Mechanism Of Penetration Enhancement Research Articles

Percutaneous penetration enhancement effect of essential oil of mint (Mentha haplocalyx Briq.) on Chinese herbal components with different lipophilicity

ObjectiveTo investigate the percutaneous penetration effect of essential oil of mint from Mentha haplocalyx Briq. on the complex active components in Chinese herbal external preparations, and assess its toxicity on the skin cells. MethodsThe cytotoxicity of mint oil on HaCaT keratinocytes and CCC-ESF-1 fibroblasts was measured using an MTT assay. Five model drugs with a wide range of lipophilicity, namely osthole, tetramethylpyrazine, ferulic acid, puerarin, and geniposide, were tested using in vitro permeation studies to investigate the percutaneous penetration enhancement effect of mint oil. Secondary structure alterations of skin stratum corneum (SC) were measured using Fourier transform infrared spectroscopy (FTIR). Saturation solubilities and SC/vehicle partition coefficients of the five model drugs with and without mint oil were also determined to understand the potential mechanisms of the essential oil. ResultsHalf maximal inhibitory concentration (IC50) values of mint oil were significantly higher in HaCaT and CCC-ESF-1 cell lines than values in the well-established and standard penetration enhancer Azone. ConclusionsMint oil at proper concentration could effectively facilitate percutaneous penetration of both lipophilic and hydrophilic drugs, and exhibit higher efficiency for moderate hydrophilic drugs. Mechanisms of penetration enhancement by mint oil could be explained with saturation solubility, SC/vehicle partition coefficient and the secondary structure change of SC.

Nanostructured lipid carriers based nanogel for meloxicam delivery: mechanistic, in-vivo and stability evaluation

Aim: Our investigation was aimed to investigate the potential suitability of meloxicam-loaded nanostructured lipid carriers (MLX-NLC) gel for topical application.Main methods: MLX-NLC gel was prepared and in vivo skin penetration ability of the NLC gel was evaluated using confocal laser scanning microscopy. We studied the effect of MLX-NLC gel on the changes in lipid profile of skin to get an insight into its skin penetration enhancement mechanism. Acetic acid induced writhing test was performed to evaluate the analgesic effect. Drug concentration-time profile of MLX in rat plasma and skin after topical and oral treatment with MLX-NLC gel and oral MLX-solution, respectively, was observed. MLX-NLC gel was subjected to primary skin irritation test, sub-acute dermal toxicity study. Storage stability of MLX-NLC gel was also assessed for 90 days.Key findings: NLC gel was effective in permeating Rhodamine 123 to deeper layers of rat skin. Changes in skin lipid prolife were observed in the rat skin on treatment with MLX-NLC gel and the results supported skin lipid extraction as a possible penetration enhancement mechanism. MLX-NLC gel demonstrated sustained pain inhibitory effect. Pharmacokinetics study established that topical application of MLX-NLC gel had the potential to avoid systemic uptake and hence the risk of systemic adverse effects. MLX-NLC gel demonstrated good skin tolerability and biosafety. Excellent physical stability of nanogel was observed at 4 ± 2 °C.Significance: The study revealed that NLC gel is a promising carrier system for the topical application of MLX without side effects.

Evaluation of Meloxicam-Loaded Cationic Transfersomes as Transdermal Drug Delivery Carriers

The aim of this study is to develop meloxicam (MX)-loaded cationic transfersomes as skin delivery carriers and to investigate the influence of formulation factors such as cholesterol and cationic surfactants on the physicochemical properties of transfersomes (i.e., particle size, size distribution, droplet surface charge and morphology), entrapment efficiency, stability of formulations and in vitro skin permeation of MX. The transfersomes displayed a spherical structure. Their size, charge, and entrapment efficiency depended on the composition of cholesterol and cationic surfactants in the formulation. Transfersomes provided greater MX skin permeation than conventional liposomes and MX suspensions. The penetration-enhancing mechanism of skin permeation by the vesicles prepared in this study may be due to the vesicle adsorption to and/or fusion with the stratum corneum. Our results suggest that cationic transfersomes may be promising dermal delivery carriers of MX.

An attempt to clarify the mechanism of the penetration enhancing effects of lipophilic vehicles with differential scanning calorimetry (DSC).

