Gene Polymorphisms and Drug-Drug Interactions Determine the Metabolic Profile of Blonanserin.

This study aimed to evaluate the effects of cytochrome P450 3A4 (CYP3A4) gene polymorphism and drug interaction on the metabolism of blonanserin. Human recombinant CYP3A4 was prepared using the Bac-to-Bac baculovirus expression system. A microsomal enzyme reaction system was established, and drug-drug interactions were evaluated using Sprague-Dawley rats. Ultra-performance liquid chromatography-tandem mass spectrometry was used to detect the concentrations of blonanserin and its metabolite. Compared with wild type CYP34A, the relative clearance of blonanserin by CYP3A4.29 significantly increased to 251.3%, while it decreased notably with CYP3A4.4, 5, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, 23, 24, 28, 31, 33, and 34, ranging from 6.09% to 63.34%. Among 153 tested drugs, nimodipine, felodipine, and amlodipine were found to potently inhibit the metabolism of blonanserin. Moreover, the inhibitory potency of nimodipine, felodipine, and amlodipine varied with different CYP3A4 variants. The half-maximal inhibitory concentration and enzymatic kinetics assay demonstrated that the metabolism of blonanserin was noncompetitively inhibited by nimodipine in rat liver microsomes and was inhibited in a mixed manner by felodipine and amlodipine in both rat liver microsomes and human liver microsomes. When nimodipine and felodipine were coadministered with blonanserin, the area under the blood concentration-time curve (AUC)(0-t), AUC(0-∞), and C max of blonanserin increased. When amlodipine and blonanserin were combined, the C max of blonanserin C increased remarkably. The vast majority of CYP3A4 variants have a low ability to catalyze blonanserin. With combined administration of nimodipine, felodipine, and amlodipine, the elimination of blonanserin was inhibited. This study provides the basis for individualized clinical use of blonanserin. SIGNIFICANCE STATEMENT: The enzyme kinetics of novel CYP3A4 enzymes for metabolizing blonanserin were investigated. Clearance of blonanserin by CYP3A4.4, 5, 7-10, 12-14, 16-18, 23-24, 28, 31, 33, and 34 decreased notably, but increased with CYP3A4.29. Additionally, we established a drug interaction spectrum for blonanserin, in which nimodipine, felodipine, and amlodipine kinetics exhibited mixed inhibition. Moreover, their inhibitory potencies decreased with CYP3A4.4 and 5 compared to CYP3A4.1. This study provides essential data for personalized clinical use of blonanserin.

Deletion of voltage-gated calcium channels in astrocytes decreases neuroinflammation and demyelination in a murine model of multiple sclerosis.

The experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis was used in combination with a Cav1.2 conditional knock-out mouse (Cav1.2KO) to study the role of astrocytic voltage-gated Ca++ channels in autoimmune CNS inflammation and demyelination. Cav1.2 channels were specifically ablated in Glast-1-positive astrocytes by means of the Cre-lox system before EAE induction. After immunization, motor activity was assessed daily, and a clinical score was given based on the severity of EAE symptoms. Cav1.2 deletion in astrocytes significantly reduced the severity of the disease. While no changes were found in the day of onset and peak disease severity, EAE mean clinical score was lower in Cav1.2KO animals during the chronic phase of the disease. This corresponded to better performance on the rotarod and increased motor activity in Cav1.2KO mice. Furthermore, decreased numbers of reactive astrocytes, activated microglia, and infiltrating lymphocytes were found in the lumbar section of the spinal cord of Cav1.2KO mice 40 days after immunization. The degree of myelin protein loss and size of demyelinated lesions were also attenuated in Cav1.2KO spinal cords. Similar results were found in EAE animals treated with nimodipine, a Cav1.2 Ca++ channel inhibitor with high affinity to the CNS. Mice injected with nimodipine during the acute and chronic phases of the disease exhibited lower numbers of reactive astrocytes, activated microglial, and infiltrating immune cells, as well as fewer demyelinated lesions in the spinal cord. These changes were correlated with improved clinical scores and motor performance. In summary, these data suggest that antagonizing Cav1.2 channels in astrocytes during EAE alleviates neuroinflammation and protects the spinal cord from autoimmune demyelination.

