Development and Evaluation of a Nanoemulsion Containing Ursolic Acid: a Promising Trypanocidal Agent

Over a hundred years after the discovery of Chagas disease, this ailment continues to affect thousands of people. For more than 40 years, only two drugs have been available to treat it. Ursolic acid is a naturally occurring terpene that has shown a good trypanocidal action. However, the hydrophobicity of this compound presents a challenge for the development of proper delivery systems. Nanostructured systems are a prominent in delivering lipophilic drugs. Thus, a nanoemulsion containing ursolic acid was developed and had its trypanocidal activity and cytotoxicity evaluated. Pseudo-ternary phase diagrams and hydrophilic-lipophilic balance (HLB) system were used in the development. The system was stable throughout 90 days of testing, as evidenced by turbidimetry analysis and measurements of the droplet size (57.3 nm) and polydispersity index (0.24). Fourier transform infrared spectroscopy and mass spectrometry evidenced drug’s integrity in the formulation. An in vitro dissolution profile showed 75% of ursolic acid release after 5 min from the nanoemulsion into the alkaline dissolution medium, while only 20% could be released from a physical mixture after 2 h. Trypanocidal activity and cytotoxicity were evaluated on the CL Brener strain and LLC-MK2 (monkey kidney) fibroblast by chlorophenol red-β-d-galactoside (CPRG) method. Biological studies showed that the developed formulation was nontoxic and effective against replicant forms of the parasite. A stable and efficient nanoemulsion could be developed to improve the delivery of a promising drug to treat a threatening illness such as Chagas disease.     

Modulating Drug Release and Enhancing the Oral Bioavailability of Torcetrapib with Solid Lipid Dispersion Formulations

The development of drug dispersions using solid lipids is a novel formulation strategy that can help address the challenges of poor drug solubility and systemic exposure after oral administration. The highly lipophilic and poorly water-soluble drug torcetrapib could be effectively formulated into solid lipid microparticles (SLMs) using an anti-solvent precipitation strategy. Acoustic milling was subsequently used to obtain solid lipid nanoparticles (SLNs). Torcetrapib was successfully incorporated into the lipid matrix in an amorphous state. Spherical SLMs with mean particle size of approximately 15–18 μm were produced with high drug encapsulation efficiency (>96%) while SLNs were produced with a mean particle size of 155 nm and excellent colloidal stability. The in vitro drug release and the in vivo absorption of the solid lipid micro- and nanoparticles after oral dosing in rats were evaluated against conventional crystalline drug powders as well as a spray dried amorphous polymer dispersion formulation. Interestingly, the in vitro drug release rate from the lipid particles could be tuned for immediate or extended release by controlling either the particle size or the precipitation temperature used when forming the drug-lipid particles. This change in the rate of drug release was manifested in vivo with changes in Tmax as well. In addition, in vivo pharmacokinetic studies revealed a significant increase (∼6 to 11-fold) in oral bioavailability in rats dosed with the SLMs and SLNs compared to conventional drug powders. Importantly, this formulation approach can be performed rapidly on a small scale, making it ideal as a formulation technology for use early in the drug discovery timeframe.Electronic supplementary materialThe online version of this article (doi:10.1208/s12249-015-0299-8) contains supplementary material, which is available to authorized users.KEY WORDS: anti-solvent precipitation, controlled release, formulation, nanoparticles, solid lipid     

Application of Ethylcellulose Coating to Hydrophilic Matrices: A Strategy to Modulate Drug Release Profile and Reduce Drug Release Variability

