Improved dissolution and cytotoxicity of camptothecin incorporated into oxidized-cellulose microspheres prepared by spray drying

Oxidized celluloses (OC) containing 7, 13, and 20% carboxylic content (OC-7, OC-13, and OC-20, respectively) have been converted into aqueous colloidal dispersions and used to prepare microspheres of the antineoplastic agent camptothecin (CPT) by spray drying. Plasticizers used were glycerin, polyethylene glycol 400 (PEG-400), and polyethylene glycol 6000 (PEG-6000). Irrespective of the carboxyiic content of OC and the nature of plasticizer employed, the size of microspheres varied from 1.25+/-0.40 to 1.52+/-0.47 microm. The release studies in pH 7.4 buffer revealed the dissolution of CPT to be faster from the microsphere formulations than from physical mixtures and free CPT. The times to release 50% CPT (T-50%) from microspheres prepared using OC-7, OC-13, and OC-20 were about 31, 37, and 19 h, respectively. The in vitro cytotoxicity results indicated OC-20/CPT microspheres to be more effective than free CPT against human-derived RPMI-8402 lymphoid and THP-1 myeloid leukemia cell lines. The ED50 values for the OC-20/CPT microspheres and free CPT were 1 x 10(-5) and 0.25 x 10(-1) microg/mL, respectively, against the RPMI-8402 line and 0.5 x 10(-2) and 0.75 microg/mL, respectively, against the THP-1 line. The higher activity of OC-20/CPT microspheres compared to that of the free drug is attributed to increased dissolution of CPT from microspheres.     

Cardiovascular effects elicited by milonine, a new 8,14-dihydromorphinandienone alkaloid

Abstract: The mechanisms underlying the cardiovascular responses evoked by milonine (i.v.), an alkaloid, were investigated in rats. In normotensive rats, milonine injections produced hypotension and tachycardia, which were attenuated after N^w‐nitro‐l ‐arginine methyl esther (l ‐NAME; 20 mg/kg, i.v.). In phenylephrine (10 μM), pre‐contracted mesenteric artery rings, milonine (10^?10 M to 3 × 10^?4 M) caused a concentration‐dependent relaxation (EC₅₀ = 1.1 × 10^?6 M, E_max = 100 ± 0.0%) and this effect was rightward shifted after either removal of the vascular endothelium (EC₅₀ = 1.6 × 10^?5, p < 0.001), or after l ‐NAME 100 μM (EC₅₀ = 6.2 × 10^?5, p < 0.001), hydroxocobalamin 30 μM (EC₅₀ = 1.1 × 10^?4, p < 0.001) or ODQ 10 μM (EC₅₀ = 1.9 × 10^?4p < 0.001). In addition, in rabbit aortic endothelial cells, milonine increased NO₃^? levels. The relaxant effect induced by milonine was attenuated in the presence of KCl (20 mM), a modulator efflux K⁺ (EC₅₀ = 1.2 × 10^?5, p < 0.001), or different potassium channel blockers such as glibenclamide (10 μM) (EC₅₀ = 6.3 × 10^?5, p < 0.001), TEA (1 mM) (EC₅₀ = 2.3 × 10^?5 M, n = 6) or Charybdotoxin (0.2 μM) plus apamin (0.2 μM) (EC₅₀ = 3.9 × 10^?4 M, n = 7). In addition, pre‐contraction with high extracellular potassium concentration prevented milonine‐induced vasorelaxation (EC₅₀ = 1.0 × 10^?4, p < 0.001). Milonine also reduced CaCl₂‐induced contraction in Ca²⁺‐free solution containing KCl (60 mM). In conclusion, using combined functional and biochemical approaches, we demonstrated that the hypotensive and vasorelaxant effects produced by milonine are, at least in part, mediated by the endothelium, likely via nitric oxide release, activation of nitric oxide‐cGMP pathway and opening of K⁺ channels.     

