Development of a CD-MEKC method for investigating the metabolism of tamoxifen by flavin-containing monooxygenases and the inhibitory effects of methimazole, nicotine and DMXAA.

A selective and low-cost CD-MEKC method under acidic conditions was developed for investigating the N-oxygenation of tamoxifen (TAM) by flavin-containing monooxygenases (FMOs). The inhibitory effects of methimazole (MMI), nicotine and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on the given FMO reaction were also evaluated; 100 mM phosphate buffer (pH 8.6) was used for performing the enzymatic reaction and the separation of TAM and its metabolite tamoxifen N-oxide (TNO) was obtained with a BGE consisting of 100 mM phosphoric acid solution adjusted to pH 2.5 with triethanolamine containing 50 mM sodium taurodeoxycholate, 20 mM carboxymethyl ß-CD and 20% ACN. The proposed method was applied for the kinetics study of FMO1 using TAM as a substrate probe. A Michaelis-Menten constant (K(m)) of 164.1 µM was estimated from the corrected peak area of the product, TNO. The calculated value of the maximum reaction velocity (V(max)) was 3.61 µmol/min/µmol FMO1; 50% inhibitory concentration and inhibition constant (K(i)) of MMI, the most common alternate substrate FMO inhibitor, were evaluated and the inhibitory effects of two other important FMO substrates, nicotine and DMXAA, a novel anti-tumour agent, were investigated.

A simple and sensitive LC-ESI-MS (ion trap) method for the determination of bupropion and its major metabolite, hydroxybupropion in rat plasma and brain microdialysates.

A specific and highly sensitive liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) method for the direct determination of bupropion (BUP) and its main metabolite hydroxybupropion (HBUP) in rat plasma and brain microdialysate has been developed and validated. The analysis was performed on a Bonus RP C18 (100 mm × 2.1mm i.d., 3.5 µm particles) column using gradient elution with the mobile phase consisting of acetonitrile and ammonium formate buffer (10mM, pH 4). Plasma samples were analyzed after a simple, one-step protein precipitation clean-up with trichloroacetic acid (TCA), however clean-up for microdialysis samples was not necessary, enabling direct injection of the samples into the LC-ESI-MS system. Signals of the compounds were monitored under the multiple reaction monitoring (MRM) mode of the LC-ESI-MS (ion trap) for quantification. The precursor to product ion transitions of m/z 240-184 and m/z 256-238 were used to measure BUP and HBUP, respectively. The method was validated in both plasma and microdialysate samples, and the obtained lower limit of quantification (LLOQ) was 1.5 ng mL(-1) for BUP and HBUP in both matrices. The intra- and inter-day assay variability was less than 15% for both analytes. This LC-ESI-MS method provided simple sampling, rapid clean-up and short analysis time (<9 min), applicable to the routine therapeutic monitoring and pharmacokinetic studies of BUP and HBUP.

The effects of vasoactive intestinal peptide on dura mater nitric oxide levels and vessel-contraction responses in sympathectomized rats.

Nitric oxide (NO) and neurogenic inflammation in dura mater due to nociceptor activation has been implicated for pathophysiology of primary headache disorders. Development of migraine has also been observed in patients treated with ganglion blockage for sympathetic reflex dystrophy. Vasoactive intestinal peptide (VIP) is an antioxidant, anti-inflammatory, and neuroprotective neuropeptide. This study is intended to investigate the effects of VIP on dura mater NO levels and vessel-contraction responses in sympathectomized rats. In the experiments, 30 male rats in five groups were used. Group 1 sympathectomized: under anesthesia, superior cervical sympathetic ganglion was removed via incision at the center line in the neck area. Group 2 sympathectomized + VIP: postoperative VIP of 25 ng/kg/day (0.2 ml) intraperitoneally administered to the rats exposed to the same operations for 5 days. Group 3 sham: ganglia and nerves were exposed but not dissected. Group 4 control: no treatment was done. Group 5 VIP: only VIP was administered for 5 days. Sympathectomy induced a significant increase in dura mater NO levels and VIP decreased NO to control levels and increased the norepinephrine vessel-contraction responses of sympathectomized rats. VIP is an efficient NO modulator in superior cervical ganglionectomized rats.

Isolation of peptide transport system-6 from brain endothelial cells: therapeutic effects with antisense inhibition in Alzheimer and stroke models.