Hydrophilic matrix tablets are commonly used for extended release dosage forms. For low aqueous-solubility drugs, there may be challenges in modulation of release profiles and achieving consistent release in physiological conditions. To evaluate potential formulation strategies, matrix tablets of a low-soluble drug, hydrochlorothiazide, were developed using hypromellose and two fillers of different solubility, lactose (soluble) or partially pregelatinized maize starch (partially soluble). Additionally, application of an insoluble barrier membrane, aqueous ethylcellulose coating system, and a hydrophilic pore former onto matrix tablets was evaluated. Drug release from uncoated matrix tablets was variable at different agitation rates. Evaluation of tablets in bio-relevant media using physiologically relevant residence time indicated variable and higher initial release rate for uncoated matrices containing lactose but more robust behavior for tablets containing partially pregelatinized starch. Such in vitro behavior may lead to erratic drug release in vivo, when comparing fed versus fasted conditions. Dissolution profiles from barrier membrane-coated tablets showed initial delay, followed by zero-order release kinetics, with reduction or elimination of variability compared to uncoated matrices. Such reduced variability may mitigate mechanical effects of post-prandial stomach. Effects of coating weight gain and inclusion levels of pore former were evaluated and found to be critical in achieving robust and stable release profiles.     

Disintegration Mediated Controlled Release Supersaturating Solid Dispersion Formulation of an Insoluble Drug: Design,Development, Optimization,and In Vitro Evaluation

The objective of this study was to develop a solid dispersion based controlled release system for drug substances that are poorly soluble in water. A wax-based disintegration mediated controlled release system was designed based on the fact that an amorphous drug can crystallize out from hydrophilic matrices. For this study, cilostazol (CIL) was selected as the model drug, as it exhibits poor aqueous solubility. An amorphous solid dispersion was prepared to assist the drug to attain a supersaturated state. Povidone was used as carrier for solid dispersion (spray drying technique), hydrogenated vegetable oil (HVO) as wax matrix former, and sodium carboxymethyl cellulose (NaCMC) as a disintegrant. The extreme vertices mixture design (EVMD) was applied to optimize the designed and developed composition. The optimized formulation provided a dissolution pattern which was equivalent to the predicted curve, ascertaining that the optimal formulation could be accomplished with EVMD. The release profile of CIL was described by the Higuchi’s model better than zero-order, first-order, and Hixson-Crowell’s model, which indicated that the supersaturation state of CIL dominated to allow drug release by diffusion rather than disintegration regulated release as is generally observed by Hixson-Crowell’s model. The optimized composition was evaluated for disintegration, dissolution, XRD, and stability studies. It was found that the amorphous state as well as the dissolution profile of CIL was maintained under the accelerated conditions of 40°C/75% RH for 6 months.KEY WORDS: cilostazol, controlled release, disintegration-mediated controlled release (DMCR), extreme vertices mixture design (EVMD), solid dispersion     

Evaluation of Drug Load and Polymer by Using a 96-Well Plate Vacuum Dry System for Amorphous Solid Dispersion Drug Delivery

It is well recognized that poor dissolution rate and solubility of drug candidates are key limiting factors for oral bioavailability. While numerous technologies have been developed to enhance solubility of the drug candidates, poor water solubility continuously remains a challenge for drug delivery. Among those technologies, amorphous solid dispersions (SD) have been successfully employed to enhance both dissolution rate and solubility of poorly water-soluble drugs. This research reports a high-throughput screening technology developed by utilizing a 96-well plate system to identify optimal drug load and polymer using a solvent casting approach. A minimal amount of drug was required to evaluate optimal drug load in three different polymers with respect to solubility improvement and solid-state stability of the amorphous drug-polymer system. Validation of this method was demonstrated with three marketed drugs as well as with one internal compound. Scale up of the internal compound SD by spray drying further confirmed the validity of this method, and its quality was comparable to a larger scale process. Here, we demonstrate that our system is highly efficient, cost-effective, and robust to evaluate the feasibility of spray drying technology to produce amorphous solid dispersions.     

