In Silico Exploration of Microtubule Agent Griseofulvin and Its Derivatives Interactions with Different Human ß-Tubulin Isotypes.

Tubulin isotypes are known to regulate microtubule stability and dynamics, as well as to play a role in the development of resistance to microtubule-targeted cancer drugs. Griseofulvin is known to disrupt cell microtubule dynamics and cause cell death in cancer cells through binding to tubulin protein at the taxol site. However, the detailed binding mode involved molecular interactions, and binding affinities with different human ß-tubulin isotypes are not well understood. Here, the binding affinities of human ß-tubulin isotypes with griseofulvin and its derivatives were investigated using molecular docking, molecular dynamics simulation, and binding energy calculations. Multiple sequence analysis shows that the amino acid sequences are different in the griseofulvin binding pocket of ßI isotypes. However, no differences were observed at the griseofulvin binding pocket of other ß-tubulin isotypes. Our molecular docking results show the favorable interaction and significant affinity of griseofulvin and its derivatives toward human ß-tubulin isotypes. Further, molecular dynamics simulation results show the structural stability of most ß-tubulin isotypes upon binding to the G1 derivative. Taxol is an effective drug in breast cancer, but resistance to it is known. Modern anticancer treatments use a combination of multiple drugs to alleviate the problem of cancer cells resistance to chemotherapy. Our study provides a significant understanding of the involved molecular interactions of griseofulvin and its derivatives with ß-tubulin isotypes, which may help to design potent griseofulvin analogues for specific tubulin isotypes in multidrug-resistance cancer cells in future.

OSMAC Strategy Integrated with Molecular Networking for Accessing Griseofulvin Derivatives from Endophytic Fungi of Moquiniastrum polymorphum (Asteraceae).

Three endophytic fungi isolated from Moquiniastrum polymorphum (Less.) G. Sancho (Asteraceae) were cultivated using the one strain many compounds (OSMAC) strategy to evaluate the production of griseofulvin derivatives. Extracts obtained were analyzed by HPLC-MS/MS and the chromatographic and spectrometric data used to elaborate a feature-based molecular network (FBMN) through the GNPS platform. This approach allowed the observation of differences such as medium-specific and strain-specific production of griseofulvin derivatives and variations of cytotoxic activity in most extracts. To evaluate the efficiency of the OSMAC approach allied with FBMN analysis in the prospection of compounds of biotechnological interest, griseofulvin and 7-dechlorogriseofulvin were isolated, and the relative concentrations were estimated in all culture media using HPLC-UV, allowing for the inference of the best strain-medium combinations to maximize its production. Malt extract-peptone broth and Wickerham broth media produced the highest concentrations of both secondary metabolites.

A photoresponsive surface molecularly imprinted polymer shell for determination of trace griseofulvin in milk.

A new photoresponsive surface molecularly imprinted polymer shell (PMIPS) was developed for determination of trace griseofulvin from milk. The PMIPS was prepared by surface imprinting technique using poly(styrene-co-methacrylic acid) (PS-co-PMMA) microspheres as the sacrificial substrate, griseofulvin as the template, a photoresponsive azobenzene derivative 4-((4-(methacryloyloxy)phenyl)diazenyl)-3,5-dimethyl benzenesulfonic acid as the functional monomer, and triethanolamine trimethacrylate as the cross-linker. The PMIPS was obtained after the removal of the sacrificial PS-co-PMMA core from the surface imprinted core-shell microspheres, PS-co-PMAA@PMIP. Compared with PS-co-PMAA@PMIP, PMIPS displayed better properties such as higher surface area and pore volume, rapid photo-isomerization rate, and higher adsorption capacities, specific binding constant and binding density. The PMIPS could efficiently detect griseofulvin in complex samples such as milk.

