Diglucosyl-glycerolipids from the marine sponge-associated Bacillus pumilus strain AAS3: their production, enzymatic modification and properties.

The marine strain Bacillus pumilus strain AAS3, isolated from the Mediterranean sponge Acanthella acuta, produced a diglucosyl-glycerolipid, 1,2-O-diacyl-3-[beta-glucopyranosyl-(1-6)-beta-glucopyranosyl)]glycerol, with 14-methylhexadecanoic acid and 12-methyltetradecanoic acid as the main fatty acid moieties (GGL11). On a 30 l scale, using artificial seawater supplemented with glucose (20 g/l), yeast extract (10 g/l), and suitable nitrogen/phosphate sources, growth-associated glycoglycerolipid production reached its maximum yield of 90 mg/l after 11 h. Lipase-catalyzed modification of the native substance led to the deacylated parent compound (GG11), which could be reacylated using the same enzyme system to afford a new dipentenoyl-diglucosylglycerol (GGL12) as the major product upon addition of 4-pentenoic acid to the medium. GGL11 decreased the surface tension of water from 72 mN/m to 29 mN/m and the interfacial tension of the water/ n-hexadecane system from 44 to 5 mN/m. Anti-tumor-promoting studies on this class of diglucosyl glycerol products showed that the carbohydrate/glycerol backbone (GG11) has a more potent inhibitory activity than the acylated compounds. The diglucosyl-glycerol GG11 strongly inhibited growth of the tumor cell lines HM02 and Hep G2 (50% inhibition at approximately 1 microg/ml), while the glycerolipids GGL11 and GGL12 were less active or had no effect.

Initiation of an Aquaculture of Sponges for the Sustainable Production of Bioactive Metabolites in Open Systems: Example, Geodia cydonium.

Among Metazoa, sponges (phylum Porifera) are the richest source for different bioactive compounds. The availability of the raw material is, however, restricted. To obtain enough of the bioactive compounds for application in human therapy, sponges have to be cultured in in vitro systems. One technique for the establishment of a long-term cell culture from sponges has recently been elaborated. Here, we present a procedure to cultivate tissue samples from sponges in an open system. The species Geodia cydonium, which produces bioactive compounds, has been selected. Tissue samples of approximately 10 g were attached to the bottoms of cultivation trays. After 2 to 3 days, the tissue samples formed a robust contact with the metal support. Subsequently, sets of trays, called tray batteries, either remained in huge aquaria at the Center for Marine Research or were transferred to the vicinity of a fish and mussel farm. The growth rates of the samples remained unchanged within the first month; however, after 3 and 6 months, they increased to 147% and 189%, respectively. In parallel, extracts were prepared from the tissue samples and tested for cytotoxicity in a mouse lymphoma cell assay system. Extracts from cultured tissue initially had a low inhibitory potency; however, after cultivation for 3 or 6 months, values comparable to those of extracts from sponges taken from the biotope were found. In addition, a molecular marker was applied to document the response (health state) of the tissue and the identity of the material in culture. The CD63 molecule was chosen because the expression of this molecule in mammalian systems changes with the age of the animals. The corresponding complementary DNA was isolated from Geodia cydonium. With this probe, the level of expression in cultured tissue samples decreased immediately after starting cultivation; after a cultivation period of 6 months, however, values were similar to those found in controls. These data show that sponge species that produce bioactive compounds can be cultivated in open systems, in which they retain their potency to produce bioactive compounds as well as their health state.

Cytoprotective effect of NMDA receptor antagonists on prion protein (PrionSc)-induced toxicity in rat cortical cell cultures.

Rat cortical cells were incubated with the Scrapie prion protein, PrionSc. At concentrations of 3 ng/ml of PrionSc and higher, the viability of the cells decreased significantly after a 12-h incubation period. Simultaneously, the degree of DNA fragmentation increased. In control experiments with antibodies against PrionSc, PrionSc lost its deleterious effect on neurons. PrionSc did not affect the viability of astrocytes. Drugs known to block NMDA receptor channels, such as memantine (1-amino-3,5-dimethyl-adamantane) (Mem), its analogue 1-N-methylamino-3,5-dimethyl-adamantane as well as (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) prevented the effect of PrionSc. Production of PrionSc in the Scrapie prion-infected subclone of N2 a cells (ScN2 a cells) was not affected by memantine. We conclude that antagonists of the NMDA receptor-channel complex (i) abolish the PrionSc-induced neuronal injury in vitro, and (ii) display no influence on the synthesis and/or the processing of PrionSc.