Hot Melt Extrusion: Development of an Amorphous Solid Dispersion for an Insoluble Drug from Mini-scale to Clinical Scale

The objective of the study was to develop an amorphous solid dispersion (ASD) for an insoluble compound X by hot melt extrusion (HME) process. The focus was to identify material-sparing approaches to develop bioavailable and stable ASD including scale up of HME process using minimal drug. Mixtures of compound X and polymers with and without surfactants or pH modifiers were evaluated by hot stage microscopy (HSM), polarized light microscopy (PLM), and modulated differential scanning calorimetry (mDSC), which enabled systematic selection of ASD components. Formulation blends of compound X with PVP K12 and PVP VA64 polymers were extruded through a 9-mm twin screw mini-extruder. Physical characterization of extrudates by PLM, XRPD, and mDSC indicated formation of single-phase ASD’s. Accelerated stability testing was performed that allowed rapid selection of stable ASD’s and suitable packaging configurations. Dissolution testing by a discriminating two-step non-sink dissolution method showed 70–80% drug release from prototype ASD’s, which was around twofold higher compared to crystalline tablet formulations. The in vivo pharmacokinetic study in dogs showed that bioavailability from ASD of compound X with PVP VA64 was four times higher compared to crystalline tablet formulations. The HME process was scaled up from lab scale to clinical scale using volumetric scale up approach and scale-independent-specific energy parameter. The present study demonstrated systematic development of ASD dosage form and scale up of HME process to clinical scale using minimal drug (～500 g), which allowed successful clinical batch manufacture of enabled formulation within 7 months.     

Assessing the In Vitro Drug Release from Lipid-Core Nanocapsules: a New Strategy Combining Dialysis Sac and a Continuous-Flow System

The in vitro assessment of drug release from polymeric nanocapsules suspensions is one of the most studied parameters in the development of drug-loaded nanoparticles. Nevertheless, official methods for the evaluation of drug release from submicrometric carriers are not available. In this work, a new approach to assess the in vitro drug release profile from drug-loaded lipid-core nanocapsules (LNC) was proposed. A continuous-flow system (open system) was designed to evaluate the in vitro drug release profiles from different LNC formulations containing prednisolone or clobetasol propionate (LNC-CP) as drug model (LNC-PD) using a homemade apparatus. The release medium was constantly renewed throughout the experiment. A dialysis bag containing 5 mL of formulation (0.5 mg mL⁻¹) was maintained inside the apparatus, under magnetic stirring and controlled temperature (37°C). In parallel, studies based on the conventional dialysis sac technique (closed system) were performed. It was possible to discriminate the in vitro drug release profile of different formulations using the open system. The proposed strategy improved the sink condition, by constantly renewing the release medium, thus maintaining the drug concentration farther from the saturated concentration in the release medium. Moreover, problems due to sampling errors can be easily overcome using this semi-automated system, since the collection is done automatically without interference from the analyst. The system proposed in this paper brings important methodological and analytical advantages, becoming a promising prototype semi-automated apparatus for performing in vitro drug release studies from drug-loaded lipid-core nanocapsules and other related nanoparticle drug delivery systems.KEY WORDS: dialysis sac technique, flow-through cell apparatus, in vitro drug release, lipid-core nanocapsules, sink condition     

Wetting Kinetics: an Alternative Approach Towards Understanding the Enhanced Dissolution Rate for Amorphous Solid Dispersion of a Poorly Soluble Drug

Developing amorphous solid dispersions of water-insoluble molecules using polymeric materials is a well-defined approach to improve the dissolution rate and bioavailability. While the selected polymer plays a vital role in stabilizing the amorphous solid dispersion physically, it is equally important to improve the dissolution profile by inhibiting crystallization from the supersaturated solution generated by dissolution of the amorphous material. Furthermore, understanding the mechanism of dissolution rate enhancement is of vital importance. In this work, wetting kinetics was taken up as an alternative approach for understanding the enhanced dissolution rate for amorphous solid dispersion of a poorly soluble drug. While cilostazol (CIL) was selected as the model drug, povidone (PVP), copovidone, and hypromellose (HPMC) were the polymers of choice. The concentrations against time profiles were evaluated for the supersaturated solutions of CIL in the presence and absence of the selected polymers. The degree of supersaturation increased significantly with increase in polymer content within the solid dispersion. While povidone was found to maintain the highest level of supersaturation for the greatest length of time both in dissolution and solution crystallization experiments, copovidone and hypromellose were found to be the less effective as crystallization inhibitor. The ability of polymers to generate and maintain supersaturated drug solutions was assessed by dissolution studies. The wetting kinetics was compared against the solid dispersion composition to establish a correlation with enhanced dissolution rate.KEY WORDS: Cilostazol, Crystallization inhibition, Solid dispersions, Supersaturated solutions, Wetting kinetics     