ISSLS PRIZE in basic science 2023: Lactate in lumbar discs-metabolic waste or energy biofuel? Insights from in vivo MRS and T2r analysis following exercise and nimodipine in healthy volunteers.

PURPOSE: To quantitatively assess the dynamic changes of Lactate in lumbar discs under different physiological conditions using MRS and T2r. METHODS: In step1, MRS and T2r sequences were standardized in 10 volunteers. Step2, analysed effects of high cellular demand. 66 discs of 20 volunteers with no back pain were evaluated pre-exercise (EX-0), immediately after targeted short-time low back exercises (EX-1) and 60 min after (EX-2). In Step 3, to study effects of high glucose and oxygen concentration, 50 lumbar discs in 10 volunteers were analysed before (D0) and after 10 days intake of the calcium channel blocker, nimodipine (D1). RESULTS: Lactate showed a distinctly different response to exercise in that Grade 1 discs with a significant decrease in EX-1 and a trend for normalization in Ex-2. In contrast, Pfirrmann grade 2 and 3 and discs above 40 years showed a higher lactate relative to proteoglycan in EX-0, an increase in lactate EX-1 and mild dip in Ex-2. Similarly, following nimodipine, grade 1 discs showed an increase in lactate which was absent in grade 2 and 3 discs. In contrast, exercise and Nimodipine had no significant change in T2r values and MRS spectrum of proteoglycan, N-acetyl aspartate, carbohydrate, choline, creatine, and glutathione across age groups and Pfirrmann grades. CONCLUSION: MRS documented changes in lactate response to cellular demand which suggested a 'Lactate Symbiotic metabolic Pathway'. The differences in lactate response preceded changes in Proteoglycan/hydration and thus could be a dynamic radiological biomarker of early degeneration.

Aging differentially affects LTCC function in hippocampal CA1 and piriform cortex pyramidal neurons.

Aging is associated with cognitive decline and memory loss in humans. In rats, aging-associated neuronal excitability changes and impairments in learning have been extensively studied in the hippocampus. Here, we investigated the roles of L-type calcium channels (LTCCs) in the rat piriform cortex (PC), in comparison with those of the hippocampus. We employed spatial and olfactory tasks that involve the hippocampus and PC. LTCC blocker nimodipine administration impaired spontaneous location recognition in adult rats (6-9 months). However, the same blocker rescued the spatial learning deficiency in aged rats (19-23 months). In an odor-associative learning task, infusions of nimodipine into either the PC or dorsal CA1 impaired the ability of adult rats to learn a positive odor association. Again, in contrast, nimodipine rescued odor associative learning in aged rats. Aged CA1 neurons had higher somatic expression of LTCC Cav1.2 subunits, exhibited larger afterhyperpolarization (AHP) and lower excitability compared with adult neurons. In contrast, PC neurons from aged rats showed higher excitability and no difference in AHP. Cav1.2 expression was similar in adult and aged PC somata, but relatively higher in PSD95- puncta in aged dendrites. Our data suggest unique features of aging-associated changes in LTCCs in the PC and hippocampus.

N-methyl-D-aspartate receptor-mediated spinal cord ischemia-reperfusion injury and its protective mechanism.

