Reduction of Maturation Phenomenon in Cerebral Ischemia with CDP-Choline-Loaded Liposomes1

Purpose. Cerebral ischemia represents a serious therapeutic challenge. We investigated the therapeutic efficacy of CDP-choline-loaded liposomes against cerebral ischemia. The determination of post-ischemic brain recovery by EEG analysis was carried out to evaluate the effect of CDP-choline-loaded liposomes with respect to the free drug on the maturation of ischemic injury. Methods. Long-circulating unilamellar liposomes were prepared by a freeze and thaw procedure followed by an extrusion through polycarbonate membranes. Wistar rats were ischemized by bilateral clamping of the common carotid arteries. Free or liposomally entrapped drug was administered (20 mg/kg) just after ischemia and thereafter once a day for six days. Post-ischemic survival, neuronal membrane peroxidation and brain recovery (EEG analysis) were evaluated. Results. The post-ischemic reperfused rats treated with CDP-choline-loaded liposomes showed a higher survival rate than animals treated with the free drug. The delayed cerebral neurodegen-erative injury due to an ischemic event, referred to as maturation phenomenon, was substantially reduced with the administration of the liposomal formulation. The liposomal carrier showed a marked protection against lipoperoxidative damage. Conclusions. Liposomes ensured a rapid recovery of the damaged membranous structure of the neuronal cells, allowing a significant improvement of brain functionality. The reduction of the maturation phenomenon may probably be of particular importance in humans, where a fundamental problem is the quality of life after an ischemic event.     

Prevention of toxicity to isolated hepatocytes by liposome entrapment of chenodeoxycholic acid

Cellular uptake and cytotoxicity of chenodeoxycholic acid, delivered free or encapsulated in liposomes, were compared in isolated rat hepatocytes. As quantified by leakage of cytoplasmic and lysosomal enzymes into the medium, cells exposed to free chenodeoxycholic acid displayed a dose-dependent cytotoxic response. Cellular uptake of the drug was either similar or less for free than for entrapped chenodeoxycholic acid at 100 to 400 microM. However, the cytolytic changes were prevented when the cells were exposed to liposome-encapsulated drug. The prevention of cytotoxicity by delivering the drug entrapped in liposomes indicates that the free drug has the capacity to injure cell membranes directly, and suggests that drug-membrane interactions play a role in the hepatotoxic potential of chenodeoxycholic acid.     

Oral bioavailability enhancement of a hydrophilic drug delivered via folic acid‐coupled liposomes in rats

Objectives A liposome preparation that is amenable to receptor‐mediated endocytosis has been developed to enhance the oral bioavailability of poorly absorbable peptidomi‐metic drugs by use of folic acid as the mediator of liposomal uptake. Methods Folic acid was physically coupled to the surface of the liposomes and cefotaxime was used as the model drug. In‐vivo evaluation was carried out on eight Sprague‐Dawley rats in a two‐way crossover study to compare the oral bioavailability of cefotaxime loaded in folic acid‐free liposomes and folic acid‐coupled liposomes. Blood samples were collected from the tail vein and plasma cefotaxime levels were determined using an HPLC method. Key findings Enhanced oral bioavailability (AUC_0‐∞) of cefotaxime was observed when administered via folic acid‐coupled liposomes. The peak plasma concentration (C_max) of cefotaxime was increased when administered via folic acid‐coupled liposomes as compared with folic acid‐free liposomes. At 90% confidence interval, the value for AUC_0‐∞ was 1.4–2‐times higher and the value for C_max was 1.2–1.8‐times higher for the folic acid‐coupled liposomes compared with folic acid‐free liposomes. Conclusions Folic acid could enhance the uptake of liposomally entrapped drug. It could be a useful candidate to supplement liposome delivery systems.     

The effect of lipid composition of small unilamellar liposomes containing melphalan and vincristine on drug clearance after injection into mice

Melphalan and vincristine together with their radiolabelled derivatives were entrapped in small unilamellar liposomes of varying choleresterol content and phospholipid composition. After intravenous injection of drug-containing egg phosphatidylcholine liposomes into mice, drug clearance rates from the blood were reduced with increasing cholesterol content. Circulating drugs were partially associated with the carrier and partly free, mostly bound to plasma proteins. The ratio of drug associated with liposomes to that circulating as free was dependent on the type of liposomes used and highest when these were cholesterol-rich. Drug clearance rates were reduced and entrapped:free drug ratios increased further when egg phosphatidylcholine in cholesterol-rich liposomes was replaced by sphingomyelin. Drug-containing cholesterol-rich liposomes injected intraperitoneally were found capable of entering the periphery intact and quantitatively to assume clearance rates similar to those observed after intravenous treatment. Such manipulations in liposomal lipid composition can alter pharmacokinetics in ways that could provide optimal conditions for drug distribution into tumours and a therapeutic effect.     

