Can artificial tears prevent Acanthamoeba keratitis? An in vitro approach.

BACKGROUND: The use of contact lenses has increased in recent years as has the incidence of Dry Eye Syndrome, partly due to their use. Artificial tears are the most common treatment option. Since these changes can facilitate Acanthamoeba infection, the present study has been designed to evaluate the effect of three artificial tears treatments in the viability of Acanthamoeba genotype T4 trophozoites. Optava Fusion™, Oculotect®, and Artelac® Splash were selected due to their formulation. METHODS: Viability was assessed using two staining methods, Trypan Blue stain and CTC stain at different time intervals (2, 4, 6, 8 and 24 h). Trypan Blue viability was obtained by manual count with light microscopy while the CTC stain was determined using flow cytometry. RESULTS: Trypan Blue staining results demonstrated a decrease in viability for Optava Fusion™ and Artelac® Splash during the first 4 h of incubation. After, this effect seems to lose strength. In the case of Oculotect®, complete cell death was observed after 2 h. Using flow cytometry analysis, Optava Fusion™ and Oculotect® exhibited the same effect observed with Trypan Blue staining. However, Artelac® Splash revealed decreasing cell respiratory activity after four hours, with no damage to the cell membrane. CONCLUSIONS: The present study uses, for the first time, CTC stain analyzed by flow cytometry to establish Acanthamoeba viability demonstrating its usefulness and complementarity with the traditional stain, Trypan Blue. Artelac® Splash, with no preservatives, and Optava Fusion TM, with Purite®, have not shown any useful amoebicidal activity. On the contrary, promising results presented by Ocultect®, with BAK, open up a new possibility for Acanthamoeba keratitis prophylaxis and treatment although in vivo studies should be carried out.

Development of a liquid chromatographic method for the quantification of paromomycin. Application to in vitro release and ex vivo permeation studies.

We have developed a reversed phase high performance liquid chromatography pulsed amperometric detection (RPHPLC-PAD) method for the determination of paromomycin. It is sensitive, repeatable, and selective without the pretreatment step. Trifluoroacetic acid-water was utilized as the eluent and detected by PAD under NaOH alkaline conditions. The paromomycin detection limit (S/N=3.3) was 2µgmL(-1) and the quantification limit (S/N=10) was 6µgmL(-1). Coefficients of linear regression were higher than 0.99 for concentrations between 6.25 and 200µgmL(-1). The intra and inter-day precision (RSD) was less than 6.5%. The average recoveries were 97.53-102.01%. The proposed HPLC-PAD method presented advantageous performance characteristics and it can be considered suitable for the evaluation of paromomycin loaded nanogel formulation in ex vivo permeation and in vitro release studies.

Bioanalytical method development, pharmacokinetics, and toxicity studies of paromomycin and paromomycin loaded in albumin microspheres.

Intracellular location of leishmania parasite in macrophages protects them from both hosts defence system as well as from antibiotics like paromomycin (PM) acting against them, thus there is a need of a formulation targeting intracellular parasites. Considering this, PM-loaded albumin microspheres (PM-MS) were prepared to target PM to macrophages where leishmania parasites resides and evaluated for their safety profile. A new bioanalytical method for quantitative determination of PM in rat plasma was developed by pre-column derivatization with 9-fluorenylmethyl chloroformate. The developed bioanalytical method was validated and applied for pharmacokinetic studies of PM administered by intramuscular and intravenous routes as well as for developed PM-MS which were administered by intravenous route. Comparative acute and subacute toxicity studies were also carried out for these formulations. The developed method was found to be very sensitive with a quantification limit of 40 ng/ml. Pharmacokinetic studies demonstrated nearly 80% reduction in C(max) of PM when administered as PM-MS, compared to other formulations at equivalent dose. Toxicity studies indicated increased level of blood urea and blood urea nitrogen in PM intramuscular injection at 90 mg/kg dose, whereas at the same dose level PM-MS showed no symptoms of toxicity. Results obtained suggest that developed PM-MS formulation is a promising alternative to the presently marketed PM intramuscular injection for the treatment of visceral leishmaniasis.

Validated chromatographic and spectrophotometric methods for analysis of some amoebicide drugs in their combined pharmaceutical preparation.