In Vitro additive effect on griseofulvin and terbinafine combinations against multidrug-resistant dermatophytes

ABSTRACT Griseofulvin (GF) and terbinafine (TF) are commonly used drugs to treat dermatophytosis, a fungal infection of the skin. Today there is an increase in drug resistance to these antifungals which highlight the need for alternative synergistic therapies. Minimum Inhibitory Concentration (MIC) of GF and TF were determined against fungi clinical isolates from local hospitals with values ranging 0.03-2.0 µg mL-1 and 0.24-4.0 µg mL-1, respectively. A checkboard test was used to determine the combination of GF:TF which could induce an additive effect against the fungi isolates Multidrug-resistant isolates showed susceptibility after treatment with 16:2 µg mL-1 GF:TF. An MTT assay further verified that GF and TF combinations have greater additive effect against pathological and multidrug-resistant isolates than antifungals alone. Herein we disclose GF:TF combinations that could constitute as a possible new anti-dermatophyte therapy.

A comparative assessment of nanocomposites vs. amorphous solid dispersions prepared via nanoextrusion for drug dissolution enhancement.

Nanoextrusion was used to produce extrudates of griseofulvin, a poorly water-soluble drug, with the objective of examining the impact of drug particle size and polymeric matrix type-size of the extrudates on drug dissolution enhancement. Hydroxypropyl cellulose (HPC) and Soluplus® were used to stabilize wet-milled drug suspensions and form matrices of the extrudates. The wet-milled suspensions along with additional polymer (HPC/Soluplus®) were fed to a co-rotating twin-screw extruder, which dried the suspensions and formed various extrudates. The extrudates were dry-milled and sieved into samples with two different sizes. A wet-milled suspension was also spray-dried in comparison to nanoextrusion. Due to differences in polymer-drug miscibility, two forms of the drug were prepared: extrudates with nano/micro-crystalline drug particles dispersed in the HPC matrix as a secondary phase (nano/microcomposites) and extrudates with amorphous drug molecularly dispersed within the Soluplus® matrix (amorphous solid dispersion, ASD). Under non-supersaturating conditions in the dissolution medium, drug nanocrystals in the HPC-based nanocomposites dissolved faster than the amorphous drug in Soluplus®-based ASD. While smaller extrudate particles led to faster drug release for the ASD, such matrix size effect was weaker for the nanocomposites. These findings suggest that nanocrystal-based formulations could outperform ASDs for fast dissolution of low-dose drugs.

qNMR for profiling the production of fungal secondary metabolites.

Analysis of complex mixtures is a common challenge in natural products research. Quantitative nuclear magnetic resonance spectroscopy offers analysis of complex mixtures at early stages and with benefits that are orthogonal to more common methods of quantitation, including ultraviolet absorption spectroscopy and mass spectrometry. Several experiments were conducted to construct a methodology for use in analysis of extracts of fungal cultures. A broadly applicable method was sought for analysis of both pure and complex samples through use of an externally calibrated method. This method has the benefit of not contaminating valuable samples with the calibrant, and it passed scrutiny for line fitting and reproducibility. The method was implemented to measure the yield of griseofulvin and dechlorogriseofulvin from three fungal isolates. An isolate of Xylaria cubensis (coded MSX48662) was found to biosynthesize griseofulvin in the greatest yield, 149 ± 8 mg per fermentation, and was selected for further supply experiments. Copyright © 2016 John Wiley & Sons, Ltd.

An in situ method to quantitatively determine dissolved free drug concentrations in vitro in the presence of polymer excipients using pulsatile microdialysis (PMD).

