Probing the particulate microstructure of the aerodynamic particle size distribution of dry powder inhaler combination products.

The in-vitro aerosol performance of two combination dry powder inhaler (DPI) products, Foster® NEXThaler® and Seretide® Diskus® were investigated with single particle aerosol mass spectrometry (SPAMS). The in-vitro pharmaceutical performance is markedly different for both inhalers. Foster® NEXThaler® generates a higher fine particle fraction (FPF <5 µm) and a much higher relative extra fine particle fraction (eFPF <2 µm). In terms of the composition of the aerodynamic particle size distribution (APSD), it could be verified with SPAMS that overall Foster® NEXThaler® emits a significantly higher number of fine and extra fine particles with a median aerodynamic diameter (MAD) of 2.1 µm while Seretide® Diskus® had a larger MAD of 3.1 µm. Additionally, the interactions between the two active pharmaceutical ingredients (APIs) in both products are different. While Seretide® Diskus® emits a significant (37%) number of co-associated API particles, only a negligible number of co-associated API particles were found in Foster® NEXThaler® (<1%). A major difference with Foster® NEXThaler® is that it contains magnesium stearate (MgSt) as a second excipient besides lactose in a so-called 'dual excipient' platform. The data generated using SPAMS suggested that nearly all of the beclomethasone dipropionate particles in Foster® NEXThaler® also contain MgSt and must therefore be co-associated with this additional excipient. This may help explain why beclomethasone dipropionate in Foster® NEXThaler® forms less particle co-associations with the second API, formoterol fumarate, shows a lower cohesive strength in respect to beclomethasone itself and why both APIs exhibit superior detachment from the carrier as evidenced by the increased eFPF and smaller MAD.

Membrane transport of nobilin conjugation products and use of the extract of Chamomillae romanae flos influence absorption of nobilin in the Caco-2 model.

The purpose of this work was to investigate the role of bioconjugation and carrier mediated efflux of conjugation products in the absorption mechanism of the sesquiterpene lactone nobilin in the Caco-2 model in vitro and to elucidate the impact of the extract of Chamomillae romanae flos and its ingredients on absorption. Transport experiments with inhibitors of P-gp, BCRP, and MRPs were performed to detect efflux and its connection to bioconversion and the effect of different ingredients of the plant extract on absorption processes was determined. Permeability, transport and bioconversion parameter values were deduced by kinetic multi-compartment modeling. Nobilin exhibited high permeability, low relative absorption and fast bioconversion producing glucuronide, cysteine conjugate, and glutathione conjugate that were transported by P-gp (the first two), apical MRP2 and basal MRP3 and possibly MRP1 out of the cell. Inhibition of efflux resulted in diminished bioconjugation and improved absorption. The extract increased the relative fraction absorbed primarily by directly inhibiting bioconversion, and by reducing efflux. Individual extract ingredients could only partly explain this effect. Extensive bioconversion, hence, limited absorption of nobilin in the Caco-2 model and the interplay between conjugation and efflux was shown to provide a possible mechanism for absorption increase. Plant extract increased absorption by this mechanism in addition to metabolic enzyme inhibition.

Biorelevant media for transport experiments in the Caco-2 model to evaluate drug absorption in the fasted and the fed state and their usefulness.

In this work we developed and characterized transport media that simulate the composition of micellar phase of intestinal fluids in the fasted and, especially, in the fed state and are appropriate for evaluating intestinal drug permeability characteristics using the Caco-2 model (FaSSIF-TM(Caco) and FeSSIF-TM(Caco), respectively). Media composition was based on FaSSIF-V2 and FeSSIF-V2 and recently reported data on total lipid concentrations in the micellar phase of contents of the upper small intestine in the fasted and the fed state and was adapted for cell culture compatibility. Permeation data were evaluated by compartmental kinetic modeling. Permeability coefficients, P, of hydrophilic drugs were not affected by media composition. In contrast, P values of a series of lipophilic compounds measured with FaSSIF-TM(Caco) and FeSSIF-TM(Caco), and reflecting transport by diffusion were smaller than those obtained with a purely aqueous reference transport medium, aq-TM(Caco), following the rank order aq-TM(Caco)>FaSSIF-TM(Caco)>FeSSIF-TM(Caco). The decline of permeability values was stronger as lipophilicity of the compounds increased. Compared with values estimated using aq-TM(Caco), permeability was reduced, depending on the compound, by more than 20- to 100-fold when measured with FeSSIF-TM(Caco) whereas compound ranking in regard to the permeability characteristics was also affected. The impact of reduced P value on flux through the mucosa, hence on drug absorption, in combination with the drug amount loaded on colloidal particles needs to be taken into consideration in PBPK modeling especially when the food effect is evaluated.

