Nanostructures in n-Octanol Equilibrated with Additives and/or Water.

Fluid fatty alcohols are believed to be nanostructured but broadly amorphous (i.e., noncrystalline) fluids and solvents, including the most popular fatty tissue mimetic, hydrated n-octanol (i.e., hydro-octanol). To check this premise, we studied dry octanol and hydro-octanol as a model of relatively short fluid n-alkanols with small-angle X-ray scattering (SAXS). We also combined this alkanol with the matching alkane (i.e., octane) and with a common anti-inflammatory pain killer (ketoprofen). This revealed that (hydro-)octanol and arguably any other short fatty alcohol form a mesophase. Its basic structural motif are regularly packed polar nanoclusters, reflected in the inner peak in the SAXS diffractogram of (hydro-)octanol and other fluid n-alkanols. The nanoclusters arguably resemble tiny, (inverse) hydrated bilayer fragments, located on a thermally smeared para-crystalline lattice. Additives to hydro-octanol can change the nanoclusters only moderately, if at all. For example, octane and the drug ketoprofen added to hydro-octanol enlarge the nanoclusters only little because of the mixture's packing frustration. To associate with and to bring more water into hydro-octanol, an additive must hence transform the nanoclusters: it expands them into irregularly distributed aqueous lacunae that form a proto-microemulsion, reflected in the previously unknown Guinier's SAXS signal. A "weak" (i.e., a weakly polar or nonpolar) additive can moreover create only size-limited lacunae. Coexistence of nanoclusters and lacunae as well as size variability of the latter in hydro-octanol subvert the concept of octanol-water partition coefficient, which relies on the studied compartment homogeneity. In turn, it opens new possibilities for interfacial catalysis. Reinterpreting "octanol-water partition coefficient" data in terms of octanol-water association or binding constant(s) could furthermore diminish the variability of molecular lipophilicity description and pave the ground toward a more precise theoretical quantification and prediction of molecular properties.

The effect of membrane softeners on rigidity of lipid vesicle bilayers: Derivation from vesicle size changes.

Deformability is not just a fundamentally interesting vesicle characteristic; it is also the key determinant of vesicle ability to cross the skin barrier; i.e. skin penetrability. Development of bilayer vesicles for drug and vaccine delivery across the skin should hence involve optimization of this property, which is controllable by the concentration of bilayer softeners in or near the vesicle bilayers. To this end, we propose a simple method for quantifying the effect of bilayer softeners on deformability of bilayer vesicles. The method derives the bending rigidity of vesicle bilayers from vesicle size dependence on softener concentration. To exemplify the method, we studied mixtures of soybean phosphatidylcholine with anionic sodium deoxycholate, non-ionic polyoxyethylene (20) sorbitan oleyl ester (polysorbate 80), or non-ionic polyoxyethylene (20) oleyl ether (C18:1EO20, Brij® 98). With each of the tested bilayer softeners, the bending rigidity of the resulting mixed-amphipat vesicle bilayers decreased quasi-exponentially as the concentration of the bilayer softener increased, as one would expect on theoretical ground. The bilayer bending rigidity reached low values, near the thermal stability limit, i.e. kBT, before vesicle transformation into non-vesicular aggregates began. For a soybean phosphatidylcholine concentration of 5.0mmolkg-1, the bilayer bending rigidity reached 1.5kBT at the total deoxycholate concentration of 4.1mmolkg-1 and 3.4kBT at the total polysorbate 80 concentration of 2.0mmolkg-1. In the case of C18:1EO20, the bilayer bending rigidity reached 1.5kBT at the bilayer surface occupancy α=0.1. The dependence of vesicle size on bilayer softener concentration thus reveals vesicle transformation into different aggregate structures (such as mixed micelles with poor skin penetrability) and practically valuable information on vesicle deformability. Our results compare favorably with results of literature measurements. We provide practical guidance on using the new analytical method to optimize deformable vesicle formulations.

Differential diagnosis and proper treatment of acute rhinosinusitis: Guidance based on historical data analysis.

