Ex-vivoand in-vitro assessment of mucoadhesive patches containing the gel-forming polysaccharide psyllium for buccal delivery of chlorhexidine base.

The aim of the present study was to evaluate the gel-forming polysaccharide psyllium in the preparation of mucoadhesive patches for the controlled release of chlorhexidine (CHX) to treat pathologies in the oral cavity, using the casting-solvent evaporation technique. A number of different film-forming semi-synthetic polymers, such as sodium carboxymethyl cellulose (SCMC) and hydroxypropylmethyl cellulose (HPMC) were evaluated for comparison. The patch formulations were characterized in terms of drug content, morphology surface, swelling and mucoadhesive properties, microbiology inhibition assay and in vitro release tests. Three ex-vivo testswere carried out using porcine mucosa: an alternative dissolution test using artificial saliva that allows contemporary measurement of dissolution and mucoadhesion, a permeation test through the mucosa and the measurement of mucoadhesion using a Nouy tensile tester, as the maximum force required for the separation of the patch from the mucosa surface. The patches were also examined for determination of the minimum inhibitory concentration in cultures of Escherichia coli and Staphylococcus aureus. All the patches incorporating psyllium were found suitable in terms of external morphology, mucoadhesion and controlled release of the drug: in the presence of psyllium the drug displays prolonged zero-order release related to slower swelling rate of the system.

Image analysis of lutrol/gelucire/olanzapine microspheres prepared by ultrasound-assisted spray congealing.

Nine systems were prepared containing Gelucire 50/13 and various amounts (9-18-36-45% w/w) of Lutrol F68 and F127 in the presence and in the absence of 10% w/w of olanzapine and formulated as a solid dispersion in the form of microspheres by ultrasound (US)-assisted spray congealing. Thermal analysis, using differential scanning calorimetry (DSC) and thermomicroscopy (HSM), suggested the presence of particles of reduced size of olanzapine precipitated inside the microspheres. The microspheres were also studied by means of electron microscopy (SEM) for their shape and aspect, by some image analysis parameters (fractal dimension) and using Energy-dispersive X-ray (X-EDS) and micro-Raman spectroscopy to qualitatively evaluate the composition of different points of the surface. The surface of the microspheres displayed a non-homogeneous distribution of the drug by the presence of wart-like protuberances, whose number increases as the Lutrol content of the systems increases. The same systems in the absence of US, obtained after cooling the molten mixtures, lack these structures and only a very few of them can be found. The blooming of the surface was hypothesized as related to crystallization or phase de-mixing or lipid component diffusion of the carrier mixture inside the cooling mass subjected to ultrasound vibration. Ultrasounds accelerate the physical changes concerning carriers and drug, outlining the importance of ultrasound to achieve stability for formulations of this type. The microspheres de-aggregate on contact with the dissolution medium and release the drug with a bimodal mode according to the Lutrol content.

Design of olanzapine/lutrol solid dispersions of improved stability and performances.

Eleven solid dispersions containing olanzapine, with carriers of different composition (Lutrol® F68, Lutrol® F127, Gelucire® 44/14), were prepared and examined by thermal (differential scanning calorimetry (DSC); thermomicroscopy (HSM)) and X-ray diffraction (XRD) analysis, both as fresh or aged (one year) samples. Drug and carriers were preliminarily selected in order to avoid problems related to the aging of the formulation, according to the solubility parameters of carriers and drug. These parameters make it possible to predict the low solubility of olanzapine in the carriers (alone or in mixtures). Systems containing only Lutrol (also in the presence of Transcutol®) contain the drug in the form of particles of reduced size and in a crystalline form. Gelucire® 44/14 apparently increases the amount of olanzapine dissolved in the solid carrier, but this is presumed to be a metastable state, probably related to the heterogeneous nature of the carrier that delays crystallization of the drug. The high hydrophilicity of the carriers proves suitable to an accelerated and quick release of the drug regardless of aging. Differences in the release profiles between Lutrol- and Gelucire-containing systems were interpreted in terms of the formation of polymer micelles by the Lutrols when in aqueous solution.

Olanzapine solvates.