Preparation and Characterization of Spray-Dried Oxidized Cellulose Microparticles

The goal of this study was to investigate the feasibility of spray drying to produce microparticles of oxidized cellulose (OC), a biocompatible and bioresorbable polymer. OCs containing 7, 13, and 20 wt% carboxylic groups were converted into stable aqueous dispersions and then spray dried using a Yamoto G-32 spray dryer, equipped with a standard fluid nozzle with an orifice of 406 μm. The following operating conditions were investigated: inlet temperature 140, 170, and 190°C; feed rate 3, 6, and 9 mL / min; and atomization air pressure 0.5, 1, and 1.5 kg f/cm². The amounts of OC used in feed were 1, 2.5, and 5%. OC microparticles produced under these conditions were shrunken spheres, ranging in size between 0.98 ± 0.47 and 2.05 ± 0.98 μm. The different operating conditions used had no significant effect on the size and shape of particles. The use of a water-soluble plasticizer (glycerin, polyethylene glycol 400, or polyethylene glycol 6000) in the dispersion yielded microparticles with a good sphericity and a smooth surface morphology, whereas no change in the shape or size of microparticles was noted with water-insoluble plasticizers, Triacetin® and dibutyl phthalate. Powder X-ray diffraction and Fourier transform infrared spectral analyses of spray-dried microparticles showed no change in the solid-state structure of OC. In conclusion, results show that OC can be converted into stable aqueous dispersions and used to produce microparticles by spray drying.     

Pharmacokinetics of ibuprofen in man—III: Plasma protein binding

Plasma protein binding of ibuprofen was measured by equilibrium dialysis on 406 plasma samples collected from 15 normal volunteers following doses of 400, 800, and 1200 mg of ibuprofen as tablets (N=102, 100, 104, respectively) and 420 mg as an aqueous solution (N=100). Individual subject bound concentration at dialysis equilibrium (C_bd) vs. free concentration at dialysis equilibrium (C_fd) were well fitted via computer to the Scatchard equation with one class of binding sites. The binding capacity averaged 1231 μM (range 848–1658 μM), and the association constant averaged 1.76 × 10⁵M^?1 (range 1.15 × 10⁵ to 2.73 × 10⁵M^?1). Distributional analysis was performed on the free fraction (f_d) and bound/free ratios (C_bd/C_fd=1/f_d?1) at dialysis equilibrium for each treatment. Using pooled data of all four treatments, distributional analysis was also performed on the free fractions (f) and bound/free ratios (C_b/C_f=1/f?1) corresponding to the plasma drug concentrations in blood as it was withdrawn from the subjects. The bound/free ratios were normally distributed, whereas the distributions of the free fractions were skewed towards higher values.     

A comparative study of the interaction of ochratoxins with bovine serum albumin

The interaction of different ochratoxins with bovine serum albumin (BSA) has been studied by equilibrium dialysis, solubility and spectrophotometric analyses. Spectrophotometric analyses revealed that the absorption maxima of ochratoxin B (OB) and ochratoxin C (OC) shift to longer wavelengths (365–377 nm for OB. 380–390 nm for OC) as a result of interaction. The spectra of ochratoxins α (Oα) and β (Oβ) were not altered in the presence of BSA. Equilibrium dialysis results indicate that 1 mole of BSA binds 1.03, 1.02, 1.93 and 3.24 moles of Oα, Oβ, OB and OC, with binding constant of 1.5 × 10⁶ M^?1, 5.1 × HF M^?1. 7.1 and 8.9 × 10⁵ M ^?1 respectively. Spectrophotometric titrations of the 1:1 molar ratio of BSA ochratoxin complexes indicate that the pK values of OB and OC decrease on binding, whereas the pK values of Oα and Oβ remain unaltered. The dissociation of the phenolic hydroxyl group in the isocoumarin ring of ochratoxins A, B and C appears responsible for the binding of the second molecule of these toxins with albumin. The overall results suggest that both ionic and hydrophobic forces are important in the binding of ochratoxins with BSA.     