Uptake of liposomally entrapped fluorescent antisense oligonucleotides in NG108-15 cells: conventional versus pH-sensitive

Antisense oligodeoxynucleotides (asODN) are novel therapeutic agents designed to alter RNA metabolism, ultimately resulting in decreased production of disease-associated gene products. To investigate internalisation of liposomally delivered asODN in NG108-15 cells, a hybrid cell line of mouse neuroblastoma and rat glioma, and assure that uptake of marker corresponds to that of antisense, we compared the cellular uptake of fluorescently labelled marker (fluorescein isothiocyanate (FITC)-dextran) and antisense oligonucleotide (FITC-asODN), entrapped either in conventional soy phosphatidylcholine (SPC) liposomes or pH-sensitive liposomes (composed of dioleoylphosphatidylethanolamine and cholesteryl hemisuccinate in a molar ratio of 3 : 2). Both SPC and pH-sensitive liposomes were prepared by a modified freeze-thawing method. Entrapment efficiencies (about 20% of the original material) did not depend on the liposome compositions and fluorescent material used. Fluorescence activated cell sorting (FACS) analysis was used to quantify the association of fluorescent material with the NG108-15 cells, whereas confocal microscopy gave insight on the location of cell associated-fluorescence. Conventional liposomes failed to deliver fluorescent material into the cells, but in contrast, pH-sensitive liposomes significantly improved the uptake of both FITC-dextran and FITC-asODN, with the uptake of liposomal FITC-dextran being greater than the uptake of liposomal FITC-asODN. These results suggest that pH-sensitive liposomes can be applied as a carrier system in the delivery of genetic material into the cells.     

The effect of entrapment of procaine hydrochloride in liposomes on its local anesthetic action

The study has shown the effect of liposomally entrapped procaine hydrochloride on local anaesthesia and its intensity after intradermal administration. The experiments of anaesthetic infiltration have been carried out on guinea pig skin applying mechanical stimulus. Liposomes were prepared by the shaking method followed by suspending in phosphate buffer pH 7.2, 0.5% methylcellulose in phosphate buffer or 5% glucose solution. The results of the study were compared with those of procaine hydrochloride in analogous solutions. The study has shown significant influence of liposomally entrapped procaine hydrochloride on the prolongation of local anaesthesia and its intensity. It has also been shown that local anaesthesia was influenced by the composition of the solutions in which liposomes were suspended. The longest effect (55 min) has been observed after administration of liposomes in phosphate buffer with methylcellulose.     

Specific binding of sterically stabilized anti-B-cell immunoliposomes and cytotoxicity of entrapped doxorubicin

Administration of doxorubicin (DXR) formulated in sterically stabilized liposomes, (SL) containing engrafted poly(ethylene glycol)-modified phosphatidylethanolamine (PEG-PE) on their surface, has been shown to increase the therapeutic index of the drug. A further improvement could be achieved through targeting of liposome-entrapped drug selectively to cancer cells. This paper describes the conjugation of the anti-B-cell lymphoma monoclonal antibody LL2 to the surface of DXR-loaded liposomes by use of a PEG-based heterobifunctional coupling agent. Competitive-binding ELISA of the resulting immunoliposomes (SIL) against the monoclonal anti-idiotype antibody, WN, indicated preserved immunological activity. The pH-sensitive probe, HPTS was used to study the binding of liposomes with target cells. The results showed a 3.8-fold increased cellular association of SIL compared to that of SL and an apparent internalization of SIL into low pH compartments. Addition of an excess of unconjugated free LL2 displaced about 72% of the HPTS-SIL association with cells. Experiments with 125I-labeled free and SIL-bound LL2 showed approximately 50% degradation for both preparations. In vitro MTT cytotoxicity tests against neoplastic B cells gave IC(50) values of 1.6, 2.9 and 0.35 microM for DXR-SIL, DXR-SL and free DXR, respectively. Leakage of drug from the liposomes apparently reduced the specificity of the cytotoxic action of DXR-SIL.     