This work is concerned with development and validation of chromatographic and spectrophotometric methods for analysis of mebeverine HCl (MEH), diloxanide furoate (DF) and metronidazole (MET) in Dimetrol® tablets - spectrophotometric and RP-HPLC methods using UV detection. The developed spectrophotometric methods depend on determination of MEH and DF in the combined dosage form using the successive derivative ratio spectra method which depends on derivatization of the obtained ratio spectra in two steps using methanol as a solvent and measuring MEH at 226.4-232.2 nm (peak to peak) and DF at 260.6-264.8 nm (peak to peak). While MET concentrations were determined using first derivative (1D) at λ = 327 nm using the same solvent. The chromatographic method depends on HPLC separation on ODS column and elution with a mobile phase consisting water: methanol: triethylamine (25: 75: 0.5, by volume, orthophosphoric acid to pH =4). Pumping the mobile phase at 0.7 ml min-1 with UV at 230 nm. Factors affecting the developed methods were studied and optimized, moreover, they have been validated as per ICH guideline and the results demonstrated that the suggested methods are reproducible, reliable and can be applied for routine use with short time of analysis. Statistical analysis of the two developed methods with each other using F and student's-t tests showed no significant difference.

In vitro effect of Acanthospermum australe (Asteraceae) extracts on Acanthamoeba polyphaga trophozoites / Efeito in vitro de extratos de Acanthospermum australe (Asteraceae) sobre trofozoitos de Acanthamoeba polyphaga

Acanthamoeba is a free-living protozoan widely distributed in the environment, occurring in vegetative trophozoite and resistance cyst stages during its life cycle. It constitutes an etiological agent of Acanthamoeba keratitis, a disease that may cause severe ocular inflammation and blindness. New drugs can be developed from molecules found in plants and thus help in its difficult treatment. Acanthospermum australe (Asteraceae), a plant used in folk medicine, had its effect tested on Acanthamoeba polyphaga. Aqueous and ethanolic extracts of A. austral were obtained from aerial parts for infusion and static maceration, respectively. Concentrations of 10, 5, 2.5, 1.25 and 0.625 mg/ml of the extract were tested against Acanthamoeba polyphaga trophozoites. The cytotoxic effect of the extracts was tested in mammalian cells using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The 10 mg/ml concentration of ethanolic extract was lethal to 100% of the A. polyphaga trophozoites in 24 h and both extracts presented cytotoxic effect against mammalian cells. These findings suggest that the A. austral ethanolic extract may have compounds with relevance to the development of new amoebicidal drugs.

Acanthamoeba é um protozoário de vida livre amplamente distribuído no ambiente, ocorrendo sob a forma trofozoítica (metabolicamente ativa) e cística (de resistência), durante seu ciclo de vida. O protozoário constitui um agente etiológico da Ceratite Amebiana, uma doença que pode causar inflamação ocular severa e cegueira. Novos fármacos podem ser desenvolvidos a partir de moléculas encontradas em plantas e assim ajudar em seu difícil tratamento. Aqui, Acanthospermum australe (Asteraceae), uma planta utilizada na medicina popular, teve seu efeito sobre trofozoítos de Acanthamoeba polyphaga testado. O extrato aquoso e etanólico de A. australe foram obtidos das partes aéreas por infusão e maceração estática, respectivamente. As concentrações 10, 5, 2,5, 1,25 e 0,625 mg/ml dos extratos foram testadas contra trofozoítos do protozoário. O efeito citotóxico dos extratos foi testado em células de mamífero utilizando o ensaio de brometo de 3-[4,5-dimetiltiazol-2-il]-2,5-difeniltetrazólio (MTT). A concentração de 10 mg/ml do extrato etanólico foi letal a 100% dos trofozoítos de A. polyphaga em 24 h e ambos os extratos apresentaram efeito citotóxico contra as células de mamífero. Estes resultados sugerem que o extrato etanólico de A. australe pode ter componentes com relevância para o desenvolvimento de novos fármacos amebicidas.

Simultaneous determination of diloxanide furoate and metronidazole in presence of diloxanide furoate degradation products.

Three methods are presented for the simultaneous determination of diloxanide furoate (DLX) and metronidazole (MTR), used for their antiprotozoal and antiamoebic effect, in the presence of DLX alkaline degradates and in pharmaceutical formulations, without previous separation. The first method is chemometric-assisted spectrophotometry, in which principal component regression and partial least squares were applied. These two approaches were successfully applied to quantify each drug in the mixture using the information included in, the absorption spectra in the range of 225-320 nm. The second method is TLC-densitometry, in which the binary mixture and degradates were separated on silica gel plates using a chloroform-acetone-glacial acetic acid (9.5 + 0.5 + 0.07, v/v/v) mobile phase and the bands were scanned at 254 nm. The last method is HPLC, in which DLX, MTR, and degradates were separated using the mobile phase acetonitrile-0.05 M dibasic potassium phosphate (25 + 75, v/v), adjusted to pH 4 with orthophosphoric acid, at a flow rate of 1 mL/min, on a C18 analytical column. Detection was at 254 nm. The proposed methods were successfully applied for the analysis of DLX and MTR in pharmaceutical formulations, and the results were statistically compared with a reported spectrophotometric method.