BACKGROUND: The time course of rhinovirus positive and negative rhinosinusitis has not been quantified yet, which aggravates proper selection and justification of the optimum treatment for this illness. Such quantitative information would facilitate an early and proper identification of the disease and its differentiation from acute bacterial rhinosinusitis, and could diminish harmful overuse of antibiotics, arguably driven by patients' want for attention and the treating physicians' inability to offer an adequate verbal comfort in its stead. OBJECTIVE: Extraction of the quantitative information needed to identify rhinovirus positive or negative rhinosinusitis and to allow selection of the most appropriate treatment from the published time dependence of individual clinical symptoms of the disease. METHODS: Scrutiny (and modeling) of temporal evolution of all noteworthy symptoms of rhinosinusitis with a simple mathematical expression that relies on two adjustable parameters per symptom (and potentially a general time offset as an extra adjustable parameter). RESULTS: Adverse effects of rhinosinusitis can be grouped according to the sequence of their exponential appearance and ∼2.6 times slower exponential disappearance, rhinovirus negative rhinosinusitis generally improving ∼25% faster and being ∼40% less severe. The major early local symptoms (throat soreness and scratchiness, headache) vanish with a half-life of ∼1.8 days, whereas further local symptoms take ∼1.6 times longer to disappear. At least 50-60% improvement of two prominent early symptoms, sore throat and sneezing (but not of nasal discharge, cough, and hoarseness) by day 5 of the disease implies a nonbacterial origin of rhinitis and should exclude use of antibiotics. CONCLUSION: Temporal evolution of all rhinosinusitis symptoms is qualitatively similar, which makes the early symptom decay a good proxy for, and predictor of, the disease perspective. Knowing a symptom intensity at just three to four time points suffices for reconstructing its entire time course and total intensity or gravity. This permits an easy and early identification of rhinosinusitis, and its plausible differentiation from acute bacterial rhinosinusitis, disease treatment optimization, and corresponding clinical trials simplification and/or shortening.

Improved protection against tuberculosis after boosting the BCG-primed mice with subunit Ag 85a delivered through intact skin with deformable vesicles.

To improve vaccination against tuberculosis (TBC) with Bacillus Calmette-Guerin (BCG), we introduce novel, non-invasive, secondary immunisations relying on epicutaneous (e.c.) applications of the TBC subunit antigen, Ag 85a, associated with deformable carrier vesicles. Immuno-boosting with such antigen-vesicles recruits more CD11c positive cells into the draining murine lymph nodes, and typically stimulates, especially the proximal, immune cells more than immunogen injections. Non-invasive antigen application also protects mice better against an infection with TBC. Subcutaneous injections of vesicular Ag 85a into BCG-primed mice mainly yield IgG1 and IgG2a, indicative of a mixed Th1 and Th2 response. Conversely, transcutaneous immuno-boosts of such mice with a deformable vesicle-Ag 85a combination mainly generate serum IgA and IgG2a, indicative of an IgA facilitated, Th1-mediated, immune response. The Ag 85a specific antibody titres are generally low, but T lymphocytes also proliferate in the immunised mice. The new, partially non-invasive, vaccination method lowers the burden of pulmonary infection with M. tuberculosis. In mice immunised with Ag85a associated with deformable vesicles we measured 116× (e.c.) to 51× (s.c.) lower colony forming units number in spleen and 9× (e.c.) to 3× (s.c.) lower such number in lungs.

Improved bioassays using a local effect, such as muscle paralysis, as an endpoint.

Drug potency testing consumes many animals, botulinum neurotoxin (BoNT) testing being perhaps the most notorious example. To avoid 50% lethal dose determination, the so-called digital abduction score (DAS) and other BoNT induced local paralysis assays were developed. This paper reveals that a simple mathematical expression - the Bateman's equation used in many pharmacokinetic data analyses - can describe in full detail the time dependence of the BoNT induced local paralysis; the equation hence allows robust interpolation and extrapolation, as well as integral effect (AUC), and its dose dependence, evaluation. The equation is moreover a convenient tool for experimental planning and for extracting, from experimental data, the parameters that characterise BoNT potency. Most important, one can generally reduce the number of animals needed to gain reliable results at least 20-33% (and possibly 50% or even 75%) by analysing and modelling the time course of a local effect (such as muscle paralysis) with the equation, rather than just by averaging the maximum observed effect size at one point in time.

