Drug Solubilization by Means of Partition/Association Equilibrium Using a Modified Nanosized Dendrimeric Biopolymer.

The objective of this study is to elucidate the combined effects of a novel type of material being investigated as a new excipient, an octenylsuccinate-modified dendrimer-like biopolymer (OS-DLB) and poloxamer (PLX), on the solubility of poorly water-soluble compounds. Phenytoin (PHT), griseofulvin (GSF), ibuprofen (IBU), and loratadine (LOR) were used as model compounds. Phase solubility measurements were conducted to determine the relative proportions of API, OS-DLB, and PLX that result in the most stable dendrimeric complexes. The solubilizing power of OS-DLB increases with increasing hydrophobicity of the solute. In the presence of PLX, the solubilization effect of OS-DLB is modestly accentuated for the most hydrophobic drugs (IBU and LOR) but has no effect on the least hydrophobic one (PHT). The maximum potentiation effect of PLX on the solubilizing properties of OS-DLB was observed for GSF, the drug of intermediate hydrophobicity. Three different types of solubilization profiles were obtained in the study. All three different profiles can be appropriately described by a single solubilization model, depending on the specific parameter values. The defining parameters of the model reflect the hydrophobicity of the drug on the one hand and, on the other hand, the inherent tendency of the drug (crystal lattice energy) toward crystallization.

Stain removal effect of novel papain- and bromelain-containing gels applied to enamel.

OBJECTIVES: The aims of the study are to prepare novel stain removal gel-based formulations containing papain or bromelain and to investigate their stain removal effect when applied to enamel. MATERIALS AND METHODS: Experimental bromelain- and papain-based stain removal gels were prepared. Next, enamel/dentin tooth samples (6 × 6 mm2, 4 mm in thickness) were obtained from bovine teeth, stained in coffee solution for 1 week, and measured with a digital spectrophotometer (Easyshade, Vita Zahnfabrik) for color assessment (baseline). The samples were then randomly allocated into four groups (n = 7), according to the stain removal agent applied: ContrastPM+ (Discus Dental, LLC), which is based on 20 wt.% carbamide peroxide (positive control); bromelain-based; papain-based; and no agent (negative control). The materials were applied once a week, three times per day, during 4 weeks, and following the directions of use from positive control. The samples were measured again with the Easyshade and using the CIEL * a * b * color system. The color change (ΔE *) results were obtained by subtracting the baseline values from the final color values obtained at each time point. The data were statistically analyzed using two-way repeated-measures analysis of variance and Student Newman Keuls's test as a post hoc test (α = 5 %). RESULTS: All stain removal agents produced greater color change than the negative control (p < .001), with the positive control demonstrating greater ΔE * values when compared to the experimental gels (p ≤ .004). The second application of all gels resulted in greater ΔE * values compared to the first application (p ≤ .025), although no color change was observed after the third application (p ≥ .051), regardless of the material evaluated. CLINICAL SIGNIFICANCE: The proposed gels containing proteolytic enzymes (bromelain or papain) of vegetal origin may hold significant clinical potential as active agents for the preparation of stain removal agents free of hydrogen/carbamide peroxide.

A dimensionless variable for the scale up and transfer of a roller compaction formulation.

Roller compaction is the most commonly employed dry granulation process in the pharmaceutical industry. While this process is increasingly used as an alternative to wet granulation, there are no parameter sets or system of equations to quickly scale up or transfer a formulation between two pieces of equipment. In this work, dimensionless variable was examined as a method to transfer the operating parameters of a formulation between two different pieces of equipment. This work was completed to establish the ground work for the development of a dimensionless relationship relating the operating parameters of the equipment to the porosity of the ribbon. The working hypothesis was three-fold, namely (i) that ribbons of the same porosity made with different equipment will have similar properties, (ii) that it is possible to establish an objective relationship between ribbon porosity and a combination of operating parameters and raw material attributes and (iii) that by expressing such parameter combination as a dimensionless variable, it will be possible to use the same relationship for different pieces of roller compaction equipment. The dimensionless variable RP/RS*HFS*True Density*D2 was found to correlate well with the ribbon porosity for the formulations and equipment used in these experiments. Depending on the formulation, the average difference in ribbon porosity between the two units varied between 0.012 and 0.024.

