A Model for the Transient Subdiffusive Behavior of Particles in Mucus.

In this study we have applied a model to explain the reported subdiffusion of particles in mucus, based on the measured mean squared displacements (MSD). The model considers Brownian diffusion of particles in a confined geometry, made from permeable membranes. The applied model predicts a normal diffusive behavior at very short and long time lags, as observed in several experiments. In between these timescales, we find that the "subdiffusive" regime is only a transient effect, MSD∝τα,α<1. The only parameters in the model are the diffusion-coefficients at the limits of very short and long times, and the distance between the permeable membranes L. Our numerical results are in agreement with published experimental data for realistic assumptions of these parameters. Finally, we show that only particles with a diameter less than 40 nm are able to pass through a mucus layer by passive Brownian motion.

Size-Limited Penetration of Nanoparticles into Porcine Respiratory Mucus after Aerosol Deposition.

We investigated the rheological properties and the penetration of differently sized carboxylated nanoparticles in pig pulmonary mucus, on different distance and time scales. Nanoparticles were either mechanically mixed into the mucus samples or deposited as an aerosol, the latter resembling a more physiologically relevant delivery scenario. After mechanical dispersion, 500 nm particles were locally trapped; a fraction of carboxylated tracer particles of 100 or 200 nm in diameter could however freely diffuse in these networks over distances of approximately 20 µm. In contrast, after aerosol deposition on top of the mucus layer only particles with a size of 100 nm were able to penetrate into mucus, suggesting the presence of smaller pores at the air-mucus interface compared to within mucus. These findings are relevant to an understanding of the fate of potentially harmful aerosol particles, such as pathogens, pollutants, and other nanomaterials after incidental inhalation, as well as for the design of pulmonary drug delivery systems.

Optical tweezers reveal relationship between microstructure and nanoparticle penetration of pulmonary mucus.

In this study, the mobility of nanoparticles in mucus and similar hydrogels as model systems was assessed to elucidate the link between microscopic diffusion behavior and macroscopic penetration of such gels. Differences in particle adhesion to mucus components were strongly dependent on particle coating. Particles coated with 2 kDa PEG exhibited a decreased adhesion to mucus components, whereas chitosan strongly increased the adhesion. Despite such mucoinert properties of PEG, magnetic nanoparticles of both coatings did not penetrate through native respiratory mucus, resisting high magnetic forces (even for several hours). However, model hydrogels were, indeed, penetrated by both particles in dependency of particle coating, obeying the theory of particle mobility in an external force field. Comparison of penetration data with cryogenic scanning EM images of mucus and the applied model systems suggested particularly high rigidity of the mucin scaffold and a broad pore size distribution in mucus as reasons for the observed particle immobilization. Active probing of the rigidity of mucus and model gels with optical tweezers was used in this context to confirm such properties of mucus on the microscale, thus presenting the missing link between micro- and macroscopical observations. Because of high heterogeneity in the size of the voids and pores in mucus, on small scales, particle mobility will depend on adhesive or inert properties. However, particle translocation over distances larger than a few micrometers is restricted by highly rigid structures within the mucus mesh.

A Novel 3D Printed Model of Infected Human Hair Follicles to Demonstrate Targeted Delivery of Nanoantibiotics.

Hair follicle-penetrating nanoparticles offer a promising avenue for targeted antibiotic delivery, especially in challenging infections like acne inversa or folliculitis decalvans. However, demonstrating their efficacy with existing preclinical models remains difficult. This study presents an innovative approach using a 3D in vitro organ culture system with human hair follicles to investigate the hypothesis that antibiotic nanocarriers may reach bacteria within the follicular cleft more effectively than free drugs. Living human hair follicles were transplanted into a collagen matrix within a 3D printed polymer scaffold to replicate the follicle's microenvironment. Hair growth kinetics over 7 days resembled those of simple floating cultures. In the 3D model, fluorescent nanoparticles exhibited some penetration into the follicle, not observed in floating cultures. Staphylococcus aureus bacteria displayed similar distribution profiles postinfection of follicles. While rifampicin-loaded lipid nanocapsules were as effective as free rifampicin in floating cultures, only nanoencapsulated rifampicin achieved the same reduction of CFU/mL in the 3D model. This underscores the hair follicle microenvironment's critical role in limiting conventional antibiotic treatment efficacy. By mimicking this microenvironment, the 3D model demonstrates the advantage of topically administered nanocarriers for targeted antibiotic therapy against follicular infections.