A molecularly imprinted monolith for the fast chiral separation of antiparasitic drugs by pressurized CEC.

Molecularly imprinted polymer (MIP) monoliths with (S)-ornidazole ((S)-ONZ) as the template molecule have been designed and prepared by the simple thermal polymerization of methacrylic acid, 4-vinylpyridine, and ethylene dimethacrylate in the presence of a binary porogenic mixture of toluene and dodecanol. The influences of polymerization mixture composition on the chiral recognition of ONZ have been evaluated, and the imprint effect in the optimized MIP monolith has been clearly demonstrated. The new monolithic stationary phase with optimized porous property and good selectivity was used for the chiral separation of ONZ by pressurized CEC. The pressurized CEC conditions were also optimized to obtain the good chiral separation. The enantiomers were rapidly separated within 9 min on the MIP-based chiral stationary phase, whereas the chiral separation was not obtained on the nonimprinted polymer. Additionally, the proposed method has been successfully applied to the chiral separation of ONZ in tablet samples by injection of the crude sample. The cross-selectivity for similar antiparasitic drug was investigated. The results indicated that the chiral separation of secnidazole could also be obtained on the optimized MIP monolith within 14 min.

HPTLC determination of cefuroxime axetil and ornidazole in combined tablet dosage form.

A new simple high-performance thin layer chromatographic method for determination of cefuroxime axetil and ornidazole in combined tablet dosage form is developed and validated. Cefuroxime axetil is second-generation cephalosporin used to treat or prevent infections that are proven or strongly suspected to be caused by bacteria. Ornidazole is used to cure protozoan infections. The separation is carried out on Merck precoated silica gel aluminium plate 60 F(254) using toluene-n-butanol-triethylamine (8.5:2:0.5, v/v/v) as mobile phase. Quantitative determination of drugs is carried out by densitometric scanning of plates at 285 nm. The retention factor for ornidazole and cefuroxime axetil is found to be 0.51 +/- 0.007 and 0.67 +/- 0.009, respectively. The method is validated with respect to linearity, accuracy, precision, and robustness. Response found to be linear in the concentration range of 100-500 ng/band for both cefuroxime axetil and ornidazole. The method has been successfully applied for the analysis of drugs in pharmaceutical formulation. The % assay is found to be 102.36 +/- 0.775 and 101.00 +/- 1.192 for cefuroxime axetil and ornidazole, respectively.

Validated HPTLC method for simultaneous estimation of levofloxacin hemihydrate and ornidazole in pharmaceutical dosage form.

A simple, rapid, and accurate high-performance thin-layer chromatography (HPTLC) method is described for the simultaneous determination of levofloxacin hemihydrate and ornidazole in tablet dosage form. The method is based on the HPTLC separation of the two drugs followed by densitometric measurements of their spots at 298 nm. The separation is carried out on Merck TLC aluminium sheets of silica gel 60 F254 using n-butanol-methanol-ammonia (5:1:1.5, v/v/v) as mobile phase. The linearity is found to be in the range of 50-250 and 100-500 ng/spot for levofloxacin hemihydrate and ornidazole, respectively. The method is successively applied to pharmaceutical formulation because no chromatographic interferences from the tablet excipients are found. The suitability of this HPTLC method for the quantitative determination of the compounds is proved by validation in accordance with the requirements laid down by International Conference on Harmonization (ICH) guidelines.

Stability indicating methods for the determination of diloxanide furoate.