INTRODUCTION: This study investigated the specific mechanism of N-methyl-D-aspartate (NMDA) receptor-mediated spinal cord ischemia-reperfusion by comparing the protective effects of the voltage-gated Ca2+ channel blocker nimodipine and the NMDA receptor blocker K-1024 on the spinal cord. MATERIAL AND METHODS: In this study, 42 SD rats were divided randomly into four groups: non-blocking (n = 6), normal saline (n = 12), K-1024 (n = 12) and nimodipine (n = 12). The rats in three groups (saline, K-1024, nimodipine) received an intraperitoneal injection 30 minutes before ischemia. In these three groups, 6 out of 12 rats were selected randomly to have their thoracic aorta blocked with a balloon to induce spinal cord ischemia for 10 minutes. Then, the spinal cord tissues were collected. The remaining six rats were evaluated for nerve function at 1, 2, 4 and 8 hours after reperfusion. The lumbar spinal cord was removed for histological examination. The release of neurotransmitter amino acids was observed by high-pressure liquid chromatography, and the protein expression level of neuronal nitric oxide synthase (nNOS) in the spinal cord was determined by immunohistochemistry. RESULTS: All the animals in the normal saline group and five in the nimodipine group were paralysed after ischemia. Compared with the normal saline and nimodipine groups, the rats in the K-1024 group had more normal motor neurons and better behavioural scores. In addition, the histopathology of the rats in the K-1024 group was significantly better than in the normal saline and nimodipine groups. After 10 minutes of ischemia, there was no significant difference in glutamate concentration in each group. The protein expression level of nNOS in the K-1024 group was significantly downregulated compared with the saline and nimodipine groups. At 8 hours after reperfusion, the protein expression level of nNOS in the K-1024 group was significantly upregulated compared with the normal saline group. CONCLUSIONS: The specific mechanism of the NMDA receptor blocker K-1024 in protection against spinal cord ischemia-reperfusion injury is related closely to the inhibition of NMDA receptors and the downregulation of the protein expression level of nNOS.

The effect of CYP3A4 genetic polymorphism and drug interaction on the metabolism of istradefylline.

AIM: This study investigated into the effect of CYP3A4 genetic polymorphism on istradefylline metabolism. Moreover, the potential drug-drug interaction with istradefylline was determined as well as underlied mechanism. METHOD: In vitro, enzymatic reaction was performed to determine the kinetic parameters of CYP3A4 and its variants on catalyzing istradefylline. Meanwhile, the rat liver microsomes incubation assay was applied to screen interacting drugs. In vivo, SD rats were used to investigate the selected drug interaction. UPLC-MS/MS was used to detect the metabolite M1. RESULT: The results demonstrated that the relative clearance rate of CYP3A4.29 decrease significantly compared with CYP3A4.1. But there is no statistically diverse in activities among CYP3A4.1, 2 and 3. The relative clearance rates of the remaining variants are significantly decreased compared with CYP3A4.1. In addition, 148 drugs were screened to determine the potential interaction with istradefylline, among which calcium channel blockers were identified. It's indicated that nimodipine has a significant inhibitory effect on metabolizing istradefylline with IC50 of 6.927 ± 0.372 µM, which via competitive and non-competitive mixed mechanism. In vivo, when istradefylline and nimodipine was co-administered to SD rats, we found the main pharmacokinetic parameters of M1 reduced remarkably, including AUC, MRT, Cmax and CLz/F. CONCLUSION: CYP3A4 genetic polymorphism and nimodipine affect the metabolism of istradefylline. Thus, the present study provided reference data for clinical individualized medicine of istradefylline.

Nimodipine Attenuates Early Brain Injury by Protecting the Glymphatic System After Subarachnoid Hemorrhage in Mice.

The glymphatic system (GS) plays an important role in subarachnoid hemorrhage (SAH). Nimodipine treatment provides SAH patients with short-term neurological benefits. However, no trials have been conducted to quantify the relationship between nimodipine and GS. We hypothesized that nimodipine could attenuate early brain injury (EBI) after SAH by affecting the function of the GS. In this study, we assessed the effects of nimodipine, a dihydropyridine calcium channel antagonist, on mice 3 days after SAH. The functions of GS were assessed by immunofluorescence and western blot. The effects of nimodipine were assessed behaviorally. Concurrently, correlation analysis was performed for the functions of GS, immunofluorescence and behavioral function. Our results indicated that nimodipine improved GS function and attenuated neurological deficits and brain edema in mice with SAH. Activation of the cAMP/PKA pathway was involved in this process. GS function was closely associated with perivascular AQP4 polarization, cortical GFAP/AQP4 expression, brain edema and neurobehavioral function. In conclusion, this study shows for the first time that nimodipine plays a neuroprotective role in the period of EBI after SAH in mice through the GS.