Preparation of chitosan microspheres using membrane emulsification and its size modelling

The chitosan microspheres were prepared by a membrane emulsification method with variations of the N₂ gas pressure and the chitosan concentration. The pressure of N₂ gas was varied within the range from 0.2?×?10⁵ to 0.8?×?10⁵?Pa at chitosan concentration 1.5?wt%. In addition, the concentration of chitosan was varied between 0.5?～?2.0?wt% at 0.4?×?10⁵?Pa of N₂ gas pressure. Using this method, it is possible to prepare stable emulsions with a very narrow droplet size distribution in comparison with conventional methods. The average size of the microspheres was dependent on the N₂ gas pressure and the concentration, that is it was increased with the pressure and the concentration. The modelling of the size for the microspheres according to the concentration was carried out using Macleod's relation and Parkins & Brown equation. The former shows the relationship between density and surface tension and the latter demonstrates the correlation between the volume of the microspheres and the interfacial tension. The modelling results were in good agreement with the experimental data to predict the microspheres size with the variation of concentration.     

Dynamics of microparticles inside lipid vesicles: movement in confined spaces

Microparticles and nanoparticles used in drug delivery frequently depend on their movement in confined spaces such as cells. Liposomes containing small numbers of 1-µm diameter polystyrene particles were used to study the dynamics of their movement within the confined space of the liposome interior. The analysis of the trajectories of single and multiple entrapped particles revealed that the particles were largely localized toward the periphery of the liposome with a rare presence in the centre. Interparticle interactions were studied by calculating interparticle distances, ranging from close to zero to around 8 µm with a mean of ～4 µm. The diffusion coefficient of a single entrapped particle was D?=?0.27?×?10^?9 cm² s^?1 when compared with 5.1?×?10^?9 cm² s^?1 free in water. When more than one particle was entrapped, the calculated diffusion coefficients were D?=?0.61?×?10^?9 cm² s^?1 for two particles, D?=?1.26?×?10^?9 cm² s^?1 for three particles, and D?=?1.3?×?10^?9 cm² s^?1 for multiple particles). Particle movement was found to be distinctly faster at the periphery (average velocity 21.4 μm s^?1) than at the centre of the vesicle (average velocity 14.2 μm s^?1). These results demonstrate the significance of particle–particle interactions as well as particle–surface interactions, which is evident here in some systems by particle aggregation close to the liposome membrane.     

Plasma Protein Binding of the Experimental Antitumour Agent Acridine-4-carboxamide in Man,Dog, Rat and Rabbit

Abstract— The plasma binding of N-[2-(dimethylamino)ethyl]acridine-4-carboxamide (AC) was investigated in-vitro by equilibrium dialysis for 3 h at 37°C against isotonic phosphate buffer (pH 7·35) using [³H]AC. There were significant species differences with the smallest % free fraction (mean ± s.d.) occurring in human plasma (3·4 ± 0·2), followed by dog (8·1 ±0·4), mouse (14·8 ± 0·8), rat (16·3 ± 0·9) and rabbit (20·2 ± 0·7). In plasma from healthy individuals (n = 5), the % free fraction ranged from 2·7 to 3·8. In physiological solutions of human proteins, the greatest binding was observed for α-acid glycoprotein (AAG) (0·75 g L^?1) with a mean free fraction of 24·1 ± 2·2%, followed by albumin (40 g L^?1) with 31·6 ± 0·7 and 39·8 ± 2·5% for fatty-acid-free and globulin-free, respectively. There was also some binding to globulins (5 g L^?1) with a mean % free fraction of 70·3 ± 1·6 and 84·8 ± 2·2 for Conn's fraction I and IV, respectively. Binding data from the displacement of [³H]AC by increasing concentrations of AC in human AAG (0·75 g L^?1) or albumin solution (40 g L^?1) indicated that AAG had 10-fold greater binding affinity for AC (K_a, 7·8 × 10⁴ m^?1) compared with albumin (K_a, 6·8 × 10³ m^?1). In human plasma enriched with AAG there was a significant negative linear correlation (r = 0·932; P < 0·001) between % AC free fraction and increasing AAG concentration over the range 0·6–4·5 g L^?1. Small but significant (P < 0·05) increases in AC free fraction occurred in the presence of various metabolites (50 and 100 μm) but, of those tested, only N-monomethyl-acridine carboxamide increased the free fraction to the same extent as parent AC.     

