Silencing brassinosteroid receptor BRI1 impairs herbivory-elicited accumulation of jasmonic acid-isoleucine and diterpene glycosides, but not jasmonic acid and trypsin proteinase inhibitors in Nicotiana attenuata.

The brassinosteroid (BR) receptor, BR insensitive 1 (BRI1), plays a critical role in plant development, but whether BRI1-mediated BR signaling is involved in plant defense responses to herbivores was largely unknown. Here, we examined the function of BRI1 in the resistance of Nicotiana attenuata (Solanaceae) to its specialist insect herbivore Manduca sexta. Jasmonic acid (JA) and JA-isoleucine conjugate (JA-Ile) are important hormones that mediate resistance to herbivores and we found that after wounding or simulated herbivory NaBRI1 had little effect on JA levels, but was important for the induction of JA-Ile. Further experiments revealed that decreased JAR (the enzyme for JA-Ile production) activity and availability of Ile in NaBRI1-silenced plants were likely responsible for the low JA-Ile levels. Consistently, M. sexta larvae gained more weight on NaBRI1-silenced plants than on the control plants. Quantification of insect feeding-induced secondary metabolites revealed that silencing NaBRI1 resulted in decreased levels of carbon-rich defensive secondary metabolites (hydroxygeranyllinalool diterpene glycosides, chlorogenic acid, and rutin), but had little effect on the nitrogen-rich ones (nicotine and trypsin proteinase inhibitors). Thus, NaBRI1-mediated BR signaling is likely involved in plant defense responses to M. sexta, including maintaining JA-Ile levels and the accumulation of several carbon-rich defensive secondary metabolites.

First derivative ratio spectrophotometric, HPTLC-densitometric, and HPLC determination of nicergoline in presence of its hydrolysis-induced degradation product.

Three methods are presented for the determination of Nicergoline in presence of its hydrolysis-induced degradation product. The first method was based on measurement of the first derivative of ratio spectra amplitude of Nicergoline at 291 nm. The second method was based on separation of Nicergoline from its degradation product followed by densitometric measurement of the spots at 287 nm. The separation was carried out on HPTLC silica gel F(254) plates, using methanol-ethyl acetate-glacial acetic acid (5:7:3, v/v/v) as mobile phase. The third method was based on high performance liquid chromatographic (HPLC) separation and determination of Nicergoline from its degradation product on a reversed phase, nucloesil C(18) column using a mobile phase of methanol-water-glacial acetic acid (80:20:0.1, v/v/v) with UV detection at 280 nm. Chlorpromazine hydrochloride was used as internal standard. Laboratory prepared mixtures containing different percentages of the degradation product were analysed by the proposed methods and satisfactory results were obtained. These methods have been successfully applied to the analysis of Nicergoline in Sermion tablets. The validities of these methods were ascertained by applying standard addition technique, the mean percentage recovery +/- R.S.D.% was found to be 99.47 +/- 0.752, 100.01 +/- 0.940, 99.75 +/- 0.740 for the first derivative of ratio spectra method, the HPTLC method and the HPLC method, respectively. The proposed methods were statistically compared with the manufacturer's HPLC method of analysis of Nicergoline and no significant difference was found with respect to both precision and accuracy. They have the advantage of being stability indicating. Therefore, they can be used for routine analysis of the drug in quality control laboratories.

Antioxidant properties of MDL and MMDL, two nicergoline metabolites, during chronic administration of haloperidol.

We evaluated the effects of 10-alpha-methoxy-9,10-dihydrolysergol (MDL) and 1-methyl-10-alpha-methoxy-9,10-dihydrolysergol (MMDL), two nicergoline metabolites, during chronic treatment with haloperidol in rats. Haloperidol induced a significant decrease in the glutathione (GSH) content in selected areas of the brain and in the liver. Prolonged administration of MDL, MMDL or nicergoline antagonized the haloperidol-induced GSH decrease. Lipid peroxidation in the cortex and striatum was suppressed by MDL, MMDL or nicergoline administration. Our results show that MDL, MMDL and nicergoline have antioxidant activity, preventing not only GSH depletion but also lipid peroxidation. These observations suggest beneficial properties of MDL and MMDL in the treatment of neuroleptic-induced side effects.

Immunoassays for the detection of nicergoline and its metabolites in human plasma.

In order to determine nicergoline pharmacokinetics after oral administration to humans, we have developed two radioimmunoassays, one directed against nicergoline and the other directed against known nicergoline metabolites. The assays were validated according to the recommendations of international regulatory agencies and their limits of quantification were 40 and 10 pg/ml, respectively. In order to further validate the methods, a chromatographic separation of immunoreactive entities was performed with samples from healthy volunteers who were given 15 mg of Sermion (nicergoline orally administered). Chromatographic determination of assay specificity showed that the metabolite radioimmunoassay recognised known nicergoline metabolites but also a new metabolite. Using the antibodies directed against nicergoline, we were unable to detect nicergoline in the human plasma. This suggests that nicergoline is absent in the circulation because of complete metabolism through its first-pass effect.