Partition coefficient vs. binding constant: How best to assess molecular lipophilicity.

Partition coefficient, P, is the preferred descriptor of molecular lipo- or hydrophilicity, and thus of relationships between a solute (S, e.g., a drug), a polar medium (W, e.g., an aqueous buffer), and an essentially apolar, organic, medium or a drug carrier (O). The coefficient is commonly identified with the linear ratio of solute quantities in the two media, P=nSO/nSW, even to characterise solute association with or binding to a surface (e.g., of a HPLC column or a drug carrier). To check the latter practice correctness-and credibility of the prevailing P definition-this paper compares an ideal solute distribution between two separate homogeneous fluid media (i.e., partitioning) to solute association with a uniform surface immersed in one such medium (i.e., binding). This reveals that solute partitioning and binding fundamentally differ and can only exceptionally be described, or analysed, with similar equations. Nonlinearised formulae that describe partitioning (Eq. (9)) and binding (Eq. (11)) can yield similar lipophilicity descriptor values only if solute preparation is relatively dilute; employing a large organic medium fraction is helpful in this respect. Additional prerequisites for partitioning and binding models match are: 1:1 stoichiometry at the association maximum and identical interfacial area of solute and organic medium molecules. If these requirements are not met, binding model yields different, potentially somewhat higher, but more often up to >10 times lower results than partitioning model. The main reason is saturation of organic medium with solute molecules. Partitioning model excludes this phenomenon, which cannot always be prevented by focussing on dilute solute preparations. The current practice of using a linear model and approximate equations to study partitioning or binding can cause large errors (>10(3)×), and is one possible reason for the notoriously high experimental logP values scattering. The latter makes logP predictions more difficult and less reliable than they could be if the measured data were evaluated with non-linearised partitioning or binding equations, as appropriate.

Non-invasive, epicutaneous immunisation with toxoid in deformable vesicles protects mice against tetanus, chiefly owing to a Th2 response.

A non-invasive, intra/transcutaneous immunisation of mice with a suitable combination of tetanus toxoid, ultradeformable vesicle (Transfersome®) carrier, and monophosphoryl lipid A adjuvant targets immuno-competent cells in a body and can protect 100% of the tested mice against an otherwise lethal (50×LD50) parenteral tetanus toxin challenge. The late immune response to the epicutaneously applied tetanus toxoid in such vesicles consists chiefly of circulating IgG1 and IgG2b antibody isotypes, indicative of a specific Th2 cellular response bias. Immunisations by subcutaneous injections moreover protect 100% of mice against a similar, otherwise lethal, dose of tetanus toxin. However, the immune response to transcutaneous and invasive immunisation differs. The latter elicits mainly IgG1 and IgG2b as well as IgG2a antibody isotypes, indicative of a mixed Th1/Th2 response. The cytokine response of the intra/transcutaneously and subcutaneously immunised mice reflects the difference in the organ-specific manner. IFN-γ concentration is appreciably increased in the draining lymph nodes and IL-10 in spleen. Since tetanus is a neutral antigen, both the Th1-specific IFN-γ and the Th-2 specific-IL-10 are observable.

'Project launch': from research finding to therapeutic product.