Interactions between tetraethylammonium and methohexitone at the chick neuromuscular junction: Experiments with the moving fluid electrode technique

The effect of methohexitone on the depolarization and contracture responses produced by tetraethylammonium (TEA), acetylcholine (ACh) and repetitive indirect stimulation were investigated, using the moving fluid electrode technique, in the chick biventer cervicis (BVC) nerve muscle preparation. TEA (4.8 × 10^?4?4.8 × 10^?2 M) produced contracture and depolarization responses which were concn-dependent. These responses were potentiated by methohexitone (8.8 × 10^?5 M). The mean ED₅₀S for the contracture responses in the control Krebs solution and with methohexitone were (mean ± S.E.M.) 6.5 ± 0.03 × 10^?3 M and 1.3 ± 0.04 × 10^?3 M (N = 6) respectively. The mean ED₅₀S for the depolarizations were (mean ± S.E.M.) 5.9 ± 0.1 × 10^?3 and 1.5 ± 0.06 × 10^?3 M (N = 6) respectively. ACh (5.5 × 10^?6?1.1 × 10^?2 M) produced contracture and depolarization responses which were concn-dependent. These responses were reduced by methohexitone (8.8 × 10^?5 M). The mean ED₅₀S for the contracture responses in the control Krebs solution and with methohexitone were (mean ± S.E.M. 2.4 ± 0.21 × 10^?4 and 2.3 ± 0.1 × 10^?3 M (N = 6) respectively. The mean (± S.E.M.) ED₅₀S for the depolarizations were 8.4 ± 0.33 × 10^?4 and 3.7 ± 0.14 × 10^?3 M (N = 6), respectively. Repetitive indirect stimulation, at 1–20 Hz, produced contraction and depolarization responses which were frequency-dependent. These responses were slightly potentiated by methohexitone (8.8 × 10^?5 M). The mean (± S.E.M.) frequency₅₀S for the contractions produced in the control Krebs solution and with methohexitone were 9.2 ± 0.1 and 8.5 ± 0.2 Hz (N = 6) respectively. The mean frequency₅₀S for the depolarizations were (mean ± S.E.M.) 7.2 ± 0.1 and 5.8 ± 0.19 Hz (N = 6) respectively. It is concluded that TEA may have a direct post-synaptic action, in addition to releasing ACh from the presynaptic nerve terminals. TEA produces more contracture tension than does ACh for a given level of membrane depolarization. Methohexitone, non-competitively, reduces the responses produced by applied ACh whereas it potentiates those produced by TEA and repetitive nerve stimulation.     

Four plasma kallikreins generated in acetone-activated human plasma

Four enzymatically active fractions were isolated from acetone-activated human plasma. These four esterases were termed KI, KII, KIII and KIV. KI, KII, KIII and KIV were found to represent free kallikrein, kallikrein-α2-macroglobulin (α2M) complex, kallikrein-α2M-high molecular weight (HMW) kininogen complex and kallikrein-HMW kininogen complex, respectively. KI, KII, KIII and KIV showed K_m, values 2.27 × 10^?3, 7.14 × 10^?3, 7.69 × 10^?3 and 3.59 × 10^?3 M for TAMe hydrolysis, respectively. Kininogenase activity in KII and KIII was much less than in KI and KIV. Soy bean trypsin inhibitor completely inhibited the esterase activity in KI and KIV but not in KII and KIII.     

Differential toxicity of carrier-bound methotrexate toward human lymphocytes,marrow and tumor cells