Cerebralcare Granule® combined with nimodipine improves cognitive impairment in bilateral carotid artery occlusion rats by reducing lipocalin-2.

AIMS: Clinically, Cerebralcare Granule® (CG) has been widely utilized to treat various types of headache, chronic cerebral insufficiency and other diseases, and the effect is significant. Clinical studies have shown that CG can significantly relieve vascular dementia (VaD), however, the molecular mechanisms haven't been established. To clear the therapeutic mechanisms of CG against VaD, a hypothesis was proposed that CG could treat neurovascular injury by inhibiting the production of lipocalin-2 (LCN 2). MAIN METHODS: 90 dementia rats were selected by water maze test and randomly divided into 6 groups, including nimodipine (NM), CG L (low dose) (0.314 g kg-1), CG H (high dose) (0.628 g kg-1), and combined group (CG + NM). And in vitro neuronal cell OGD modeling to evaluate the effect of CG on JAK2/STAT3. KEY FINDINGS: CG could significantly shorten the escape latency of two-vessel occlusion (2-VO) rats, increase their exploratory behavior, alleviate the symptoms of VaD and improve the ultrastructural pathological damage of neurovascular unit and accelerate the recovery of cerebral blood perfusion. CG combined with NM is better than NM alone. It was further showed that CG could inhibit the pathogenicity of LCN 2 through JAK2/STAT3 pathway and suppress the production of inflammatory cytokines. It plays a role in the protection of cerebral microvasculature and BBB in 2-VO rats. SIGNIFICANCE: Taken together, there data has supported notion that CG can protect the integrity of cerebral blood vessels and BBB and improve cognitive impairment through mainly inhibiting LCN 2, which provides scientific evidence for clinical application.

Newer Non-ionic A2 B2 -Type Enzyme-Responsive Amphiphiles for Drug Delivery.

A new series of nonionic gemini amphiphiles have been synthesized in a multi-step chemoenzymatic approach by using a novel A2 B2 -type central core consisting of conjugating glycerol and propargyl bromide on 5-hydroxy isophthalic acid. A pair of hydrophilic monomethoxy poly(ethylene glycol) (mPEG) and hydrophobic linear alkyl chains (C12 /C15 ) were then added to the core to obtain amphiphilic architectures. The aggregation tendency in aqueous media was studied by dynamic light scattering, fluorescence spectroscopy and cryogenic transmission electron microscopy. The nanotransport potential of the amphiphiles was studied for model hydrophobic guests, that is, the dye Nile Red and the drug Nimodipine by using UV/Vis and fluorescence spectroscopy. Evaluation of the viability of amphiphile-treated A549 cells showed them to be well tolerated up to the concentrations studied. Being ester based, these amphiphiles exhibit stimuli-responsive sensitivity towards esterases, and a rupture of amphiphilic architecture was observed in the presence of immobilized Candida antarctica lipase (Novozym 435), thus facilitating release of the encapsulated guest from the aggregate.

Microdialysis combined with liquid chromatography-tandem mass spectrometry for the determination of nimodipine in the guinea pig hippocampus.