Only 0.1-0.5% of new therapy candidates gains marketing approval; just 10-20% of the approved ones ultimately recoup the ~0.6-0.9$USbn invested into their R&D until marketing authorisation. One reason is the high inherent risk of new therapeutic products development. Further reasons are suboptimal decisions during R&D and, too often, lack of adequate experience. To bridge the latter gap, this article succinctly reviews identification of new product opportunities and their patent protection, the resulting commercial opportunity and portfolio valuation, planning and conduct of the ensuing preclinical and clinical tests, as well as therapeutic product registration and price reimbursement, covering risk management as an aside. The article also clarifies the key terms, identifies the main pit falls, highlights the essential requirements for and the goals of different product development steps, to facilitate communication between researchers and developers. By combining public information with personal experience and recommendations the article aims at informing more broadly those who are familiar mainly with some of the (strictly regulated) activities involved in design, development and launch of new therapeutic products, be it that they are medicinal products or medical devices. Taken together, this should support initiation and evolution of new therapeutic products and assist researchers in finding-and better and more smoothly co-operating with-consultants or partners in development and marketing.

A multicentre, randomized, placebo- and active-controlled trial comparing the efficacy and safety of topical ketoprofen in Transfersome gel (IDEA-033) with ketoprofen-free vehicle (TDT 064) and oral celecoxib for knee pain associated with osteoarthritis.

OBJECTIVE: To assess the efficacy and safety of 12-week treatment with ketoprofen in ultradeformable phospholipid vesicles in patients with OA knee pain and to compare the efficacy with that of ketoprofen-free vehicle and celecoxib. METHODS; A multicentre, double-blind controlled study in which patients with knee OA and moderate pain were randomized to one of the six arms: topical ketoprofen 50 or 100 mg in ultradeformable vesicles (IDEA-033), 2.2 or 4.4 g ketoprofen-free vehicle (TDT 064), oral celecoxib 100 mg or matching oral placebo, all bd. The primary outcome was change from baseline in the WOMAC pain subscale at week 12. RESULTS: A total of 1395 patients received treatment. Baseline mean WOMAC pain scores ranged from 4.7 to 4.8 across groups. The mean reduction in WOMAC pain score at week 12 was -1.9 (-40.8%) for ketoprofen 50 mg, -1.9 (-40.9%) for ketoprofen 100 mg, -1.9 (-39.8%) for 2.2 g TDT 064, -1.8 (-37.8%) for 4.4 g TDT 064, -1.9 (-40.4%) for celecoxib and -1.4 (-29.3%) for oral placebo. IDEA-033 was not statistically superior to TDT 064. All topical treatments were statistically superior to oral placebo and non-inferior to celecoxib. The most frequent types of treatment-related adverse events reported were gastrointestinal for oral (15.9% for celecoxib) and dermal for topical applications (12.2% for ketoprofen 100 mg). CONCLUSION: IDEA-033 was not superior to ketoprofen-free vehicle, but both formulations were superior to oral placebo and non-inferior to celecoxib in reducing OA knee pain. TRIAL REGISTRATION: ClinicalTrials.gov, http://clinicaltrials.gov/, NCT00716547.

Rational design of new product candidates: the next generation of highly deformable bilayer vesicles for noninvasive, targeted therapy.

Amphipat bilayer vesicles are a subgroup of "fat-and-water" mixtures useful as drug carriers. Scrutinising amphipat aggregation in terms of the popular molecular descriptors (esp. the Israelachvili's form-factor or HLB number) is "too static" to foretell reliably and quantitatively bilayer vesicle formation. A better predictor introduced in this work is the effective area per lipid chain (cross-section of a "tail", A(c)), which also correlates, quasi-exponentially, with the ease of bilayer vesicle formation and bilayer deformability. The latter is highest near an uppermost, bilayer-compatible but nearly headgroup independent, A(c)-value reachable on different paths to bilayer solubilisation. The deformable bilayer vesicles class is thus more diverse than had previously been recognised. It includes phospholipid or phospholipid-surfactant blends (1st generation), synergistic phospholipid-amphipat or drug mixtures (2nd generation), and novel (non-phospholipid) amphipat combinations with appropriate effective tail(s) cross-section (3rd generation). Typically, vesicularisation ability and bilayer adaptability of such preparations is proportional, and arguably depends upon, the dynamic and stress-dependent molecular re-arrangement during aggregate formation and bilayer adaptation. In the previously described formulations such re-arrangement took place within or across the mixed lipid bilayer. This work shows that water-soluble molecules redistribution near a bilayer surface can be similarly effective. The new mechanism for bilayer properties modulation thus potentially avoids using harsher molecules in the adaptable vesicles, and can utilise buffers, microbicides, etc., in their stead. A plethora of amphipats can comprise hyper-adaptable vesicles of the new generation, including some that are more stable than the previously recognised ones. Encompassing well-chosen hydrophilic additive(s) and/or drug(s), such hyper-adaptable vesicles can be blended into fluid or semisolid preparations suitable for non-invasive, and potentially parenteral, applications. Pharmacologically relevant examples include, but are not limited to, the composite adaptable phospholipid-free vesicles loaded with anti-mycosis drugs (such as terbinafine), surfactant-free preparations of non-steroidal anti-inflammatory drugs (such as indomethacin or ketoprofen), etc. Further interesting implementations of the new technology contain hyper-adaptable drug-free vesicles that suppress human skin inflammation after local application better than hydrocortisone and broadly similar to conventional topical NSAIDs. The carriers described in this work thus provide unprecedented options for cutaneous or targeted subcutaneous deposition of drugs and/or for the sustained delivery of the corresponding carrier associated therapeutic agents.