Five new selective, precise and accurate methods are described for the determination of diloxanide furoate (DI) in presence of its degradation products. Method A utilizes the first and second derivative spectrophotometry at 270 and 280 nm, respectively. Method B is a RSD(1) spectrophotometric method based on the simultaneous use of the first derivative of ratio spectra and measurement at 270 nm. Method C is a pH-induced difference spectrophotometry using UV measurement at 295 nm. Method D is a densitometric one, after separation on silica gel plate using chloroform: methanol as mobile phase and the spots were scanned at 258 nm. Method E is reversed phase high performance liquid chromatography using methanol: water (80:20% v/v) as mobile phase at a flow rate of 1 ml/min and UV detection at 258 nm. Regression analysis showed good correlation in the concentration ranges 5-30, 5-25, 10-40 microg/ml, 100-500 ng/spot, 2-50 microg/ml with percentage recoveries of 99.92+/-0.56 and 99.79+/-0.47, 99.23+/-0.38, 99.96+/-0.06, 99.03+/-0.51, 98.81+/-0.68 for methods A, B, C, D and E, respectively. These methods are suitable as stability indicating methods for the determination of DI in presence of its degradation products either in bulk powder or in pharmaceutical formulations.

Spectrophotometric determination of diloxanide furoate in its dosage forms.

A simple and sensitive spectrophotometric method has been developed for the determination of diloxanide furoate in its dosage forms. The method is based on the reaction of the drug with potassium permanganate in the presence of sodium hydroxide to produce a bluish green coloured species measurable at 610 nm. The absorbance-concentration plot is linear over the range 2.5-20 microg/ml with correlation coefficient (n = 8) of 0.9998 and minimum detectability of 0.2 microg/ml (6.1 x 10(-7) M). The molar absorptivity was 1.1 x 10(4) l/mol cm. The different experimental parameters affecting the development and stability of the colour were carefully studied and optimised. The proposed method was applied successfully for the determination of diloxanide furoate in its tablet form. The results obtained were in good agreement with those obtained using the official method. The proposed method could be applied to the determination of diloxanide furoate in presence of some co-formulated drugs. The effect of sensitisers and surfactants on the performance of the proposed method was also studied. A proposal of the reaction pathway was presented.

Solid-phase extraction and liquid chromatographic quantitation of quinfamide in biological samples.

This paper describes a high-performance liquid chromatographic method for the assay of quinfamide and its main metabolite, 1-(dichloroacetyl)-1,2,3,4,-tetrahydro-6-quinolinol, in plasma, urine and feces. It requires 1 ml of biological fluid, an extraction using Sep-Pack cartridges and acetonitrile for drug elution. Analysis was performed on a CN column (5 microm) using water-acetonitrile-methanol (40:50:10) as a mobile phase at 269 nm. Results showed that the assay was linear in the range between 0.08 and 2.0 microg/ml. The limit of quantitation was 0.08 microg/ml. Maximum assay coefficient of variation was 14%. Recovery obtained in plasma, urine and feces ranged from 82% to 98%.

Application of the bivariate spectrophotometric method for the determination of metronidazole, furazolidone and di-iodohydroxyquinoline in pharmaceutical formulations.

The bivariate calibration algorithm was applied to the spectrophotometric determination of metronidazole, furazolidone and di-iodohydroxyquinoline in pharmaceutical dosage forms. The results obtained were compared with the results of derivative spectrophotometry. The statistical evaluation of method bias was carried out, and it was shown that the proposed procedure may be competitive with commonly used first-derivative spectrophotometry. The advantage of the bivariate calibration is its simplicity, and the fact that there is no need to use the derivatization procedures.

Fluorimetric determination of chloroxine using manual and flow-injection methods.

A reliable and highly sensitive method is described for the determination of chloroxine in pharmaceutical preparations. It involves the formation of a complex between chloroxine and aluminum(III) in a micellar medium. The complex is a very fluorescent species, and there is a linear relationship between chloroxine concentration and fluorescence intensity over the range 2.0 x 10(-8)-5.1 x 10(-5) mol l-1. The limit of detection is 5 x 10(-9) mol l-1. The method can be easily adapted to a flow system using a three-channel manifold, the peak height being proportional to the chloroxine concentration over the range 5.6 x 10(-7)-5.6 x 10(-5) mol l-1. Manual and flow-injection procedures permit the determination of chloroxine in the presence of chlorquinaldol, and have been successfully applied to the determination of chloroxine in pharmaceutical preparations.

Search for new antiamebic agents. I. Synthesis and bioassay of new Schiff bases of 1-phenyl-2-morpholinoethylidene-1-urea.

As potential agents against E. histolytica 19 new Schiff bases of 1-phenyl-2-morpholinoethylidene-1-urea were synthesized by the condensation of the parent compounds with various carbonyl compounds. Thirteen of them were tested against the axenic culture of E. histolytica at concentration 125 microgram/cm3 but no significant amoebicidal activity was observed.