Development of a UPLC-MS/MS method for the determination of narciclasine and 7-deoxynarciclasine in mouse blood and its application in pharmacokinetics.

In this study, we used ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to measure the concentration of narciclasine and 7-deoxynarciclasine in mouse blood after intravenous (i.v.) and oral administration (p.o.), and we used this method to investigate their pharmacokinetics profiles in mice. Chromatographic separation of the analytes was achieved using a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 µm) with a mobile phase consisting of acetonitrile-water (0.1% formic acid) by gradient elution. Electrospray ionization (ESI positive-ion mode)-tandem mass spectrometry in multiple reaction monitoring (MRM) mode was employed for quantitative analysis of the analytes in mouse blood samples. Twelve mice were administered narciclasine and 7-deoxynarciclasine (2 mg/kg) intravenously (iv), while the other twelve mice were administered narciclasine and 7-deoxynarciclasine (10 mg/kg) orally. The mouse blood was withdrawn from the caudal vein to be processed, after which the blood was analyzed by UPLC-MS/MS, and the corresponding data were fitted using the Drug and Statistics (DAS) software. Standard curves of narciclasine and 7-deoxynarciclasine were generated over the concentration range of 5-5000 ng/mL. The intra-day accuracy of narciclasine and 7-deoxynarciclasine was 90-105%, and the corresponding inter-day accuracy was 87-108%. The intra-day precision was less than 13%, while the inter-day precision was less than 14%. Matrix effects were also observed (between 94% and 104%), and the recovery calculated was higher than 70%. The developed and validated UPLC-MS/MS method was then successfully applied in determining the mouse pharmacokinetics of narciclasine and 7-deoxynarciclasine. From this, thebioavailabilityofnarciclasine and 7-deoxynarciclasinewasdetermined to be 10.3%and35.4%, respectively.

Lycorine Suppresses Endplate-Chondrocyte Degeneration and Prevents Intervertebral Disc Degeneration by Inhibiting NF-κB Signalling Pathway.

BACKGROUND/AIMS: Cartilaginous endplate (CEP) degeneration is an important cause for intervertebral disc (IVD) degeneration that leads to low-back pain. The identification of compounds that may prevent CEP degeneration is of interest for the prevention of IVD degeneration. METHODS: Catabolic protease expression in the CEP of disc degeneration patients was first assessed. The toxicity, function and underlying mechanism of lycorine (LY) on CEP-derived chondrocytes degeneration were assessed in vitro by flow cytometry analysis and western blotting. The concentration and function of LY in rat-tail disc-degeneration models were also assessed by HPLC (High Performance Liquid Chromatography) quantification and histological analysis. RESULTS: In CEP cells, Interleukin (IL)-1ß upregulated the expression of matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 that is critical for the degradation of cartilage extracellular matrix. Interestingly, LY suppressed the expression of these enzymes via the inhibition of nuclear factor-κB (NFκB) signalling and thus prevented IL-1ß-induced endplate cell degeneration in vitro. More importantly, LY also reduced the expression of MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 in CEP and exerted a protective effect on both CEP and nucleus pulposus (NP) degeneration. In addition to its inhibitory effect on matrix-degrading protease expression, LY treatment also reduced positive regulators of proinflammatory cytokines, such as MIF, which can be secreted by CEP cells and subsequently target NP cells. CONCLUSION: LY could serve as a potential drug for treating IVD disease.

The metabolites of Ambinine, a benzo[c]phenanthridine alkaloid, in rats identified by ultra-performance liquid chromatography-quadrupoletime-of-flight mass spectrometry (UPLC/Q-TOF-MS/MS).

Ambinine (AM), one of the major hexahydrobenzo[c]phenanthridine alkaloids from Corydalis ambigua Cham. et Schlecht. var. amurensis Maxim, is considered to be an important compound because of its special content and activity. However, there are few published studies on AM metabolism. In this research, the metabolism of AM in vivo was comprehensively studied for the first time. A total 44 metabolites (including 13 phase I and 31 phase II metabolites) as well as its parent drug in plasma, bile, urine and feces of rats were identified and 41 of them were reported for the first time. The results obtained indicated that demethylation, sulfation and glucuronidation were the major metabolic pathways of AM in vivo. Furthermore, this study provides valuable and new information about the metabolism of AM, which will be very helpful for understanding the safety and efficacy of AM, as well as its analogues.