Nimodipine is a dihydropyridine calcium-channel blocker that has been recently shown to be effective on the function of central nervous system. It has been reported that treatment against deficits of learning and memory in animals and human by maintain the calcium homeostasis in hippocampus with nimodipine may be promising therapeutic strategies. A rapid and sensitive liquid chromatography-tandem mass spectrometric method was developed to determination the nimodipine in hippocampus using microdialysis technique. The separation was accomplished on an Agilent Zorbax SB-C18 column (100mm×2.1mm ID, 3.5µm) with the mobile phase composed of methanol-water (80:20, v/v) containing 0.2% formic acid at a flow rate of 0.2ml/min. Multiple reaction monitoring of the precursor-product ion transitions 419→343 for nimodipine and 361→315 nitrendipine (IS) was used for quantitation. The method was sensitive with a lower limit of quantitation (LLOQ) of 0.2ng/ml for nimodipine, with good linearity in the range of 0.2-20ng/ml. All the validation data, such as accuracy, precision, intra- and inter-day repeatability and stability were within the required limits. The method was successfully applied to p harmacokinetic study of the nimodipine in the guinea pig hippocampus.

Absorption, elimination and cerebrospinal fluid concentrations of nimodipine in healthy beagle dogs receiving human intravenous and oral formulation.

Nimodipine is an L-type calcium channel blocker and is used to treat vasospasm in patients with subarachnoid hemorrhage. Its putative mechanism of action is relaxation of smooth muscle cells in cerebral arteries. In addition, nimodipine may have pleiotropic effects against vasospasm. Systemic hypotension is an adverse effect when patients are treated with oral or intravenous nimodipine. Intracranial administration of nimodipine formulations may produce higher concentration of nimodipine in the cerebrospinal fluid (CSF) than is possible to achieve orally or intravenously, while resulting in lower incidence of systemic hypotension. The aim of this study was to provide information on plasma and CSF levels of nimodipine in beagle dogs as a comparative data for development of experimental intracranial treatment modalities. Plasma levels of nimodipine were measured after current 30 and 60 mg single oral dose of nimodipine (Nimotop(®) 30 mg tablets), a single intravenous bolus 0.72 mg/dog of nimodipine (Nimotop(®) 0.2 mg/ml infusion solution) and CSF levels after 60 mg single oral dose of nimodipine. CSF/Plasma concentration ratio of nimodipine after oral administration of 60 mg at 1 h was 0.013 ± 0.0005. The mean terminal elimination half-life of nimodipine after i.v. bolus dose 0.72 mg was 1.8 h and mean plasma clearance was 40.3 and 3.4 l/h/kg. Absolute bioavailability was 22 %. Maximum plasma concentration and area under the plasma concentration-time curve from time of administration until the last measurable plasma concentration increased in a dose-proportional manner comparing the exposure parameters at oral doses of 30 and 60 mg. Individual variation in the kinetic profile of nimodipine was measured.

Contribution of both olfactory and systemic pathways for brain targeting of nimodipine-loaded lipo-pluronics micelles: in vitro characterization and in vivo biodistribution study after intranasal and intravenous delivery.

Nimodipine (NM) is the only FDA-approved drug for treating subarachnoid hemorrhage induced vasospasm. NM has poor oral bioavailability (5-13%) due to its low aqueous solubility, and extensive first pass metabolism. The objective of this study is to develop radiolabeled NM-loaded LPM and to test its ability prolong its circulation time, reduce its frequency of administration and eventually target it to the brain tissue. NM was radiolabeled with 99mTc by direct labeling method using sodium dithionite. Different reaction conditions that affect the radiolabeling yield were studied. The in vivo pharmacokinetic behavior of the optimum NM-loaded LPM formulation in blood, heart, and brain tissue was compared with NM solution, after intravenous and intranasal administration. Results show that the radioactivity percentage (%ID/g) in the heart of mice following administration of 99mTc-NM loaded LPM were lower compared with that following administration of 99mTc-NM solution, which is greatly beneficial to minimize the cardiovascular side effects. Results also show that the %ID/g in the blood, and brain following intravenous administration of 99mTc-NM-loaded LPM were higher at all sampling intervals compared with that following intravenous administration of 99mTc-NM solution. This would be greatly beneficial for the treatment of neurovascular diseases. The drug-targeting efficiency of NM to the brain after intranasal administration was calculated to be 1872.82%. The significant increase in drug solubility, enhanced drug absorption and the long circulation time of the NM-loaded LPM could be promising to improve nasal and parenteral delivery of NM.