Olanzapine was crystallized from 12 organic solvents alone or in mixture, by cooling in the freezer, by slow evaporation of the solvent, or by suspending olanzapine powder for some time in the solvent. All the samples thus obtained were examined by thermal analysis (differential scanning calorimetry-DSC and thermogravimetry-TG) to certify the formation of a solvate, the presence of polymorph (form 1 or 2) in the desolvated olanzapine, comparing the different profile of the thermograms, and to calculate the stoichiometry of the possible solvate. According to the DSC thermogram, the solvents can be divided into four classes: those that do not form solvates and leave olanzapine form 1 (ethyl acetate, toluene, diethyl ether, and acetone); those that form solvate and leave form 1 of olanzapine after desolvation (methanol, 1- and 2-propanol); those that after desolvation of the solvate show a polymorph transition in the thermogram indicating the presence of form 2 of olanzapine (ethanol); other solvents (tetrahydrofuran, chloroform, acetonitrile) give solvate thermograms, where this last thermal trace is only poorly evident. With few exceptions, each solvent forms solvate both when pure and in mixture (10%, v/v, in ethyl acetate). Methanol monosolvate displays complex thermogram and thermogravimetric desolvation profiles, depending on the crystallization experimental conditions, used to prepare the solvates. Dichloromethane solvate was found by X-ray diffraction analysis to be amorphous and, on heating during DSC analysis, allowed the crystallization of both form 1 and 2, with different weight ratio, according to the experimental conditions of the solvate preparation.

Mucoadhesive multiparticulate patch for the intrabuccal controlled delivery of lidocaine.

The aim of the present study was to prepare and evaluate patches for the controlled release of lidocaine in the oral cavity. Mucoadhesive buccal patches, containing 8 mg/cm(2) lidocaine base, were formulated and developed by solvent casting method technique, using a number of different bio-adhesive and film-forming semi-synthetic and synthetic polymers (Carbopol, Poloxamer, different type Methocel) and plasticizers (PEG 400, triethyl citrate); the patches were evaluated for bioadhesion, in vitro drug release and permeation using a modified Franz diffusion cell. A lidocaine/Compritol solid dispersion in the form of microspheres, embedded inside the patch, alone or together with free lidocaine, was also examined to prolong the drug release. The effects of the composition were evaluated considering a number of technological parameters and the release of the drug. All the formulations tested offer a variety of drug release mechanisms, obtaining a quick or delayed or prolonged anesthetic local activity with simple changes of the formulation parameters.

Diclofenac salts, part 7: are the pharmaceutical salts with aliphatic amines stable?

Eight cyclic aliphatic amines, pyrrolidine (Py), piperidine (Pp), morpholine (M), piperazine (Pz), and the N-hydroxyethyl (HE) analogues, were employed to prepare a salt with acidic diclofenac (D). These salts were examined by thermal [differential scanning calorimetry (DSC), thermogravimetric analysis, and hot-stage microscopy (HSM)] and spectroscopic [Fourier transform infrared (FTIR), Raman, (1) H NMR, and ultraviolet] analysis. The results show the thermal instability of these salts: the thermal dissociation leaves the starting acidic D, evidenced by the FTIR and Raman spectra inside the molten mass of the salts with M and HEM. The nature of the salt with Pz (1:1 or 1:2) and HEPy (anhydrous or hydrate polymorph), but not for the salt with HEPz and Py, depends on the polarity of the solvent used for the preparation of the salt. Incomplete dehydration of the hydrate Py and Pz salts progressively modifies the thermogram profiles and originates false information. Melting of the salts with Pp, M, and HEM could be demonstrated by HSM, but not with DSC. The difficulty of providing a description of these salts in a simple way originates doubts on the utility of a wide application of aliphatic amines to prepare pharmaceutical salts with D, whose solubility in water does not significantly differ from that of the common sodium D.

Diclofenac Salts, VIII. Effect of the Counterions on the Permeation through Porcine Membrane from Aqueous Saturated Solutions.