In drug formulations containing polymer excipients, the effects of the polymer on the dissolved free drug concentration and resulting dissolution or release can be important, especially for poorly soluble drugs. In this study, an in vitro method based on pulsatile microdialysis (PMD) was developed to quantitatively determine dissolved free concentrations of drugs in the presence of polymers in aqueous media in situ (e.g., in place within the system being characterized). Formulations were made by dissolving various ratios of the drug griseofulvin and polymer PVP K30 in water and allowing the mix to equilibrate. A PMD probe was immersed in each mixture and the dissolved free drug concentrations were determined in the PMD samples. The experimental procedure and the equations used for data analysis are presented. To assess the consistency of data, a binding model was fit to the data obtained using PMD by calculating the dissolved free drug fraction fD for each drug-polymer ratio in solution, and obtaining the product of the binding stoichiometry and binding constant (νK per mole of polymer) from the slope of a plot of (1-fD)/fD vs. the molar polymer concentration. For comparison, equilibrium binding experiments were also performed at 23C, and the determined value of νK was similar to the value found using PMD. Experiments were performed at three temperatures, and a plot of ln (νK) vs. 1/T was linear and a binding enthalpy of -110.9±4.4J/mol of monomer was calculated from its slope. It was concluded that PMD can be used to determine the dissolved free drug concentrations in situ, which allows characterization of the drug-polymer interaction, even for low drug concentrations. This information may be important in modeling the dissolution or release of drugs from formulations containing polymers.

Evaluation of in-line Raman spectroscopic monitoring of size reduction during wet media milling of Biopharmaceutics Classification System Class II drugs.

The aim of this study was to evaluate in-line Raman spectroscopy for monitoring the progress of particle size reduction in real time during wet-stirred media milling of two Biopharmaceutics Classification System (BCS) Class II drugs, griseofulvin and naproxen. To develop a validated online Raman method, Raman analyses were carried out offline by taking samples from the mill at various milling times. A multivariate linear model (partial least squares, PLS) was fitted to the raw data obtained from the Raman measurements and good linearity between online and offline Raman spectra was found. Line intensities (I) of the in-line spectra obtained during the wet media milling were processed by multivariate analyses and correlated with both the median size (d50) and the 90% passing particle size (d90), which were measured offline by laser diffraction. A two-parameter exponential growth model of the form d = exp[A(I - I0)] was found to establish a good correlation (R(2) > 0.90) as a statistically significant model with statistically significant parameters (P < 10(-4)). The correlations were applicable to milled suspensions with particles in the approximate size range of 0.1-6 µm for griseofulvin and 0.1-8 µm for naproxen. These results suggest that in-line Raman spectroscopy can be used to successfully monitor the progress of particle size reduction during wet media milling.

Kinetic performance comparison of fully and superficially porous particles with a particle size of 5 µm: intrinsic evaluation and application to the impurity analysis of griseofulvin.

After the great commercial success of sub-3 µm superficially porous particles, vendors are now also starting to commercialize 5 µm superficially porous particles, as an alternative to their fully porous counterparts which are routinely used in pharmaceutical analysis. In this study, the performance of 5 µm superficially porous particles was compared to that of fully porous 5 µm particles in terms of efficiency, separation performance and loadability on a conventional HPLC instrument. Van Deemter and kinetic plots were first used to evaluate the efficiency and performance of both particle types using alkylphenones as a test mixture. The van Deemter and kinetic plots showed that the superficially porous particles provide a superior kinetic performance compared to the fully porous particles over the entire relevant range of separation conditions, when both support types were evaluated at the same operating pressure. The same observations were made both for isocratic and gradient analysis. The superior performance was further demonstrated for the separation of a pharmaceutical compound (griseofulvin) and its impurities, where a gain in analysis time of around 2 could be obtained using the superficially porous particles. Finally, both particle types were evaluated in terms of loadability by plotting the resolution of the active pharmaceutical ingredient and its closest impurity as a function of the signal-to-noise ratio obtained for the smallest impurity. It was demonstrated that the superficially porous particles show better separation performance for griseofulvin and its impurities without significantly compromising sensitivity due to loadability issues in comparison with their fully porous counterparts. Moreover these columns can be used on conventional equipment without modifications to obtain a significant improvement in analysis time.

Atomic force microscope infrared spectroscopy of griseofulvin nanocrystals.