Freeze-drying as a preserving preparation technique for in vitro testing of human skin.

In vitro testing of drugs with excised human skin is a valuable prerequisite for clinical studies. However, the analysis of excised human skin presents several obstacles. Ongoing drug diffusion, microbial growth and changes in hydration state influence the results of drug penetration studies. In this work, we evaluate freeze-drying as a preserving preparation method for skin samples to overcome these obstacles. We analyse excised human skin before and after freeze-drying and compare these results with human skin in vivo. Based on comprehensive thermal and spectroscopic analysis, we demonstrate comparability to in vivo conditions and exclude significant changes within the skin samples due to freeze-drying. Furthermore, we show that freeze-drying after skin incubation with drugs prevents growth of drug crystals on the skin surface due to drying effects. In conclusion, we introduce freeze-drying as a preserving preparation technique for in vitro testing of human skin.

3D bioprinting ofE. coliMG1655 biofilms on human lung epithelial cells for building complexin vitroinfection models.

Biofilm-associated infections are causing over half a million deaths each year, raising the requirement for innovative therapeutic approaches. For developing novel therapeutics against bacterial biofilm infections, complexin vitromodels that allow to study drug effects on both pathogens and host cells as well as their interaction under controlled, physiologically relevant conditions appear as highly desirable. Nonetheless, building such models is quite challenging because (1) rapid bacterial growth and release of virulence factors may lead to premature host cell death and (2) maintaining the biofilm status under suitable co-culture requires a highly controlled environment. To approach that problem, we chose 3D bioprinting. However, printing living bacterial biofilms in defined shapes on human cell models, requires bioinks with very specific properties. Hence, this work aims to develop a 3D bioprinting biofilm method to build robustin vitroinfection models. Based on rheology, printability and bacterial growth, a bioink containing 3% gelatin and 1% alginate in Luria-Bertani-medium was found optimal forEscherichia coliMG1655 biofilms. Biofilm properties were maintained after printing, as shown visually via microscopy techniques as well as in antibiotic susceptibility assays. Metabolic profile analysis of bioprinted biofilms showed high similarity to native biofilms. After printing on human bronchial epithelial cells (Calu-3), the shape of printed biofilms was maintained even after dissolution of non-crosslinked bioink, while no cytotoxicity was observed over 24 h. Therefore, the approach presented here may provide a platform for building complexin vitroinfection models comprising bacterial biofilms and human host cells.

Influence of the application area on finite dose permeation in relation to drug type applied.

For finite dose skin absorption experiments, a homogeneous donor distribution over the skin surface is usually assumed. However, the influence of the surface distribution on skin absorption is still unknown. The aim of this study was to evaluate the influence of the application area on the permeation of drugs during finite dose skin absorption experiments in static Franz diffusion cells. Permeation experiments with stained aqueous drug formulations were conducted, and the application area was determined by a suitable, objective, automated computational approach. The permeation of caffeine is strongly dependent on the application area. The variability between single experiments decreased when including the application area. For the lipophilic flufenamic acid, this was not the case. The variability highly increased after inclusion of the application area. Thus, a correction of the area is misleading. In summary, depending on the drug's physicochemical characteristics, the application area may influence skin absorption.

Uptake of nanoparticles by alveolar macrophages is triggered by surfactant protein A.

Understanding the bio-nano interactions in the lungs upon the inhalation of nanoparticles is a major challenge in both pulmonary nanomedicine and nanotoxicology. To investigate the effect of pulmonary surfactant protein A (SP-A) on the interaction between nanoparticles and alveolar macrophages, we used magnetite nanoparticles (110-180 nm in diameter) coated with different polymers (starch, carboxymethyldextran, chitosan, poly-maleic-oleic acid, phosphatidylcholine). Cellular binding and uptake of nanoparticles by alveolar macrophages was increased for nanoparticles treated with SP-A, whereas albumin, the prevailing protein in plasma, led to a significant decrease. A significantly different adsorption pattern of SP-A, compared to albumin was found for these five different nanomaterials. This study provides evidence that after inhalation of nanoparticles, a different protein coating and thus different biological behavior may result compared to direct administration to the bloodstream. FROM THE CLINICAL EDITOR: In this nano-toxicology study of inhaled nanoparticles, the authors investigated the effect of pulmonary surfactant protein A on the interaction between nanoparticles and alveolar macrophages utilizing magnetite nanoparticles coated with different polymers (starch, carboxymethyldextran, chitosan, poly-maleic-oleic acid, phosphatidylcholine). Cellular binding and uptake of nanoparticles increased for nanoparticles treated with SP-A, whereas albumin, the prevailing protein in plasma, led to a significant decrease.