Influences of tromantadine and nonoxinol 9 on the stability of red cell membrane.

The antiviral compound tromantadine (ViruMerz) and the detergent nonoxinol 9 have been investigated in their effects on biophysical parameters of red cell membranes. Up to a maximum ratio of 1 mol per 800 mol of phospholipids tromantadine enhances the membrane phase transition/separation break at 16-20 degrees C measured by 1-anilinonaphthalene-sulfonate (ANS) fluorescence. It also increases order parameters obtained from spin labeling experiments with 5-doxyl stearic acid. Nonoxinol 9 interacts with the membrane at a maximum ratio of 1 mol per 40 mol of phospholipids determined by UV spectrophotometry. The substance decreases the intensity of the above phase transition/separation break and the order parameters of the spin label 5-doxyl stearic acid. These experiments indicate that tromantadine probably stabilizes the membrane whereas nonoxynol 9 exhibits opposite effects. Combination of both compounds equalizes the influences of the single substances on the above biophysical parameters of red cell membrane with predominance of the nonoxinol 9 effect.

Interaction of memantine with cholecystokinin receptors in mouse brain.

The effect of memantine on CCK receptors in mouse brain has been investigated using particles of dissected cortex and striatum. Total binding of radio-labelled CCK33 was one-half maximal within 10 min of incubation and reached a maximum after 30 to 60 min when either cortex or striatum was used. Non-specific binding (presence of 100 microM unlabelled CCK8) was 50 to 80% of total binding at steady state conditions. CCK8 inhibited specific binding of radiolabelled CCK33 in a dose-dependent manner; the IC50 (half-maximal inhibitory concentration) was in the range 3 to 4 nM. Memantine increased CCK binding in a concentration-dependent manner, though at high concentrations. The EC50 (half-maximal effective concentration) of this effect was less than 100 microM. The memantine effect is not due to an inhibition of labelled CCK degradation in the medium. The effect of memantine on CCK binding is unique for brain since it was not observed in pancreatic acinar membranes. These data, therefore, suggest a modulatory effect of memantine on CCK receptors in mouse brain (cortex and striatum) particles.

Radioelectroencephalographic comparison of memantine with receptor-specific drugs acting on dopaminergic transmission in freely moving rats.

Chronic implantation of four bipolar concentric electrodes into frontal cortex, hippocampus, striatum and reticular formation allows repetitive recordings of field potentials from freely moving rats. After radiotransmission the EEG signals are submitted to a quantitative spectral power analysis. Drug-induced changes in single frequency bands as obtained from the power spectra from different brain areas lead to a drug-specific pattern which can be compared with those of various standard compounds known to influence dopaminergic transmission in the central nervous system. With regard to receptor specificity the research compounds SK & F 38393 (D-1 agonist), SCH 23390 (D-1 antagonist), quinpirole (D-2 agonist) as well as haloperidol (D-2 antagonist, less specific) are tested under identical conditions. Memantine (1-6 mg/kg, i.p.) induces power decreases in a dose-dependent manner in nearly all frequency bands thus resembling the action of apomorphine. Less similar but still comparable is the action of amphetamine, whereas among the receptor-specific dopaminergic drugs only SK & F 38393 can be regarded as similar in as much as mainly delta, alpha-2 and beta-1 frequencies decrease at the same time. Thus memantine develops its own specific pattern of changes in the EEG which can be used to discriminate it from other drugs. The results are in accordance with the drug's proposed action of enhancing the dopaminergic transmission, which probably implies an indirect mechanism of action as biochemically no direct interaction with any of the dopaminergic receptors has been described for memantine so far.

