In vitro effects of tropisetron and granisetron against Echinococcus granulosus (s.s.) protoscoleces by involvement of calcineurin and calmodulin.

BACKGROUND: Cystic echinococcosis (CE) is a disease caused by the larval stage of Echinococcus granulosus sensu lato  (s.l.). The treatment of CE mainly relies on the use of benzimidazoles, which can commonly cause adverse side effects. Therefore, more efficient treatment options are needed. Drug repurposing is a useful approach for advancing drug development. We have evaluated the in vitro protoscolicidal effects of tropisetron and granisetron in E. granulosus sensu stricto (s.s.) and assessed the expression of the calcineurin (CaN) and calmodulin (CaM) genes, both of which have been linked to cellular signaling activities and thus are potentially promising targets for the development of drugs. METHODS: Protoscoleces (PSC) of E. granulosus (s.s.) (genotype G1) obtained from sheep hepatic hydatid cysts were exposed to tropisetron and granisetron at concentrations of 50, 150 and 250 µM for various periods of time up to 10 days. Cyclosporine A (CsA) and albendazole sulfoxide were used for comparison. Changes in the morphology of PSC were investigated by light microscopy and scanning electron microscopy. Gene expression was assessed using real-time PCR at the mRNA level for E. granulosus calcineurin subunit A (Eg-CaN-A), calcineurin subunit B (Eg-CaN-B) and calmodulin (Eg-CaM) after a 24-h exposure at 50 and 250 µM, respectively. RESULTS: At 150 and 250 µM, tropisetron had the highest protoscolicidal effect, whereas CsA was most effective at 50 µM. Granisetron, however, was less effective than tropisetron at all three concentrations. Examination of morphological alterations revealed that the rate at which PSC were killed increased with increasing rate of PSC evagination, as observed in PSC exposed to tropisetron. Gene expression analysis revealed that tropisetron at 50 µM significantly upregulated Eg-CaN-B and Eg-CaM expression while at 250 µM it significantly downregulated both Eg-CaN-B and Eg-CaM expressions; in comparison, granisetron decreased the expression of all three genes at both concentrations. CONCLUSIONS: Tropisetron exhibited a higher efficacy than granisetron against E. granulosus (s.s.) PSC, which is probably due to the different mechanisms of action of the two drugs. The concentration-dependent effect of tropisetron on calcineurin gene expression might reflect its dual functions, which should stimulate future research into its mechanism of action and evaluation of its potential therapeutical effect in the treatment of CE.

Formulation and evaluation of taste mask pellets of granisetron hydrochloride as oro dispersible tablet

Orally disintegrating systems have carved a niche amongst the oral drug delivery systems due to the highest compliance of the patients, especially the geriatrics and pediatrics. In addition, patients suffering from dysphagia, motion sickness, repeated emesis and mental disorders prefer these medications because they cannot swallow large quantity of water. Further, drugs exhibiting satisfactory absorption from the oral mucosa or intended for immediate pharmacological action can be advantageously formulated in these dosage forms. However, the requirements of formulating these dosage forms with mechanical strength sufficient to withstand the rigors of handling and capable of disintegrating within a few seconds on contact with saliva are inextricable. The purpose of this research was to mask the bitter taste of granisetron hydrochloride. To mask the taste Kollicoat(r) Smartseal 30D was used as coating polymer for pellet coating. The coated pellets of the drug was directly compressed with different superdisintegrant as AC-Di-Sol, Explotab and Kollidon CL in different concentration 5.0-7.5% w/w into an ODT. The prepared tablets were evaluated for hardness, friability, weight variation, wetting time, wet absorption ratio, in-vitro disintegration time and in vitro dissolution studies. Tablets exhibited quick disintegration characteristics with Kollidon CL in concentration 7.5% w/w i.e., within 20 seconds, which is characteristic of orally disintegrating dosage forms. More than 98% of drug was released from the formulations within 15 minutes. Formulations subjected to stability testing as per the ICH guidelines for 3 months, indicated stability with no change in taste, hardness, drug content, disintegration time and dissolution profiles. Thus, the results conclusively demonstrated successful masking of taste and rapid disintegration of the formulated dosage forms in the oral cavity.