Chitosan matrix with three dimensionally ordered macroporous structure for nimodipine release.

Three dimensionally ordered macroporous (3DOM) chitosan (3D-CS) matrix with interconnected pores in the nanometer range was developed as a drug carrier for the first time. 3D-CS was prepared using a template-assisted assembly and characterized by SEM, TGA, N(2) adsorption and FT-IR. As a model drug, nimodipine (NMDP) was incorporated into the pores of 3D-CS matrix. The solid state properties of NMDP-loaded samples were characterized by SEM, XRD, DSC and FT-IR. Dissolution studies showed that release behavior of the drug was markedly affected by the particle size of the matrix. With a relatively small matrix particle size, formulations of NMDP-3D-CS-0.5 and NMDP-3D-CS-1 exhibited rapid release patterns. However, on increasing the amount of carrier, release rate of the drug decreased. The pH-dependent slow-release characteristic of 3D-CS matrix delivery system was demonstrated by investigating the release behavior of NMDP at different pH values.

Species differences in intestinal metabolic activities of cytochrome P450 isoforms between cynomolgus monkeys and humans.

The oral bioavailability of some drugs is markedly lower in cynomolgus monkeys than in humans. One of the reasons for the low bioavailability in cynomolgus monkeys may be the higher metabolic activity of intestinal CYP3A; however, the species differences in intestinal metabolic activities of other CYP isoforms between cynomolgus monkeys and humans are not well known. In the present study, we investigated the intrinsic clearance (CL(int)) values in pooled intestinal microsomes from cynomolgus monkeys and humans using 25 substrates of human CYP1A2, CYP2J2, CYP2C, and CYP2D6. As in humans, intestinal CL(int) values of human CYP1A2 and CYP2D6 substrates in cynomolgus monkeys were low. On the other hand, intestinal CL(int) values of human CYP2J2 and CYP2C substrates in cynomolgus monkeys were greatly higher than those in humans. Using immunoinhibitory antibodies and chemical inhibitors, we showed that the higher intestinal CL(int) values of the human CYP2J2 and CYP2C substrates in cynomolgus monkeys might be caused by monkey CYP4F and CYP2C subfamily members, respectively. In conclusion, there is a possibility that the greatly higher metabolic activity of CYP2C and CYP4F in cynomolgus monkey intestine is one of the causes of the species difference of intestinal first-pass metabolism between cynomolgus monkeys and humans.

Characterization of the zebrafish (Danio rerio) mineralocorticoid receptor.

Comparison between evolutionarily distant receptors can provide critical insights into both structure and function. Sequence comparison between the mineralocorticoid receptors (MR) of the zebrafish (zMR) and human (hMR) reveals a high degree of sequence conservation in the major functional domains. We isolated a zMR cDNA to contrast the transcriptional response to a range of ligands and to establish whether a teleost MR exhibits the amino/carboxyl-terminal interaction (N/C-interaction) previously reported for the hMR. Aldosterone, deoxycorticosterone (DOC) and cortisol induced zMR transcriptional activity with similar efficacy to that observed with the hMR. The hMR antagonist, spironolactone, acted as an agonist with the zMR. The zMR exhibited an N/C-interaction in response to aldosterone but, in contrast to the hMR, cortisol and DOC predominantly stimulated the interaction in the zMR. Conservation of the N/C-interaction between evolutionarily distant MR provides evidence of functional significance.

Differences in the quantal release of catecholamines in chromaffin cells of rat embryos and their mothers.