Involvement of CYP2D6 but not CYP2C19 in nicergoline metabolism in humans.

1. Nicergoline, an ergot derivative previously used as a vasodilator, has gained a new indication in treating the symptoms of senile dementia. 2. Nicergoline is rapidly hydrolysed to an alcohol derivative, 1-methyl-10-alpha-methoxy-9,10-dihydrolysergol (MMDL), which is further N-demethylated to form 10-alpha-methoxy-9,10-dihydrolysergol (MDL). A few individuals display aberrant metabolism of this drug, as shown by their diminished capacity to form the MDL metabolite. The aim of this study was to determine whether defective nicergoline metabolism is associated with the debrisoquine and/or the S-mephenytoin hydroxylation polymorphisms. 3. After a single, oral 30 mg dose of nicergoline, the plasma concentrations of its two metabolites were studied in 15 subjects, divided into three groups with respect to their debrisoquine and S-mephenytoin hydroxylation phenotypes. 4. The pharmacokinetic parameters of MMDL and MDL were similar in the ten subjects who were extensive metabolisers of debrisoquine (five of whom were poor metabolisers of S-mephenytoin) (mean MMDL Cmax 59 nmol l-1 and AUC (0, th) 144 nmol l-1h, mean MDL Cmax 183 nmol l-1 and AUC 2627 nmol l-1h) but were markedly different from the five subjects who were poor metabolisers of debrisoquine (mean MMDL Cmax 356 nmol l-1 and AUC 10512 nmol l-1h, MDL concentrations below limit of quantitation). 5. We conclude that the formation of MDL from MMDL in the metabolism of nicergoline is catalysed to a major extent by CYP2D6 and that the observed interindividual variation in the metabolic pattern of the drug is related to the debrisoquine hydroxylation polymorphism.

Determination of 10 alpha-methoxy-9,10-dihydrolysergol, a nicergoline metabolite, in human urine by high performance liquid chromatography.

A specific method for the determination of 10 alpha-methoxy-9,10-dihydrolysergol, a nicergoline metabolite (metabolite 2), in urine is described. Metabolite 2 was well separated from the urine components on a reversed phase column, Hypersil ODS 5 microns, using an acetonitrile:pH 3.5 phosphate buffer (40:60, v/v) as the mobile phase at a flow rate of 1 mL/min. UV detection was set up at 220 nm. After addition of a known amount of lysergamide as the internal standard, the compounds were extracted from alkalysed urine on a pre-packed glass column (Extrelut 1) with dichloromethane. With 0.5 mL urine, concentrations down to 0.56 mumol/L could be determined.

Assay of nicergoline and three metabolites in human plasma and urine by high-performance liquid chromatography-atmospheric pressure ionization mass spectrometry.

A method for the simultaneous determination of nicergoline and three of its metabolites in human plasma and urine has been developed using high-performance liquid chromatography-atmospheric pressure ionization mass spectrometry. Nicergoline and its metabolites were extracted from the plasma and urine samples with chloroform and separated on a reversed-phase ODS column. The eluents were led to the atmospheric pressure ionization interface and then analysed in the selected-ion monitoring mode. The detection limits of nicergoline and three of its metabolites were ca. 2 ng/ml in plasma and ca. 10 ng/ml in urine, at a signal-to-noise ratio of 4.

Pharmacological effects of nicergoline and its metabolites, decomposition products and impurities in animals.