Cytosolic Delivery of Liposomally Targeted Proteins Induced by Photochemical Internalization

Purpose The application of therapeutic proteins is often hampered by limited cell entrance and lysosomal degradation, as intracellular targets are not reached. By encapsulation of proteins into targeted liposomes, cellular uptake via endocytosis can be enhanced. To prevent subsequent lysosomal degradation and promote endosomal escape, photochemical internalization (PCI) was studied here as a tool to enhance endosomal escape. PCI makes use of photosensitising agents which localize in endocytic vesicles, inducing endosomal release upon light exposure. Materials and Methods The cytotoxic protein saporin was encapsulated in different types of targeted liposomes. Human ovarian carcinoma cells were incubated with the photosensitiser TPPS2a and liposomes. To achieve photochemical internalization, the cells were illuminated for various time periods. Cell viability was used as read-out. Illumination time and amount of encapsulated proteins were varied to investigate the influence of these parameters. Results The cytotoxic effect of liposomally targeted saporin was enhanced by applying PCI, likely due to enhanced endosomal escape. The cytotoxic effect was dependent on the amount of encapsulated saporin and the illumination time. Conclusion PCI is a promising technique for promoting cytosolic delivery of liposomally targeted saporin. PCI may also be applicable to other liposomally targeted therapeutic proteins with intracellular targets.     

Design, development and optimization of nimesulide-loaded liposomal systems for topical application

Nimesulide, a non-steroidal anti-inflammatory drug, was incorporated into multilamellar liposomes to improve its performance on topical administration. The drug was loaded onto liposomes employing thin film hydration technique. Various process and formulation variables were investigated to obtain the liposomal products of desired quality. Liposomes were monitored for percent drug entrapment, after separating the unentrapped drug by mini column centrifugation, for vesicular properties (such as size distribution profile, morphological attributes and agglomeration tendency), drug diffused through synthetic semipermeable membrane, and drug leakage. Systematic optimization studies were carried out using 3(2) factorial design to select the optimized liposomal composition with reference to percent drug entrapment, drug diffusion and leakage. The optimized batch of liposomes was subjected to drug permeation and drug retention studies employing rat skin and human cadaver skin. In comparison to methanolic solution of pure nimesulide, liposomal formulations were found to retain higher amounts of nimesulide in the skin. Anti-inflammatory studies, using carragenan-induced rat paw edema model, indicated significantly better performance of liposomally entrapped nimesulide in comparison to the marketed gel formulation and the Carbopol gel containing nimesulide.     

Effect of Cyclodextrin Complexation on the Liposome Permeability of a Model Hydrophobic Weak Acid

Purpose  This study examines the effect of a chemically modified β-cyclodextrin on the liposome bilayer permeability of a liposomally entrapped model hydrophobic weak acid, DB-67 (7-t-butyldimethylsilyl-10-hydroxycamptothecin). Materials and Methods  Permeability studies were conducted in liposomes prepared by hydration–extrusion in the presence or absence of entrapped hydroxypropyl-β-cyclodextrin (HPβCD). A gradient HPLC method with evaporative light scattering detection was developed for analysis of HPβCD. DB-67 was analyzed by HPLC with fluorescence detection. Results  HPβCD entrapped in the aqueous compartment of liposomes was found to be membrane impermeable. Gel phase liposomes were stable in the presence of HPβCD. HPβCD complexation did not significantly alter the apparent permeability of DB67 lactone, due to its high membrane binding. However, lactone ring-opening and ionization significantly decreased the apparent permeability and improved the liposomal retention of DB-67, an effect that was amplified in the presence of 50 mM HPβCD. Conclusions  In liposomes, cyclodextrin complexation competes with liposomal membrane binding which may temper the potential benefit of complexation in prolonging hydrophobic drug retention. Cyclodextrin complexation combined with drug ionization may nevertheless significantly enhance the retention of ionizable hydrophobic drugs in liposomes as complexation may compete more favorably with membrane binding when the drug is ionized.     