Turbidity spectroscopy for characterization of submicroscopic drug carriers, such as nanoparticles and lipid vesicles: size determination.

PURPOSE: To apply UV/Vis spectrometry for characterization of submicroscopic drug carriers, such as nanoparticles and lipid vesicles. METHODS: We first investigated theoretically, within the framework of the Rayleigh-Gans-Debye approximation (RGDA), parameters affecting turbidity spectrum, τ(λ), of nanosized light scatterers. We then analyzed, within the framework of the RGDA, experimental turbidity spectra (λ = 400-600 nm) of extruded unilamellar vesicle (70 nm ≤ 2r ≤ 110 nm) suspensions to derive vesicle size, using dynamic light scattering results for comparison. We similarly studied the preparations polydispersity and lamellarity and monitored vesicle size changes. RESULTS: Turbidimetry suffices for accurate, fast, and viscosity-independent characterization of submicroscopic particles. Analysis of turbidity spectra, or more precisely wavelength exponent spectra (derivatives of logarithmic turbidity spectra), yielded similar average radii (r = 54.2 ± 0.2 nm; 46.0 ± 0.2 nm; 35.5 ± 0.1 nm) as dynamic light scattering (r = 55.9 ± 1.5 nm; 46.1 ± 0.4 nm; 36.1 ± 0.4 nm). Both methods also revealed similar suspension polydispersity and cholate-induced vesicle size changes in a few nanometer range. CONCLUSION: Despite its experimental simplicity, the widely accessible turbidimetric method provides accurate size values and is suitable for (continuous) monitoring size stability, or sameness, of submicroscopic drug carriers.

The vesicle-to-micelle transformation of phospholipid-cholate mixed aggregates: a state of the art analysis including membrane curvature effects.

We revisited the vesicle-to-micelle transformation in phosphatidylcholine-cholate mixtures paying special attention to the lipid bilayer curvature effects. For this purpose, we prepared unilamellar vesicles with different starting sizes (2r(v)=45-120nm). We then studied mixtures of the unilamellar vesicles (1-8mmol kg(-1)) and sodium cholate (0-11.75mmolkg(-1)) by static and dynamic light scattering. The transformation generally comprises at least two, largely parallel phenomena; one increases and the other decreases the average mixed aggregate size. In our view, cholate first induces bilayer fluctuations that lead to vesicle asphericity, and then to lipid bilayer poration followed by sealing/reformation (or fusion). The cholate-containing mixed bilayers, whether in vesicular or open form, project thread-like protrusions with surfactant enriched ends even before complete bilayer solubilisation. Increasing cholate concentration promotes detachment of such protrusions (i.e. mixed micelles formation), in parallel to further softening/destabilising of mixed amphipat bilayers over a broad range of concentrations. Vesicles ultimately fragment into mixed thread-like micelles. Higher cholate relative concentrations yield shorter thread-like mixed micelles. Most noteworthy, the cholate-induced bilayer fluctuations, the propensity for large aggregate formation, the transformation kinetics, and the cholate concentration ensuring complete lipid solubilisation all depend on the starting mean vesicle size. The smallest tested vesicles (2r(v)=45nm), with the highest bilayer curvature, require ~30% less cholate for complete solubilisation than the largest tested vesicles (2r(v)=120nm).