Sistemas de desintegração oral têm um nicho entre os sistemas de administração de medicamentos por via oral devido à maior aceitação dos pacientes, especialmente os de geriatria e pediatria. Além disso, pacientes que sofrem de disfagia, enjoo de movimento, emese repetida e distúrbios mentais preferem estes medicamentos porque não podem engolir grande quantidade de água. Além disso, os fármacos que exibem absorção satisfatória a partir da mucosa oral ou que se destinam a ação farmacológica imediata podem ser vantajosamente formulados nestas formas de dosagem. No entanto, a formulação destas formas farmacêuticas exige-lhes resistência mecânica suficiente para suportar os rigores do manuseio e capacidade de desintegrar dentro de alguns segundos em contato com a saliva. O objetivo desta pesquisa foi o de mascarar o gosto amargo de cloridrato de granisetrona. Para mascarar o sabor, utilizou-se Kollicoat smartseal 30D como polímero para io revestimento dos péletes. Os péletes revestidos do fármaco foram diretamente comprimidos com superdesintegrante diferente como Ac-Di-Sol, Explotab e Kollidon CL, em diferentes concentrações 5.0-7.5% m/m em comprimidos de dispersão oral (ODT). Os comprimidos preparados foram avaliados quanto à dureza, friabilidade, variação de peso, ao tempo de umedecimento, à razão de absorção de umidade, ao tempo de desintegração in vitro e em estudos de dissolução in vitro. Os comprimidos apresentaram características de desintegração rápida com Kollidon CL, em concentração de 7,5% m/m, ou seja, dentro de 20 segundos, o que é característico para formas farmacêuticas de desintegração oral. Mais do que 98% do fármaco foi liberado a partir das formulações no prazo de 15 minutos. Formulações submetidas a testes de estabilidade de acordo com as diretrizes da ICH por 3 meses indicaram estabilidade sem alteração no sabor, dureza, teor de fármaco, tempo de desintegração e perfis de dissolução. Assim, os resultados demonstraram que o mascaramento de gosto foi bem-sucedido e atingiu-se rápida desintegração das formas de dosagem na cavidade oral.

Study on the interactions of antiemetic drugs and 12-tungstophosphoric acid by absorption and resonance Rayleigh scattering spectra and their analytical applications.

In 0.1 mol L(-1) HCl medium, antiemetic drugs (ATM), such as granisetron hydrochloride (GS) and tropisetron hydrochloride (TS), reacted with H(3)PW(12)O(40)·nH(2)O and formed 3:1 ion-association complex of [(ATM)(3)PW(12)O(40)], then self-aggregated into nanoparticles-[(ATM)(3)PW(12)O(40)](n) with an average size of 100 nm. The reaction resulted in the enhancement of resonance Rayleigh scattering (RRS) and the absorption spectra. The increments of scattering intensity (ΔI(RRS)) and the change of absorbance (ΔA) were both directly proportional to the concentrations of ATM in certain ranges. Accordingly, two new RRS and spectrophotometric methods were proposed for ATM detection. The detection limits (3σ) of GS and TS were 3.2 ng mL(-1) and 4.0 ng mL(-1)(RRS method), 112.5 ng mL(-1) and 100.0 ng mL(-1)(spectrophotometric method). These two methods were applied to determine GS in orally disintegrating tablets and the results were in good agreement with the official method. The ground-state geometries and electronic structures of GS and TS were optimized by the hybrid density functional theory (DFT) method and the shape of [(ATM)(3)PW(12)O(40)](n) was characterized by atomic force microscopy (AFM). Take the RRS method with higher sensitivity as an example, the reaction mechanism and the reasons for enhancement of scattering were discussed.

First derivative spectrophotometric determination of granisetron hydrochloride in presence of its hydrolytic products and preservative and application to pharmaceutical preparations.

Granisetron is a selective 5-HT3 receptor antagonist used in prevention and treatment of chemotherapy-induced nausea and vomiting. The drug is available in tablet dosage form and parenteral dosage form containing benzyl alcohol as a preservative. The main route of degradation of granisetron is through hydrolysis. The present work describes the development of a simple, rapid, and reliable first derivative spectrophotometric method for the determination of granisetron in presence of its hydrolytic products as well as the formulations adjuvant and benzyl alcohol. The method is based on the measurement of the first derivative response of granisetron at 290 nm where the interference of the hydrolytic products, the co-formulated adjuvant and benzyl alcohol is completely eliminated. The proposed method was validated with respect to specificity, linearity, selectivity, accuracy, precision, robustness, detection, and quantification limits. Regression analysis showed good correlation between the first derivative response and the concentration of granisetron over a range of 8-16 µg ml(-1) . Statistical analysis proved the accuracy of the proposed method compared with a reference stability indicating high performance liquid chromatography method. The described method was successfully applied to the determination of granisetron in different batches of tablets and ampoules. The assay results obtained in this study strongly encourage us to apply the validated method for the quality control and routine analysis of tablets and parenteral preparations containing granisetron.