The goal of this work was to evaluate the ability of photothermal-induced resonance (PTIR) to measure the local infrared absorption spectra of crystalline organic drug nanoparticles embedded within solid matrices. Herein, the first reports of the chemical characterization of sub-100 nm organic crystals are described; infrared spectra of 90 nm griseofulvin particles were obtained, confirming the chemical resolution of PTIR beyond the diffraction limit. Additionally, particle size distributions via dynamic light scattering and PTIR image analysis were found to be similar, suggesting that the PTIR measurements are not significantly affected by inhomogeneous infrared absorptivity of this system. Thus as medical applications increasingly emphasize localized drug delivery via micro/nanoengineered structures, PTIR can be used to unambiguously chemically characterize drug formulations at these length scales.

Metabolite profiling using liquid chromatography/quadrupole time-of-flight mass spectrometry for the identification of a suitable marker and target matrix of griseofulvin use in bovines.

RATIONALE: Griseofulvin is an antifungal agent with potential for misuse in food-producing animals. Little is known about its metabolism in ruminants and hence what are suitable marker residues and target matrices for monitoring purposes. METHODS: Tissues harvested from cattle treated with the antifungal agent griseofulvin were screened using liquid chromatography coupled to positive and negative electrospray ionization (ESI) quadrupole time-of-flight mass spectrometry (qToFMS) operated in ToF mode. RESULTS: Twenty-five possible metabolites were detected across all tissue types, but two isomeric compounds with accurate masses corresponding to loss of a methyl group from parent griseofulvin were considered to be the best candidate markers. Data from fragmentation experiments enabled a tentative assignment of the structures of the two compounds as 4-demethylgriseofulvin and 6-demethylgriseofulvin. These assignments were confirmed by matching the product ion spectra of incurred residues to those of custom synthesized reference standards. CONCLUSIONS: 4-Demethyl- and 6-demethylgriseofulvin have been identified as potential marker compounds of griseofulvin use in cattle. Liver was identified as the target matrix. Hair was shown to have potential for non-invasive testing.

Development and validation of a reversed phase liquid chromatographic method for analysis of griseofulvin and impurities.

A simple and robust reversed phase liquid chromatographic method was developed and validated for the quantitative determination of griseofulvin (GF) and its impurities in drug substances and drug products (tablets). Chromatographic separation was achieved on a Discovery C18 (250mm×4.6mm, 5µm) column kept at 30°C. The mobile phase consisted of a gradient mixture of mobile phase A (water-0.1% formic acid pH 4.5, 80:20, v/v) and B (ACN-water-0.1% formic acid pH 4.5, 65:15:20, v/v/v) pumped at a flow rate of 1.0mL/min. UV detection was performed at 290nm. The method was validated for its robustness, sensitivity, precision, accuracy and linearity based on ICH guidelines. The robustness study was performed by means of an experimental design and multivariate analysis. Satisfactory results were obtained from the validation studies. The use of volatile mobile phases allowed for the identification of three main impurities present above the identification threshold using mass spectrometry (MS). The developed LC method has been applied for the assay and impurity determination of GF drug substances and tablets. The method could be very useful for the quality control of GF and its impurities in bulk and formulated dosage forms.

HPLC-NMR revisited: using time-slice high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance with database-assisted dereplication.

Time-based trapping of chromatographically separated compounds onto solid-phase extraction (SPE) cartridges and subsequent elution to NMR tubes was done to emulate the function of HPLC-NMR for dereplication purposes. Sufficient mass sensitivity was obtained by use of a state-of-the-art HPLC-SPE-NMR system with a cryogenically cooled probe head, designed for 1.7 mm NMR tubes. The resulting (1)H NMR spectra (600 MHz) were evaluated against a database of previously acquired and prepared spectra. The in-house-developed matching algorithm, based on partitioning of the spectra and allowing for changes in the chemical shifts, is described. Two mixtures of natural products were used to test the approach: an extract of Carthamus oxyacantha (wild safflower), containing an array of spiro compounds, and an extract of the endophytic fungus Penicillum namyslowski, containing griseofulvin and analogues. The database matching of the resulting spectra positively identified expected compounds, while the number of false positives was few and easily recognized.