LC-MS/MS method for the determination of haemanthamine in rat plasma, bile and urine and its application to a pilot pharmacokinetic study.

Evidence gathered in various studies points to the fact that haemanthamine, an isoquinoline alkaloid, has multiple medicinally interesting characteristics, including antitumor, antileukemic, antioxidant, antiviral, anticonvulsant and antimalarial activity. This work presents, for the first time, a universal LC-MS/MS method for analysis of haemanthamine in plasma, bile and urine which has been verified in a pilot pharmacokinetic experiment on rats. Chromatographic separation was performed on a pentafluorophenyl core-shell column in gradient elution mode with a mobile phase consisting of acetonitrile-methanol-ammonium formate buffer. A sample preparation based on liquid-liquid extraction with methyl tert-butyl ether was employed with ambelline used as an internal standard. Quantification was performed using LC-MS-ESI(+) in Selected Reaction Monitoring mode. The method was validated according to the European Medicines Agency guideline in a concentration range of 0.1-10 µmol/L in plasma, bile and urine. The concentration-time profiles of haemanthamine in plasma, bile and urine after a single i.v. bolus of 10 mg/kg have been described for the first time. The presented study addresses the lack of information on haemanthamine pharmacokinetics and also introduces a new universal method of haemanthamine analysis in complex biological matrices. Copyright © 2015 John Wiley & Sons, Ltd.

Rapid and sensitive HPLC-MS/MS method for quantitative determination of lycorine from the plasma of rats.

A simple, rapid and sensitive high-performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method was developed and validated for determining lycorine in rat plasma. Plasma samples were prepared by a simple protein precipitation with methanol containing dextrorphan as internal standard. The chromatographic separation was performed on a Kromasil 60-5CN column (3 µm, 2.1 mm × 150 mm) with the mobile phase of methanol/water (containing 0.1% formic acid) (40:60, v/v) at a flow rate of 0.2 mL/min, the total analytical runtime was 5 min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with Electronic Spray Ion by selected reaction monitoring (SRM) of the transitions at m/z 288.1→147.1 for lycorine and m/z 258.1→157.2 for dextrorphan, respectively. The calibration curve was linear over the range of 1-1000 ng/mL with the lower limit of quantification of 1 ng/mL for lycorine. The intra- and inter-day precision (R.S.D.%) were less than 8.5% and accuracy (R.E.%) was within ±7.0%. Lycorine was sufficiently stable under all relevant analytical conditions. This method was successfully applied to the pharmacokinetic study of lycorine in rats after intraperitoneal administration with different doses of 5, 10 and 20 mg/kg.

Study on pharmacokinetic and tissue distribution of lycorine in mice plasma and tissues by liquid chromatography-mass spectrometry.

A fast and simple liquid chromatography-mass spectrometry method for the determination of lycorine in mice plasma and tissues was developed and well used in the pharmacokinetic and tissue distribution study of lycorine after tail vein injection and intraperitoneal administration. Biological samples were processed with ethyl acetate by liquid-liquid extraction, and evodiamine was used as the internal standard. Chromatographic separation was performed on an Amethyst C18 column (4.6 × 150 mm) with a mobile phase consisting of methanol and water. Quantification was performed by selected ion monitoring with m/z 288 [M+H](+) for lycorine and m/z 304 [M+H](+) for the internal standard. Good linearity was observed over the concentration ranges. Limits of quantification were low up to 10.0 ng/mL in plasma samples, 9.0 ng/g for lung, 12.0 ng/g for heart, 18.0 ng/g for spleen and 6.5 ng/g for other tested tissues. The intraday accuracy and precision in plasma and tissues ranged from -7.4% to 9.1%. Recoveries in plasma and tissue were more than 80%. The method was rapid, accurate and fully validated. It was successfully applied to the investigation of the pharmacokinetics and tissue distribution of lycorine in mice.