Studies on the bulk catecholamine release from fetal and neonatal rat adrenals, adrenal slices, or isolated chromaffin cells stimulated with high K(+), hypoxia, hypercapnia, or acidosis are available. However, a study analyzing the kinetics of quantal secretion is lacking. We report here such a study in which we compare the quantal release of catecholamines from immature rat embryo chromaffin cells (ECCs) and their mothers' (MCCs). Cell challenging with a strong depolarizing stimulus (75 mM K(+)) caused spike bursts having the following characteristics. ECCs released more multispike events and wave envelopes than MCCs. This, together with narrower single-spike events, a faster decay, and a threefold smaller quantal size suggest a faster secretory machinery in ECCs. Furthermore, with a milder stimulus (25 mM K(+)) enhanced Ca(2+) entry by L-type Ca(2+) channel activator BAY K 8644 did not change the kinetic parameters of single spikes in ECCs; in contrast, augmentation of Ca(2+) entry increased spike amplitude and width, quantal size, and decay time in MCCs. This suggests that in mature MCCs, the last exocytotic steps are more tightly regulated than in immature ECCs. Finally, we found that quantal secretion was fully controlled by L-type voltage-dependent Ca(2+) channels (VDCCs) in ECCs, whereas both L- and non-L VDCCs (N and PQ) contributed equally to secretion control in MCCs. Our results have the following physiological, pharmacological, and clinical relevance: 1) they may help to better understand the regulation of adrenal catecholamine release in response to stress during fetal life and delivery; 2) if clinically used, L-type Ca(2+) channel blockers may augment the incidence of sudden infant death syndrome (SIDS); and 3) so-called Ca(2+) promotors or activators of Ca(2+) entry through L-type VDCCs may be useful to secure a healthy catecholamine surge upon violent stress during fetal life, at birth, or to prevent the SIDS in neonates at risk.

Poly(L-lactide)-b-poly(ethylene oxide) copolymers with different arms: hydrophilicity, biodegradable nanoparticles, in vitro degradation, and drug-release behavior.

Biodegradable and amphiphilic poly(L-lactide)-b-poly(ethylene oxide) copolymers with different arms (PLLA-b-PEO having one, two, four, and six arms) were successfully synthesized via a two-step synthetic strategy. The hydrophilicity-hydrophobicity balance of these copolymers was mainly controlled by both the arm number of copolymers (i.e., macromolecular architecture) and the poly(ethylene oxide) (PEO) composition. Biodegradable nanoparticles could be generated by direct injection of these PLLA-b-PEO copolymers solutions into distilled water, and their critical micelles concentrations decreased with the increasing arm number of copolymers. Moreover, both the hydrophilic PEO composition and the arm number of copolymers controlled the average size of PLLA-b-PEO nanoparticles, and the nanoparticles with adjustable sizes (20-85 nm) completely meet the size prerequisite (less than 100 nm) for targeted drug delivery. In vitro degradation of PLLA-b-PEO nanoparticles showed that the PLLA composition gradually increased over the degradation time, and the degree of crystallinity of PLLA block within copolymers increased simultaneously. Furthermore, the nimodipine drug loading efficiency of the PLLA-b-PEO copolymers was apparently higher than that of PLLA homopolymers. The drug-release experiments demonstrated that these biodegradable nanoparticles might be used for a short-time controlled release system. Consequently, this will provide a facile method not only to design new PLLA-based biomaterials from both the macromolecular architecture and the hydrophilicity-hydrophobicity balance, but also to fabricate biodegradable nanoparticles with adjustable sizes for drug delivery.

Chronic benzodiazepine-induced reduction in GABA(A) receptor-mediated synaptic currents in hippocampal CA1 pyramidal neurons prevented by prior nimodipine injection.