On the polymorphism of griseofulvin: identification of two additional polymorphs.

In this paper, we present an investigation of the polymorphism of griseofulvin. In addition to the only reported crystalline form (form I), two new polymorphic forms (II and III) have been identified and characterized by differential scanning calorimetry and powder X-ray diffraction. Reasons why these two polymorphs were isolated during the present study, but not detected during the numerous previous studies on this drug, are also discussed.

Crystallization kinetics of amorphous griseofulvin by pattern fitting procedure using X-ray diffraction data.

PURPOSE: A pattern fitting procedure using X-ray powder diffraction patterns was applied to study the crystallization kinetics of amorphous griseofulvin. From the optimized parameters obtained by pattern fitting, a change in the quantity and quality of griseofulvin crystals with crystallization was also investigated. MATERIALS AND METHODS: Amorphous griseofulvin was prepared by cooling the melts followed by pulverization. X-ray diffraction patterns of amorphous griseofulvin were repeatedly measured every 20 h. The observed pattern was separated into crystalline diffraction intensity and amorphous scattering intensity by the nonlinear least-squares procedure. RESULTS: The fitting between the observed and simulated diffraction patterns was satisfactorily independent of the degree of crystallinity. Since a good linear relationship was found in a plot of amorphous scattering intensity against crystalline diffraction intensity, the degree of crystallinity can be determined according to Hermans' method. The diffraction peak width increased with higher diffraction angles with crystallization. The crystallization was biphasic: fast and slow crystallization with the growth of low disordered crystals and disordered crystals, respectively. CONCLUSION: The pattern fitting procedure is a powerful tool to analyze the X-ray diffraction patterns of semicrystalline materials. This procedure can simultaneously analyze the degree of crystallinity and crystal disorder in semicrystalline samples during crystallization.

Preparation and characterization of poly-epsilon-caprolactone nanoparticles containing griseofulvin.

Griseofulvin is an antifungal agent with poor solubility and low bioavailability. The aim of this work was to prepare poly-epsilon-caprolactone nanospheres and nanocapsules of griseofulvin by nanoprecipitation and to characterize them. Nanoparticles of griseofulvin were obtained with high encapsulation efficiency. The particle size was about 250-326 nm for nanospheres and 390-400 nm for nanocapsules. The dissolution rate of griseofulvin nanoparticles was higher than that of micronized griseofulvin therefore recourse to nanoencapsulation of griseofulvin should enhance its bioavailability and possibly its efficiency for the treatment of dermatomycosis.

[The dynamics of the griseofulvin levels in the blood and skin of guinea pigs during its combined use with alpha-tocopherol]. / Dinamika soderzhaniia grizeoful'vina v krovi i kozhe morskikh svinok pri ego kompleksnom primenenii s al'fa-tokoferolom.

Experiments with 120 guinea pigs have revealed that skin griseofulvin levels depend on blood griseofulvin level and reduce after a prolonged (for 3-4 weeks) administration of the antibiotic in a dose of 30 mg/kg. Reduction of P-450 system enzymes activities achieved with alpha-tocopherol slows down griseofulvin biotransformation rate and significantly elevates its blood serum and skin concentrations.

In vitro dissolution profile of water-insoluble drug dosage forms in the presence of surfactants.

The determination of the in vitro release profile of water-insoluble drug products requires dissolution media different from those used for water-soluble drug products. Since the relevance of drug dissolution in organic solvents is questionable, we investigated the use of surfactants to determine the dissolution profiles of water-insoluble drug products. In most cases, the drug dissolution rate and extent increased as the surfactant concentration in the aqueous dissolution medium increased. Suitable dissolution profiles were obtained in the presence of sodium lauryl sulfate (SLS) for water-insoluble drug products, such as griseofulvin, carbamazepine, clofibrate, medroxyprogesterone, and cortisone acetate. These findings recommend the use of surfactants for determining the aqueous dissolution of water-insoluble drug products rather than adding organic solvents to the dissolution medium.