Dose-dependent emetic effects of the Amaryllidaceous alkaloid lycorine in beagle dogs.

Ingestions of plant material from Amaryllidaceae, especially the bulbs of daffodils, are known to be toxic, representing a persistent cause of poisoning in human and animals. Empiric data from case reports suggested, that the alkaloid lycorine could be the toxic constituent of the multi-component mixture responsible for symptoms like nausea and emesis. Systematic studies of the in vivo effects of the amaryllidaceaeous-type alkaloids are not available. Therefore, in an open, prospective, randomized and controlled trial we studied the dose-effect relationship of lycorine-induced nausea and emesis and the toxicokinetics of lycorine in beagle dogs. Subcutaneously administered lycorine-induced nausea and emesis starting at 0.5 mg/kg body weight reaching statistical significance at 1.0 mg/kg. The maximum emetic dose of lycorine (ED(100)) was 2 mg/kg body weight. There was a correlation between dose and nausea score as well as between dose and number of the induced emetic events. Nausea and emesis were short-lasting and occurred not later than 2.5 h post dose. Lycorine showed linear plasma kinetics with a mean elimination half-life of 0.67 and 0.3 h after single s.c. and i.v. administration, compatible with the clinical course of nausea and emesis. The mean oral bioavailability was calculated to be about 40%. Biochemical and haematological parameters of safety showed no pathological signs. The results provide evidence that lycorine can be considered as a main, if not the crucial constituent responsible for nausea and emesis in human and animals in poisoning due to ingestion of plant material of the Amaryllidaceae.

Metabolism of isometamidium in hepatocytes isolated from control and inducer-treated rats.

Little is known about the metabolism and mechanism of action of the trypanocide, isometamidium (ISM), the major drug used for prophylaxis of trypanosomiasis. We have investigated its metabolism and distribution in isolated rat hepatocytes using liquid chromatography-mass spectrometry and confocal laser scanning microscopy (CLSM). Two putative metabolites were formed, which were proposed to be a mono-acetyl derivative and an oxidized metabolite (SII). This is the first demonstration of the hepatic metabolism of ISM, as previous in vivo studies were hampered by dose-limiting toxicity and insensitive analytical methods. The intrinsic fluorescence of the drug enabled its intracellular uptake to be followed by CLSM. It is taken up rapidly into the nucleolus, nuclear membrane and endoplasmic reticulum within 5 min, and retained in the nucleus for at least 24 h. Persistent binding of ISM to cellular macromolecules may contribute to its prophylactic effect in vivo. Pretreatment of rats with 3-methylcholanthrene, phenobarbitone (PB) or the widely used pyrethroid pesticide, deltamethrin, resulted in an increase in metabolism of ISM to the proposed SII after 1 h incubation with hepatocytes. 3-methylcholanthrene was the most potent inducer, causing a maximal 19.5-fold induction of SII formation after exposure of hepatocytes to ISM for 1 h compared with formation by control hepatocytes. In comparison, at the 1 h timepoint deltamethrin pre-treatment caused a 10.2-fold induction, and PB only 8.2 fold.

Monitoring the correct use of isometamidium by farmers and veterinary assistants in Eastern Province of Zambia using the isometamidium-ELISA.