The influence of API concentration on the roller compaction process: modeling and prediction of the post compacted ribbon, granule and tablet properties using multivariate data analysis.

OBJECTIVE: While previous research has demonstrated roller compaction operating parameters strongly influence the properties of the final product, a greater emphasis might be placed on the raw material attributes of the formulation. There were two main objectives to this study. First, to assess the effects of different process variables on the properties of the obtained ribbons and downstream granules produced from the rolled compacted ribbons. Second, was to establish if models obtained with formulations of one active pharmaceutical ingredient (API) could predict the properties of similar formulations in terms of the excipients used, but with a different API. MATERIALS AND METHODS: Tolmetin and acetaminophen, chosen for their different compaction properties, were roller compacted on Fitzpatrick roller compactor using the same formulation. Models created using tolmetin and tested using acetaminophen. The physical properties of the blends, ribbon, granule and tablet were characterized. Multivariate analysis using partial least squares was used to analyze all data. RESULTS: Multivariate models showed that the operating parameters and raw material attributes were essential in the prediction of ribbon porosity and post-milled particle size. The post compacted ribbon and granule attributes also significantly contributed to the prediction of the tablet tensile strength. CONCLUSIONS: Models derived using tolmetin could reasonably predict the ribbon porosity of a second API. After further processing, the post-milled ribbon and granules properties, rather than the physical attributes of the formulation were needed to predict downstream tablet properties. An understanding of the percolation threshold of the formulation significantly improved the predictive ability of the models.

Investigation of Film with ß-Galactosidase Designed for Stabilization and Handling in Dry Configuration.

Gelatin-based films with an immobilized enzyme designed for extending the stability of the protein in dry, non-powder configuration with precise dosing attributes were subjected to stress conditions of temperature and relative humidity. ß-galactosidase was used as model functional protein. The film configuration preserved the activity of the enzyme under the different storage conditions investigated, which include room temperature under low (ambient) and high (75%) relative humidity, and 36 °C under low (oven) and high relative humidity conditions for a period of 46 days. The influence of the enzyme and plasticizer (glycerol) on the physical and mechanical properties of the films was investigated using DMA (dynamic mechanical analysis). Films containing 5% ß-galactosisdase and glycerol concentrations of 14% or greater exhibited greater tensile strength, Young's modulus, and elongation at break than films with equal concentrations of plasticizer but devoid of any enzyme. The surface texture of the films was analyzed using scanning electron microscopy (SEM). ß-galactosidase and glycerol have opposite effects on the surface morphology of the films. Increasing concentrations of the enzyme result in rougher film surface, whereas increasing the concentration of glycerol leads to films with denser and smoother surface. The results obtained suggest that the dry film configuration approach can help in facilitating the stabilization, handling, storage, and transportation of functional proteins in a cost effective manner.

A novel method for determining the solubility of small molecules in aqueous media and polymer solvent systems using solution calorimetry.

PURPOSE: To explore the application of solution calorimetry for measuring drug solubility in experimentally challenging situations while providing additional information on the physical properties of the solute material. METHODS: A semi-adiabatic solution calorimeter was used to measure the heat of dissolution of prednisolone and chlorpropamide in aqueous solvents and of griseofulvin and ritonavir in viscous solutions containing polyvinylpyrrolidone and N-ethylpyrrolidone. RESULTS: Dissolution end point was clearly ascertained when heat generation stopped. The heat of solution was a linear function of dissolved mass for all drugs (<10% RSD, except for chlorpropamide). Heats of solution of 9.8 ± 0.8, 28.8 ± 0.6, 45.7 ± 1.6 and 159.8 ± 20.1 J/g were obtained for griseofulvin, ritonavir, prednisolone and chlorpropamide, respectively. Saturation was identifiable by a plateau in the heat signal and the crossing of the two linear segments corresponds to the solubility limit. The solubilities of prednisolone and chlopropamide in water by the calorimetric method were 0.23 and 0.158 mg/mL, respectively, in agreement with the shake-flask/HPLC-UV determined values of 0.212 ± 0.013 and 0.169 ± 0.015 mg/mL, respectively. For the higher solubility and high viscosity systems of griseofulvin and ritonavir in NEP/PVP mixtures, respectively, solubility values of 65 and 594 mg/g, respectively, were obtained. CONCLUSION: Solution calorimetry offers a reliable method for measuring drug solubility in organic and aqueous solvents. The approach is complementary to the traditional shake-flask method, providing information on the solid properties of the solute. For highly viscous solutions, the calorimetric approach is advantageous.