Topical bioavailability of triamcinolone acetonide: effect of occlusion.

BACKGROUND/AIMS: Occlusion by covering the skin with an impermeable wrap enhances skin hydration, affects drug absorption and can induce the formation of a drug reservoir within the stratum corneum. This is desired in local therapy with topical corticosteroids. The aim of the study was to investigate the effect of occlusion before (experiment 1) and after (experiment 2) application on the penetration of triamcinolone acetonide (TACA) into the stratum corneum. METHODS: The experiments were conducted on the forearms of 10 healthy volunteers. In experiment 1, 100 microg/cm(2) TACA in acetone were applied on 3 sites per arm, one arm having been pre-occluded for 16 h. In experiment 2, the same dose was applied on 2 sites per arm, and one arm was occluded after application until skin sampling. Stratum corneum samples were removed by tape stripping at 0.5, 4 and 24 h (experiment 1) and 4 and 24 h (experiment 2) after application. Corneocytes and TACA were quantified by ultraviolet-visible spectroscopy and HPLC, respectively. The total TACA amount penetrated into the stratum corneum was evaluated by multifactor ANOVA. RESULTS: TACA penetration into the stratum corneum with and without pre-occlusion (experiment 1) showed no significant difference and decreased with time. Occlusion after application (experiment 2) produced a marked TACA accumulation within the stratum corneum, which persisted for 24 h. CONCLUSION: Pre-occlusion showed no effect on the topical bioavailability of TACA in the stratum corneum. In contrast, post-occlusion enhanced the TACA penetration by a factor of 2, favouring the development of a drug reservoir.

Interaction of liposome formulations with human skin in vitro.

The interaction of liposome formulations consisting of Phospholipon 80 and sphingomyelin with human skin was investigated. These formulations were shown previously to have a composition-dependent effect on the penetration of Heparin into the skin. Fluorescence labelled phosphatidylethanolamine (PE-NBD) was incorporated in the liposomes and the depth in which the fluorescent phospholipid label enters into epidermal membrane and full thickness skin was studied by confocal laser scanning microscopy (CLSM). Confocal sections parallel to the surface of the skin were recorded in heat separated epidermis. An even distribution of phospholipid in the lipid matrix of the stratum corneum surrounding the corneocytes was observed with Phospholipon 80 but not when sphingomyelin was included in the formulation. The addition of Heparin which formed a coating around the liposomes, caused a strong localization of fluorescence within the epidermis. For full thickness skin, mechanical cross sections of skin were made and optical sections were recorded parallel to the plane of cut. Phospholipid penetrated and was distributed fairly homogeneously in the lower dermis layers within 30 min of application regardless of liposome composition and the presence of Heparin. This rather quick penetration process seemed to follow distinct pathways along the epidermis and the upper dermis, notably the hair follicle route. Thus, a strong and in some respects composition-dependent interaction of phospholipids with skin is evident. These observations, however, are limited to the level of phospholipid molecules, rather than of entire liposomes interacting with skin.

Heparin penetration into and permeation through human skin from aqueous and liposomal formulations in vitro.

The transport of unfractionated (UH) and low molecular weight Heparin (LMWH) in human skin was investigated in vitro using heat separated epidermal membrane and dermis and the effect of liposomal formulations with Phospholipon(R) 80 (PL80) and Sphingomyelin (SM) was assessed. The distribution of Heparin within skin tissue was studied by the tape stripping method. Heparin concentrations were measured with a biological assay. Transepidermal water loss was determined to characterize barrier properties of skin. No consistent permeation of Heparin through epidermal membrane was detected. Penetration into the epidermal membrane was for LMWH significantly greater than for UH. Accumulation of UH was largely restricted to the outermost layers of the stratum corneum while LMWH penetrated into deeper epidermal layers. UH penetration into epidermis was detected for the PL80 liposomal formulation only. The extent of LMWH penetration was independent of the formulation, LMWH, however, showed a trend to accumulate in deeper epidermal layers for the PL80 compared to the aqueous formulation. Thus, molecular weight and liposomal formulations influenced the penetration pattern of Heparin in the epidermis. It can not be concluded whether the concentration of LMWH achieved at the blood capillaries is sufficient to exert a pharmacological effect. UH permeated readily through dermis irrespectively of formulation and its accumulation in the dermis was significantly enhanced and its lag time of permeation increased in the presence of SM liposomes.