Human Skin Permeation Enhancement Using PLGA Nanoparticles Is Mediated by Local pH Changes.

The steady improvement and optimization of transdermal permeation is a constant and challenging pharmaceutical task. In this study the influence of poly(lactide-co-glycolide) (PLGA) nanoparticles on the dermal permeation of the anti-inflammatory drug flufenamic acid (FFA) was investigated. For this aim, different vehicles under non-buffered and buffered conditions and different skin models (human heat separated epidermis and reconstructed human epidermis equivalents) were tested. Permeation experiments were performed using static Franz diffusion cells under infinite dosing conditions. Already the presence of drug-free nanoparticles increased drug permeation across the skin. Drug permeation was even enhanced when applying drug-loaded nanoparticles. In contrast, buffered vehicles with different pH values (pH 5.4-7.4) revealed the influence of the pH on the permeation of FFA. The change of the surrounding pH of the biodegradable nanoparticulate system was demonstrated and visualized using pH-sensitive fluorescent probes. While a potential contribution of hair follicles could be ruled out, our data suggest that the enhanced permeation of FFA through human skin in the presence of PLGA nanoparticles is mediated by a locally decreased pH during hydrolytic degradation of this polymer. This hypothesis is supported by the observation that skin permeation of the weak base caffeine was not affected.

Vitamin E TPGS P-glycoprotein inhibition mechanism: influence on conformational flexibility, intracellular ATP levels, and role of time and site of access.

Previous work conducted in our laboratories established the notion that TPGS 1000 (d-alpha-tocopheryl polyethylene glycol 1000 succinate), a nonionic surfactant, modulates P-glycoprotein (P-gp) efflux transport via P-gp ATPase inhibition. The current in vitro research using Caco-2 cells was conducted to further explore the P-gp ATPase inhibition mechanism. Using a monoclonal CD243 P-gp antibody shift assay (UIC2), we probed P-gp conformational changes induced via TPGS 1000. In the presence of TPGS 1000, UIC2 binding was slightly decreased. TPGS 1000 does not appear to be a P-gp substrate, nor does it function as a competitive inhibitor in P-gp substrate efflux transport. The reduction in UIC2 binding with TPGS 1000 was markedly weaker than with orthovanadate, data ruling out trapping P-gp in a transition state by direct interaction with one or both of the P-gp ATP nucleotide binding domains. An intracellular ATP depletion mechanism could be ruled out in the UIC2 assay, and by monitoring intracellular ATP levels in the presence of TPGS 1000. Indicating slow distribution of TPGS 1000 into the membrane, and in agreement with an intramembranal or intracellular side of action, Caco-2 cell monolayer experiments preincubated with TPGS 1000 produce stronger substrate inhibitory activity than those conducted by direct substrate and surfactant coapplication.

The Pharmaceutical Aerosol Deposition Device on Cell Cultures (PADDOCC) in vitro system: design and experimental protocol.

The development of aerosol medicines typically involves numerous tests on animals, due to the lack of adequate in vitro models. A new in vitro method for testing pharmaceutical aerosol formulations on cell cultures was developed, consisting of an aerosolisation unit fitting a commercial dry powder inhaler (HandiHaler(c), Boehringer Ingelheim, Germany), an air-flow control unit (Akita(c), Activaero, Germany) and a custom-made sedimentation chamber. This chamber holds three Snapwell(c) inserts with monolayers of pulmonary epithelial cells. The whole set-up, referred to as the Pharmaceutical Aerosol Deposition Device On Cell Cultures (PADDOCC) system, aims to mimic the complete process of aerosol drug delivery, encompassing aerosol generation, aerosol deposition onto pulmonary epithelial cells and subsequent drug transport across this biological barrier, to facilitate the investigation of new aerosol formulations in the early stages of development. We describe here, the development of the design and the protocol for this device. By testing aerosol formulations of budesonide and salbutamol sulphate, respectively, reproducible deposition of aerosol particles on, and the integrity of, the pulmonary cell monolayer could be demonstrated.