The following bases: monoethylamine (EtA), diethylamine (DEtA), triethylamine (TEtA), monoethanolamine (MEA), diethanolamine (DEA), triethanolamine (TEA), pyrrolidine (Py), piperidine (Pp), morpholine (M), piperazine (Pz) and their N-2-hydroxyethyl (HE) analogs were employed to prepare 14 diclofenac salts. The salts were re-crystallized from water in order to obtain forms that are stable in the presence of water. Vertical Franz-type cells with a diffusional surface area of 9.62 cm2 were used to study the permeation of these diclofenac salts from their saturated solutions through an internal pig ear membrane. The receptor compartments of the cells contained 100 mL of phosphate buffer (pH 7.4); a saturated solution (5 mL) of each salt was placed in the donor compartment, thermostated at 37 °C. Aliquots were withdrawn at predetermined time intervals over 8 h and then immediately analyzed by HPLC. Fluxes were determined by plotting the permeated amount, normalized for the membrane surface area versus time. Permeation coefficients were obtained dividing the flux values J by the concentration of the releasing phase-that is, water solubility of each salt. Experimental results show that fluxes could be measured when diclofenac salts with aliphatic amines are released from a saturated aqueous solution. Different chemical species (acid, anion, ion pairs) contribute to permeation of the anti-inflammatory agent even though ion-pairs could be hypothesized to operate to a greater extent. Permeation coefficients were found higher when the counterion contains a ring; while hydroxy groups alone do not appear to play an important role, the ring could sustain permeation, disrupting the organized domains of the membrane.

Diclofenac salts, part 6: release from lipid microspheres.

The release of diclofenac (20%, w/w) was studied from lipidic solid dispersions using three different chemical forms (acid, sodium salt, and pyrrolidine ethanol salt) and two different lipid carriers (Compritol 888 ATO or Carnauba wax) either free or together with varying amounts (10%-30%, w/w) of stearic acid. Microspheres were prepared by ultrasound-assisted atomization of the molten dispersions and analyzed by scanning electron microscopy, differential scanning calorimetry, and hot stage microscopy. The effects of different formulations on the resulting drug release profiles as a function of pH were studied and the results were discussed. The formulation of the 18 systems and the chemical form of the drug were found to strongly affect the mode of the drug release. The solubility of the chemical forms in the lipid mixture is in the following order: pyrrolidine ethanol salt ≫ acid > sodium salt (according to the solubility parameters), and the nature of the systems thus obtained ranges from a matrix, for mutually soluble drug/carrier pairs, to a microcapsule, for pairs wherein mutual solubility is poor. Drug release from microspheres prepared by pure lipids was primarily controlled by diffusion, whereas the release from microspheres containing stearic acid was diffusion/erosion controlled at pH 7.4.

Theophylline-loaded compritol microspheres prepared by ultrasound-assisted atomization.

Nine solid dispersions were prepared by the melting method in the form of particles containing theophylline at 10%, 20%, and 30% (w/w) in three Compritols (Compritol 888 ATO, HD5 ATO, E ATO) to compare their efficiency in controlling theophylline release. After solidification the mass was ground and granules were evaluated by thermal [differential scanning calorimetry, hot stage microscopy (HSM)] and spectroscopic [Fourier transform infrared (FTIR), Raman, X-ray powder diffraction (XRD)] analysis and the solubility parameters. Another nine samples of the same composition were obtained as microspheres by ultrasound-assisted (US) atomization. XRD confirmed the presence of crystalline theophylline inside the solid dispersions. FTIR and Raman microspectroscopy revealed that crystals of the drug were present on the granule surface. On the contrary, the surface of the final microspheres did not present free drug crystals. The granules do not work so efficiently as microspheres in controlling the release of theophylline: 888 ATO ≈ HD5 ATO > E ATO represents the order of the ability of the Compritols to control the theophylline release from microspheres. HSM revealed that, on aging, the dissolved drug crystallizes, considerably modifying the granule formulation and that US vibration, speeding up the crystallization of the drug during the preparation of microspheres, greatly reduces the changes associated with aging.

Mucoadhesive gels designed for the controlled release of chlorhexidine in the oral cavity.

This study describes the in vitro/ex vivo buccal release of chlorhexidine (CHX) from nine mucoadhesive aqueous gels, as well as their physicochemical and mucoadhesive properties: CHX was present at a constant 1% w/v concentration in the chemical form of digluconate salt. The mucoadhesive/gel forming materials were carboxymethyl- (CMC), hydroxypropylmethyl- (HPMC) and hydroxypropyl- (HPC) cellulose, alone (3% w/w) or in binary mixtures (5% w/w); gels were tested for their mucoadhesion using the mucin method at 1, 2 and 3% w/w concentrations. CHX release from different formulations was assessed using a USP method and newly developed apparatus, combining release/permeation process in which porcine mucosa was placed in a Franz cell. The combination of HPMC or HPC with CMC showed slower drug release when compared to each of the individual polymers. All the systems proved suitable for CHX buccal delivery, being able to guarantee both prolonged release and reduced transmucosal permeation. Gels were compared for the release of previously studied tablets that contained Carbopol and HPMC, alone or in mixture. An accurate selection and combination of the materials allow the design of different pharmaceutical forms suitable for different purposes, by simply modifying the formulation compositions.