Nanotechnology and the transdermal route: A state of the art review and critical appraisal.

The skin is refractive to most molecules, especially hydrophilic ones, despite the existence of trans-barrier pathways. It is essential to maintain this protective barrier even after breaching skin surface for purposes of transdermal drug delivery to cope with cutaneous microbiota. Mechanical abraders or local energy dischargers (porators) or else hard, sharp objects (perforators, micro-needles) can punch a limited number (approximately 10(2)cm(-2)) of relatively wide (> or =10(3)nm) openings in the skin barrier, which then lets transiently (approximately 1 day) small drug quantities (< or =10 mg) and even large molecules (and pathogens?) pass. Microscopic (> or =10(3)nm) ballistic droplets or particles also insert small drug amounts (approximately 1mg) into the upper skin through the > or =10(6)cm(2) pores they create. The latter "skin breaching" method is approved for use in humans, whereas the hard nano-sized (5 nm-10 microm) skin perforators are still in development for transdermal drug delivery. Alternatively, controlled and reliable drug delivery across skin barrier can be achieved with sufficiently deformable and stable nano-sized carriers. Such "soft" skin penetrators are typically composite colloids. As such they must obtain, or retain, their ability to act as drug carriers on, in, and ideally below skin barrier(s). If properly designed and applied, such self-regulating, ultra-adaptable, and stable hetero-aggregates can open spontaneously and carry drugs through < or =10(9)cm(-2) cutaneous pores in the primary skin barrier and minimise cutaneous drug clearance; this permits deep/targeted deposition and prolonged action of the carrier-transported drugs. Therapeutic products based on ultra-adaptable, self-regulating, nano-sized (approximately 10(2)nm) carriers are under development. The first one is already approved in Switzerland.

Accurate potentiometric determination of lipid membrane-water partition coefficients and apparent dissociation constants of ionizable drugs: electrostatic corrections.

PURPOSE: Potentiometric lipid membrane-water partition coefficient studies neglect electrostatic interactions to date; this leads to incorrect results. We herein show how to account properly for such interactions in potentiometric data analysis. MATERIALS AND METHODS: We conducted potentiometric titration experiments to determine lipid membrane-water partition coefficients of four illustrative drugs, bupivacaine, diclofenac, ketoprofen and terbinafine. We then analyzed the results conventionally and with an improved analytical approach that considers Coulombic electrostatic interactions. RESULTS: The new analytical approach delivers robust partition coefficient values. In contrast, the conventional data analysis yields apparent partition coefficients of the ionized drug forms that depend on experimental conditions (mainly the lipid-drug ratio and the bulk ionic strength). This is due to changing electrostatic effects originating either from bound drug and/or lipid charges. A membrane comprising 10 mol-% mono-charged molecules in a 150 mM (monovalent) electrolyte solution yields results that differ by a factor of 4 from uncharged membranes results. CONCLUSION: Allowance for the Coulombic electrostatic interactions is a prerequisite for accurate and reliable determination of lipid membrane-water partition coefficients of ionizable drugs from potentiometric titration data. The same conclusion applies to all analytical methods involving drug binding to a surface.

Adaptability and elasticity of the mixed lipid bilayer vesicles containing non-ionic surfactant designed for targeted drug delivery across the skin.