Synthesis and Biopharmaceutical Characterization of Amphiphilic Squalenyl Derivative Based Versatile Drug Delivery Platform.

Limited drug loading capacity (LC), mostly below 5% w/w, is a significant drawback of nanoparticulate drug delivery systems (DDS). Squalenoylation technology, which employs bioconjugation of squalenyl moiety and drug, allows self-assemble of nanoparticles (NPs) in aqueous media with significantly high LC (>30% w/w). The synthesis and particle preparation of squalenoylated prodrugs are, however, not facile for molecules with multiple reactive groups. Taking a different approach, we describe the synthesis of amphiphilic squalenyl derivatives (SqDs) as well as the physicochemical and biopharmaceutical characterizations of their self-assembled NPs as DDSs. The SqDs included in this study are (i) cationic squalenyl diethanolamine (ii) PEGylated SqD (PEG 750 Da), (iii) PEGylated SqD (PEG 3,000 Da), and (iv) anionic squalenyl hydrogen sulfate. All four SqDs self-assemble into NPs in a size range from 100 to 200 nm in an aqueous solution. Furthermore, all NP derivatives demonstrate appropriate biocompatibility and adequate colloidal stability in physiological relevant pH environments. The mucoprotein binding of PEGylated NPs is reduced compared to the charged NPs. Most importantly, this technology allows excellent LC (at maximum of 45% w/w) of a wide range of multifunctional compounds, varying in physicochemical properties and molecular weight. Interestingly, the drug release profile can be tuned by different loading methods. In summary, the SqD-based NPs appear as versatile drug delivery platforms.

Tofacitinib Loaded Squalenyl Nanoparticles for Targeted Follicular Delivery in Inflammatory Skin Diseases.

Tofacitinib (TFB), a Janus kinase inhibitor, has shown excellent success off-label in treating various dermatological diseases, especially alopecia areata (AA). However, TFB's safe and targeted delivery into hair follicles (HFs) is highly desirable due to its systemic adverse effects. Nanoparticles (NPs) can enhance targeted follicular drug delivery and minimize interfollicular permeation and thereby reduce systemic drug exposure. In this study, we report a facile method to assemble the stable and uniform 240 nm TFB loaded squalenyl derivative (SqD) nanoparticles (TFB SqD NPs) in aqueous solution, which allowed an excellent loading capacity (LC) of 20%. The SqD NPs showed an enhanced TFB delivery into HFs compared to the aqueous formulations of plain drug in an ex vivo pig ear model. Furthermore, the therapeutic efficacy of the TFB SqD NPs was studied in a mouse model of allergic dermatitis by ear swelling reduction and compared to TFB dissolved in a non-aqueous mixture of acetone and DMSO (7:1 v/v). Whereas such formulation would not be acceptable for use in the clinic, the TFB SqD NPs dispersed in water illustrated a better reduction in inflammatory effects than plain TFB's aqueous formulation, implying both encouraging good in vivo efficacy and safety. These findings support the potential of TFB SqD NPs for developing a long-term topical therapy of AA.

The role of corneocytes in skin transport revised--a combined computational and experimental approach.

PURPOSE: To investigate mechanisms of compound-corneocyte interactions in a combined experimental and theoretical approach. MATERIALS AND METHODS: Experimental methods are presented to investigate compound-corneocyte interactions in terms of dissolution within water of hydration and protein binding and to quantify the extent of the concurrent mechanisms. Results are presented for three compounds: caffeine, flufenamic acid, and testosterone. Two compartmental stratum corneum models M1 and M2 are formulated based on experimentally determined input parameters describing the affinity to lipid, proteins and water. M1 features a homogeneous protein compartment and considers protein interactions only via intra-corneocyte water. In M2 the protein compartment is sub-divided into a cornified envelope compartment interacting with inter-cellular lipids and a keratin compartment interacting with water. RESULTS: For the non-protein binding caffeine the impact of the aqueous compartment on stratum corneum partitioning is overestimated but is successfully modeled after introducing a bound water fraction that is non-accessible for compound dissolution. For lipophilic, keratin binding compounds (flufenamic acid, testosterone) only M2 correctly predicts a concentration dependence of stratum corneum partition coefficients. CONCLUSIONS: Lipophilic and hydrophilic compounds interact with corneocytes. Interactions of lipophilic compounds are probably confined to the corneocyte surface. Interactions with intracellular keratin may be limited by their low aqueous solubility.