Mucoadhesive chitosan-coated liposomes: characteristics and stability

Lecithin liposomes, empty or containing FITC-dextran, were prepared by the ethanol injection method. Three different types of chitosans with different molecular weight and degrees of deacetylation were used (Seacure 113, 210 and 311). Chitosan coating was carried out by mixing the liposomal suspension with the chitosan solution followed by incubation. The size of liposomes was measured before and after polymer coating by an image analysis technique. The mean diameter of liposomes containing FITC-dextran was in the size range 250-280nm, whereas the size after coating was 300-330nm, regardless of chitosan type. All chitosan-coated liposomes were of spherical shape and no morphological differences between uncoated and coated liposomes were observed. Liposomes with FITC-dextran, originally entrapping 50% of the marker substance taken in the preparation and coated in the presence of unentrapped marker substance, contained 60-65%of the marker substance. The highest entrapment was found for liposomes coated with medium molecular weight chitosan. The stability of chitosan-coated liposomes in simulated gastric fluid was significantly higher as compared to uncoated liposomes. One can conclude that chitosan is stabilizing the original liposomal structure and protecting liposomally entrapped drug.     

Inhibitory effect of the neuroprotective agent idebenone on arachidonic acid metabolism in astrocytes.

Idebenone, a compound with protective efficacy against neurotoxicity both in in vitro and in in vivo models, exists in two different oxidative states: the ubiquinol-derivative (reduced idebenone) and the ubiquinone-derivative (oxidised idebenone). In the present study, we have observed that both the redox forms of idebenone have a dose-dependent inhibitory effect on the enzymatic metabolism of arachidonic acid in astroglial homogenates (IC50 reduced idebenone: 1.76 +/- 0.86 microM; IC50 oxidised idebenone: 16.65 +/- 3.48 microM), while in platelets, they are apparently less effective (IC50 reduced idebenone: 18.28 +/- 4.70 microM; IC50 oxidised idebenone: > 1 mM). We have also observed that the oxidised form preferentially inhibited cyclooxygenase vs. lipoxygenase metabolism (IC50 ratio lipoxygenase/cyclooxygenase: 3.22), while the reduced form did not discriminate between the two pathways (IC50 ratio lipoxygenase/cyclooxygenase: 1.38). In this respect, the inhibitory action of reduced idebenone resembled that of the antioxidant nordihydroguaiaretic acid, while oxidised idebenone behaved similarly as indomethacin and piroxicam--two typical anti-inflammatory agents. Our results suggest the existence of two distinct mechanisms of action for the two redox forms of idebenone and a preferential action of the drug on arachidonic acid metabolism in the central nervous system.     

Cell injury and its protection in astrocytes

Incubation of cultured astrocytes in Ca(2+)-containing medium after exposure to Ca(2+)-free medium causes Ca2+ influx followed by delayed cell death. Here, we summarize the mechanisms underlying the Ca(2+)-mediated injury of cultured astrocytes and the protective effects of drugs against Ca(2+)-reperfusion injury. Our results show that Ca(2+)-reperfusion injury of astrocytes appears to be mediated by apoptosis as evidenced by DNA fragmentation and nuclear condensation. Calpain, reactive oxygen species (ROS) production, calcineurin, caspase-3, and NF-kappa B activation are involved in Ca(2+)-reperfusion injury. Several drugs including T-588 and idebenone protect astrocytes against Ca(2+)-reperfusion injury. The protective effect of idebenone is mediated by nerve growth factor production, whereas that of T-588 is mediated mainly by the mitogen-activated protein/extracellular signal-regulated kinase signal cascade.     

Effect of idebenone and various nootropic drugs on lipid peroxidation in rat brain homogenate in the presence of succinate

The effects of idebenone and various nootropic drugs on lipid peroxidation in rat brain homogenate were examined. Idebenone inhibited lipoperoxide (LPO) production in brain homogenate in a concentration-dependent manner, with an IC50 of 38 microM. The inhibition was strongly enhanced (about 100-fold) by adding succinate, a substrate in the mitochondrial respiration. The optimal concentration of succinate was 0.5 mM. Inhibition of lipid peroxidation in brain homogenate by various nootropic drugs in the presence or absence of succinate was then examined. Drugs added to the brain homogenate at 100 microM in the absence of succinate inhibited LPO production in the order: idebenone greater than vinpocetine greater than bifemelane greater than indeloxazine greater than calcium hopantenate. However, when the drugs were added at 1 microM in the presence of succinate, only idebenone demonstrated inhibition. These results suggest that although almost all of the drugs tested inhibit lipid peroxidation in brain homogenate, only idebenone is activated by succinate, the other drugs being insensitive to this compound.     