不同剂量熊果酸干预糖尿病小鼠视网膜病变的作用及机制研究

目的:探究不同剂量熊果酸(UA)干预糖尿病小鼠视网膜病变的作用及机制。方法:选取雄性健康C57BL/6小鼠60只,其中50只按50 mg/kg的剂量一次性往小鼠尾静脉注射新鲜配置的四氯嘧啶生理盐水溶液构建小鼠糖尿病视网膜病变模型,随机分为5组,每组10只,分别为模型组、阳性对照组(小鼠玻璃体注射3μL 40 mg/m L的曲安奈德),低剂量UA干扰组(小鼠玻璃体注射3μL剂量为0.5μg/μL的UA)、中剂量UA干扰组(小鼠玻璃体注射3μL剂量为1.0μg/μL的UA),高剂量UA干扰组(小鼠玻璃体注射3μL剂量为2.0μg/μL的UA),余下10只小鼠作为正常对照组。观察各组小鼠对胰岛素敏感性、视网膜内糖代谢情况、小鼠视网膜神经节细胞(RGCs)凋亡情况,比较各组小鼠视网膜组织中血管内皮生长因子(VEGF)、环氧化酶-2(COX-2)、基质金属蛋白酶2(MMP-2)蛋白及其mRNA的表达情况。结果:建模后,正常对照组胰岛素抵抗指数(HOMA-IR)、视网膜含糖量、葡萄糖转运体-1(GLUT-1)与葡萄糖转运体-3(GLUT-3)含量、RGCs凋亡率、视网膜组织中VEGF、COX-2、MMP-2蛋白及其mRNA的表达量低于模型组(P0.05);经干扰后,阳性对照组、不同剂量UA干扰组HOMA-IR、视网膜含糖量、GLUT-1、GLUT-3的含量、RGCs凋亡率、视网膜组织中VEGF、COX-2、MMP-2蛋白及其mRNA的表达量低于模型组(P0.05),且随UA干扰剂量的升高而降低(P0.05)。结论:UA能够降低HOMA-IR和视网膜糖代谢能力,抑制RGCs的凋亡,对VEGF、COX-2、MMP-2蛋白及其mRNA的表达具有一定的抑制作用,高剂量UA对糖尿病小鼠视网膜病预防治疗效果较好。     

Release of a Poorly Soluble Drug from Hydrophobically Modified Poly (Acrylic Acid) in Simulated Intestinal Fluids

A large part of new pharmaceutical substances are characterized by a poor solubility and high hydrophobicity, which might lead to a difference in drug adsorption between fasted and fed patients. We have previously evaluated the release of hydrophobic drugs from tablets based on Pemulen TR2 and showed that the release can be manipulated by adding surfactants. Here we further evaluate the possibility to use Pemulen TR2 in controlled release tablet formulations containing a poorly soluble substance, griseofulvin. The release is evaluated in simulated intestinal media that model the fasted state (FaSSIF medium) or fed state (FeSSIF). The rheology of polymer gels is studied in separate experiments, in order to gain more information on possible interactions. The release of griseofulvin in tablets without surfactant varied greatly and the slowest release were observed in FeSSIF. Addition of SDS to the tablets eliminated the differences and all tablets showed a slow linear release, which is of obvious relevance for robust drug delivery. Comparing the data from the release studies and the rheology experiment showed that the effects on the release from the different media could to a large extent be rationalised as a consequence of the interactions between the polymer and the surfactants in the media. The study shows that Pemulen TR2 is a candidate for controlled release formulations in which addition of surfactant provides a way to eliminate food effects on the release profile. However, the formulation used needs to be designed to give a faster release rate than the tablets currently investigated.     