Cooperative interaction of monoterpenes and phenylpropanoids on the in vitro human skin permeation of complex composed essential oils.

For essential oils, several biological effects such as antibacterial, anti-inflammatory, expectorant, and blood-circulation-enhancing properties have been described. The method of application depends on the pathophysiology, the desired outcome, safety, and toxicity data. For treating respiratory symptoms and nervous disorders, inhalation may be the best means of application, whereas topical application is the best way for treating skin diseases. For dermal application, percutaneous absorption of essential oil and oil components is of great interest. Essential oils are complex mixtures of different volatile substances. So, the question is raised whether all components of a complex composed essential oil are equivalent with respect to their human skin permeation. By means of artificial mixtures of different essential oil ingredients, we investigated the cooperative effect of monoterpenes and phenylpropanoids on the permeation through heat-separated human skin epidermis in static Franz-Diffusion Cells. Limonene showed an enhancing effect on the permeation of citronellol and eugenol. Both alpha-pinene and myrcene increased the apparent permeability ( P(app)) value of phenylethanol. beta-Pinene had an enhancing effect on the permeation behaviour of methyleugenol but not of geraniol. The investigations clearly show that cooperative effects of single essential oil components may influence percutaneous essential oil absorption.

In-silico model of skin penetration based on experimentally determined input parameters. Part II: mathematical modelling of in-vitro diffusion experiments. Identification of critical input parameters.

This work describes a framework for in-silico modelling of in-vitro diffusion experiments illustrated in an accompanying paper [S. Hansen, A. Henning, A. Naegel, M. Heisig, G. Wittum, D. Neumann, K.-H. Kostka, J. Zbytovska, C.M. Lehr, U.F. Schaefer, In-silico model of skin penetration based on experimentally determined input parameters. Part I: experimental determination of partition and diffusion coefficients, Eur. J. Pharm. Biopharm. 68 (2008) 352-367 [corrected] A mathematical model of drug permeation through stratum corneum (SC) and viable epidermis/dermis is presented. The underlying geometry for the SC is of brick-and-mortar character, meaning that the corneocytes are completely embedded in the lipid phase. The geometry is extended by an additional compartment for the deeper skin layers (DSL). All phases are modelled with homogeneous diffusivity. Lipid-donor and SC-DSL partition coefficients are determined experimentally, while corneocyte-lipid and DSL-lipid partition coefficients are derived consistently with the model. Together with experimentally determined apparent lipid- and DSL-diffusion coefficients, these data serve as direct input for computational modelling of drug transport through the skin. The apparent corneocyte diffusivity is estimated based on an approximation, which uses the apparent SC- and lipid-diffusion coefficients as well as corneocyte-lipid partition coefficients. The quality of the model is evaluated by a comparison of concentration-SC-depth-profiles of the experiment with those of the simulation. Good agreements are obtained, and by an analysis of the underlying model, critical parameters of the models can be identified more easily.

In-silico model of skin penetration based on experimentally determined input parameters. Part I: experimental determination of partition and diffusion coefficients.

Mathematical modeling of skin transport is considered a valuable alternative of in-vitro and in-vivo investigations especially considering ethical and economical questions. Mechanistic diffusion models describe skin transport by solving Fick's 2nd law of diffusion in time and space; however models relying entirely on a consistent experimental data set are missing. For a two-dimensional model membrane consisting of a biphasic stratum corneum (SC) and a homogeneous epidermal/dermal compartment (DSL) methods are presented to determine all relevant input parameters. The data were generated for flufenamic acid (M(W) 281.24g/mol; logK(Oct/H2O) 4.8; pK(a) 3.9) and caffeine (M(W) 194.2g/mol; logK(Oct/H2O) -0.083; pK(a) 1.39) using female abdominal skin. K(lip/don) (lipid-donor partition coefficient) was determined in equilibration experiments with human SC lipids. K(cor/lip) (corneocyte-lipid) and K(DSL/lip) (DSL-lipid) were derived from easily available experimental data, i.e. K(SC/don) (SC-donor), K(lip/don) and K(SC/DSL) (SC-DSL) considering realistic volume fractions of the lipid and corneocyte phases. Lipid and DSL diffusion coefficients D(lip) and D(DSL) were calculated based on steady state flux. The corneocyte diffusion coefficient D(cor) is not accessible experimentally and needs to be estimated by simulation. Based on these results time-dependent stratum corneum concentration-depth profiles were simulated and compared to experimental profiles in an accompanying study.