Novel potential carriers for non-invasive drug delivery were prepared from polyoxyethylene(20) oleyl ether (C(18:1)EO(20)) and soybean phosphatidylcholine (SPC) in different relative molar ratios, R(e); this produced stiff SPC liposomes (2r(ves) approximately 120 nm) at one end and much smaller (2r(mic) or= R(e)(sat) = 0.25 in the bilayer. The surfactant-saturated bilayers exhibit bending rigidity of kappa(c) approximately 2.1 k(B)T, as determined with an improved vesicle adaptability assay involving analysis of normalised flux density through a nano-porous barrier as an activated transport process. Pore penetrability vs. driving pressure data measured with the mixed amphiphat vesicles resemble results of computer simulation of deformable vesicles penetrating a constriction [Gompper G, Kroll DM. 1995. Driven transport of fluid vesicles through narrow pores. Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics. 52:4198-4208], confirming basic similarity of both processes. The improved assay can reveal partial lipid solubilisation at R(e)>R(e)(sat), which is linearly proportional to R(e) - R(e)(sat). C(18:1)EO(20)-SPC mixed vesicles that can cross narrow pores are arguably suitable for targeted drug delivery across intact skin.

Occlusion effect on transcutaneous NSAID delivery from conventional and carrier-based formulations.

We studied skin occlusion effects in vitro and in vivo on local and systemic delivery of ketoprofen across the organ, using the drug in a conventional non-occlusive topical gel (Togal Mobil-Gel), an occlusive tape (Mohrus), and the new targeted analgesic (Diractin), comprising ultradeformable, hydrophilic carriers in the form of a Transfersome vesicle. In vitro occluded skin permeability to ketoprofen from the tape (0.086cmh(-1)) marginally exceeds the value for the drug from carriers in a gel (0.058cmh(-1)), which resembles conventional gel on open excised skin (0.057cmh(-1)); smallness of occlusion-induced permeation enhancement ( approximately 1.5x) may be due to the high tested applied dose. In contrast, open skin permeability to the drug from the carriers in vitro is approximately 15xlower (0.004cmh(-1)). The benefit of ketoprofen association with the carriers for targeted transcutaneous delivery only shows-up in vivo after an non-occlusive epicutaneous application: the area under the curve (AUC) in peripheral deep muscle for the carrier-based gel then exceeds AUC for conventional gel approximately 35-fold. The AUC for occluded ultradeformable, hydrophilic carriers measured in living pigs is conversely approximately 10x lower, being 1.4-2.2x below that of the tape that is inferior to non-occluded carriers formulation (normalised cmax: approximately 200x). Occlusion thus disables ultradeformable, hydrophilic carriers by eliminating transcutaneous hydration gradient that normally drives the carriers across the skin. Compared with other non-steroidal anti-inflammatory agents (NSAIDs) for local usage, Diractin is thus evidently well differentiated and innovative.

Preclinical characterisation of NSAIDs in ultradeformable carriers or conventional topical gels.

We compared in vivo transport and biodistribution of ketoprofen applied on the skin in ultradeformable carriers (Diractin) or a conventional topical gel (Gabrilen) with oral drug (Oruvail); for reference we used in vitro study data. The drug from Gabrilen diffuses into body with low bioavailability (<10%) and limited regio-selectivity (AUC(deep muscle/plasma) approximately 45/0.8 (t=0-8h), reaching maximum concentration in subcutaneous tissues and plasma at similar time (t(max) approximately 3-4h). The apparent drug elimination half-life is then similar to oral ketoprofen (t1/2,a) approximately 2 h). In contrast, Diractin containing ultradeformable carriers (Transfersome vesicles) delivers the drug more efficiently (>50%) and more directly into peripheral muscles (AUC(deep muscle/plasma) approximately 447/0.7 (652/1.4) for t=0-8 (0-24)h; tmax approximately 1 h), arguably in non-diffusive fashion. Ketoprofen from Diractin moreover disappears from body periphery slower (t1/2,a) approximately 4-6 h), owing to sustained drug release from the carriers in target tissue. Final clearance always proceeds via plasma (tmax approximately 4 h). Epicutaneous application of ketoprofen in conventional gels or the carrier-based formulation thus leads to different local accumulations and clearances. Ketoprofen from Diractin achieves more desirable biodistribution and clearance, arguably due to spontaneous carrier-mediated drug transport across the skin, which ensures local and relatively long-lasting drug deposition into peripheral target tissues.