Development and application of a validated stability-indicating high-performance liquid chromatographic method using photodiode array detection for simultaneous determination of granisetron, methylparaben, propylparaben, sodium benzoate, and their main degradation products in oral pharmaceutical preparations.

A simple, rapid, and sensitive RP-HPLC method using photodiode array detection was developed and validated for the simultaneous determination of granisetron hydrochloride, 1-methyl-1H-indazole-3-carboxylic acid (the main degradation product of granisetron), sodium benzoate, methylparaben, propylparaben, and 4-hydroxybenzoic acid (the main degradation product of parabens) in granisetron oral drops and solutions. The separation of the compounds was achieved within 8 min on a SymmetryShield RP18 column (100 x 4.6 mm id, 3.5 microm particle size) using the mobile phase acetonitrile--0.05 M KH2PO4 buffered to pH 3 using H3PO4 (3+7, v/v). The photodiode array detector was used to test the purity of the peaks, and the chromatograms were extracted at 240 nm. The method was validated, and validation acceptance criteria were met in all cases. The robust method was successfully applied to the determination of granisetron and preservatives, as well as their degradation products in different batches of granisetron oral drops and solutions. The method proved to be sensitive for determination down to 0.04% (w/w) of granisetron degradation product relative to granisetron and 0.03% (w/w) 4-hydroxybenzoic acid relative to total parabens.

Development and validation of a selective and rapid LC-MS-MS method for the quantification of abacavir in human plasma.

A simple, precise, and rapid liquid chromatography-tandem mass spectrometry method is developed and validated for the quantification of abacavir, a nucleoside reverse transcriptase inhibitor. Abacavir and granisetron (internal standard) were isolated from 100 muL human plasma by liquid-liquid extraction in ethyl acetate and dichloromethane (90:10, v/v). The chromatographic separation is achieved on Gemini C(18) analytical column (150 mm x 4.6mm, 5-mum particle size) under isocratic conditions at a flow rate of 1.0 mL/min. The parent --> product ion transitions for abacavir (m/z 287.2-->191.2) and internal standard (m/z 313.1-->138.2) were monitored on a triple quadrupole mass spectrometer operating in the multiple reaction monitoring (MRM) and positive ion mode. The linearity of the method for abacavir is established in the range of 29.8-9318 ng/mL with an analysis time of 2.0 min. Acceptable precision and accuracy were obtained for concentrations over the standard curves studied. The mean recovery and process efficiency of analyte obtained at three quality control levels was 86.8% and 87.9%, respectively. The application of this method for routine measurement of plasma abacavir concentration is demonstrated by a pharmacokinetic and/or bioequivalence study conducted in 28 healthy volunteers for a 300 mg tablet formulation under fasting condition.

Development and application of a validated stability-indicating HPLC method for simultaneous determination of granisetron hydrochloride, benzyl alcohol and their main degradation products in parenteral dosage forms.

A simple, rapid and sensitive reversed phase high performance liquid chromatographic method using photodiode array detection was developed and validated for the simultaneous determination of granisetron hydrochloride, benzyl alcohol, 1-methyl-1H-indazole-3-carboxylic acid (the main degradation product of granisetron) and benzaldehyde (the main degradation product of benzyl alcohol) in granisetron injections. The separation was achieved on Hypersil BDS C8 (250 mm x 4.6 mm i.d., 5 microm particle diameter) column using a mobile phase consisted of acetonitrile:0.05 M KH(2)PO(4):triethylamine (22:100:0.15) adjusted to pH 4.8. The column was maintained at 25 degrees C and 20 microL of solutions was injected. Photodiode array detector was used to test the peak purity and the chromatograms were extracted at 210 nm. Naphazoline hydrochloride was used as internal standard. The method was validated with respect to specificity, linearity, accuracy, precision, limit of quantitation and limit of detection. The validation acceptance criteria were met in all cases. Identification of the pure peaks was carried out using library match programmer and wavelengths of derivative optima of the spectrograms of the peaks. The method was successfully applied to the determination of the investigated drugs and their degradation products in different batches of granisetron injections. The method was proved to be sensitive for the determination down to 0.03 and 0.01% of granisetron degradation product and benzaldehyde, respectively, which are far below the compendia limits for testing these degradation products in their corresponding intact drugs.

