Extracts and constituents of Hypericum perforatum inhibit the binding of various ligands to recombinant receptors expressed with the Semliki Forest virus system.

Extracts, fractions and constituents of Hypericum perforatum were studied for in vitro receptor binding with various ligands to recombinant CNS receptors expressed with the Semliki Forest virus expression system. For this purpose we have prepared membranes of CHO cells with high density of several opioid, serotonin, estrogen, histamine, GABAA, neurokinin and metabotropic glutamate receptors, respectively. A lipophilic Hypericum fraction revealed relatively potent inhibition to the binding of the mu-, delta- and kappa-opioid and the 5-HT6 and 5-HT7 receptors. Moreover, Hypericum constituents such as the naphthodianthrones, hypericin and pseudohypericin, and the phloroglucinole hyperforin inhibited both binding to the opioid and serotonin receptors in the lower micromolar range. Estrogen binding was 50% inhibited by the biflavonoid I3,II8-biapigenin at micromolar concentration. The lipophilic Hypericum fraction provided a less potent inhibition of the neurokinin-1 receptor binding compared to the opioid and serotonin receptors. A total ethanolic Hypericum extract potently inhibited GABAA binding at approximately 3 micrograms/ml. This inhibition is however not specific to Hypericum, since extracts of plants like Valeriana officinalis and Passiflora incarnata showed similar inhibitions. Binding to neither histamine nor metabotropic glutamate receptors was affected by Hypericum extracts. These results support the hypothesis that several active constituents of Hypericum might in a synergistic way contribute to its antidepressant effect in the central nervous system.

Hypericum perforatum inhibits the binding of mu- and kappa-opioid receptor expressed with the Semliki Forest virus system.

The effects of Hypericum perforatum extracts on in vitro [3H]naloxone binding to the human mu- and rat kappa-opioid receptors were studied in chinese hamster ovary (CHO) cells using the Semliki Forest virus (SFV) expression system. Binding of [3H]naloxone to the mu- and kappa-opioid receptor was inhibited in the presence of Hypericum extracts showing IC50 values of approximately 25 and 90 micrograms/ml, respectively. In contrast, extracts of Valeriana officinalis did not inhibit binding to the mu-opioid receptor. Also, single constituents of H. perforatum like the flavonoids quercetin and kaempferol and the glycosilated flavonoid quercitrin did not inhibit [3H]naloxone binding to the mu-opioid receptor up to a concentration of 10 microM. The present in vitro data may suggest a new possible mechanism for the anti-depressant effect of H. perforatum.

Inhibition of benzodiazepine binding in vitro by amentoflavone, a constituent of various species of Hypericum.

Flower extracts of Hypericum perforatum, Hypericum hirsutum, Hypericum patulum and Hypericum olympicum efficiently inhibited binding of [3H]flumazenil to rat brain benzodiazepine binding sites of the GABAA-receptor in vitro with IC50 values of 6.83, 6.97, 13.2 and 6.14 micrograms/ml, respectively. Single constituents of the extracts like hypericin, the flavones quercetin and luteolin, the glycosylated flavonoides rutin, hyperoside and quercitrin and the biflavone 13, II8-biapigenin did not inhibit binding up to concentrations of 1 microM. In contrast, amentoflavone revealed an IC50 = 14.9 +/- 1.9 nM on benzodiazepine binding in vitro. Comparative HPLC analyses of hypericin and amentoflavone in extracts of different Hypericum species revealed a possible correlation between the amentoflavone concentration and the inhibition of flumazenil binding. For hypericin no such correlation was observed. Our experimental data demonstrate that amentoflavone, in contrast to hypericin, presents a very active compound with regard to the inhibition of [3H]-flumazenil binding in vitro and thus might be involved in the antidepressant effects of Hypericum perforatum extracts.

[The combined use of photodynamic therapy with ionizing radiation on breast carcinoma cells in vitro]. / Kombinierte Anwendung der photodynamischen Therapie mit ionisierenden Strahlen bei Mammakarzinomzellen in vitro.