Cyclodextrin-Crosslinked Poly(Acrylic Acid): Adhesion and Controlled Release of Diflunisal and Fluconazole from Solid Dosage Forms

The controlled release of diflunisal and fluconazole from tablets made of novel polymers, poly(acrylic acid) (PAA) crosslinked with either β-cyclodextrin (βCD) or hydroxypropyl-βCD (HPβCD), was investigated and Carbopol 934P (Carbopol) was used as a highly crosslinked PAA for comparison. Diflunisal strongly associates with βCD-PAA and HPβCD-PAA polymers (K_a of 486 and 6,055 M⁻¹ respectively); thus, it was physically mixed into the conjugates and also precomplexed to identify whether decomplexation has any influence on release kinetics. Fluconazole has poor complexing ability (K_a of 34 M⁻¹ with HPβCD-PAA); thus, it was only tested as a physical mixture. Swelling and adhesion studies were conducted on all tablet combinations and adhesivity of the CD-PAA polymer tablets was maintained. Diflunisal release was much slower from HPβCD-PAA tablets than from βCD-PAA, suggesting that a higher degree of complexation retards release. The precomplexed diflunisal release was also slower than the physically mixed diflunisal of the corresponding conjugate. The release closely followed zero-order kinetics for HPβCD-PAA, but was more sigmoidal for βCD-PAA and especially Carbopol. Conversely, poorly associating fluconazole released in almost exactly the same way across both polymers and Carbopol, indicating that the release kinetics of poorly associating drugs are not influenced by the presence of cyclodextrins. In view of the varying profiles and release rates shown with diflunisal for the different polymers, the fluconazole data support the concept that adequate complexation can indeed modulate the release kinetics of drugs.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-012-9903-3) contains supplementary material, which is available to authorized users.Key words: controlled release, cyclodextrin, diflunisal, fluconazole, poly(acrylic acid)     

Release and Activity of Bound beta-Amylase in a Germinating Barley Grain

In resting grains of Triumph barley (Hordeum vulgare L. cv Triumph) about 40% of the β-amylase could be extracted with a saline solution, the remaining 60% being in a bound form. During seedling growth (20°C), the bound form was released mainly between days 1 and 3. When a preparation containing bound β-amylase was incubated with an extract made of endosperms separated from germinating grains, release of bound β-amylase took place and could be studied in vitro. The release was almost completely prevented by leupeptin and antipain, specific inhibitors of a group of SH-proteinases, but it was not inhibited by pepstatin A or EDTA, which inhibit some other barley proteinases. It is thus very likely that in a whole grain, at least the bulk of the bound β-amylase is released by the proteolytic action of one or several SH-proteinases. When the bound β-amylase was released by papain, its molecular weight was about 5000 daltons smaller than that of β-amylase released by dithiothreitol. This indicates that the release is due to removal of a sequence of β-amylase itself. A similar decrease in size took place during seedling growth. Bound β-amylase showed some activity against native starch and it hydrolyzed maltotetraose at a rate that was about 70% of the rate the same amount of bound β-amylase gave after release. Bound β-amylase is thus not inactive and it is likely that the slower rate of hydrolysis is due to steric hindrances which prevent substrates from reaching the active site.     

Microbial Activity and 13C/12C Ratio as Evidence of N-Hexadecane and N-Hexadecanoic Acid Biodegradation in Agricultural and Forest Soils