In a previous in-vivo skin penetration study, it was observed that certain lipophilic liquid vehicles enhanced drug penetration, whilst others did not. To clarify the mechanism of skin penetration enhancement, isolated sheets of human stratum corneum were measured by differential scanning calorimetry (DSC), either untreated or after pretreatment with various lipophilic liquids (highly purified light mineral oil, isopropyl myristate, caprylic/capric acid triglycerides containing 5% phospholipids, dibutyl adipate, dimethicone 100, cetearyl iso-octanoate, caprylic/capric acid triglycerides), commonly used in ointment bases. All samples were analysed over a heating range of at least--10-130 degrees C. All DSC curves were evaluated with regard to the phase-transition enthalpies (peak areas) and peak maximum temperatures of the lipid-phase transitions at ca 75 and 85 degrees C. With the exception of dimethicone 100, cetearyl iso-octanoate and caprylic/capric acid triglycerides, all vehicles showed characteristic alterations of the phase-transition temperatures and enthalpies of the stratum corneum lipids. Mineral oil and isopropyl myristate caused a reduction of the enthalpy and a decrease of the phase-transition temperatures. These two vehicles are thought to fluidize the lamellar-gel phase of the stratum corneum lipids, and possibly partially dissolve the lipids. Dibutyl adipate and caprylic/capric acid triglycerides containing 5% phospholipids decreased the phase-transition enthalpy only, probably due to dissolution or extraction of the stratum corneum lipids. These DSC results provide an explanation for the in-vivo penetration-enhancing effects observed previously.

Visualization, dermatopharmacokinetic analysis and monitoring the conformational effects of a microemulsion formulation in the skin stratum corneum

The use of nano-systems such as the microemulsions is considered as an increasingly implemented strategy in order to enhance the percutaneous transport into and across the skin barrier. The determination of the major pathway of penetration and the mechanisms by which these formulations work remains crucial. In this study, laser confocal scanning microscopy was used to visualize the penetration and the distribution of a fluorescently-labelled microemulsion (using 0.1% w/v Nile red) consisting of (%, w/w) 15.4% oleic acid, 30.8% Tween 20, 30.8% Transcutol® and 23% water. The surface images revealed that the microemulsion accumulated preferentially in the intercellular domains of the stratum corneum. Additionally, by analysis of the images taken across the whole stratum corneum (SC), the penetration was found to occur along its whole depth. The latter result was confirmed using tape stripping and the subsequent sensitive analysis using liquid chromatography mass spectroscopy. Dermatopharmacokinetic parameters were obtained for the microemulsion different components. These values proved the breakage of the microemulsion during its penetration across the stratum corneum. Moreover, the mechanisms of penetration enhancement and the micro molecular effects on the skin stratum corneum were investigated using attenuated Fourier transform infra-red spectroscopy. The results revealed the penetration of all the microemulsion components in the stratum corneum and demonstrated the microemulsion interaction with the skin barrier perturbing its architecture structure.

Effect and mechanism of penetration enhancement of organic base and alcohol on Glycyrrhetinic acid in vitro

The aim of the present study was to explore the effects of organic bases and alcohols on the percutaneous absorption of Glycyrrhetinic acid (GA). GA is a metabolite of Glycyrrhizic acid (GL), a major active ingredient of Glycyrrhizae (Gancao) Radices. Skin penetration parameters of GA were obtained via in vitro penetration experiments using intact and stripped mice back skin. Non-aqueous solvent comprising isopropyl myristate (IPM) and alcohols (ethanol, butanol, octanol and dodecanol) loaded with organic base (triethanolamine or triethylamine) were applied to improve the penetration of GA. In order to further confirm the mechanism by which the organic bases enhanced the penetration of GA, conductivity measurement, 1H NMR spectroscopy and FT-IR analysis were used to observe the formation of ion pair between GA and organic base. The formation of ion pair increased the solubility of GA in the stratum corneum (SC) and its partition into the viable skin, and therefore enhanced the penetration of GA in skin.