PURPOSE: The photodynamic therapy is a technique by which the tumor cells are selectively sensitized to destruction by light of an appropriate wavelength. The aim of this work is to analyze the biological effectiveness of photochemical reactions induced by laser light in tumor cells exposed to photosensitizers. MATERIAL AND METHODS: The toxicity of the 2 photosensitizers zinc phthalocyanine (ZnPC) and meso-tetrahydroxyphenylchlorine (m-THPC) as well as the biological effect of the combination of sensitizers with laser light were tested in vitro by means of a colony forming assay. In addition, the influence on the photodynamic reaction of a previous exposure of the tumor cells to ionizing radiation has been tested. RESULTS: For both sensitizers doses of 5 micrograms per milliliter of culture medium showed low toxicity, i.e. the survival of the treated cells exceeded 90%. For laser treatments the dose permitting 90% survival was determined to be around 10 J/cm2. With these doses, the combined application of photosensitizers and laser light proved to be very effective and resulted in a nearly complete reduction of survival. As expected, irradiation of the cells with doses of 1 and 2 Gy of X-rays reduced the survival to 66 and 47%, respectively, compared to untreated controls. Cells surviving such treatment showed no changes either in the response to treatments with photosensitizers or to combined applications of photosensitizers and laser light. CONCLUSION: The effects of photodynamic treatment by ionizing radiation seem to be additive and independent of each other. So, our preliminary results are quite encouraging and point out the need of further detailed studies in view of the intended clinical application of this new kind of a local treatment.

Pharmacology of moclobemide.

Moclobemide is a reversible inhibitor of monoamine oxidase (MAO) with clear preference for the A type (so-called RIMA). The enzyme inhibition shows complex kinetics, and the molecular mechanism of interaction with the enzyme is not yet clear. Moclobemide increases the extracellular concentration of the monoamines in rat brain and decreases the level of their metabolites. Neither a loss nor a cumulation of activity has been observed after chronic treatment. Reversibility of MAO-A inhibition was demonstrated in vitro as well as in vivo. In various animal behavioral models, in particular in a novel model of stress-induced anhedonia, moclobemide was as effective as standard antidepressants. Moclobemide improves cognitive functions that are impaired in experimental situations. A neuroprotective action is seen in rats subjected to transient global ischemia/-hypoxia. Moclobemide lacks anticholinergic and other effects and only slightly increases the pressor effect of orally administered tyramine. Possible links between MAO-A inhibition and the various effects of moclobemide on brain function are discussed.

Interaction of moclobemide and tricyclic antidepressants with the tyramine pressor effect in rats.

Tyramine at high doses (20 mg/kg) increased arterial blood pressure in freely moving rats. This increase was completely prevented by pretreatment with inhibitors of neuronal membrane carriers for noradrenaline (e.g. desipramine or oxaprotiline). Pretreatment with moclobemide induced a mild potentiation of this tyramine-pressor effect which could also be attenuated dose-dependently by co-administration of oxaprotiline.

Biochemistry and pharmacology of moclobemide, a prototype RIMA.

RIMA is a term for reversible inhibitors of monoamine oxidase (MAO) with preference for MAO-A; moclobemide is a prototype of this new class of antidepressants and is a highly selective inhibitor of MAO-A in vitro. This inhibition is reversible by dialysis in vitro, which accounts for the dose-dependent duration of in vivo enzyme inhibition of 12-24 h. Moclobemide increases the content of serotonin, noradrenaline and dopamine in the brain, and decreases that of their deaminated metabolites. Its biochemical, neurological and behavioural effects indicate that it increases the extracellular concentration of the classic monoamine neurotransmitters/neuromodulators - in particular 5-HT. Potentiation of the cardiovascular effects of tyramine is less pronounced after taking moclobemide than after irreversible MAO-A inhibitors. Understanding of the physiological role of MAO and of the events that link inhibition of the enzyme with modulation of neuronal activities in the CNS remains incomplete. A major physiological role of intraneuronal MAO is to keep cytosolic amine concentration very low, to enable the neuronal monoamine carriers to produce a net inward transport of monoamines, and thereby to act as the first step in the termination of action of extracellular monoamines. MAO is likely to have a similar function in non-monoaminergic cells with respect to the monoamine carriers they contain. In addition to the classic monoamines, "trace" amines may become functionally active after MAO inhibition. An alternative role for MAO is that of a scavenger, preventing natural substrates from accumulating in monoaminergic neurons and interacting with storage, release, uptake and receptor function of monoamines.

Comparison of monoamine oxidase-A inhibition by moclobemide in vitro and ex vivo in rats.