Development and validation of a high performance liquid chromatographic method for the separation of exo and endo isomers of granatamine (9-methyl-9-azabicyclo[3.3.1]nonan-3-amine); a key intermediate of granisetron.

A simple and accurate high-performance liquid chromatographic method was developed for the determination of exo-9-methyl-9-azabicyclo[3.3.1]nonan-3-amine in endo-9-methyl-9-azabicyclo[3.3.1]nonan-3-amine, commercially known as grantamine and used as a key intermediate in the preparation of granisetron bulk drug. Chromatographic separation of the exo and endo isomers of 9-methyl-9-azabicyclo[3.3.1]nonan-3-amine was achieved on an Inertsil C8 column using a mobile phase containing 0.3% trifluoroacetic acid. The resolution between the two isomers was found to be more than 4. The limit of detection (LOD) and limit of quantification (LOQ) of exo isomer were 0.8 and 2.5 microg x mL(-1) respectively, for a 10 microL injection volume. The percentage recovery of exo-isomer ranged from 99 to 102% w/w in the endo-9-methyl-9-azabicyclo[3.3.1]nonan-3-amine sample. The test solution and mobile phase were observed to be stable up to 48 h after preparation. The validated method yielded good results for precision, linearity, accuracy, robustness and ruggedness. The proposed method was found to be suitable and accurate for the quantitative determination of exo-isomer in bulk samples of endo-9-methyl-9-azabicyclo[3.3.1]nonan-3-amine.

[Determination of dimethyl sulphate residual in granisetron hydrochloride by headspace gas chromatography].

OBJECTIVE: To develop a headspace gas chromatography method for determining dimethyl sulphate residual in granisetron hydrochloride. METHODS: An Angilent INNOWAX capillary column with nitrogen gas as carrier and FID as detector was applied in this study. Dimethyl sulphate was tested under a constant column temperature. RESULT: Dimethyl sulphate had different retention time from other organic solvents such as alcohol,acetoacetate, isopropanol, dichlormethane and chloroform, which might exist in granisetron hydrochloride. The detection limit of dimethyl sulphate;s was 0.0016%. CONCLUSION: The method can be used for the determination of dimethyl sulphate residual in granisetron hydrochloride.

Development and validation of a selective and robust LC-MS/MS method for high-throughput quantifying rizatriptan in small plasma samples: application to a clinical pharmacokinetic study.

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection (LC-MS/MS) was developed for the determination of a potent 5-HT(1B/1D) receptor agonist, rizatriptan in human plasma using granisetron as the internal standard. The analyte and internal standard were isolated from 100 microL plasma samples by liquid-liquid extraction (LLE) and chromatographed on a Lichrospher C18 column (4.6mm x 50mm, 5 microm) with a mobile phase consisting of acetonitrile-10mM aqueous ammonium acetate-acetic acid (50:50:0.5, v/v/v) pumped at 1.0 mL/min. The method had a chromatographic total run time of 2 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 270-->201 (rizatriptan) and 313.4-->138 (granisetron) used for quantitation. The assay was validated over the concentration range of 0.05-50 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was above 98%. The intra-day accuracy of the assay was within 12% of nominal and intra-day precision was better than 13% C.V. Following a 10mg dose of the compound administered to human subjects, mean concentrations of rizatriptan ranged from 0.2 to 70.6 ng/mL in plasma samples collected up to 24h after dosing. Inter-day accuracy and precision results for quality control samples run over a 5-day period alongside clinical samples showed mean accuracies of within 12% of nominal and precision better than 9.5% C.V.

Simultaneous measurement of blood and brain microdialysates of granisetron in rat by high-performance liquid chromatography with fluorescence detection.