The dynamics of microbial degradation of exogenous contaminants, n-hexadecane and its primary microbial oxidized metabolite, n-hexadecanoic (palmitic) acid, was studied for topsoils, under agricultural management and beech forest on the basis the changes in O₂ uptake, CO₂ evolution and its associated carbon isotopic signature, the respiratory quotient (RQ) and the priming effect (PE) of substrates. Soil microbial communities in agricultural soil responded to the n-hexadecane addition more rapidly compared to those of forest soil, with lag-periods of about 23 ± 10 and 68 ± 13 hours, respectively. Insignificant difference in the lag-period duration was detected for agricultural (t_lag = 30 ± 13 h) and forest (t_lag = 30 ± 14 h) soils treated with n-hexadecanoic (palmitic) acid. These results demonstrate that the soil microbiota has different metabolic activities for using n-hexadecane as a reductive hydrocarbon and n-hexadecanoic acid as a partly oxidized hydrocarbon. The corresponding δ¹³C of respired CO₂ after the addition of the hydrocarbon contaminants to soils indicates a shift in microbial activity towards the consumption of exogenous substrates with a more complete degradation of n-hexadecane in the agricultural soil, for which some initial contents of hydrocarbons are inherent. It is supposed that the observed deviation of RQ from theoretically calculated value under microbial substrate mineralization is determined by difference in the time (Δt_i) of registration of CO₂ production and O₂ consumption. Positive priming effect (PE) of n-hexadecane and negative PE of n-hexadecanoic (palmitic) acid were detected in agricultural and forest soils. It is suggested that positive PE of n-hexadecane is conditioned by the induction of microbial enzymes that perform hydroxylation/oxygenation of stable SOM compounds mineralized by soil microbiota to CO₂. The microbial metabolism coupled with oxidative decarboxylation of n-hexadecanoic acid is considered as one of the most probable causes of the revealed negative PE value.     

Swelling/Floating Capability and Drug Release Characterizations of Gastroretentive Drug Delivery System Based on a Combination of Hydroxyethyl Cellulose and Sodium Carboxymethyl Cellulose

The aim of this study was to characterize the swelling and floating behaviors of gastroretentive drug delivery system (GRDDS) composed of hydroxyethyl cellulose (HEC) and sodium carboxymethyl cellulose (NaCMC) and to optimize HEC/NaCMC GRDDS to incorporate three model drugs with different solubilities (metformin, ciprofloxacin, and esomeprazole). Various ratios of NaCMC to HEC were formulated, and their swelling and floating behaviors were characterized. Influences of media containing various NaCl concentrations on the swelling and floating behaviors and drug solubility were also characterized. Finally, release profiles of the three model drugs from GRDDS formulation (F1-4) and formulation (F1-1) were examined. Results demonstrated when the GRDDS tablets were tested in simulated gastric solution, the degree of swelling at 6 h was decreased for each formulation that contained NaCMC in comparison to those in de-ionized water (DIW). Of note, floating duration was enhanced when in simulated gastric solution compared to DIW. Further, the hydration of tablets was found to be retarded as the NaCl concentration in the medium increased resulting in smaller gel layers and swelling sizes. Dissolution profiles of the three model drugs in media containing various concentrations of NaCl showed that the addition of NaCl to the media affected the solubility of the drugs, and also their gelling behaviors, resulting in different mechanisms for controlling a drug’s release. The release mechanism of the freely water-soluble drug, metformin, was mainly diffusion-controlled, while those of the water-soluble drug, ciprofloxacin, and the slightly water-soluble drug, esomeprazole, were mainly anomalous diffusion. Overall results showed that the developed GRDDS composed of HEC 250HHX and NaCMC of 450 cps possessed proper swelling extents and desired floating periods with sustained-release characteristics.     

Relation of Lipopolysaccharide and Fatty Acid Ester Release to the Ethylenediaminetetraacetic Acid Alteration of Permeability in Enterobacteriaceae

Escherichia coli subjected to cold osmotic shock released 30 to 40% of their fatty acid esters and 42% of their cellular hexosamine. In contrast, Enterobacter, although they released 40% of fatty acid esters, release only 25% of hexosamine. Proteus released less than 15% of either fatty acid esters or hexosamine. These differences are taken to explain the differences among the Enterobacteriaceae in releasing surface enzymes after osmotic shock. It is felt that the release of additional lipopolysaccharide after osmotic shock is necessary for the release of surface enzymes that are not freed by ethylenediaminetetraacetic acid-tris(hydroxymethyl)aminomethane exposure.     