A survey to monitor the use of trypanocidal drugs by cattle breeders was conducted in Zambia. Use was made of a questionnaire and of the isometamidium-ELISA technique. One hundred and twenty-two farmers and 50 veterinary assistants were interviewed. The isometamidium-ELISA was used to monitor the isometamidium serum concentration in 72 cattle, 1 week after unsupervised treatment by 56 farmers and 16 veterinary assistants. Although there was no clear indication of underestimation of the weight of the animals and although farmers had adequate knowledge of the correct usage of isometamidium, the results suggest frequent underdosing when considering isometamidium serum concentrations 1 week after treatment. In 76% of the cases, the expected protection period was equal or shorter than 28 days and equal or shorter than 33 days in 90% of the treated cattle.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect isometamidium chloride in Oncorhynchus spp.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure isometamidium chloride in the plasma of Oncorhynchus tshawytscha and O. mykiss. Isometamidium-ovalbumin conjugate and anti-isometamidium antibodies were used to coat polystyrene plates. The peroxidase saturation technique was used to optimize the coating antigen concentration; it demonstrated low affinity of the isometamidium-ovalbumin conjugate but high affinity of the anti-isometamidium antibodies for polystyrene surface sites. The optimal conditions of antiisometamidium antibodies to coat plates was at pH 7.3 and a 1:1000 dilution (0.0012 mg ml(-1) protein). The ELISA was sensitive as it detected 0.0006 mg ml(-1) of isometamidium in fish plasma. Isometamidium diluted with saline could not be detected at concentrations less than 0.05 mg ml(-1). The results indicate that this ELISA is much more sensitive when isometamidium is bound to plasma than unbound isometamidium in saline.

The therapeutic use of isometamidium chloride against Cryptobia salmositica in rainbow trout Oncorhynchus mykiss.

Rainbow trout Oncorhynchus mykiss injected intramuscularly with isometamidium chloride (0.01 or 0.1 mg kg-1) at 3 wk post-infection and given a booster 2 wk later had significantly lower parasitaemias than infected controls. Packed cell volume increased after treatment and remained higher than in infected controls. The concentration of isometamidium in plasma was highest at 2 wk after injection and then declined. An intramuscular dose of 1.0 mg kg-1 of isometamidium chloride at 1, 2 and 3 wk postinfection (preclinical) significantly reduced the parasitaemia in rainbow trout 2 wk after treatment. A booster at 9 wk postinfection (chronic disease phase) reduced the parasitaemia further in all fish. The packed cell volume in these fish was higher than in infected controls. Treatment at 5, 6, and 7 wk postinfection (acute disease) had no effects and parasitaemias in treated fish were higher than in infected controls; also, anti-Cryptobia salmositica antibodies and titres of complement-fixing antibody were higher in these than in infected controls. Incubation of immune plasma or complement with isometamidium for 3 h did not affect the lytic titres of complement-fixing antibodies nor rainbow trout complement.

Concentrations of isometamidium chloride (Samorin) in sera of Zebu cattle which showed evidence of hepatotoxicity following frequent trypanocidal treatments.

The concentrations of isometamidium circulating in poorly nourished Zebu cattle which showed morbidity, mortality, and biochemical and histopathological evidence of hepatotoxicity, following frequent treatments with isometamidium chloride and diminazene aceturate were investigated using the isometamidium-ELISA. As few as two isometamidium treatments one month apart were associated with significant weight loss, and cattle treated with diminazene aceturate after three or four isometamidium treatments suffered a 50% mortality. Although there were no obvious, marked elevations in isometamidium concentration which might have allowed the use of the ELISA as a predictor of a potential toxicity problem, concentrations did increase significantly with the number of monthly treatments administered, suggesting drug accumulation, and the increases were significantly higher in cattle to which diminazene had also been administered. In cattle treated with both trypanocides, weight loss and serum glutamate dehydrogenase levels were correlated with isometamidium concentrations. These observations, together with the histopathological findings, support the hypothesis that the morbidity and mortality observed were related to the repeated treatment with isometamidium in conjunction with diminazene aceturate, and that the pathogenesis involved a component of hepatic damage. It is therefore recommended that cattle, particularly those under nutritional stress, are not subjected to repeated treatments with isometamidium at intervals as short as one month, and particularly not with concurrent administration of diminazene.

Concentrations of isometamidium in the sera of cattle challenged with drug-resistant Trypanosoma congolense.