Enhancing the Bioavailability of Poorly Soluble Drugs.

The poor aqueous solubility of drugs is such a frequent challenge to drug absorption, bioavailability, and drug delivery that it is occasionally spoken about as an "unwritten rule" in drug product development [...].

Albumin is an important factor in the control of serum free fatty acid flux in both male and female mice.

Albumin knockout (Alb-/-) mice exhibit a low plasma free fatty acid (FFA) concentration, but it was not known if the suppressed concentration reflects a lower rate of appearance (Ra) of FFA in the circulation (i.e., lower FFA flux) or if the absence of albumin alters the relationship between FFA flux and concentration. For understanding the role of albumin in FFA transport through the bloodstream, it is not sufficient to rely on FFA concentration data alone. Therefore, we developed a method to study FFA kinetics in Alb-/- mice. Using an albumin-free formulation of [U-13C]palmitate tracer, serum FFA kinetics were tested in Alb-/- and wild-type (WT) mice. Results indicate that the flux of FFA in serum of Alb-/- mice was significantly lower than in WT mice (P < 0.05), while albumin deficiency did not alter the relationship between FFA flux and concentration. Next, to test if suppressed lipolysis might have also been involved in the suppressed FFA kinetics, gene expression of a lipolytic enzyme (adipose triglyceride lipase, Atgl) and a marker of lipolysis (phosphorylation of hormone-sensitive lipase, p-HSL) were measured in adipose tissue. In contrast to the low FFA flux in Alb-/-, both Atgl gene expression and p-HSL protein were significantly higher in adipose tissue of Alb-/- than in WT mice (P < 0.05). Thus, the low FFA flux in Alb-/- appeared to be driven by the absence of albumin's FFA binding functions rather than through regulation of lipolysis, indicating that albumin is an important factor in determining the flux of FFA in circulation.NEW & NOTEWORTHY To improve understanding of the albumin protein's function in vivo, we tested plasma free fatty acid kinetics in albumin knockout mice compared with wild-type mice. Using a new tracer formulation strategy, it was discovered that the appearance rate of free fatty acids in serum is lower in albumin knockout mice than in wild-type mice. The results indicate that albumin is a major controller of free fatty acid kinetics.

Tunable Drug Release Rate Using Modular Oral Dosage Forms.

Oral dosage forms with adjustable drug release profiles were prepared using progesterone (PGR) as a poorly-soluble model drug. The dosage forms were made as stack assemblies of functional modules. The modules were made as PGR-carrying HPMC films cut into wafer-like circular pieces. Two types of modules were used in the study; one exhibited comparatively fast drug release and the other slow release. The fast vs. slow release of each type of film utilized resulted from the grade of HPMC used in each case. Drug loading in the assembly was controlled through the total number of modules. By adjusting the proportions of the two types of modules, it is possible to fine-tune the drug release rate of the multi-layer assemblies to a wide range of profiles, bracketed between a high and low end, corresponding to the inherently fastest or slowest release obtainable with the specific materials and procedures employed. This procedure is suitable for adjusting the spring-and-parachute parameters for enhancing/optimizing the bioavailability of poorly-soluble drugs, and for developing patient-centric formulations.

Improvement of the dissolution rate of poorly soluble drugs by solid crystal suspensions.

We present a novel extrusion based approach where the dissolution rate of poorly soluble drugs (griseofulvin, phenytoin and spironolactone) is significantly accelerated. The drug and highly soluble mannitol are coprocessed in a hot melt extrusion operation. The obtained product is an intimate mixture of the crystalline drug and crystalline excipient, with up to 50% (w/w) drug load. The in vitro drug release from the obtained solid crystalline suspensions is over 2 orders of magnitude faster than that of the pure drug. Since the resulting product is crystalline, the accelerated dissolution rate does not bear the physical stability concerns inherent to amorphous formulations. This approach is useful in situations where the drug is not a good glass former or in cases where it is difficult to stabilize the amorphous drug. Being thermodynamically stable, the dissolution profile and the solid state properties of the product are maintained after storage at 40 °C, 75% RH for at least 90 days.