Bimodal release of olanzapine from lipid microspheres.

Olanzapine was formulated as 10% (w/w) mixture with cutina to which stearic acid was added, ranging from 10% to 90% (w/w) of the total mass to control the drug release. The molten mixtures were processed by ultrasound-assisted spray-congealing technique, obtaining solid microspheres. The drug is stable under these conditions and only a partial miscibility in the solid state was observed by DSC between the two fatty materials with two separated melting endotherms in the thermograms: this can be due to the presence of two phases inside the solid dispersion. Olanzapine is distributed into the two phases according to its partition coefficient: two phases make the system less suitable to crystallization of the drug; the loading of the drug could reach saturation with difficulty and the rate of the olanzapine release is differentiated, since the drug is released from two different carriers. Dissolution profiles suggest occurrence of a bimodal release, where each portion of the release profile is linear and the slope increases with a higher content of stearic acid in the carrier mixture, that behaves as a release promoter. Tests were also carried out with palmitic and lauric acids for comparison and also for systems in the absence of ultrasound.

Diclofenac Salts. V. Examples of Polymorphism among Diclofenac Salts with Alkyl-hydroxy Amines Studied by DSC and HSM.

Nine diclofenac salts prepared with alkyl-hydroxy amines were analyzed for their properties to form polymorphs by DSC and HSM techniques. Thermograms of the forms prepared from water or acetone are different in most cases, suggesting frequent examples of polymorphism among these salts. Polymorph transition can be better highlighted when analysis is carried out by thermo-microscopy, which in most cases made it possible to observe the processes of melting of the metastable form and re-crystallization of the stable one. Solubility values were qualitatively related to the crystal structure of the salts and the molecular structure of the cation.

Synthesis, structural characterization and antibacterial activity of novel 7beta-{[3-(substituted phenyl)-2-propenoyl]amino}-3-[(2,5-dihydro-6-hydroxy-2-methyl)-5-oxo-cis-triazin-3-yl]-thiomethyl-cefalosporins.

A series of 3-[(2,5-dihydro-6-hydroxy-2-methyl)-5-oxo-cis-triazin-3-yl]-thiomethyl-cefalosporins with various 3-phenyl-2-propenoyl substituted groups at the 7beta-position were synthesized, structurally characterized and evaluated for antibacterial activity in vitro. To prepare these derivatives by the Vilsmeier's reagent method, it was necessary to carefully control the reaction conditions in order to avoid the formation of the biologically inactive alpha epimer. The NMR studies showed that the 3-phenyl-2-propenoyl moiety has little effect on chemical shifts of cephem nucleus protons and carbon atoms. Some of these cephalosporin derivatives showed good in vitro activity against methicillin sensible strains of Staphylococcus aureus (MSSA) and coagulase negative Staphylococcus (MSCoNS). Particularly effective were the compounds carrying a 3-(2'-chlorophenyl)-2-propenoyl or 2-methyl-3-phenyl-2-propenoyl moiety at 7beta-position, both with an antibacterial potency close to cefazoline and higher than cefuroxime. All the synthesized cephalosporins were inactive against methicillin resistant strains of Staphylococcus aureus (MRSA) and coagulase negative Staphylococcus (MRCoNS).

ATR/Raman and fractal characterization of HPBCD/progesterone complex solid particles.