The relationship between serum concentrations of the prophylactic trypanocidal drug isometamidium chloride and protection against tsetse challenge with two populations of Trypanosoma congolense was investigated in Boran (Bos indicus) cattle, using an isometamidium-ELISA. Isometamidium chloride (Samorin) was administered to cattle at a dose rate of 1.0 mg/kg body weight by deep intramuscular injection. Thereafter, the animals were challenged at monthly intervals with either a drug-sensitive clone (T. congolense IL 1180) or a clone expressing a moderate level of resistance to isometamidium (T. congolense IL 3343). Untreated control cattle were used to confirm the infectivity of each challenge. Of ten drug-treated cattle that were challenged with T. congolense IL 3343, all were refractory to infection at the first challenge. 1 month after drug administration. However, all ten animals succumbed to infection at either the second (seven cattle) or third (three cattle) monthly challenges. By contrast, all five drug-treated cattle challenged with T. congolense IL 1180 resisted four monthly challenges. The mean isometamidium concentration at the time of the first, 1 month, challenge was 5.6 +/- 2.8 ng/ml. At the time of the second monthly challenge the mean concentration was 2.0 +/- 0.86 ng/ml: at this time, concentrations were not significantly different between those cattle refractory to challenge with T. congolense IL 3343 and those cattle that were not. Thus, differences in susceptibility to challenge at this time would appear to be due to differences in the drug sensitivity of the parasite challenge. Finally, the mean isometamidium concentration in uninfected cattle at the time of the fourth monthly challenge was 0.4 +/- 0.18 ng/ml. These results indicate that when T. congolense infection occurs in cattle under isometamidium prophylaxis, the parasites may be considered at least moderately drug resistant if the concentration of isometamidium in serum is 2.0 ng/ml. At concentrations between 0.4 and 2.0 ng/ml a low level of drug resistance may be inferred. Below 0.4 ng/ml, however, no inference regarding drug resistance should be made.

Isometamidium concentrations in the sera of cattle maintained under a chemoprophylactic regime in a tsetse-infested area of Zimbabwe.

An experiment was carried out to determine the concentrations of the trypanocidal drug isometamidium chloride in the sera of cattle maintained under a chemoprophylactic regimen at Rekomitjie, Zimbabwe, an area of high tsetse challenge in the Zimbabwe valley. In February 1993, 24 cattle at this site were treated intramuscularly with isometamidium chloride at a dose of 1.0 mg/kg body weight. Thereafter all animals were monitored regularly for 6 months for the presence of trypanosomes and sera were collected to determine the concentrations of isometamidium using an ELISA. Isometamidium treated cattle appeared to be protected against trypanosome infections for at least 18 weeks following treatment. Thereafter, three trypanosome infections were detected, between 20 and 22 weeks following treatment. In contrast, in 18 untreated control cattle at the same site, 9 trypanosome infections were detected over the first 18 weeks of the experiment. Quantification of the isometamidium concentration in sera from the drug treated cattle indicated that the apparent half-life of isometamidium in these animals was 23 days. This was similar to the half-life observed previously in cattle treated under laboratory conditions. The isometamidium ELISA was shown to be capable of quantifying drug levels in 20 out of 23 cattle for at least 70 days after treatment. There was no evidence of drug resistant trypanosomes at this site.

A simple competitive enzyme immunoassay for the detection of the trypanocidal drug isometamidium.

A new competitive enzyme immunoassay technique has been developed for the determination of concentrations of the trypanocidal drug isometamidium chloride (Samorin) in bovine serum. The method has been shown to be highly repeatable and reproducible, and it has several advantages over previous immunoassay techniques for the drug. There are fewer incubation steps overall; microtitre plates may be of coated in batches and stored frozen for future use; and the competition incubation is overnight and is followed only by a brief colour development stage of 10 min. Coefficients of variation (CVs) of duplicate samples were approximately 5%, and mean response variances of untreated cattle (n = 57) were small (CV, 10%). Partitioning of variance showed 77% of this variability to be intrinsic to the samples, and the remaining 23% was due to the procedure. The limit of detection was approximately 0.5 ng ml-1, which was considered to be satisfactory for the intended use of the method. The drug could be detected in serum of treated cattle for up to 10 weeks following treatment, and determinations showed a high level of reproducibility.

