ASO administration to the brain for the treatment of CNS disorders.     

Systemic Brain Delivery of Antisense Oligonucleotides across the Blood–Brain Barrier with a Glucose‐Coated Polymeric Nanocarrier

Current antisense oligonucleotide (ASO) therapies for the treatment of central nervous system (CNS) disorders are performed through invasive administration, thereby placing a major burden on patients. To alleviate this burden, we herein report systemic ASO delivery to the brain by crossing the blood–brain barrier using glycemic control as an external trigger. Glucose‐coated polymeric nanocarriers, which can be bound by glucose transporter‐1 expressed on the brain capillary endothelial cells, are designed for stable encapsulation of ASOs, with a particle size of about 45 nm and an adequate glucose‐ligand density. The optimized nanocarrier efficiently accumulates in the brain tissue 1 h after intravenous administration and exhibits significant knockdown of a target long non‐coding RNA in various brain regions, including the cerebral cortex and hippocampus. These results demonstrate that the glucose‐modified polymeric nanocarriers enable noninvasive ASO administration to the brain for the treatment of CNS disorders.     

Determination of Pharmacokinetics of Tanshinone II A in Mouse Plasma and Brain by High Performance Liquid Chromatography

High performance liquid chromatography (HPLC) method was developed to measure the concentration of tanshinone IIA (Ts IIA) in mouse plasma and brain. The method was applied to preliminary study on pharmacokinetics of Ts IIA in mouse plasma and brain. After an administration of 8.0 mg kg⁻¹ of Ts IIA by an intravenous injection, plasma and brain samples were collected and extracted by liquid-liquid extraction with ethyl acetate and determined by HPLC. This method has a linear range from 0.05 to 7.12 mg l⁻¹ with correlation coefficients of 0.9984 in plasma and a linear range from 0.022 to 2.37 mg l⁻¹ with correlation coefficients of 0.9988 in brain. The limits of quantitation in plasma and brain were 0.050 and 0.022 mg l⁻¹, respectively, and the limits of detection were 0.026 and 0.017 mg l⁻¹, respectively. The intra-day and inter-day precisions were less than 10.3%. The developed method was selective, accurate, and sensitive and can be applied to determine the concentration of Ts IIA in mouse plasma and brain quantitatively after intravenous administration of Ts IIA. It was suitable for the pharmacokinetic study of Ts IIA. The plasma concentration-time curve was fitted as three-compartment model. The peak concentration of Ts IIA in mouse plasma was 1.58 mg l⁻¹, and the value of the areas under the plasma concentration-time curve (AUC 0-t) was 68.18 mg l⁻¹ min⁻¹. The concentration of Ts IIA in mouse brain achieved the peak value of 0.17 mg l⁻¹ 5 min after mainlined, and Ts IIA could be still detected in brain 480 min after mainlined. The results indicated that Ts IIA readily penetrated the blood-brain barrier and could stay in the brain for a long time.     

Determination of dopamine and its relativity of baicalin in rat nuclei after intravenous administration of flavonoids from Scutellariae radix

Baicalin is an active component of Scutellariae radix extracts. It can cross the blood-brain barrier and distribute in cerebral nuclei. However, its mechanism and the effects on the nuclei where it accumulates remain unclear. We used an HPLC-electrochemical detection method to determine the dopamine in cerebral nuclei after intravenous administration of flavonoids from Scutellariae radix and analyzed the relativity between baicalin and DA in cerebral nuclei. We found that the dopamine system is another target system of brain function which baicalin acts on.     

Cognitive impairment related changes in the elemental concentration in the brain of old rat

In order to evaluate the elemental concentration as a function of learning and memory deficiency, six different structures of the brain were analyzed by total reflection X-ray fluorescence spectrometry with synchrotron radiation (SR-TXRF). To evaluate the cognitive processes, the animals were tested in an adaptation of the Morris water maze. After the test, the animals were divided into two groups: cognitively healthy (control group) and cognitively impaired. The measurements were carried out at XRF beam line at Light Synchrotron Brazilian laboratory, Campinas, Brazil. The following elements were identified: Al, P, S, Cl, K, Ca, Ti, Cr, Fe, Cu, Zn, Br and Rb. K concentration was higher in all regions of the brain studied for control group than the cognitively impaired group. Moreover, the control group presented higher levels for P and Fe in the entorhinal cortex, in the temporal cortex (only P), in the hypothalamus and in the thalamus, than the cognitively impaired group. Br concentration in the animals which presented cognitive impairment was three times larger in the hypothalamus and thalamus, twice larger in temporal cortex and higher in visual cortex than the cognitively healthy group. Cu was more remarkable in the hippocampus and hypothalamus from the animals with cognitive impairment than the control group.We observed that the cognitively impaired group presented highest concentrations of Br and Cu in certain areas than the control group, on the other hand, this group presented highest levels of K for all brain areas studied.     