Inhibition of MAO activity was measured in rat brain homogenates using 5-HT as MAO-A substrate and phenylethylamine as MAO-B substrate. Moclobemide rather selectively inhibited MAO-A. Its inhibitory potency is rather low, like that of toloxatone, whereas clorgyline, harmaline, cimoxatone and brofaromine were all found to be at least 100 times more potent. Phenelzine, isocarboxazid and tranylcypromine were nonspecific, inhibiting MAO-A and MAO-B to about the same extent. The same drugs were also tested ex vivo. Here again moclobemide preferentially inhibited MAO-A; it was equipotent to clorgyline and brofaromine in these tests, and 2-4 times as potent as cimoxatone and harmaline. Moclobemide is a relatively weak MAO-A inhibitor in vitro and yet more potent in vivo than other reversible inhibitors, suggesting that the compound may be converted in vivo to an active form. Nevertheless, it has not been possible so far to identify activated derivatives, and recent findings that moclobemide markedly inhibits liver MAO-A within 5 min of an intravenous injection strongly suggests that the compound itself is responsible for the inhibition.

Short-lasting and reversible inhibition of monoamine oxidase-A by moclobemide.

In ex vivo experiments, the time course of monoamine oxidase-A (MAO-A) inhibition after 10 mg/kg oral moclobemide was virtually the same for whole rat brains and liver: the onset was rapid, the maximum effect was obtained in 15 min, and the duration of action was short (about 16 h) compared with several days for the irreversible MAO inhibitors. The time course of MAO-B inhibition was peculiar: almost complete inhibition was obtained in the time only between the first and second hours, whereas in the whole brain, or various brain areas, maximum MAO-B inhibition (40%) did not occur until 2 h after drug administration. Inhibition in other peripheral organs, such as kidney and small intestine, was of even shorter duration (3 h). After even marked MAO-A inhibition in the liver by moclobemide, the activity recovered within about 4 h when the homogenate was dialysed against water or buffer at 37 degrees C. This rapid recovery is temperature-dependent and does not occur at 18 degrees C. The substance causing the marked inhibition of MAO-A activity when moclobemide is given is probably itself inactivated by the enzyme (reversibility by metabolism). It is expected that more insight into the mechanism of action of moclobemide will be obtained from the use of highly purified preparations of the MAO-A isoenzyme.

Hypotensive action and weak potentiation of tyramine effect by moclobemide in rats.

Moclobemide belongs to a new class of reversible, selective monoamine oxidase-A (MAO-A) inhibitors; it is clinically well tolerated and has little liability to potentiate tyramine pressor effects. Measurement of blood pressure and heart rate in conscious, freely moving rats showed only a slight, nonsignificant decrease in mean arterial pressure in normotensive animals. However, in spontaneously hypertensive rats, moclobemide significantly decreased blood pressure by 20 mmHg within 30 min of oral intake of 30 mg/kg. In the same animals, heart rate was decreased by 20%; normal values returned after 2-3 h. Tyramine alone in oral doses up to 15 mg/kg had no effect on blood pressure in normotensive rats, and after treatment with 30 mg/kg moclobemide, tyramine at 5 mg/kg did not alter mean arterial pressure, whereas there was a significant increase after doses of tranylcypromine, toloxatone and brofaromine. Higher doses of tyramine (10-20 mg/kg) following moclobemide led to a rise of 30-40 mmHg in pressure, but this had disappeared within 20 min. This effect was almost completely eliminated by desipramine, suggesting that coadministration of a norepinephrine uptake inhibitor with a reversible MAO inhibitor is likely to reduce the risk of tyramine-induced hypertensive crisis. Thus, the authors conclude that moclobemide exerts only a slight hypotensive action in hypertensive rats, and differs from other MAO inhibitors in potentiating the pressor effect of tyramine only weakly.

Some basic aspects of reversible inhibitors of monoamine oxidase-A.

A novel class of antidepressants is emerging with considerable therapeutic potential: reversible inhibitors of monoamine oxidase type A (RIMA). Moclobemide (Aurorix) is a representative RIMA. It is a fully and rapidly reversible inhibitor of MAO-A with a correspondingly intermediate duration of action in vivo. It is free of hepatotoxicity and produces a much weaker potentiation of the tyramine pressor effect than the classical irreversible MAO inhibitors. Interaction of MAO inhibitors and monoamine reuptake inhibitors with tyramine is discussed on the basis of experiments in conscious rats. The issue of tyramine content of foods and beverages has been reinvestigated and its relevance for treatment with RIMA antidepressants is discussed. Recently observed antihypoxic (neuroprotective) effects of moclobemide suggest new indications for this compound.