PURPOSE: Characterization of hydroxypropyl-beta-cyclodextrin/progesterone (HPBCD/P) complex solid particles obtained from an aqueous solution, by three different technological processes, with the aim of preparing ready-to-dissolve powders for injectable as well as solid oral formulations in progestinic therapy. METHODS: HPBCD/P complex in the 2:1 molar ratio was prepared in aqueous solution and obtained as dry solid particles by freeze-drying, by spray-drying and by fluid-bed evaporation of the solvent. The particles were characterized by mu-FT-IR, mu-Raman and X-ray spectroscopy, by thermal analysis (differential scanning calorimetry-DSC and thermogravimetry-TGA), by Karl Fischer (KF) titration, by image and fractal analysis and by BET specific surface area analysis. The structure of the complex was also defined by comparison of FT-IR and Raman spectra of progesterone with those of pregnenolone and testosterone, structurally related. Dissolution tests were also performed. RESULTS: Powders of the complex obtained by the three different methods are different in size and shape. Particles obtained by freeze-drying are flat and angular, irregularly shaped without any relation to known geometrical solid figures. Particles obtained by spray-drying are spherically shaped and display a very small size (5-10 microm), with evident deformations and depression of the external surface, due to the rapid evaporation of the solvent. Particles obtained by fluid bed technique have intermediate sizes, display a tri-dimensional structure and irregular surface, with small and rounded protuberances. Fractal dimension of the particle contour was found close to one unit for the microspheres obtained by spray-drying. FT-IR and Raman spectra confirm the occurrence of the complexation by the shift of representative bands of the two carbonyl groups in positions 3 and 20 of the complexed progesterone. X-ray diffractograms indicate the amorphous nature of all the types of particles, also suggested by the absence of any melting peak of the drug in DSC thermograms. The samples contain different amounts of humidity: particles obtained by fluid-bed method demonstrated non-porous in BET analysis. Dissolution of different types of particles is complete after 3 min and only negligible differences could be appreciated among the three powders. CONCLUSIONS: - mu-FT-IR, mu-Raman and X-ray spectroscopy, and the dissolution test did not reveal defined differences among the three different types of particles, confirming occurrence of the complex in the solid state. The spherical shape, the very small size and the low value of the contour fractal dimension allows better technological performance of the particles obtained by spray-drying: this drying process appears the most promising one to prepare dry particles of the HPBCD/P complex, in view of its formulation in the fast preparation of extemporaneous injectable solutions and solid oral formulations intended for sublingual delivery.

Raman and thermal analysis of indomethacin/PVP solid dispersion enteric microparticles.

Indomethacin (IMC) and three types of poly-(vinylpyrrolidone) (PVP 12PF, PVP K30 and PVP K90) were studied in the form of solid dispersion, prepared with the solvent evaporation method, by spectroscopic (Raman, FT-IR, X-ray diffraction), thermal (differential scanning calorimetry, thermogravimetry, hot-stage microscopy), fractal and image analysis. Raman and FT-IR micro-spectroscopy indicated the occurrence of drug/polymer interaction and the presence of an amorphous form of IMC, as also resulting from X-ray diffractometry. Hot-stage microscopy suggested that the interaction between IMC and the polymer occurring on heating of a physical mixture, is common to other acidic compounds and causes a depression of the temperature of the appearance of a molten phase. Co-evaporated particles were coated by spray-congealing process with molten stearic acid for gastroprotection, but also for stabilization of the amorphous structure of the drug: the final particles were spherically shaped. Dissolution tests carried out on the final microparticles showed that the coating with stearic acid prevents IMC release at acidic pH and also protects against recovery of the IMC crystallinity, at least after 9 months of aging: the extent and mode of the release, before and after aging, overlap perfectly. The test revealed a notable improvement of the drug release rate from the solid dispersion at suitable pH, with respect to pure IMC. The comparison of the present solid dispersion with IMC/PVP (surface) solid dispersion obtained by freeze-drying of an aqueous suspension, where IMC maintained its crystalline state, revealed that there was no difference concerning the release rate, but suggested a superior quality of this last process as a mean of improving IMC availability for the easiness of preparation and stability, due to the absence of the amorphous state of the drug, as a possible instability source of the system. Finally, the coating with stearic acid is discussed as a determining process for the practical application of solid dispersions.

Control of transdermal permeation of hydrocortisone acetate from hydrophilic and lipophilic formulations.