Methotrexate that was covalently linked to poly-l-lysine (mol. wt 3,000 and 60,000) (MTX-PLL 3K and 60K) was more inhibitory to the growth of five cell lines from human solid tumors (IC₅₀ 5?10 × 10^?8 M and 1?2.6 × 10^?8 M respectively) than to the growth of five lines of human lymphocytes (IC₅₀ 5?8 × 10^?7 M and 2?5 × 10^?7 M). In contrast, both methotrexate that was covalently linked to human serum albumin (MTX-HSA), and the free drug, were equally toxic to the two classes of cells, with IC₅₀ of 3?15 × 10^?7 M and 2?7 × 10^?8 M, respectively, for the cell types. Uptake studies showed that, whereas MTX and MTX-HSA were transported equally well into WI-L2 lymphocytes, human bone marrow cells, and an astrocytoma tumor line, uptake of MTX-PLL by the astrocytoma cells at 37° was three to four times greater than uptake by WI-L2 lymphocytes or marrow cells. [³H]Deoxyuridine ([³H]-Urd) incorporation studies indicated that low concentrations of MTX-PLL 60K (5 × 10^?7 M) resulted in inhibition of the target enzyme dihydrofolate reductase (DHFR) in the astrocytoma cells, but no iinhibition of DHFR occurred in WI-L2 lymphocytes or marrow cells until concentrations were reached where the carrier itself became toxic (5 × 10^?6 M). Two inhibitors of the lysosomal enzymes, chloroquine and lupuptin, were able to reverse the toxicity of MTX-PLL 60K against the astrocytoma cell line, increasing its IC₅₀ from 2 × 10^?8 to 2 × 10^?7 M. Both lysosomal inhibitors had no effect on the toxicity of MTX-PLL 60K against the WI-L2 lymphocytes or of MTX or MTX-HSA against either cell type, indicating that the increased toxicity of MTX-PLL 60K against the tumor cells was due, in part, to the ability of the lysosomes of these cells to convert MTX-PLL 60K either to the free drug or to a derivative that was effective in inactivating DHFR. These results suggest that comparable differential toxicity between marrow and tumor cells might also be achieved in vivo if MTX-PLL is infused over long periods at a rate that would maintain a constant serum concentration sufficient to kill tumor cells without affecting bone marrow cells.     

Interaction of myo-inositol hexaphosphate (MIHP) with β-globulin from Sesamum indicum L.

β-Globulin, the low molecular weight protein fraction from Sesamum indicum L., interacts with myo-inositol hexaphosphate (MIHP) maximally at pH 3.0, with concomitant precipitation up to 85 ± 2% at an MIHP concentration of 8 × 10^?4m . The kinetics of interaction as followed by stopped-flow spectrophotometry suggested the reaction to be of pseudo first-order, having an initial fast step followed by a relatively slow step of rate constants 1.9 × 10^?2 s^?1 and 1.2 × 10^?2 s^?1 respectively at 1 × 10^?4m MIHP concentration. The analysis of the complex indicated the presence of polymer as seen in sedimentation velocity experiment. This was accompanied by conformational change of a three-fold decrease in β-structure and also an increase in fluorescence emission intensity accompanied with a red shift from 330 to 334 nm. Stoichiometric analysis of MIHP binding suggested four independent binding sites for MIHP, with a free energy change, ΔG^o= -5.1 kcal mol^?1 resulting from a binding constant of 3.6 × 10³m -1.     

In vitro hydroxylation of diphenylhydantoin and its inhibition by other commonly used anticonvulsant drugs

Using a crude 9,000 g rat liver musomal preparation, in vitro studies were carried out on (a) the metabolism (hydroxylation) of diphenylhydantoin (DPH), (b) the effect of other commonly used anticonvulsants on this hydroxylation. DPH hydroxylation exhibited saturation kinetics at a DPH substrate concentration of approximately 10^?4M. Mean K_m and V_m values for the reaction were 9.3 × 10^?5 M and 23.3 μg/m1 respectively. The linear Hill plot with an interaction coefficient of 1.0 suggests that there is no cooperativity between different DPH molecules during DPH receptor binding process. The anticonvulsants ethosuximide, sodium phenobarbitone, sodium valproate and sulthiame all exhibited inhibition of DPH hydroxylation to varying degrees. K_i, inhibition constants for the four anticonvulsants were respectively 1.1 × 10^?2, 9 × 10^?4, 1.8 × 10^?2 and 8.8 × 10^?4M. Inhibition of DPH hydroxylation by sodium phenobarbitone and sulthiame was strong and competitive in nature. Ethosuximide showed a weak competitive type of inhibition and sodium valproate a weak uncompetitive type of inhibition.     