Liposomal formulations of Cyclosporin A: a biophysical approach to pharmacokinetics and pharmacodynamics

There are about 20 publications about liposomal formulations of Cyclosporin A (CyA) in the pharmaceutical and preclinical literature. Liposomal formulations were developed in order to reduce the nephrotoxicity of CyA and to increase pharmacological effects. However, conflicting results have been published as to the therapeutic properties of these formulations. This is also true for the change in pharmacokinetics and organ distribution of the liposomally encapsulated CyA as compared to conventionally formulated CyA. Using biophysical methods, it could be shown that CyA is not tightly entrapped in liposomal membranes, despite its high lipophilicity. CyA shows retardation only at high lipid concentrations in blood, following a massive injection of liposomes.This effect may diminish nephrotoxicity, as could be demonstrated by in vitro studies using a model tubule system. The results of these studies can be used to predict the formulation behavior in vivo and to optimize liposomal formulations. When applied in an early phase of the drug formulation process, these types of biophysical experiments can also help minimize animal experiments. However, these basic interaction studies cannot cover all physiological, pharmacological, and toxic effects in animals and humans.     

Mucoadhesive chitosan-coated liposomes: characteristics and stability

Lecithin liposomes, empty or containing FITC-dextran, were prepared by the ethanol injection method. Three different types of chitosans with different molecular weight and degrees of deacetylation were used (Seacure 113, 210 and 311). Chitosan coating was carried out by mixing the liposomal suspension with the chitosan solution followed by incubation. The size of liposomes was measured before and after polymer coating by an image analysis technique. The mean diameter of liposomes containing FITC-dextran was in the size range 250-280 nm, whereas the size after coating was 300-330 nm, regardless of chitosan type. All chitosan-coated liposomes were of spherical shape and no morphological differences between uncoated and coated liposomes were observed. Liposomes with FITC-dextran, originally entrapping 50% of the marker substance taken in the preparation and coated in the presence of unentrapped marker substance, contained 60-65% of the marker substance. The highest entrapment was found for liposomes coated with medium molecular weight chitosan. The stability of chitosan-coated liposomes in simulated gastric fluid was significantly higher as compared to uncoated liposomes. One can conclude that chitosan is stabilizing the original liposomal structure and protecting liposomally entrapped drug.     

Drug-induced lipid peroxidation in mice—III: Glutathione content of liver,kidney and spleen after intravenous administration of free and liposomally entrapped glutathione

The half-life of extracellular glutathione was found to be 1.9 min in fed mice with a hepatic glutathione content of 44 ± 10 nmol glutathione per mg protein. It was 4.9 min in animals that had been fed for 48 hr a liquid sucrose diet resulting in a decreased hepatic glutathione of 25 ± 7 nmol/mg. A single intravenous injection of 16.2 μmol liposomally entrapped glutathione led to an increase in hepatic glutathione to 45 nmol/mg in the sucrose-fed mice after 2 hr and had no effect in the fed group. The spleen glutathione content reached a maximum at 30 min after injection in both groups. The maximum uptake into liver was 21% of the applied dose, into the spleen 7% and into the kidneys 2.4%. Injection of glutathione in solution led to a similar increase of hepatic glutathione as observed with GSH- containing liposomes, while liposomes filled with the constituent amino acids had only a marginal effect. The spleen took up only liposomal GSH. In contrast, the kidney glutathione content increased within 10 min up to 150% upon injection of free glutathione. The findings are consistent with a rapid hydrolysis of extracellular free glutathione followed by an interorgan turnover utilizing the constituent amino acids for resynthesis in the liver. Pretreatment of the animals with the glutathione synthesis inhibitor buthionine sulfoximine essentially abolished the hepatic glutathione increase upon treatment with GSH-liposomes or with the free compound. The finding that only liposomally entrapped glutathione protects mice against liver necrosis induced by highly dosed paracetamol is discussed with respect to differential uptake and distribution of GSH-liposomes in the liver.     

Traumatic brain injury opens blood–brain barrier to stealth liposomes via an enhanced permeability and retention (EPR)-like effect

The opening of the tight junctions in the blood–brain barrier (BBB) following traumatic brain injury (TBI) is hypothesized to be sufficient to enable accumulation of large drug carriers, such as stealth liposomes, in a similar manner to the extravasation seen in tumor tissue via the enhanced permeability and retention (EPR) effect. The controlled cortical impact model of TBI was used to evaluate liposome accumulation in mice. Dual-radiolabeled PEGylated liposomes were administered either immediately after induction of TBI or at increasing times post-TBI to mimic the likely clinical scenario. The accumulation of radiolabel in the brain tissue ipsilateral and contralateral to the site of trauma, as well as in other organs, was evaluated. Selective influx of liposomes occurred at 0–8?h after injury, while the barrier closed between 8 and 24?hr after injury, consistent with reports on albumin infiltration. Significantly enhanced accumulation of liposomes occurred in mice subjected to TBI compared to anaesthetized controls, and accumulation was greater in the injured versus the contralateral side of the brain. Thus, stealth liposomes show potential to enhance drug delivery to the site of brain injury with a wide range of encapsulated therapeutic candidates.     