Bioavailability studies of etofibrate in rhesus monkeys.

Etofibrate (Lipo-Merz) is a newer antilipaemic agent that contains the nicotinic and clofibric acid moieties joined together through a diester link with ethylene glycol. The bioavailability of these moieties in etofibrate was compared to that from equimolar amounts of these drugs administered alone to rhesus monkeys (clofibric acid 354 mg, nicotinic acid 203 mg). For this comparative purpose, analytical methods were chosen which converted etofibrate and its initial metabolites into clofibric and nicotinic acid. Following this treatment, the rate and extent of bioavailability of unchanged nicotinic acid from etofibrate was significantly lower (P less than 0.01) than that from nicotinic acid alone. Mean peak whole blood levels of 11.9 micrograms/ml at 2.8 h and 35.3 micrograms/ml at 1.3 h, respectively, occurred after administration of etofibrate and nicotinic acid alone. The extent of bioavailability of clofibric acid hydrolysed from etofibrate was not significantly different (P greater than 0.05) from that from clofibric acid alone. However, mean peak plasma levels of 127.3 micrograms/ml at 3.6 h and 170.8 micrograms/ml at 2.4 h, respectively, occurred after administration of etofibrate and clofibric acid alone and were significantly different (P less than 0.01). The rates and extent of bioavailability of nicotinic acid or clofibric acid administered as a mixture were similar to that from these drugs administered alone. Thus either drug did not affect the absorption of the other in rhesus monkeys. The half-lives of nicotinic acid (in whole blood) and clofibric acid (in plasma) in rhesus monkeys when these drugs were administered alone were 9.9 h and 1.8 h, respectively.

Relative bioavailability of etofibrate. A comparison of an acute and a new sustained release formulation.

The relative bioavailability of 2-(p-chlorophenoxy)-2-methylpropionic acid [2-(nicotinyloxy)-ethyl]-ester (etofibrate) from Lipo-Merz retard (500 mg) with respect to Lipo-Merz (600 mg) has been determined in 10 health volunteers in a crossover study. Etofibrate was determined in plasma after hydrolysis to clofibrinic acid. Pharmacokinetic parameters were derived to describe the plasma concentration-time profiles using a minimisation of least squares technique. The absorption was apparently delayed following the sustained release formulation with a longer time to maximum plasma concentration, which was significantly lower following the retard form. No significant differences were found in the mean apparent elimination half-lives nor areas under the plasma concentration-time curves indicating that the two formulations can be considered bioequivalent.

[Atherosclerosis treatment with etofibrate retard. New perspectives]. / Atherosklerose-Behandlung mit Etofibrat retard. Neue Perspektiven.

In a multicentric general-practice-study 2504 hyperlipoproteinemic patients were treated with etofibrate retard for 4 weeks. The drug was administered once daily in the evening. A highly significant decrease of the mean values of cholesterol around 18.4% and 27.6% resp., and of the mean triglycerides between 31.3% and 16.5% resp. were observed. The atherogenic index was reduced by 28%. The simultaneous, highly significant reduction of blood glucose and of uric acid levels as well as of blood pressure showed the comprising effects of etofibrate retard against the atherosclerosis and its risk factors. The excellent tolerance of the drug was stated by more than 99% of the patients treated.

[On a novel Fomocaine synthesis/10th communication: on syntheses of new compounds with local anaesthetic activity (author's transl)]. / Uber eine neue Fomocain-Synthese. 10. Mitt.: Uber Synthesen neuer Verbindungen mit lokalanästhetischer Wirkung.

4-[3-(4-phenoxymethyl-phenyl)-propyl]-morpholine (fomocaine, Erbocain) is a very active topical local anaesthetic with low toxicity. A new synthesis starting from 4-phenoxy-methyl-benzonitrile, via a modified Willgerodt-Kindler reaction, is reported. The yield is of the same order as with the known technical process starting from 3-phenylpropanol. The distinct advantage of the new procedure is the avoidance of the C-chloromethylation of 3-phenylpropylchloride during which process the o-chloromethyl derivative is also formed, and this cannot be separated by fractional distillation.