Application of an improved proteomics method, fluorogenic derivatization-liquid chromatography-tandem mass spectrometry, to differential analysis of proteins in small regions of mouse brain

To identify age-related proteins in small regions of mouse brain, we improved a proteomics approach, fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS), and applied the method to the differential proteome analysis of aging in cerebral cortex, hippocampus and brainstem. The method showed good accuracy with RSDs <10% for between-day protein peak heights, and much better sensitivity for the detection of proteins compared to other proteomics approaches. The existence of 28 regionally specific age-related proteins in mouse brain was demonstrated. These results verified that the small brain regions could be the targets for proteome analysis by the FD-LC-MS/MS method.     

Regional cerebral blood flow measurement in brain tumors

The regional cerebral blood flow (CBF) was determined on seventeen patients with brain tumors. Ring type single photon emission CT (SPECT) was used following intravenous injection of 133Xe. Case materials included eleven meningiomas and six malignant gliomas. Evaluation was performed with emphasis on the following points; Correlation of the flow data within tumors to the angiographic tumor stains, Influence of tumors on the cerebral blood flow of the normal brain tissue, Correlation between degree of peripheral edema and the flow data of the affected hemispheres. There was significant correlation between flow data within tumors and angiographic tumor stains in meningiomas. Influence of tumors on cerebral blood flow of the normal tissue was greater in meningiomas than in gliomas. There was negative correlation between the degree of peripheral edema and the flow data of the affected hemisphere. It has been concluded that the measurement of CBF in brain tumors is a valuable method in evaluation of brain tumors.     

Determination of dehydrodiisoeugenol in rat tissues using HPLC method

Dehydrodiisoeugenol (DDIE) is a bioactive neolignan from the seeds of Myristica fragrans Houtt., which exhibits good anti-inflammatory activity. A rapid and simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of DDIE in rat tissues after intravenous administration. The tissue samples were processed by liquid-liquid extraction. The analyses were successfully carried out on a Diamonsiltrade mark ODS C18 column (250 x 4.6 mm i.d., 5 microm) equipped with a C18 guard column (8 x 4.6 mm i.d., 5 microm). The mobile phase was the system of methanol-water (4:1, v/v). The UV detection was set at 270 nm. The calibration curves were linear from 0.4 to 200.0 microg/g with the correlation coefficients (r(2)) greater than 0.998. The intra- and inter-day precisions in quality control samples were less than 10% and the accuracies were in the range 85.4-110.3%. The average recoveries from all the tissues were between 84.4 and 106.0%. This assay method has been successfully used to study the tissues distribution of DDIE in rats after intravenous administration. The result suggests that DDIE is distributed to rat tissues rapidly with possibly greater initial concentrations in liver and brain than in other tissues.     

In vivo behavior of 111In-DTPA in rat and mouse after intra-ventricular administration (author's transl)

In vivo behavior of 111In-DTPA in rat and mouse after intra-ventricular administration was studied. Thus, 50 muCi and 35 muCi of 111In-DTPA was injected intra-ventricularly to rat and mouse respectively. At specific time intervals, the animals were sacrificed, then distribution in organs was determined by radioactivity counting and autoradiographic method. Urinary and fecal excretion were separately collected and excretion rates were estimated. Metabolites in urine of rat were examined with chromatography. A part of 111In-DTPA injected intra-ventricularly to the animals migrated to subarachnoid space, then radioactivity in cerebrospinal fluid effused into blood with about 1 hr initial half-life. Blood clearance was also rapid, about 1 hr after administration the blood level reached maximum and then decreased showing an initial half-life of about 1 hr. The predominant excretion route in rat was urinary and about 90% and 5% of administered dose were excreted within 48 hr through urine and feces respectively. Judging from the Rf-value of radioactivity peak on chromatograms, 111In-DTPA seems to be excreted without suffering any metabolic change. Concerning to the behavior of 111In-DTPA in male and female rat, no difference was observed, and the distribution pattern of 111In-DTPA in mouse was similar to that of rat.     

侧脑室注射氯化镧对大鼠血清生长素和甲状腺素的影响

西方研究了侧脑室注入ＬａＣｌ３对大鼠血清中生长系,甲状腺素,促甲状腺素和下丘脑中生长抑素的影响。侧脑室注射０．００１和０．０１ｍｏｌ．ｌ＾－１ＬａＣｌ３,血清中Ｔ４和ＧＨ含量明显高于对照组,０．１和０．５ｍｏｌ．ｌ＾－１ＬａＣｌ３组血清Ｔ４和ＧＨ未见明显变化。     

Fibrinolytic effect of tissue plasminogen activator on cerebral embolism in stroke-prone spontaneously hypertensive rats.