Extra- and intracellular sphingosylphosphorylcholine promote spontaneous transmitter release from frog motor nerve endings

Similar to phosphatidylinositol bisphosphate, sphingomyelin breakdown generates several lipids, including sphingosylphosphorylcholine (SPC), that are putative signaling molecules. The present study was undertaken to evaluate the involvement of SPC in transmitter release process. Intracellular recordings were made from isolated frog sciatic-sartorius nerve-muscle preparations, and the effects of SPC on neurosecretion in the form of miniature endplate potentials (MEPPs) were assessed. Extracellular application of SPC mixture (D,L-SPC) at 1, 10, and 25 microM increased the MEPP frequency by 68, 96, and 127%, respectively. D-erythro-SPC (dissolved in dimethyl sulfoxide but not coupled to bovine serum albumin), but not L-threo-SPC, was active extracellular; the former (at 10 microM) increased the MEPP frequency by 143%. D-erythro-SPC treatment did not significantly change the median amplitude or frequency-distribution of MEPPs. Intracellular delivery via liposomes, in which 10, 100, or 1000 microM SPC mixture was entrapped in liposomal aqueous phase, induced a concentration-dependent increase in MEPP frequency of 45, 91, and 100%, respectively. D-erythro-SPC and L-threo-SPC at the concentration of 100 microM increased the MEPP frequency by 117 and 67%, respectively, or 91 and 61%, respectively, when coupled to bovine serum albumin. Pretreatment with thapsigargin significantly reduced but did not abolish the effects of extracellular D-erythro-SPC (10 microM) or liposomes containing 100 microM D-erythro-SPC. Liposomes filled with 100 microM D-myo-inositol 1,4,5-trisphosphate (IP3) enhanced the MEPP frequency to the same magnitude as 100 microM D-erythro-SPC entrapped in liposomes. However, administration of 100 microM D-erythro-SPC and IP3 entrapped in the same liposomes enhanced the MEPP frequency by 70%, which was less than that produced by these two compounds alone. The result provides the first electrophysiological evidence that SPC can modulate transmitter release by an extra- or intracellular action at the frog motor nerve ending.     

Decursinol and decursin protect primary cultured rat cortical cells from glutamate-induced neurotoxicity

We previously reported six neuroprotective decursinol derivatives, coumarins from Angelica gigas (Umbelliferae) roots. To elucidate the action patterns of decursinol derivatives, we investigated the neuroprotective effects of decursinol and decursin, which showed highly significant activity and were major constituents of A. gigas, using primary cultures of rat cortical cells in-vitro. At concentrations of 0.1-10.0 microM, both decursinol and decursin exerted a significant neuroprotective activity pretreatment and throughout treatment. In addition, decursin had a neuroprotective impact in the post-treatment paradigm implying that decursin might possess different action mechanisms from that of decursinol in the protection of neurons against glutamate injury. Both decursinol and decursin effectively reduced the glutamate-induced increased intracellular calcium ([Ca(2+)](i)) in cortical cells, suggesting that these two coumarins may exert neuroprotection by reducing calcium influx by overactivation of glutamate receptors. This suggestion was supported by the result that decursinol and decursin protected neurons against kainic acid (KA)-induced neurotoxicity better than against that induced by N-methyl-D-aspartate (NMDA). Moreover, both decursinol and decursin significantly prevented glutamate-induced decreases in glutathione, a cellular antioxidant, and glutathione peroxidase activity. In addition, both compounds efficiently reduced the overproduction of cellular peroxide in glutamate-injured cortical cells. These results suggested that both decursinol and decursin protected primary cultured rat cortical cells against glutamate-induced oxidative stress by both reducing calcium influx and acting on the cellular antioxidative defence system. Moreover, decursin is considered to probably have a different action mechanism from that of decursinol in protecting cortical cells against glutamate injury.