A comparative evaluation of corneal epithelial cell cultures for assessing ocular permeability.

The purpose of this study was to evaluate the potential value of different epithelial cell culture systems as in vitro models for studying corneal permeability. Transformed human corneal epithelial (HCE-T) cells and Statens Serum Institut rabbit corneal (SIRC) cells were cultured on permeable filters. SkinEthic human corneal epithelium (S-HCE) and Clonetics human corneal epithelium (C-HCE) were received as ready-to-use systems. Excised rabbit corneas (ERCs) and human corneas (EHCs) were mounted in Ussing chambers, and used as references. Barrier properties were assessed by measuring transepithelial electrical resistance, and by determining the apparent permeability of markers with different physico-chemical properties, namely, fluorescein, sodium salt; propranolol hydrochloride; moxaverine hydrochloride; timolol hydrogenmaleate; and rhodamine 123. SIRC cells and the S-HCE failed to develop epithelial barrier properties, and hence were unable to distinguish between the permeation markers. Barrier function and the power to differentiate compound permeabilities were evident with HCE-T cells, and were even more pronounced in the case of C-HCE, corresponding very well with data from ERCs and EHCs. A net secretion of rhodamine 123 was not observed with any of the models, suggesting that P-glycoprotein or similar efflux systems have no significant effects on corneal permeability. Currently available corneal epithelial cell culture systems show differences in epithelial barrier function. Systems lacking functional cell-cell contacts are of limited value for assessing corneal permeability, and should be critically evaluated for other purposes.

Delivery of ethinylestradiol from film forming polymeric solutions across human epidermis in vitro and in vivo in pigs.

Film forming polymeric solutions may present an alternative to the common transdermal dosage forms such as patches or gels. To evaluate the potential of these systems for transdermal drug delivery the permeation of ethinylestradiol from four formulations with different polymers was tested across heat separated human epidermis. The formulation with the best results was then modified by incorporating chemical enhancers to further increase the efficiency of the delivery system. Finally, drug delivery from the developed film forming systems was compared to a commercially available transdermal patch in vitro as well as in vivo in pigs. Among the tested preparations the formulation with polyurethane-14-AMP-acrylates copolymer (DynamX) showed the highest ethinylestradiol permeation. The drug transport was further increased with the incorporation of oleic acid as penetration enhancer, especially when used in combination with propylene glycol. The enhancing effect of oleic acid/propylene glycol was concentration-dependent and increased disproportionately with rising enhancer content. The film forming solution showed a higher ethinylestradiol permeation through heat separated human epidermis than the commercial EVRA patch in vitro and achieved measurable plasma concentrations of ethinylestradiol in vivo in pigs. These promising results encourage the further development of film forming polymeric solutions as novel transdermal dosage form.

Development and characterization of film forming polymeric solutions for skin drug delivery.

Film forming polymeric solutions as a novel approach for skin drug delivery were developed and characterized concerning their mechanical properties and water vapor permeability. They were developed by varying type and content of the film forming polymer as well as nature and content of the plasticizer. The resulting formulations were evaluated according to five criteria: drying time, cosmetic attractiveness, outward stickiness, integrity on skin (after 18 h) and viscosity. Among the 14 tested polymers 10 film formers yielded formulations with a positive evaluation in all five test criteria. Selected formulations were then investigated for tensile strength and elongation at break in vitro and for water vapor permeability in vitro (WVP) and in vivo (TEWL). Their mechanical properties determined in vitro were found to be not predictive for the flexibility and abrasion resistance observed on living skin. Similar to this, the results derived from the WVP and the TEWL methods were not in accordance with each other. Obviously, the investigated in vitro methods do not characterize the properties of the thin films on living skin satisfactorily. Nevertheless, the identified film forming solutions are a promising approach and will provide the basis for the further development of this novel dosage form.