Two mechanisms of adriamycin-DNA interaction in L1210 cells

Among the effects exerted by adriamycin (ADR), interaction with DNA is closely related to cytotoxicity. The interaction results in the formation of protein-associated DNA single-strand breaks (PA-SSB) and, at drug levels ? 2.8 × 10^?6 M, also in “direct” (nonenzymatic) DNA single-strand breaks (D-SSB). To characterize the two types of DNA lesions, euoxic mouse leukemia L1210 cells were treated with various antioxidant agents in the presence of 2.8 × 10^?6, × 10^?5, or × 10^?4 M concentrations of ADR. The enzymes superoxide dismutase (200 μg/ml) or catalase (250 μg/ml), the OH scavengers dimethyl sulfoxide (70 mM) or ethanol (70 mM), and an inhibitor of superoxide production, 2-deoxy-glucose (1 and 10 mM), reduced the frequency of D-SSB to 18.3 to 68.2% of its level in ADR-treated controls, while the frequency of PA-SSB remained unchanged. These observations seem to indicate that ADR-mediated free radicals cause discernible DNA damage in euoxic cells only at very high drug concentrations, greater than the peak plasma level achievable clinically following i.v. bolus. At lower ADR levels, relevant to clinical use, another type of interaction between the drug and DNA prevails, which apparently does not involve a free-radical mechanism.     

Simple HPLC Determination of the Concentrations of Epiroprim in the Serum and Brains of Mice

Epiroprim, an analogue of trimethoprim, has been shown to potentiate the efficacy of dapsone in experimental parasitic infections. A simple and accurate HPLC method has been developed to estimate epiroprim in serum and brain Blood and brains from mice were sampled 0, 30, 75, 120 and 240 min after 50 or 100 mg kg^?1 oral gavage. The drug and added internal standard metoprine in serum and brain supernatant were isolated by solid-phase extraction (Supelclean LC-SCX). The HPLC system consisted of a 150 × 4.6 mm Hypersil 5 μm ODS column. The mobile phases contained various proportions of acetonitrile, methanol and phosphate buffer (0.1 M). Peaks were detected by UV absorbance at 210 run. Serum concentrations (mean ± s.e.m.) of epiroprim were highest at 30 min for both 50 and 100 mg kg^?1 doses, 173 ± 20 and 207 ± 25 ng mL^?1, respectively, falling to 8 ± 5 and 18 ±6 ng mL^?1, respectively, at 240 min. Epiroprim concentrations in the brain correlated well with those in the serum, with levels of 223 ±69 and 265 ±21 ng g^?1 falling to 10 ± 10 and 31 ± 11 ng g^?1, respectively. Epiroprim is rapidly absorbed and distributed to the brain.     

5-Fluorouracil-loaded microspheres prepared by spray-drying poly(D,L-lactide) and poly(lactide-co-glycolide) polymers: Characterization and drug release

5-Fluorouracil (5-FU), a hydrosoluble anti-neoplastic drug, was encapsulated in microspheres of poly(D,L-lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) polymers using the spray-drying technique, in order to obtain small size microspheres with a significant drug entrapment efficiency. Drug-loaded microspheres included between 47?±?11 and 67?±?12?µg 5-FU?mg^?1 microspheres and the percentage of entrapment efficiency was between 52?±?12 and 74?±?13. Microspheres were of small size (average diameter: 0.9?±?0.4–1.4?±?0.8?µm microspheres without drug; 1.1?±?0.5–1.7?±?0.9?µm 5-FU-loaded microspheres) and their surface was smooth and slightly porous, some hollows or deformations were observed in microspheres prepared from polymers with larger T_g. A fractionation process of the raw polymer during the formation of microspheres was observed as an increase of the average molecular weight and also of T_g of the polymer of the microspheres. The presence of 5-FU did not modify the T_g values of the microspheres. Significant interactions between the drug and each one of the polymers did not take place and total release of the included drug was observed in all cases. The time needed for the total drug release (28–129?h) was in the order PLA?>?PLGA 75/25?>?PLGA 50/50. A burst effect (17–20%) was observed during the first hour and then a period of constant release rate (3.52?±?0.82–1.46?±?0.26?µg 5-FU?h^?1 per milligram of microspheres) up to 8 or 13?h, depending on the polymer, was obtained.     