One week oral flurazepam (FZP) administration in rats results in reduced GABA(A) receptor-mediated synaptic transmission in CA1 pyramidal neurons associated with benzodiazepine tolerance in vivo and in vitro. Since voltage-gated calcium channel (VGCC) current density is enhanced twofold during chronic FZP treatment, the role of L-type VGCCs in regulating benzodiazepine-induced changes in CA1 neuron GABA(A) receptor-mediated function was evaluated. Nimodipine (10 mg/kg, i.p.) or vehicle (0.5% Tween 80, 2 ml/kg) was injected 1 day after ending FZP treatment and 24 h prior to hippocampal slice preparation for measurement of mIPSC characteristics and in vitro tolerance to zolpidem. The reduction in GABA(A) receptor-mediated mIPSC amplitude and estimated unitary channel conductance measured 2 days after drug removal was no longer observed following prior nimodipine injection. However, the single nimodipine injection failed to prevent in vitro tolerance to zolpidem's ability to prolong mIPSC decay in FZP-treated neurons, suggesting multiple mechanisms may be involved in regulating GABA(A) receptor-mediated synaptic transmission following chronic FZP administration. As reported previously in recombinant receptors, nimodipine inhibited synaptic GABA(A) receptor currents only at high concentrations (>30 muM), significantly greater than attained in vivo (1 muM) 45 min after a single antagonist injection. Thus, the effects of nimodipine were unlikely to be related to direct effects on GABA(A) receptors. As with nimodipine injection, buffering intracellular free [Ca(2+)] with BAPTA similarly prevented the effects on GABA(A) receptor-mediated synaptic transmission, suggesting intracellular Ca(2+) homeostasis is important to maintain GABA(A) receptor function. The findings further support a role for activation of L-type VGCCs, and perhaps other Ca(2+)-mediated signaling pathways, in the modulation of GABA(A) receptor synaptic function following chronic benzodiazepine administration, independent of modulation of the allosteric interactions between benzodiazepine and GABA binding sites.

In-vivo and in-vitro studies on the effect of Huang-Lian-Jie-Du-Tang on nimodipine transport across rat blood-brain barrier.

Huang-Lian-Jie-Du-Tang (HLJDT), an aqueous extract of Rhizoma Coptidis, Radix Scutellariae, Cortex Phellodendri and Fructus Gardeniae (3:2:2:3) is an important multi-herb remedy in traditional Chinese medicine (TCM). The aim of this study was to evaluate the effect of HLJDT on nimodipine transport across rat blood-brain barrier (BBB). It was found that in-vivo the brain concentrations of nimodipine significantly increased when rats were pretreated with HLJDT. In-vitro, the serum of HLJDT-treated rats increased the accumulation of nimodipine in primary cultured rat brain microvessel endothelial cells (rBMECs) and decreased the expression of P-glycoprotein (P-gp) on rBMECs. Our previous study showed that the peak concentration of baicalin and berberine in rats after administration of HLJDT was 5 mug mL(-1) and 10 ng mL(-1), respectively. This study showed that 5 mug mL(-1) baicalin significantly increased the accumulation of nimodipine in rBMECs, while 10 ng mL(-1) berberine had no effect on the accumulation of nimodipine in rBMECs. Both the in-vivo and in-vitro experimental findings indicated that HLJDT pretreatment may alter the transport of nimodipine across rat BBB.

Effect of baicalin and berberine on transport of nimodipine on primary-cultured, rat brain microvascular endothelial cells.

AIM: To investigate whether baicalin and berberine affects the transport of nimodipine (NMD) across the blood-brain barrier (BBB). METHODS: Primary-cultured, rat brain microvascular endothelial cells (rBMEC) were used as an in vitro model of the BBB. When cells became confluent, the steady-state uptake of NMD by rBMEC with or without baicalin and berberine was measured. The effects of baicalin and berberine on the efflux of NMD from rBMEC were also studied. RESULTS: Baicalin (2-5 microg/mL) increased the uptake of NMD, and baicalin (10-20 microg/mL) decreased the uptake. The steady-state uptake of NMD was higher than that of control group in the presence of 0.01-1 microg/mL berberine, but was lower in the presence of 2-10 microg/mL berberine. CONCLUSION: The bidirectional effect of baicalin and berberine on the uptake of NMD by rBMEC was found. Higher concentration showed an inhibitory effect, and lower concentration demonstrated an increasing effect.