Insulin-Loaded Nanoparticles Based on N-Trimethyl Chitosan: In Vitro (Caco-2 Model) and Ex Vivo (Excised Rat Jejunum, Duodenum, and Ileum) Evaluation of Penetration Enhancement Properties

The aim of this paper was to evaluate the penetration enhancement properties of nanoparticles (NP) based on N-trimethyl chitosan (TMC 35% quaternization degree) loaded with insulin. The permeation performances of TMC NP were compared with those of chitosan (CS) NP and also with TMC and CS solutions. To estimate the mechanism of penetration enhancement, two different approaches have been taken into account: an in vitro study (Caco-2 cells) and an ex vivo study (excised rat duodenum, jejunum, and ileum). Insulin-loaded CS and TMC NP had dimensions of about 250 nm and had high yield and high encapsulation efficiency. The in vitro study evidenced that TMC and CS were able to enhance insulin permeation to the same extent. Penetration enhancement properties of TMC NP seem to be prevalently related to endocytosis while the widening of tight junctions appeared more important as mechanism in the case of CS NP. The ex vivo study put in evidence the role of mucus layer and of its microclimate pH. In duodenum (pH 5-5.5), CS and TMC solutions were more effective than NP while TMC NP were more efficient towards jejunum tissue (pH 6-6.5) for their high mucoadhesive potential. Confocal laser scanning microscopy study supported the hypothesis that penetration enhancement due to TMC NP was mainly due to internalization/endocytosis into duodenum and jejunum epithelial cells. The good penetration enhancement properties (permeation and penetration/internalization) make TMC NP suitable carriers for oral administration of insulin.

Histological evaluation of buccal penetration enhancement properties of chitosan and trimethyl chitosan

The aim of the present work was to compare the penetration enhancement properties of chitosan hydrochloride (HCS) both as a polymeric solution and as a nanoparticulate system with that of trimethyl chitosan hydrochloride (TMC) on buccal mucosa. The hydrophilic high molecular weight fluorescein isothiocyanate dextran (FD4; 4400 Da) was used as a macromolecule model. The mechanism involved in the HCS (solution and nanoparticles) and TMC solution penetration enhancement was investigated on pig buccal mucosa, characterized by having stratified epithelium and lacking in tight junctions. The permeation/penetration of FD4 and the change in morphology and histology of the mucosa after contact with the polymers were assessed: the experiments were performed ex-vivo by applying the formulations on excised porcine buccal tissue. For the morphology and the histology studies, the epithelial cell layers from freshly excised pig buccal mucosa were analysed with light microscopy by means of routine histopathology analysis (haematoxylin and eosin staining and Toluidine blue staining) and immunohistochemistry reactions. The organization of desmosomal junctions was assessed by means of an immunochemical reaction on desmosomes and transmission electron microscopy. Confocal laser scanning microscopy (CLSM) was used to find evidence of the location of FD4 in the tissue. Furthermore, the increase of the FD4 apparent permeability coefficient was quantified by means of Franz diffusion cells using isolated buccal epithelium to demonstrate the penetration enhancement properties of the polymer systems. Morphological analysis, performed by light microscopy, transmission electron microscopy and CLSM, suggests a similar mechanism of penetration enhancement for both HCS and TMC solutions and for HCS nanoparticles. Such a mechanism probably involves a repackaging of the epithelial cells up to the basal membrane and a partial disarrangement of desmosomes. The cell viability and the nuclear integrity indicated on the semi-thin section stained with Toluidine blue and by CLSM analysis, respectively, suggest that HCS as a polymer solution and a nanoparticulate system, and TMC polymer solution, do not cause cell damage. Trimethyl chitosan and chitosan nanoparticulate systems were able to increase FD4 permeation across buccal epithelium to a greater extent than the chitosan solution.

Study on the mechanisms of chitosan and its derivatives used as transdermal penetration enhancers