The purpose of this research was the preparation of four formulations containing hydrocortisone acetate (HCA) for topical application, including two aqueous systems (hydrophilic microemulsion and aqueous gel) and two systems with dominant hydrophobicity (hydrophobic microemulsion and ointment). The formulations were tested for the release and permeation of HCA across an animal membrane. The release of HCA was found comparable for the four systems. The two microemulsions promote permeation across an ex-vivo membrane, examined by means of a Franz cell. Hydrophobic microemulsion guarantees the highest solubility (2,370 microg/ml) and flux (133 microg/cm(2).h) of the drug, since it contains almost 40% Transcutol, a permeation enhancer. Gel and ointment provide lower solubility and flux, being the values, related to the ointment, the lowest ones (562 microg/ml and 0.4 microg/cm(2).h). Experimental results allow the conclusion that gel and ointment can be suitable when it is desirable to minimize absorption of topically applied HCA as to keep the drug restricted to the diseased area and prevent side effects of the systemic presence of HCA.

Fast dispersible/slow releasing ibuprofen tablets.

Eight formulations were developed containing ibuprofen in the form of orally disintegrating tablets. To prevent bitter taste and side effects of the drug, the drug was associated with Phospholipon 80H, a saturated lecithin, by wet granulation. The granules were then coated using different film forming agents (Kollicoat SR 30, Amprac 01, Kollidon 90F, Eudragit RD 100) obtaining four lots 1-4. Coated granules were then formulated with a sweetener (Aspartame), a mannitol-based diluent (Pearlitol SD 200) and Kollidon CL (1-4K) or Explotab (1-4E) were added as superdisintegrants and compacted under low compression force. The eight lots of tablets, 1-4K and 1-4E, were assessed if suitable as oral disintegrating tablets by determination of a range of technological parameters. Wetting and disintegregation time matched with the requirements of EP IV Ed., for almost all these formulations. Dissolution profiles suggested that the combined action of the hydrophobic lecithin and the coating delay the release of the drug from tablets with respect to when it is free or in the form of simple granules. By an appropriate combination of excipients it was thus possible to obtain orally disintegrating tablets and a delayed release of ibuprofen using simple and conventional techniques.

Effect of vehicles on topical application of aloe vera and arnica montana components.

In this study two types of gels and microemulsions are investigated for their ability to dissolve, release, and induce the permeation of helenalin, a flavonoid responsible for the anti-inflammatory activity of arnica montana extract, and aloin, an anthrone-C-glucosyls with antibacterial activity present in aloe vera extract. The release of these agents from each vehicle was followed by HPLC, and transcutaneous permeation was examined using a modified Franz cell and a porcine skin membrane. The study showed that a microemulsion can be a good vehicle to increase the permeation of helenalin, while the gel formulation, containing Sepigel 305, proved able to reduce the release and permeation of aloin, with a consequent activity limited to the surface of application, without any permeation. This is in accordance with the necessity to avoid this process, since human skin fibroblasts can metabolize absorbed aloin into a structurally related compound that increases the sensitivity of skin to ultraviolet light.

Solid state characterization of progesterone in a freeze dried 1:2 progesterone/HPBCD mixture.

Progesterone rapidly dissolves in an aqueous solution containing hydroxypropyl-beta-cyclodextrin (HPBCD) at 1:2 molar ratio, forming a soluble inclusion complex. After filtration and freeze-drying of the final solution, the final powder was examined by SEM, DSC, TGA, XRD, Raman, and FTIR spectroscopy. Experimental results confirm that an inclusion complex exists in the solid state and possible structure of the complex is briefly discussed.

Ex vivo study of transdermal permeation of four diclofenac salts from different vehicles.

The ex vivo permeation of diclofenac was studied using four different salts (sodium, potassium, diethylamine, and epolamine) dissolved in four different solvents (water, propylene glycol (PG), Transcutol, and oleic acid (OA)) as donor phases through a human skin membrane. The four salts show different solubility values and different behavior in the four solvents, which are also permeation enhancers and this fact further is connected to the permeation results. The same order of magnitude of fluxes through the membrane as those previously reported for acidic diclofenac released from buffer solutions of pH >7 were found, taking into account differences originated by different membranes and other parameters tested in the experiments. Saturation concentration for the four salts in different solvents, necessary to calculate permeation coefficients, was critically evaluated; a short discussion made it possible to explain that corrections in the solubility values must be considered, related to the complex behavior in solution of these salts. Statistical processing of the experimental data suggests that differences between the four salts in promoting absorption of the drug is unproven; while differences are evident between the solvents, water is the most effective enhancing vehicle. Aqueous formulations containing diclofenac salt with an organic base appear to be the best combination to promote permeation in topical applications.