Simultaneous microdialysis probes in the blood and brain and sensitive high-performance liquid chromatography with fluorescence detection were used to examine the granisetron concentration in the jugular vein and frontal cortex of rats after drug administration. Two microdialysis probes were inserted into the right jugular vein and frontal cortex of male Sprague-Dawley rats to which granisetron (6 mg/kg, i.v.) had been administered. Dialysates were automatically collected using a microfraction collector. Samples were eluted with a mobile phase containing 25 mM acetate buffer (pH 4.8)-acetonitrile (72:28, v/v). Excitation and emission wavelengths were set at 305 and 360 nm, respectively, on a scanning fluorescence detector. The limit of quantification for granisetron was 0.5 ng/ml. The in vitro recovery of granisetron was 29.7+/-1.2% (n=6) for the jugular vein microdialysis probe and 6.1+/-0.5% (n=6) for the frontal cortex microdialysis probe. The increasing brain/blood concentration ratio of granisetron suggests that granisetron penetrates the blood-brain barrier.

A rapid assay of granisetron in biological fluids from cancer patients.

A convenient high-performance liquid chromatography (HPLC) was developed for the rapid assay of granisetron (GRN) in biological fluids, such as serum, urine, and pleural effusion, from cancer patients. Extrelut-1 was used for the solid-phase extraction. HPLC was carried out using a LiChroCART cartridge column packed with Lichrospher 100 CN and a mobile phase consisting of 0.1 M acetate buffer (pH 3.5) and acetonitrile (7:3). A fluorescence detector of 290 nm for excitation and 365 nm for emission was used. The standard curve was linear over the range of 2 to 100 ng/ml of GRN. Assay precision, expressed as a coefficient of variation (C.V.), was in the range of 0.9-5.4% in the within-day assay and 2.5-6.9% in the between-day assay, respectively. GRN was well separated on the HPLC chromatogram from drugs such as etoposide, metclopramide, ondansetron, and domperidone which are often used together with GRN. It was suggested that the present method is useful for the rapid monitoring of GRN in the serum, urine, and pleural effusion of patients undergoing cancer chemotherapy.

Retention behaviour of anti-emetic serotonin antagonists in reversed phase high performance liquid chromatography.

Granisetron, Ondansetron and Tropisetron, three 5-HT3 antagonists showing anti-emetic activity, were analysed by HPLC on lipophilic stationary phases. The addition of an amine or quaternary ammonium salt to the eluents was a powerful tool in the analysis of these basic substances. The influence on chromatographic parameters of pH, ionic strength and various counter-ions in the aqueous phase as well as of different organic modifiers is discussed. Some of the proposed experimental conditions allow a more strictly partition-based separation mechanism and can produce chromatographic parameters suitable for structure-activity studies. These experimental conditions are also suitable for analysis of the considered compounds in pharmaceutical dosage forms or in biological fluids.

Metabolism and disposition of 14C-granisetron in rat, dog and man after intravenous and oral dosing.

1. The disposition and metabolic fate of 14C-granisetron, a novel 5-HT3 antagonist, was studied in rat, dog, and male human volunteers after intravenous and oral administration. 2. Complete absorption occurred from the gastrointestinal tract following oral dosing, but bioavailability was reduced by first-pass metabolism in all three species. 3. There were no sex-specific differences observed in radiometabolite patterns in rat or dog and there was no appreciable change in disposition with dose between 0.25 and 5 mg/kg in rat and 0.25 and 10 mg/kg in dog. Additionally, there were no large differences in disposition associated with route of administration in rat, dog and man. 4. In rat and dog, 35-41% of the dose was excreted in urine and 52-62% in faeces, via the bile. Metabolites were largely present as glucuronide and sulphate conjugates, together with numerous minor polar metabolites. In man, about 60% of dosed radioactivity was excreted in urine and 36% in faeces after both intravenous and oral dosing. Unchanged granisetron was only excreted in urine (5-25% of dose). 5. The major metabolites were isolated and identified by MS spectroscopy and nmr. In rat, the dominant routes of biotransformation after both intravenous and oral dosing were 5-hydroxylation and N1-demethylation, followed by the formation of conjugates which were the major metabolites in urine, bile and plasma. In dog and man the major metabolite was 7-hydroxy-granisetron, with lesser quantities of the 6,7-dihydrodiol and/or their conjugates.