Effect of concentration and degree of saturation of topical fluocinonide formulations on in vitro membrane transport and in vivo availability on human skin.

PURPOSE: The thermodynamic activity of drugs in topical vehicles is considered to significantly influence topical delivery. In vitro diffusion across a synthetic membrane was shown to be correlated to the degree of saturation of the drug in the applied vehicle and therefore offers a potential for increased topical drug delivery. Fluocinonide a topical corticosteroid, was chosen as a model compound to investigate in vitro and in vivo availability from formulations with different degrees of saturation. METHODS: Sub-, as well as, supersaturated drug solutions were prepared using PVP as an antinucleant agent. In vitro membrane diffusion experiments across silicone membrane and in vivo pharmacodynamic activity assessments, using the human skin blanching assay, were carried out. RESULTS: Over the concentration range studied, the in vitro membrane transport of fluocinonide was proportional to the degree of saturation of the respective formulations. The in vivo pharmacodynamic response in the human skin blanching assay was related to the concentration of the drug in the vehicle irrespective of the degree of saturation. CONCLUSIONS: From the membrane permeation experiment it can be concluded, that the drug flux might be increased supra-proportionally with increasing donor concentration, drug (super-)saturation (proportional), beyond what would be anticipated based on ideal donor concentration and partition coefficient considerations only. These findings could not be confirmed in the in vivo investigation, probably due to additional vehicle effects (e.g., enhancement, irritation, drug binding) which have to be expected and could have altered the integrity of the stratum corneum and therewith topical bioavailability of the drug.

Utilizing vehicle imbibition by a microporous membrane and vehicle viscosity to control release rate of salbutamol.

The possibility to control the release rate of salbutamol through the hydrophobic Celgard 2500 polypropylene membrane by varying the composition and the viscosity of hydrophilic drug vehicles was investigated. The use of the polypropylene membrane as a control membrane for reservoir-type drug delivery systems was envisaged and water, glycerol, isopropyl alcohol and ethanol, pure or as binary mixtures were studied as vehicles. With varying composition of the vehicle, a sharp change of its imbibition by the membrane from practically none to a complete filling of the membrane pores occurred, which coincided with a steep rise of the drug permeability for the membrane. From this was inferred that the vehicle-filled pores were the dominant permeation pathway, while when no vehicle was imbibed, transport took place by way of the polymer domain of the membrane. In case of imbibition, the permeation rate could be modulated in a predictable fashion by adjusting the viscosity of the vehicle. This demonstrated that drug release could be controlled by utilizing the in situ interaction of the vehicles with this membrane, leading to imbibition and establishment of a permeation pathway with pre-determined viscosity in the pores of the membrane.

Effect of the amphoteric properties of salbutamol on its release rate through a polypropylene control membrane.

The permeation of salbutamol from aqueous vehicles with different pH values through the Celgard 2500 polypropylene membrane was studied, the goal being to assess the effect of the amphoteric properties of the drug on its release by the membrane. Permeation rates were generally low, which was related to the fact that purely aqueous vehicles were not imbibed into the pores of the membrane and therefore permeation took place through the amorphous polypropylene domains. Permeability coefficients were not proportional to the fraction of uncharged drug at different bulk pH values, indicating that either a pH gradient between the bulk and the membrane surface exists and/or charged drug species can permeate the hydrophobic membrane. Calculated hypothetical pH values of the membrane surface, assuming permeation of the uncharged drug only, failed to provide a consistent explanation of the experimental permeabilities. Permeability coefficients of the different ionization forms of the drug assuming no pH gradient were calculated from a system of linear equations, each one of them corresponding to a specific bulk pH. These were for the anionic and the cationic species one to two orders of magnitude smaller than for the combined uncharged and zwitterionic species. It is possible that both, a pH difference between bulk and membrane surface and permeation of ionized molecules were simultaneously responsible for the observed permeation rates.

Vehicle-dependent in situ modification of membrane-controlled drug release.