Sucrose and Hexose Release by Excised Stem Segments of Vicia faba L.: The Sucrose-Specific Stimulating Influence of Cuscuta on Sugar Release and the Activity of Acid Invertase

By washing out solutes in 0.5 mM CaSO⁴at 25 °C during aperiod of 5–6 h, the release of sugars by excised stemsegments of Vicia faba L. was measured. The stem parasite Cuscutaeuropaea strongly stimulated the release of sucrose into theefflux medium; this effect was most marked during the last hoursof each experiment but this stimulating effect of the parasitecould not be detected for glucose and fructose. The fact thatparasitized stem segments released higher than normal hexoseamounts during the last hours of several experiments, couldbe explained as the result of extracellular hydrolysis of sucroseby free space invertase. A high free space acid invertase activitywas present in young stem segments of Vicia faba and in tissuesof Cuscuta. The stimulating influence of Cuscuta on sugar releaseby cells of stem segments appears sucrose-specific, supportingthe idea that the stimulating influence of Cuscuta on sugarrelease is restricted to the sieve-tube system. When metabolic inhibitors were added to the washing solutionor when segments were incubated at low temperature, no cleareffect of the parasite could be observed and for all segments(parasitized and non-parasitized) a strongly enhanced releaseof sucrose into the efflux medium was found during the lasthours of an experiment. These data support the idea that anintensive resorption of sucrose occurs within stem segments,after its release into the apoplast. Key words: Cuscuta europaea, Parasitic relationship, Phloem unloading     

Cysteine Sulfinic Acid in the Central Nervous System: Uptake and Release of Cysteine Sulfinic Acid by a Rat Brain Preparation

Abstract: Uptake and release of cysteine sulfinic acid by synaptosomal fractions (P₂) and slices of rat cerebral cortex were investigated. The P₂ fraction had a Na⁺-dependent high-affinity uptake system for cysteine sulfinic acid (K_m, 12μM), which was restricted to the synaptosomes. High-affinity uptake of cysteine sulfinic acid was competitively inhibited by glutamate, aspartate, and cysteic acid. None of the various centrally acting drugs tested specifically inhibited this transport system. Release of [¹⁴C]cysteine sulfinic acid from preloaded cortical slices or P₂ fractions was examined by a superfusion method, which avoided reuptake of released [¹⁴C]cysteine sulfinic acid. High K⁺ (56 m M ) and veratridine (10μM) stimulated the release of cysteine sulfinic acid from slices and the P₂ fraction in a partly Ca²⁺-dependent manner. Diazepam at concentrations of 10 and 100 μM markedly inhibited the stimulated release, but not the spontaneous release, by cortical slices. On the contrary, it had no effect on the stimulated release of cysteine sulfinic acid from the P₂ fraction.     

Immobilization of Lipase from Rhizopus Niveus: A Way to Enhance its Synthetic Activity in Organic Solvent

Lipase (EC 3.1.1.3) from Rhizopus niveus was immobilized by physical adsorption on various carriers, including different types of Celite, Spherosil and Duolite. After the enzyme immobilization, the recovered hydrolytic and synthetic activities on the different carriers were then determined. The results showed that the highest synthetic activity was obtained when Duolite XAD 761 was used as the carrier. However the recovered hydrolytic activity after the immobilization on this resin was relatively low although this carrier showed the best protein loading capacity. The highest recovered hydrolytic activity was observed when the lipase was immobilized on Celite Hyflo-Supercel using an immobilization buffer adjusted to pH 4. The comparison of the free and immobilized lipase specific activities suggest that the immobilization on Celite Hyflo-Supercel, Spherosil XOA 200 and silica has enhanced the lipase hydrolytic activity. On the other hand, the use of the lipase immobilized on Duolite XAD 761 as biocatalyst of synthetic reaction, compared to that of the free enzyme, allows the reaction initial velocity to be increased 12.2-fold. In addition, the synthetic activity of the lipase immobilized on Duolite XAD 761 was shown to be maximum at a water activity in the range of 0.32-0.52.