Modular solid dosage form design - Application to pH-independent release of a weak-base API.

A novel approach to solid dosage form design is investigated, whereby instead of blending the ingredients and subsequently compacting the mixture, the dosage form is made by assembling prefabricated components, each with a specific function. The approach was used to formulate a weak-base API (active pharmaceutical ingredient), such that the modular dosage forms exhibited pH-independent drug release. Tablet-like dosage forms of ciprofloxacin (CPR), used as model weak-base drug, were prepared in order to generate dosage forms exhibiting pH-independent drug release. The dosage forms were made by assembling two types of prefabricated modules onto 3D stacks. The modules were hydroxypropyl methylcellulose circular film wafers, loaded with either CPR or citric acid (CA). CA-wafers served the function of pH-modifier modules in the microenvironment of the dosage form during the dissolution process. In vitro drug release from dosage forms consisting of CA- and CPR-wafers stacked in alternate sequence was compared with the release from assemblies containing CPR-wafers only, under pH = 1.2 and pH = 6.8 conditions. In the absence of CA-wafers, CPR release was ~25-fold slower at pH = 6.8 compared to pH = 1.2. Inclusion of CA-wafers in the dosage form assembly accelerated and decelerated drug release at pH = 6.8 and pH = 1.2, respectively, which resulted in overlapping drug release profiles under the two pH conditions. The two drug release profiles met the criteria for sameness as assessed by the f1 (difference) and f2 (similarity) factors. Modeling of drug release kinetics pointed toward polymer erosion as the primary mechanism of drug release for the overlapping pH = 1.2 and pH = 6.8 profiles. In terms of their drug release properties, the multi-modular dosage form assemblies exhibited the attributes and behavior of single bodies, rather than the combined contributions from multiple individually-operating modules. The initial geometry of the dosage form, characterized by the surface area (SA), volume (V) and SA/V ratio accounted for drug release kinetics in the same fashion as for traditional tablet compacts.

Benzoic acid complexes with Eudragit E100®: New alternative antimicrobial preservatives.

Given that the use of some preservatives in cosmetics has been restricted, novel alternative preservatives are needed. The aim of this study was to characterize the physicochemical and antimicrobial properties of two polyelectrolyte complexes (EuB100 and EuB75Cl25), which were developed through hot melt extrusion (HME) using benzoic acid (BA) and Eudragit E100. Based on phase diagrams and an experimental statistical design, the solubility of the acid in the polymer and the HME conditions were established. Intermolecular interactions were evaluated through Fourier-transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and X-ray powder diffraction (XRPD). Release behavior was determined for the systems. Antibacterial activity and ζ-potential were determined on Escherichia coli. FTIR revealed acid-base interaction, and XPS showed that the percentages of protonated nitrogen N1s were 13.5% for EuB100 and 20.3% for EuB75Cl25. The BA released showed a non-Fickian behavior, and a satisfactory antibacterial activity against E. coli was demonstrated at pH 6.9. The complexes modified ζ-potential, destabilizing the membrane functionality of E. coli. These complexes are potential antimicrobial preservatives with a greater spectrum of action, with bactericidal activity against E. coli in a wider pH range than uncomplexed BA, even at pH 6.9.

Molecular and physical characterization of octenyl succinic anhydride-modified starches with potential applications in pharmaceutics.

Five commercially available starches modified with octenyl succinic anhydride (OSA) are characterized at a molecular, physicochemical and bulk level providing useful data for designing pharmaceutical products. The degree of substitution (DS) of the starches range from 0.017 to 0.032 and their molecular weights (Mw) and radius of gyration (Rz) are lower than those of native starch, suggesting additional modification processes besides the chemical treatment with OSA. The ability of the starches to reduce the water surface tension keeps a direct relationship with the DS and an inverse association with the Mw. Thermal properties, crystallinity assays and morphology evidence that most modified starches characterize by amorphous aggregated structures, possibly generated by gelatinization processes, which favor the flow properties of the powders. Water sorption and surface energy behaviors seem to be related to the number of octenyl succinate (OS) moieties. After dispersion in water, shear-thinning and Newtonian behaviors also depend on the type of OS-starch.

Development of an antibacterial and anti-metalloproteinase dental adhesive for long-lasting resin composite restorations.