The possibility of utilizing the interaction between the drug vehicle and the polymeric control membrane of reservoir-type drug delivery systems to modulate the release rate of these systems was investigated. Lipid vehicles common in dermal formulations, with varying composition and viscosity and the Celgard polypropylene membrane were used to study the release of a model drug (salicylic acid). The release kinetics were investigated taking into account two consecutive transport processes, diffusion within the donor reservoir and permeation through the membrane. Membrane permeation was found to be the rate determining step for mass transport and convection appeared to play a significant role in the donor compartment, even though this compartment was not mechanically stirred. The lipid vehicles were imbibed by the membrane, quantitatively replacing the air from its pores. Drug permeation through the membrane was dominated by the vehicle-filled pore pathway, the pathway of the amorphous polymer domain of the membrane generally contributing a small fraction to the total permeation. Permeability coefficients for different vehicles varied within one order of magnitude. This effect was chiefly accounted for by the differences in viscosity of the vehicles occupying the pores of the membrane. Thus, based on the in situ imbibition of the lipid vehicles by the membrane, a controlled variation of the drug release rate could be achieved.

An improved diffusion cell design for determining drug transport parameters across cultured cell monolayers.

An improved two-chamber diffusion cell was developed for the study of drug transport across cultured cell monolayers. The cell monolayer was grown on a horizontal support membrane of polycarbonate, which could be rotated providing theoretically predictable thicknesses of the diffusion boundary layer in the donor and the receiver solution as a function of rotation rate. Permeation measurements were performed using the support membrane with and without a cell monolayer. The Madin-Darby-Bovine-Kidney (MDBK) cell line was employed, and permeability coefficients of model solutes (salicylic acid, mannitol, testosterone) across the three distinct mass transport barriers (i.e., the cell monolayer, the support membrane, and the diffusion boundary layer) were determined. The permeability of the diffusion boundary layer followed the theoretical dependence on the rotation rate; absolute values, however, deviated from predictions. Permeability coefficients for all three transport barriers varied substantially between solutes. This variation was the strongest for the permeability coefficient for the cell monolayer and resulted in a varying relative significance of these three barriers in controlling permeation kinetics. This improved diffusion apparatus permits the measurement of unbiased permeability values of solutes across the cell monolayer, notably when the cell monolayer is not absolutely the rate-determining barrier.

Estimation of skin target site acyclovir concentrations following controlled (trans)dermal drug delivery in topical and systemic treatment of cutaneous HSV-1 infections in hairless mice.

The use of controlled transdermal delivery of acyclovir (ACV) in the treatment of cutaneous herpes simplex virus type 1 infections in hairless mice was investigated. Using an in vivo animal model (A. Gonsho, et al. Int. J. Pharm. 65:183-194 (1990)) made it possible to quantify both, the topical and the systemic antiviral efficacy of ACV transdermal patches as a function of the drug delivery rate of the patches. Drug delivery rates required to attain systemic efficacy were found to be higher than the rates required to attain the same magnitude of topical efficacy. The ACV concentrations in the basal cell layer of the epidermis for 50% topical efficacy and 50% systemic efficacy were estimated. The basal epidermis layer was considered to be the site of antiviral drug activity (skin target site). Systemic plasma levels were obtained from pharmacokinetic studies and were used to estimate the ACV concentration achieved systemically in the basal epidermis layer. A computational model for drug permeation across skin was employed to estimate the ACV concentration achieved topically in the basal epidermis layer. Equal topical and systemic efficacies were found to correspond to equal drug concentrations at the site of antiviral activity. The length of the effective diffusion pathway of drug molecules in the dermis prior to entering the blood circulation was assumed to be approximately equal to 1/20 of the anatomical dermis thickness because of dermis vascularization.

Design of 9-beta-D-arabinofuranosyladenine (ara-A) transdermal delivery system for animal studies: regulation of drug concentration in vivo.

Transdermal delivery systems of 9-beta-D-arabinofuranosyladenine (ara-A), having controlling membranes of various permeabilities, were developed and applied to Azone-pretreated hairless mouse abdominal skin. It was confirmed that the blood concentrations of ara-A and its metabolite 9-beta-D-arabinofuranosylhypoxanthine (ara-H) in hairless mice are controlled by the permeability of the controlling membrane in the transdermal patch. Furthermore, these blood concentrations were found to closely agree with the values obtained from theoretical model calculations. Finally, but importantly, the "micropharmacokinetic" behavior of ara-A in cutaneous tissue could also be predicted. These results suggest that the transdermal patch may be employed in dermal and transdermal ara-A efficacy studies in the treatment of cutaneous herpes virus infections in hairless mice.