To study the thrombolytic effect of tissue plasminogen activator (t-PA) on cerebral emboli, we characterized cerebral embolization in stroke-prone spontaneously hypertensive rats (SHRSPs) and Wistar Kyoto rats (WKYs). [125I]Fibrin clot particles (20-100 microns diameter) were injected twice at an interval of 90 min into the left internal carotid artery of WKYs and SHRSPs. After each injection, spontaneous embolus dissolution was monitored with a gamma-ray detector placed on the head of the embolic rats. Embolus dissolution was spontaneously generated in 15 min after the injection of fibrin clots. In WKYs, 21% and 42% of the clots were dissolved 30 and 90 min after the second embolization, respectively. On the other hand, the spontaneous embolus dissolution in SHRSPs was significantly lower than that of WKYs, indicating that the endogenous fibrinolytic ability of SHRSPs is less potent than that of normotensive rats. The intravenous administration of t-PA at doses of 75, 250 and 750 micrograms/kg caused a dose-dependent embolus dissolution in SHRSPs. Furthermore, systematically applied t-PA produced embolus dissolution without causing systemic plasminogen activation, fibrinogen breakdown or bleeding. In conclusion, the intravenous administration of t-PA produces selective embolus dissolution without systemic fibrino(geno)lysis in a cerebral embolic SHRSP.     

A prodrug of NMDA/glycine site antagonist, L-703,717, with improved BBB permeability: 4-acetoxy derivative and its positron-emitter labeled analog

4-Acetoxy derivative (1) of L-703,717, a high-affinity (IC50=4.5 nM) antagonist for the glycine site of NMDA receptors, was synthesized and its brain uptake was examined using a carbon-11 labeled analog ([11C]1). Initial radioactivity in the brain after intravenous injection of [11C]1 was a 2-fold that of [11C]L-703,717 in mice. Rapid bioconversion of [11C]1 into [11C]L-703,717 was demonstrated by metabolite analyses of rat brain after [11C]1 injection. Ex vivo autoradiography of [11C]1 in rat brain showed the same cerebellar localization of radioactivity as [11C]L-703,717. These results indicate that 1 is a promising pharmacological tool as a prodrug of L-703,717 with improved BBB permeability.     

Targeting polysaccharide–methotrexate conjugates to the rat brain

Delivery of drugs to brain tumors is difficult due to the impermeability, and variable nature of the blood/tumor barrier. Of the various strategies designed to improve drug uptake-intracellularly and into the brain, cationic carriers seem to offer an advantage. In the current investigation cationic polysaccharide–methotrexate conjugates were examined in the rat. Conjugates were prepared from N,O-carboxymethyl chitosan (NOCC) and ³H-methotrexate (MTX) by two different synthetic schemes, resulting in NOCCMTX-1 and NOCCMTX-2. NOCCMTX-1 appeared to have a higher degree of cross-polymerization than NOCCMTX-2. Each conjugate and free MTX were administered intra-arterially in a retrograde manner in the external carotid artery at a MTX dose of 1 mg/kg as a constant rate infusion over 30 min. Animals were sacrificed at various times after administration, and blood and tissue samples collected, processed in a sample oxidizer, and then measured for radioactivity. Brain MTX concentrations were 18- and 12-fold greater at 15 min and 3 hr, respectively, following NOCCMTX-1 administration compared to free MTX treatment. However, NOCCMTX-1 resulted in animal death at about 12 hr after administration. NOCMTX-2 was found to be non-toxic, yet did not increase brain MTX concentrations compared to free drug administrations after correction for MTX in residual blood. Further investigations are planned to combine the positive drug targeting effect of NOCCMTX-1 with the safety of NOCCMTX-2.     

Quantification and Metabolite Identification of Sulfasalazine in Mouse Brain and Plasma Using Quadrupole-Time-of-Flight Mass Spectrometry

Sulfasalazine (SAS), an anti-inflammatory drug with potent cysteine/glutamate antiporter system xc-(SXC) inhibition has recently shown beneficial effects in brain-related diseases. Despite many reports related to central nervous system (CNS) effect of SAS, pharmacokinetics (PK) and metabolite identification studies in the brain for SAS were quite limited. The aim of this study was to investigate the pharmacokinetics and metabolite identification of SAS and their distributions in mouse brain. Using in vivo brain exposure studies (neuro PK), the PK parameters of SAS was calculated for plasma as well as brain following intravenous and oral administration at 10 mg/kg and 50 mg/kg in mouse, respectively. In addition, in vivo metabolite identification (MetID) studies of SAS in plasma and brain were also conducted. The concentration of SAS in brain was much lower than that in plasma and only 1.26% of SAS was detected in mouse brain when compared to the SAS concentration in plasma (brain to plasma ratio (%): 1.26). In the MetID study, sulfapyridine (SP), hydroxy-sulfapyridine (SP-OH), and N-acetyl sulfapyridine (Ac-SP) were identified in plasma, whereas only SP and Ac-SP were identified as significant metabolites in brain. As a conclusion, our results suggest that the metabolites of SAS such as SP and Ac-SP might be responsible for the pharmacological effect in brain, not the SAS itself.