Anticoagulant, anti-aggregation and antithrombotic effects of a novel hexapeptide

Objectives Hexapeptide is a novel synthetic oligopeptide with a structure similar to that of eptifibatide. This study was designed to investigate the anticoagulant, anti‐aggregation, disaggregation and anti‐thrombogenesis effects of hexapeptide. Methods The effects of antiplatelet aggregation induced by adenosine diphosphate (ADP), arachidonic acid (AA) and thrombin, and the effect of disaggregation of platelet aggregation induced by ADP were determined. The anticoagulation indexes were determined by different kits. Key findings Hexapeptide 1 × 10^?5–1 × 10^?4m could significantly prolong rabbit blood clotting time, thrombin time, prothrombin time and activated partial thromboplastin enzyme time, and reduce the length, wet weight, dry weight and the index of thrombus in a concentration‐dependent manner. Hexapeptide 1 × 10^?4m decreased platelet adhesion rate by 40.2%. The platelet aggregation inhibition of hexapeptide in dogs and humans was more obvious than in rabbits and rats. The aggregation inhibition rate of 1 × 10^?5m hexapeptide in dogs, rabbits, rats and humans induced by ADP was 93.9 ± 1.3%, 66.2 ± 1.4%, 76.1 ± 3.2% and 99.8 ± 0.2%, respectively; the 50% inhibitory concentration (IC50) of hexapeptide was 7.24 × 10^?8, 3.24 × 10^?6, 6.61 × 10^?6 and 8.91 × 10^?8m , respectively. For the aggregation inhibition rate of hexapeptide in dogs, rabbits and humans induced by AA, the IC50 was 1.29 × 10^?9, 1.32 × 10^?6 and 9.33 × 10^?8m , respectively; the IC50 of aggregation inhibition rates induced by thrombin was 2.88 × 10^?6, >1 × 10^?5 and 4.17 × 10^?6m , respectively. The disaggregation rate of 1 × 10^?4m hexapeptide in dog induced by ADP was 68.8 ± 7.4%. Conclusions Hexapeptide has anticoagulant, antiplatelet aggregation, disaggregation and antithrombotic effects in vitro.     

Indomethacin and the role of prostaglandins in adipose tissue.

Indomethacin (2 or 10 μg/ml) or meclofenamic acid (4 μg/ml), two potent inhibitors of prostaglandin biosynthesis, did not affect basal or noradrenaline (10^?6 M) stimulated lipolysis when added to isolated rat fat cells. Indomethacin (5 μg/ml blood) similarly was without effect on blood flow, on lipolysis or on ³H-noradrenaline overflow before, during and after nerve stimulation (4 Hz) in perfused canine subcutaneous adipose tissue in situ. Indomethacin given to rats 5 × 5 mg/kg at 10–14 hr intervals p.o. had no effect on arterial glycerol concentration, but caused a significant hypoglycemia. Fat cells or fat pads extracted from such rats had an unchanged basal lipolytic rate but a lowered responsiveness to high concentrations of noradrenaline (4 × 10^?7-2 × 10^?6 M), compared with controls. However, 10^?7 M noradpresumbaly caused a higher lipolytic response in fat cells from indomethacin-treated rats than from controls, presumably because the former caused a smaller degradation of naradrenaline during the incubation period. Indomethacin (0·2–20 μg/ml) had no effect on cAMP binding to protein kinase, but apparently caused membrane stabilization, since erythrocytes from indomethacin-treated rats were more resistant to hypotonic lysis than red cells from control animals. Our results suggest that indomethacin has a multitude of biological effects, some of which may be unrelated to inhibition of prostaglandin biosynthesis. When considered together with previous results the findings also suggest that endogenous prostaglandins are of minor importance as feed back inhibitors of lipolysis in adipose tissue.