The efficacy of chitosan (CS) and its derivatives used as transdermal penetration enhancers has been confirmed in our previous research. This study investigated the mechanisms of penetration enhancement by CS and its derivatives, i.e., N-trimethyl chitosan (TMC) with different degree of quaternization (DQ) and mono-N-carboxylmethyl chitosan (MCC). After treatment with CS, TMCs or MCC, the secondary structure changes of keratin in stratum corneum (SC) from mice were examined by an Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) combined with the application of the second-order derivative, deconvolution and curve-fitting. The water content in the SC was also studied by ATR-FTIR. HaCaT cell lines were employed as the cell models in the study. HaCaT cells were first treated with blank D-Hanks solution, CS or its derivatives, and were then fluorescent labeled with DiBAC 4 (3). The change of membrane potential was measured by a flow cytometer (FCM). Alternatively, the treated HaCaT cells were labeled with NBD-C 6-HPC and the change of membrane fluidity was examined under a Confocal Laser Scanning Microscope (CLSM). It was found that CS, TMCs and MCC could significantly affect the secondary structure of keratin in SC in different ways. Although the amide II absorption peak of keratin moved to a lower wave number following treatment with CS, TMCs, or MCC, the β-turning structure of keratin was converted to β-sheeting and random coiling after treatment with TMCs and was converted to β-sheeting and α-helix following treatment with MCC and CS. At the same time, CS and its derivatives all could increase the water content of SC, decrease HaCaT cells membrane potentials and enhance HaCaT cells membrane fluidity significantly. The effect of TMCs appeared to be independent of their DQ. The results suggest that the mechanisms of transdermal enhancement of CS, TMCs and MCC are closely related to their effects on the secondary structure of keratin and water content in SC, cell membrane potential and fluidity.

Role of complexes formation between drugs and penetration enhancers in transdermal delivery

The use of chemical penetration enhancers (CPE) is growing due to their ability to improve drug delivery through the skin. A possible mechanism of penetration enhancement could involve the complex formation between drug and components in the pharmaceutical formulation, thus altering the physicochemical properties of the active substance. Here, modelling studies indicate that hydrocarbon and oxygen-containing terpenes (penetration enhancers) could form complexes with drugs. Satisfactory correlations have been obtained between the predicted molecular properties of enhancers and their enhancement effects.

Formulation optimization of meloxicam sodium gel using response surface methodology

The influences of a combination of different mechanisms of penetration enhancers on the penetration absorption properties of meloxicam sodium formulations through rat skin were investigated using response surface methodology. A uniform design was applied to prepare model formulations systematically that were composed of four independent variables: the content of ethanol ( x 1), propylene glycol ( x 2), menthol ( x 3), and azone ( x 4). The penetration rate (flux) of meloxicam sodium gel through rat skin was chosen as the response which had to be higher than 400 μg/h cm 2 the required flux of meloxicam gel to maintain a therapeutic concentration. The result showed optimal formulation could be obtained from this response surface methodology. Menthol had the greatest potential influence on the penetration absorption of meloxicam sodium, followed by azone, ethanol and PG, respectively. By in vivo study, meloxicam could be determined 1 h after topical administration and reached steady-state concentration at about 12 h. The bioavailability (%) of the optimal meloxicam sodium gel was about 50.1%.

Effects of Enantiomer and Isomer Permeation Enhancers on Transdermal Delivery of Ligustrazine Hydrochloride

Enantiomers and isomers, such as D-limonene, L-limonene, and α-terpinene, were selected as enhancers. The effects and mechanisms of penetration enhancers on in vitro transdermal delivery of ligustrazine hydrochloride (LH) across hairless porcine dorsal skin were investigated. Transdermal fluxes of LH through porcine skin were determined in vitro by Franz-type diffusion cells. D-limonene, L-limonene, and α-terpinene could significantly promote the transdermal fluxes of LH, but no statistical difference (p > 0.05) between them was found. The lag time of L-limonene and α-terpinene were 2.55 and 2.20 times compared with that of D-limonene. Fourier transform-infrared (FTIR) was carried out to analyze the effects of enhancers on the biophysical natures of the stratum corneum (SC) and the permeation enhancement mechanism. FTIR spectra revealed that the changes of peak shift and peak area due to C-H stretching vibrations in the SC lipids were associated with the selected enhancers. All of them could perturb and extract the SC lipids to different extent and L-limonene showed obvious changes. Morphological changes of the skin treated with enhancers were monitored by a scanning electron microscope (SEM). The extraction of the SC lipids by the enhancers led to the disruption of SC and the desquamated SC flake. Apparent density (AD) was newly proposed to estimate the desquamated extent of SC flake. The results showed that the enantiomers and isomers enhanced the permeation of LH by pleiotropic mechanisms.