Despite all the advances in adhesive dentistry, dental bonds are still fragile due to degradation events that start during application of adhesive agents and the inherent hydrolysis of resin-dentin bonds. Here, we combined two outstanding processing methods (electrospinning and cryomilling) to obtain bioactive (antimicrobial and anti-metalloproteinase) fiber-based fillers containing a potent matrix metalloproteinase (MMP) inhibitor (doxycycline, DOX). Poly(ε)caprolactone solutions containing different DOX amounts (0, 5, 25, and 50 wt%) were processed via electrospinning, resulting in non-toxic submicron fibers with antimicrobial activity against Streptococcus mutans and Lactobacillus. The fibers were embedded in a resin blend, light-cured, and cryomilled for the preparation of fiber-containing fillers, which were investigated with antibacterial and in situ gelatin zymography analyzes. The fillers containing 0, 25, and 50 wt% DOX-releasing fibers were added to aliquots of a two-step, etch-and-rinse dental adhesive system. Mechanical strength, hardness, degree of conversion (DC), water sorption and solubility, bond strength to dentin, and nanoleakage analyses were performed to characterize the physico-mechanical, biological, and bonding properties of the modified adhesives. Statistical analyses (ANOVA; Kruskal-Wallis) were used when appropriate to analyze the data (α = 0.05). DOX-releasing fibers were successfully obtained, showing proper morphological architecture, cytocompatibility, drug release ability, slow degradation profile, and antibacterial activity. Reduced metalloproteinases (MMP-2 and MMP-9) activity was observed only for the DOX-containing fillers, which have also demonstrated antibacterial properties against tested bacteria. Adhesive resins modified with DOX-containing fillers demonstrated greater DC and similar mechanical properties as compared to the fiber-free adhesive (unfilled control). Concerning bonding performance to dentin, the experimental adhesives showed similar immediate bond strengths to the control. After 12 months of water storage, the fiber-modified adhesives (except the group consisting of 50 wt% DOX-loaded fillers) demonstrated stable bonds to dentin. Nanoleakage was similar among all groups investigated. DOX-releasing fibers showed promising application in developing novel dentin adhesives with potential therapeutic properties and MMP inhibition ability; antibacterial activity against relevant oral pathogens, without jeopardizing the physico-mechanical characteristics; and bonding performance of the adhesive.

Elucidating raw material variability--importance of surface properties and functionality in pharmaceutical powders.

The purpose of this study is to illustrate, with a controlled example, the influence of raw material variability on the excipient's functionality during processing. Soluble starch was used as model raw material to investigate the effect of variability on its compaction properties. Soluble starch used in pharmaceutical applications has undergone a purification procedure including washing steps. In this study, a lot of commercially available starch was divided into two parts. One was left intact and the other was subjected to an extra washing step. The two resulting lots were subjected to a series of physical characterization tests typical of those used to qualify raw materials. The two resulting lots gave virtually identical results from the tests. From the physical testing point of view, the two lots can be considered as two equivalent lots of the same excipient. However, when tested for their functionality when subjected to a compaction process, the two lots were found to be completely different. The compaction properties of the two lots were distinctly different under all environmental and processing conditions tested. From the functionality point of view, the two lots are two very different materials. The similar physical testing results but different functionality can be reconciled by considering the surface properties of the powders. It was found that the washing step significantly altered the surface energetic properties of the excipient. The washed lot consistently produced stronger compacts. These results are attributable to the measurably higher surface energy of induced by the additional washing step.

Azopolymer based nanoparticles for phototriggered drug delivery.

Controlled release by stimulus-responsive nanoparticles is oriented to increase the specificity of drug delivery, to improve the therapy effectiveness and minimizing side effects. This work presents the synthesis of photosensitive-polymeric nanoparticles as a potential system for localized drug delivery. First, the photoisomerizable amphiphilic-copolymer poly2-[4-phenylazophenoxy]ethyl acrylate-co-acrylic acid (PPAPE), was synthesized. Then, PPAPE was employed to prepare micellar nanoparticles by the nanoprecipitation method. Characterizations of the polymer were performed by proton nuclear magnetic resonance, X-ray photoelectron spectroscopy and FTIR spectroscopy. The morphology of the nanoparticles was analyzed by dynamic light scattering and transmission electron microscopy. Also, photostimulation response was confirmed by UV-VIS spectroscopy. Results indicate that the obtained photoresponsive nanoparticles have the size and photoisomerization necessary to perform the specific release of drugs.