The main pharmacological effects and acute toxicity of nicergoline (NCG, ester type), its 5 metabolites [1-DN (ester type), 1-MMDL, 1-OHMMDL, MDL (ergoline-alcohol type) and 5-BNA (bromonicotinic acid)], 2 decomposition products (1-MMDL and 5-BNA) and 2 impurities [1-DN and 5-CN (ester type)] were studied in animals. In mice, the acute intraperitoneal toxicity of 1-MMDL, 1-OHMMDL and 5-CN was similar to that of NCG (LD50 197.6 mg/kg). The toxicity of 1-DN, MDL and 5-BNA was approximately 2-fold, 2- fold and one half of NCG, respectively. These substances caused no delayed death. In protective effects against KCN- and adrenaline-induced death in mice when given i.p., 1-DN and 1-MMDL were less potent than NCG, and 1-CN was similar in potency to NCG. In competitive inhibitory effects on phenylephrine-induced contraction of isolated guinea pig vas deferens, 1-DN was less potent, 1-MMDL was much less potent and 5-CN was more potent than NCG. In an inhibitory effect on collagen-induced platelet aggregation in vitro using rat platelet-rich plasma, 1-DN and 5-CN were similar in activity to NCG and 1-MMDL was less active than NCG. On the other hand, 1-OHMMDL, MDL and 5-BNA were inactive in these experiments. In experiments on gross behavior, motor function and barbital anesthesia using mice, when given i.p., 5-CN as well as NCG moderately induced depressive effects on the central nervous systems. 1-DN and 1-MMDL were less potent in the effects than NCG. In anesthetized rats, when given i.v., 1-DN and 5-CN dose-dependently caused lowering effects on blood pressure similar to NCG. 1-MMDL and 1-OHMMDL were less potent than NCG in the lowering effect. 1-DN and 5-CN moderately decreased the contractile force in the isolated guinea pig heart when given i.a. 5-CN alone decreased urine volume in rats when given i.p. and all the substances had no effect on the intestinal transit ability in mice when given i.p. These results suggest that the pharmacological effects of NCG are due to NCG per se and in part to its metabolites. As regards structure-activity relationships, NCG, 1-DN and 5-CN with an ester linkage were found to be more potent in pharmacological activities than ergoline-alcohol compounds.(ABSTRACT TRUNCATED AT 400 WORDS)

Specific binding of 3H-nicergoline in rat brain: comparison with the selective alpha 1-antagonist 3H-prazosin.

In rat cerebral cortex, the 3H-Nicergoline (an ergot alkaloid derivative) binding was rapid, reversible, saturable and of high affinity. In various structures of the central nervous system except midbrain and cerebellum, the maximum binding capacity (B max) for 3H-Nicergoline is greater that for 3H-Prazosin. The specificity studies of 3H-Nicergoline binding show interactions with alpha 1-adrenergic and serotoninergic receptors. These results are confirmed after irreversible blockade with phenoxybenzamine. In conclusion, as nicergoline is known to cross the blood-brain barrier, this study shows that it exerts its central action on alpha 1-adrenoceptors as well as serotoninergic receptors.

[Evidence for an alpha 2-blocking property of nicergoline by using antilipolytic alpha-adrenoceptors from adipose tissue]. / Mise en évidence d'une potentialité alpha 2-bloquante de la nicergoline par utilisation du récepteur alpha-adrénergique du tissu adipeux.

alpha-Adrenergic receptor which inhibit lipolysis in human and hamster fat cells are known to be of the alpha 2-type. By measuring the lipolytic activity on isolated fat cells and by studying the [3H]-yohimbine binding on the fat cell membranes, we tried to determine the alpha-adrenergic potency of the alpha-adrenolytic drug nicergoline. 1. Clonidine inhibits, in a concentration-dependent manner, the theophylline-stimulated lipolysis. This antilipolytic effect is suppressed by various alpha 2-adrenolytic agents but not by alpha 1-adrenolytic agents. Nicergoline is found to partially and competitively inhibit the antilipolytic effect of clonidine. 2. Specific binding of [3H]-yohimbine on fat cell membranes was a rapid and saturable process and was of high affinity (KD = 3.1 +/- 0.3 nM, Bmax = 615 +/- 90 f.mol./mg protein). 3. At low concentration, nicergoline displaced the specific binding of [3H]-yohimbine (EC50 = 0.440 microM). Compared to other alpha-adrenolytic agents, the affinity decreased in the following order: yohimbine greater than or equal to phentolamine greater than piperoxane greater than nicergoline much greater than prazosin. 4. The results show that the alpha-adrenergic antagonists capacity to suppress the antilipolytic effect of clonidine on isolated adipocytes is directly correlated with the ability to inhibit [3H]-yohimbine binding on fat cell membranes. Moreover we conclude that, on this model, nicergoline exhibits alpha 2-adrenolytic properties.

[A review of pharmacological studies on nicergoline]. / Ubersicht über pharmokologische Studien mit Nicergolin.

10-Methoxy-1,6-dimethyl-ergoline-8 beta-methanol-(5-bromonicotinate) (nicergoline, Sermion) shows a strong alpha-blocking activity both in vitro and in vivo. Various studies (dog, cat, rabbit, rat, mouse, guinea-pig) show that nicergoline affects only slightly blood pressure and heart rate and increases the blood flow in brain and hind limb without affecting the splanchnic and aortic flow in normal animals. Nicergoline does not interfere with CNS functions unless applied in high doses. It stimulates the muscle oxidative metabolism and function and lacks any emetic and hallucinogenic activity. Its acute and chronic toxicity in different animal species is very low.