An opioid agonist with preference for kappa-opioid receptors: (-)-N-cyclopropylmethylmorphinan-6-one.

N-Cyclopropylmethyl- and N-cyclobutylmethylmorphinan-6-one (3 and 4, respectively) were prepared from N-methylmorphinan-6-one. The pharmacological studies showed 3 and 4 to be potent opioid agonists. Compound 3 was found to have preference for kappa rather than mu opioid receptors.

Pre-clinical pharmacology of moclobemide. A review of published studies.

The novel antidepressant, moclobemide, is a reversible inhibitor of monoamine oxidase (MAO) preferentially of monoamine oxidise-A (MAO-A); it emerged for study out of a series of lipid-lowering agents. In spite of its weak MAO-A inhibition in vitro, moclobemide is a potent inhibitor of MAO-A, in vivo; its in vivo activity is of short duration, in contrast to the extremely long-lasting inhibition, e.g. by tranylcypromine. Moclobemide only slightly potentiates the pressor effect of oral tyramine in freely moving rats, again in contrast to tranylcypromine; it is not anti-cholinergic and is free of hepatotoxicity. Published evidence on the preclinical pharmacology is reviewed.

In vitro pharmacological properties of a series of isoprenaline derivatives.

The p-trifluoromethylanilide congener of isoprenaline, tert-butyl N-[(S)[( 4-[(R)-6-[2-(3,4-dihydroxyphenyl-2- hydroxyethyl]amino]heptanamido]phenyl]methyl][(N-methylcarbamoy l) methyl]carbamoyl]methyl]carbamate (1S,4R)-4,7,7-trimethyl-3-oxo-2- oxabicyclo[2.2.1]heptane-1-carboxylate (1:1) (Ro 17-2218) was investigated for its effects in various pharmacological tests in vitro and compared to the parent compound. As Ro 17-2218 represented a mixture of four diastereomers, the pure isomers were synthesized. They had a purity of 97-98%. By pharmacological testing of the diastereomers the highest potency was found in the 6R,2'R-isomer Ro 17-8648, while the potency of the 6S,2'S-isomer, Ro 17-9651 was lower by three orders of magnitude. The amorphous hydrochloride Ro 17-8648/001 had 1/10 the potency of the respective crystalline camphanate Ro 17-8648/003. (R)-6-[(R)-[2-(3,4-Dihydroxyphenyl)-2-hydroxyethyl]amino]-N-[4- (trifluoromethyl) phenyl]heptan amide (Ro 17-8648/003) was found to have potent beta-agonist properties with clear beta 1-selectivity in radioligand binding studies. It exerted an extremely tight binding to membrane receptors. As a full beta-agonist it elicited positive inotropic effects in isolated cardiac tissues, with a potency 10-360 times that of isoprenaline and an extremely long duration of action. Electrophysiological studies in isolated guinea-pig papillary muscles confirmed the beta 1-receptor-mediated effects of the compound.

[Hyperthermia--a new element in cancer treatment]. / Hyperthermie--ein neues Element in der Krebsbehandlung.

The efficacy of radiation therapy combined with local hyperthermia is demonstrated by three case studies. Mode of action and problems are discussed and published results reported. Human tumour cells obtained of biopsies from our patients before the onset of treatments were investigated. The short time cell cultures were treated either with hyperthermia or irradiation alone or with combinations under the same conditions as the clinical treatments. We could clearly demonstrate that cell cultures of human origin respond to irradiation or hyperthermia. But the effect was limited and only cultures treated with combined modalities did not relapse. There is hope that mega-voltage radiation complemented with hyperthermia have a similar effect as high-LET-radiation. Side effects have been moderate so far using an extended clinical use.

Synthesis and biological evaluation of 14-alkoxymorphinans. 2. (-)-N-(cyclopropylmethyl)-4,14-dimethoxymorphinan-6-one, a selective mu opioid receptor antagonist.

(-)-N-(Cyclopropylmethyl)-4,14-dimethoxymorphinan-6-one (2) was synthesized with 4,14-dimethoxy-N-methylmorphinan-6-one (1) as starting material. In vivo and in vitro experiments show 2 (cyprodime) to be a pure opioid receptor antagonist. Some of these tests (opioid receptor binding assays, guinea pig ileal longitudinal muscle preparation, rat and mouse vas deferens preparation, acetic acid writhing antagonism test) indicate that 2 is a selective mu opioid receptor antagonist.