Isometamidium concentrations in the sera of Boran cattle: correlation with prophylaxis against tsetse-transmitted Trypanosoma congolense.

Fifteen Boran cattle from a trypanosomiasis-free area were injected intramuscularly with isometamidium chloride at a dose of 1 mg/kg body weight. Thereafter, the cattle were challenged at monthly intervals with Glossina morsitans centralis infected with one of three populations of Trypanosoma congolense (IL 3893, IL 3889 or IL 1180) until all animals became infected. Isometamidium concentrations in the sera of these cattle were measured using a competitive enzyme-linked immunosorbent assay over the first 105 days following treatment. All cattle challenged with IL 3893 or IL 3889 developed infection following the first challenge, at which time the mean serum drug concentration in all treated cattle was 6 ng/ml. Cattle challenged with IL 1180 became infected following 6 to 8 monthly challenges. The mean serum drug concentration in these cattle at the time of their third monthly challenge with IL 1180 was 0.75 ng/ml. Trypanosome populations IL 3893 and IL 3889 were considered to be highly resistant to isometamidium, while IL 1180, relatively sensitive. It was therefore concluded that T. congolense persisting at serum isometamidium concentrations greater than 0.75 ng/ml can be considered moderately resistant, while those persisting at concentrations greater than 6 ng/ml can be considered markedly resistant. These results will be most valuable in the investigation of isometamidium resistance of T. congolense in the field.

Evaluation and improvement of an enzyme-linked immunosorbent assay for the detection of isometamidium in bovine serum.

The control of bovine trypanosomiasis in Africa continues to rely heavily on the chemoprophylactic drug isometamidium (ISMM) chloride. However, despite many years of use, no methods are available that are sufficiently sensitive to measure drug levels in treated cattle. An enzyme-linked immunosorbent assay (ELISA) for the detection of ISMM in the serum of treated cattle has been developed and evaluated. Liquid-phase ISMM (sample) competes with solid-phase bound ISMM-protein conjugate for biotinylated sheep anti-ISMM IgG. The specific IgG is detected by streptavidin-peroxidase, using tetramethylbenzidine for colour development. Assay calibration is by four-parameter logistic curve-fitting. Factors contributing to absorbance variance were considered in assay optimization and improvement of precision and the lower limit of detection (approximately 0.1 ng/ml in serum). The ELISA was shown to detect serum ISMM for several months after treatment of cattle in a trypanosomiasis endemic country. The potential uses of this assay include the development of rational prophylactic drug regimens, and the indirect detection of drug-resistant trypanosomes.

Development of an enzyme-linked immunosorbent assay for the detection and measurement of the trypanocidal drug isometamidium chloride in cattle.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure accurately levels of the trypanocidal drug isometamidium in the serum of treated cattle. The assay requires only 5 microliters of test serum, is sensitive to a level of 0.5 pg ml-1 and is highly specific. Cross reactivity does not occur with the two other widely used trypanocidal drugs diminazene aceturate and homidium bromide. Serum drug levels are detectable for up to six months in cattle after a single dose of 1 mg kg-1 intramuscularly, the maximum period under field conditions for which effective prophylaxis can be maintained against tsetse challenge. Application of the assay will aid the rationalisation of treatment campaigns and assist in assessing the occurrence of drug-resistant trypanosome populations.

Solid-phase extraction and ion-pair reversed-phase HPLC of isometamidium in bovine serum and tissues.

An analytical method has been developed for the determination of isometamidium in bovine serum and tissues. Samples were enzymatically hydrolysed and cleaned up on a solid-phase system (C8 Bond Elut column). The drug was chromatographed by an ion-pair reversed-phase technique using heptane sulphonate as a pairing-ion and triethylamine as a counter-ion reagent. Detection was by fluorescence at 593 nm (excitation = 380 nm). The method is more sensitive and specific than existing methods and it is currently being used in evaluating the pharmacokinetics of isometamidium in cattle.