Solubility screening on a series of structurally related compounds: cosolvent-induced changes on the activity coefficient of hydrophobic solutes.

A homologous series of solutes was chosen as a model for a group of structurally related compounds with different physicochemical properties, as is commonly the case during the screening of potential drug candidates. Thermal properties of the crystalline solutes and solubility determinations were used to quantify the two independent factors that determine the solubility of organic compounds: crystallinity and hydrophobicity. A solubility screening study was conducted on the series. By expressing the obtained solubility enhancement expressed as changes in the activity coefficient, it is possible to visually compare the effect of different cosolvents. The results show the importance of solute-solvent polarity match. Polarity match between water miscible cosolvents and hydrophobic compounds is not truly attainable, but comparison of the screening results points out the closest matches (optimal effect), facilitating the systematic evaluation of solubilization approaches.

Utility of multivariate analysis in modeling the effects of raw material properties and operating parameters on granule and ribbon properties prepared in roller compaction.

This article aimed to model the effects of raw material properties and roller compactor operating parameters (OPs) on the properties of roller compacted ribbons and granules with the aid of principal component analysis (PCA) and partial least squares (PLS) projection. A database of raw material properties was established through extensive physical and mechanical characterization of several microcrystalline cellulose (MCC) and lactose grades and their blends. A design of experiment (DoE) was used for ribbon production. PLS models constructed with only OP-modeled roller compaction (RC) responded poorly. Inclusion of raw material properties markedly improved the goodness of fit (R(2) = .897) and model predictability (Q(2) = 0.72).

Investigation of the variability of NIR in-line monitoring of roller compaction process by using Fast Fourier Transform (FFT) analysis.

The purpose of this research was to investigate the variability of the roller compaction process while monitoring in-line with near-infrared (NIR) spectroscopy. In this paper, a pragmatic method in determining this variability of in-line NIR monitoring roller compaction process was developed and the variability limits were established. Fast Fourier Transform (FFT) analysis was used to study the source of the systematic fluctuations of the NIR spectra. An off-line variability analysis method was developed as well to simulate the in-line monitoring process in order to determine the variability limits of the roller compaction process. For this study, a binary formulation was prepared composed of acetaminophen and microcrystalline cellulose. Different roller compaction parameters such as roll speed and feeding rates were investigated to understand the variability of the process. The best-fit line slope of NIR spectra exhibited frequency dependence only on the roll speed regardless of the feeding rates. The eccentricity of the rolling motion of rollers was identified as the major source of variability and correlated with the fluctuations of the slopes of NIR spectra. The off-line static and dynamic analyses of the compacts defined two different variability of the roller compaction; the variability limits were established. These findings were proved critical in the optimization of the experimental setup of the roller compaction process by minimizing the variability of NIR in-line monitoring.

Drug Solubilization by Means of a Surface-Modified Edible Biopolymer Enabled by Hot Melt Extrusion.

A coprocessing/formulation approach for increasing the solubility of poorly soluble drugs using solid dispersions is presented, whereby the active pharmaceutical ingredients (API) retains its crystalline state. The approach uses a biopolymer naturally produced as dendrimeric nanoparticles that has been surface-modified to act as a solubilizing agent. The solubilizing agent is enabled by hot melt extrusion to produce the solid dispersions. Four APIs, phenytoin (PHT), griseofulvin, ibuprofen, and loratadine were used as model compounds to evaluate solubility enhancement. The rank order in solubility enhancement follows that of the hydrophobicity of the APIs. The APIs remained predominantly crystalline after hot melt extrusion processing. However, APIs with weak crystal structure (ibuprofen and loratadine) underwent measurable crystallinity loss. The solubilizing power of the modified biopolymer increases with increasing hydrophobicity and strength of the crystal structure. The solubility is described in terms of a parallel liquid-phase partition-association. For one API (PHT), solubility enhancement was minimal. The dissimilar behavior of PHT is discussed in terms of the polarity match between the API and the hydrophobic microenvironment in the solubilizing agent. This approach is expected to apply to a large number of poorly soluble drugs, offering a complementary approach to existing processing and formulation drug solubilization methods.