The effect of penetration enhancers on drug delivery through skin: a QSAR study

Skin penetration enhancers are used to allow formulation of transdermal delivery systems for drugs that are otherwise insufficiently skin-permeable. A full understanding of the mode of action could be beneficial for the design of potent enhancers and for the choice of the enhancer to be used in the topical formulation of a special drug. In this study, the structural requirements of penetration enhancers have been investigated using the Quantitative Structure–Activity Relationship (QSAR) technique. Activities of naturally occurring terpenes, pyrrolidinone and N-acetylprolinate derivatives on the skin penetration of 5-fluorouracil, diclofenac sodium (DFS), hydrocortisone (HC), estradiol and benazepril have been considered. The resulting QSARs indicated that for 5-fluorouracil and diclofenac sodium, less hydrophobic enhancers were the most active. More precisely, molecular descriptors in the corresponding QSARs indicated the possible involvement of intermolecular electron donor–acceptor interactions. This was in contrast to the skin permeation promotion of hydrocortisone, estradiol and benazepril by enhancers, where a linear relationship between enhancement activity and n-octanol/water partition coefficients of enhancers was evident. The possible mechanisms of penetration enhancement as suggested by the QSARs will be discussed.

Influence of molecular dipoles on human skin permeability: Use of 6-ketocholestanol to enhance the transdermal delivery of bacitracin

In the present work, we report the possibility of modifying the electrostatic properties of the skin by treating human epidermis with compounds whose structures possess a large molecular dipole moment. Data are presented showing that such a modification can be used to enhance dermal drug delivery. Inclusion of such compounds in biological membranes affects the so‐called membrane dipole potential, an electrical potential originating from molecular dipoles present on the lipid molecules. Modifications in the magnitude of this potential are known to affect the interaction of hydrophobic ions and peptides with model membranes. Using fluorescein‐labeled bacitracin and confocal microscopy, we show that the penetration of the antibiotic peptide bacitracin into the epidermis is enhanced when the skin has been pretreated with liposomes loaded with 30 mol % 6‐ketocholestanol, a compound known to increase the magnitude of the membrane dipole potential. Studies using the fluorescent indicators fluoresceinphosphatidylethanolamine and 1‐(3‐sulfonatopropyl)‐4‐[β [2‐(di‐n‐octylamino)‐6‐naphthyl] vinyl] pyridinium betaine show that the interaction of bacitracin with model membranes is also enhanced by the presence of 6‐ketocholestanol in the bilayer and offers some indication to the mechanism of penetration enhancement. © 2003 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:1018–1027, 2003

Evaluation of percutaneous absorption and skin irritation of ketoprofen through rat skin: in vitro and in vivo study

The influences of different mechanisms of penetration enhancers (such as menthol, azone, ethanol and nonivarnide) regarding the percutaneous absorption and skin irritation of ketoprofen formulations through rat skin were investigated by in vitro and in vivo study. The skin irritation degree at the end of the experiment (10 h) was deterinined by pathologic biopsy and colorimetry methods. In vitro, the menthol showed the most potent enhancing effect. Furthermore, the enhancement effect of a combination of menthol and nonivamide was higher than that of their individual use alone. In vivo the formulation containing 0.05% nonivantide, 5% menthol and 20% ethanol showed a higher penetration rate and an acceptable degree of skin irritation compared to a commercial product (Formax plus® gel containing 3% ketoprofen), indicating that it could be used in the clinical situation.

Analysis of skin permeation-enhancing mechanism of iontophoresis using hydrodynamic pore theory

The effects of constant DC iontophoresis (0–1.5 mA/0.966 cm 2) on the permeation of three hydrophilic compounds, antipyrine (ANP, M.W. 188.23), sucrose (SR, M.W. 342.30) and 1-kestose (KT, M.W. 506.73), through excised hairless rat skin were evaluated using hydrodynamic pore theory. The electro-osmotic flow caused by iontophoresis was measured using deuterium oxide (D 2O). The penetration-enhancing mechanism of iontophoresis was found to increase solvent flow through electro-osmosis and pore enlargement and/or new pore production in the skin barrier, together with enhancement of electrochemical potential difference across the skin. These effects were closely related to the strength of the current applied. The electro-osmotic flow of D 2O ( J D 2O ) greatly enhanced the skin permeation clearance of all hydrophilic penetrants (CL drug). Pore production was classified into reversible and irreversible processes, which resulted from lower (0–0.5 mA/0.966 cm 2) and higher (0.5–1.5 mA/0.966 cm 2) currents, respectively. Thus, the enhancing effects of iontophoresis on skin permeation of nonionic hydrophilic compounds can be explained by increase in pore size and higher solvent flow.