By isolating for the first time ever a peptide transporter from the blood-brain barrier (BBB) and developing an antisense that selectively targets the brain-to-blood efflux component, we were able to deliver a therapeutic concentration of the neurotrophic peptide pituitary adenylate cyclase-activating polypeptide (PACAP) 27 to brain in animal models of Alzheimer's and stroke. Efflux pumps at the BBB are major causes of BBB impermeability to peptides. PACAP is neuroprotective in vitro in femtomole amounts, but brain uptake of PACAP27 is limited by an efflux component of peptide transport system-6 (PTS-6). Here, we characterized, isolated, and sequenced this component of PTS-6, identifying it as beta-F1 ATPase, and colocalized it with PACAP27 on BBB endothelial cells. Antisenses targeting the BBB inhibited PACAP27 efflux, thus increasing brain uptake of PACAP27. Treatment with antisense+PACAP27 improved cognition in a mouse model of Alzheimer's disease and reduced infarct size after cerebral ischemia. This represents the first isolation from BBB tissue of a peptide transporter and shows that inhibition of peptide efflux pumps is a potential strategy for drug delivery to brain.

Passage of VIP/PACAP/secretin family across the blood-brain barrier: therapeutic effects.

In recent years, VIP/PACAP/secretin family has special interest. Family members are vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, glucagon, glucagon like peptide-1 (GLP(1)), GLP(2), gastric inhibitory peptide (GIP), growth hormone releasing hormone (GHRH or GRF), and peptide histidine methionine (PHM). Most of the family members present both in central nervous system (CNS) and in various peripheral tissues. The family members that are released into blood from periphery, especially gut, circulate the brain and they can cross the blood brain barrier. On the other hand, some of the members of this family that present in the brain, can cross from brain to blood and reach the peripheral targets. VIP, secretin, GLP(1), and PACAP 27 are transported into the brain by transmembrane diffusion, a non-saturable mechanism. However, uptake of PACAP 38 into the brain is saturable mechanism. While there is no report for the passage of GIP, GLP(2), and PHM, there is only one report that shows, glucagon and GHRH can cross the BBB. The passage of VIP/PACAP/secretin family members opens up new horizon for understanding of CNS effects of peripherally administrated peptides. There is much hope that those peptides may prove to be useful in the treatment of serious neurological diseases such as Alzheimer's disease, amyotropic lateral sclerosis, Parkinson's disease, AIDS related neuropathy, diabetic neuropathy, autism, stroke and nerve injury. Their benefits in various pathophysiologic conditions undoubtly motivate the development of a novel drug design for future therapeutics.

Passage of vasoactive intestinal peptide across the blood-brain barrier.

We investigated the ability of vasoactive intestinal peptide (VIP) to cross the blood-brain barrier (BBB), the interface between the peripheral circulation and central nervous system (CNS). VIP labeled with 131I (I-VIP) and injected intravenously into mice was taken up by brain as determined by multiple-time regression analysis. Excess unlabeled VIP was unable to impede the entry of I-VIP, indicating that passage is by nonsaturable transmembrane diffusion. High pressure liquid chromatography (HPLC) showed the radioactivity entering the brain to be intact I-VIP. After intracerebroventricular (i.c.v.) injection, I-VIP was sequestered by brain, slowing its efflux from the CNS. In summary, VIP crosses the BBB unidirectionally from blood to brain by transmembrane diffusion.

The antioxidants alpha-lipoic acid and N-acetylcysteine reverse memory impairment and brain oxidative stress in aged SAMP8 mice.

Oxidative stress may play a crucial role in age-related neurodegenerative disorders. Here, we examined the ability of two antioxidants, alpha-lipoic acid (LA) and N-acetylcysteine (NAC), to reverse the cognitive deficits found in the SAMP8 mouse. By 12 months of age, this strain develops elevated levels of Abeta and severe deficits in learning and memory. We found that 12-month-old SAMP8 mice, in comparison with 4-month-old mice, had increased levels of protein carbonyls (an index of protein oxidation), increased TBARS (an index of lipid peroxidation) and a decrease in the weakly immobilized/strongly immobilized (W/S) ratio of the protein-specific spin label MAL-6 (an index of oxidation-induced conformational changes in synaptosomal membrane proteins). Chronic administration of either LA or NAC improved cognition of 12-month-old SAMP8 mice in both the T-maze footshock avoidance paradigm and the lever press appetitive task without inducing non-specific effects on motor activity, motivation to avoid shock, or body weight. These effects probably occurred directly within the brain, as NAC crossed the blood-brain barrier and accumulated in the brain. Furthermore, treatment of 12-month-old SAMP8 mice with LA reversed all three indexes of oxidative stress. These results support the hypothesis that oxidative stress can lead to cognitive dysfunction and provide evidence for a therapeutic role for antioxidants.