The influence of lecithin and urea on the in vitro permeation of hydrocortisone acetate through skin from hairless mouse

Many workers have attempted to modify the permeability characteristics of skin. In the present work, the in vitro permeation of a model lipophilic drug (hydrocortisone acetate) was studied through hairless mouse skin. The drug was applied in poloxamer 407 gels containing known concentrations of lecithin or urea as penetration enhancers. The results of permeation studies showed that the flux and the retention of the drug were dependent on the concentration of the penetration enhancers. Lecithin (8.0%, w/v) caused a retention of seven times more than that of urea (12.0% w/v). The mechanism of penetration enhancement was investigated by differential scanning calorimetry (DSC) and FTIR spectroscopy of the stratum corneum (SC) from hairless mouse treated with solution of lecithin and urea. The lecithin was found to have a significant influence on the lipid matrix of the SC, suggesting a disruption of the intercellular lipid lamellar structure.

Effects of Four Penetration Enhancers on Corneal Permeability of Drugs in Vitro

The usefulness of penetration enhancers in promoting drug permeation across the cornea was investigated for drugs varying from hydrophilic to lipophilic. Four purported penetration enhancers [Azone (laurocapram), hexamethylenelauramide, hexamethyleneoctanamide, and decylmethylsulfoxide] were employed. Corneal permeability coefficients of drugs that were either hydrophilic (acetazolamide, cimetidine, guanethidine, and sulfacetamide), moderately lipophilic (bunolol and prednisolone), or lipophilic (flurbiprofen and its amide analogue) were measured in the absence or in the presence of various Azone concentrations. The effects of penetration enhancers on the corneal penetration of cimetidine were also compared. The corneal penetration of hydrophilic compounds was enhanced by at least 20-fold at 0.1% Azone. For prednisolone and bunolol, the maximal enhancement was at 0.025-0.1% Azone and was marginal (two- to 5-fold), whereas Azone inhibited rather than enhanced the corneal penetration of the lipophilic flurbiprofen and its amide analogue. All four enhancers behaved similarly in enhancing corneal penetration of cimetidine and corneal hydration after incubation in vitro. Possible mechanisms of penetration enhancers on corneal drug penetration were discussed. Penetration enhancers may have clinical benefits in improving ocular drug delivery of hydrophilic compounds, however, their utility may depend on the toxicological profiles.

The effect of polyethylene glycol molecular weight on corneal transport and the related influence of penetration enhancers

In order to understand the mechanism of olecular weight dependence on corneal transport of solutes, it is important to separate charge type and molecular size, two major parameters for peptide delivery. The transport characteristics of a series of different molecular weight polyethylene glycols (PEGs), which are hydrophilic and uncharged, were measured in vitro. The absorption of PEGs exhibited a dependence on the molecular weight with a cutoff between PEG 400 and PEG 600. The effect of penetration enhancers, including a bile salt, digitonin, and cytochalasin B, on the molecular weight permeability profile can be used to identify mechanisms of penetration enhancement. Bile salt did not alter the molecular weight permeability profile. In contrast, digitonin greatly increased absorption of the PEGs and selective molecular weight effects were seen with cytochalasin B. Correlation of mucosal integrity with the effectiveness of the penetration enhancer indicates that cytochalasin B acts on the tight junction by opening intercellular spaces. These results indicate that the paracellular pathway may play an important role in hydrophilic, large molecule transport through the cornea.

Mechanisms of penetration enhancement for transbuccal delivery of salicylic acid

The process of in vitro permeation across rabbit buccal mucosa was examined using salicylic acid as a model compound. The promoting action of penetration enhancers on salicylic acid flux was studied using differential scanning calorimetry, flux, electrophysiology, and microscopy techniques. The enhancers, sodium deoxycholate and sodium lauryl sulfate, decrease the electrical resistance and increase the permeability of salicylic acid across rabbit buccal mucosa. The results of these techniques appear to support the premise that the superficial layers and protein domain of the epithelium may be responsible for maintaining the barrier function of the buccal mucosa. This work provides additional insight in identification of the permeability barrier and the mechanism of enhancer activity.