Spatial distribution of cutaneous microvasculature and local drug clearance after drug application on the skin.

We analysed quantitatively blood microvessels distribution in normal skin. We conclude that the segmental area of blood vessels peaks approximately 0.1 mm below the skin surface, where the upper cutaneous blood plexus resides. Total blood vessels area then decreases quasi-exponentially to a depth of approx. -0.75 mm, with a decay length of approximately 0.1 mm, which is site and skin condition dependent, but at greater depths the decrease is approx. 6-times less steep. The corresponding permeability sink exhibits a similar, but superficially steeper, depth-profile. The lateral localisation of superficial blood vessels is such that ensures maximum diffusion from and into the capillaries, which affects transdermal drug delivery: each hairpin-like loop is in the centre of a papilla that corresponds to a cluster of corneocytes surrounded by main diffusion pathways. The aggregate area of blood vessels in the skin is >or=2.5-fold greater than total organ surface area under normal physiological conditions. The molecules diffusing through the skin barrier are thus largely cleared in outermost 20% of the organ, which may create a drug concentration maximum in the dermis, if clearance increases significantly with time. Skin microdialysis data are therefore extremely sensitive to cutaneous blood flow (distribution) and sampling. Skin microvasculature and its distribution must consequently be considered in all topical or transdermal drug transport studies, for example, by including suitably formulated clearance term into generalised diffusion equation.

Hydrocortisone and dexamethasone in very deformable drug carriers have increased biological potency, prolonged effect, and reduced therapeutic dosage.

We characterised biological properties of novel formulations of two low-potency glucocorticosteroids, dexamethasone and hydrocortisone, which have an equivalent dose ratio of 1:50 in vasoconstriction tests. The rate of such carrier-mediated, mainly non-diffusive glucocorticosteroids transport with very deformable lipid vesicles (Transfersomes) through the skin, and the corresponding cutaneous drug biodistribution data, were complemented with the drug bio-efficacy studies. The minimum effective drug dose that reduces arachidonic acid-induced murine ear oedema by 50% was used as one bioactivity indicator. The minimum drug amount ensuring such an effect in mouse skin decreases appreciably when a corticosteroid is applied epicutaneously with very deformable vesicles rather than a lotion or a crème. Specifically, the minimum effective dose for hydrocortisone in very deformable carriers is 2-3 microg cm(-2) whereas for the crème- or lotion-like preparations at least 10 microg cm(-2) is required. Such three- to fivefold relative increase of hydrocortisone potency is accompanied by at least 13%, and more often >20%, absolute drug potency enhancement. The delivery of hydrocortisone with very deformable carriers moreover prolongs the suppression of the drug-induced oedema nearly 2-fold (to approximately 24 h per application). The effective dose of dexamethasone delivered with very deformable vesicles into murine skin is reduced >10 times compared with the crème- or lotion-based products. Specifically, less than 0.1 microg cm(-2) dexamethasone in very deformable vesicles suppresses the arachidonic acid-induced murine ear oedema >50%, on the average. Dexamethasone use on the skin in such vesicles extends the duration of drug action fourfold, compared with a commercial crème, i.e. to >48 h per application. Epicutaneous use of glucocorticosteroids in very deformable vesicles also diminishes such drug's abrasion sensitivity and may increase the general robustness of drug effect. Lower frequency of skin treatment, which ensures adequate biological response, is a result of this. Topical corticosteroid delivery with very deformable vesicles, Transfersomes, thus improves the therapeutic risk-benefit ratio, arguably due to better targeting into and longer drug presence in the skin.

Lipid vesicles and other colloids as drug carriers on the skin.

Colloids from an aqueous suspension can cross the skin barrier only through hydrophilic pathways. Various colloids have a different ability to do this by penetrating narrow pores of fixed size in the skin, or the relevant nano-pores in barriers modelling the skin. Such ability is governed by colloid adaptability, which must be high enough to allow penetrant deformation to the size of a pore in such barrier: for a 100 nm colloid trespassing the skin this means at least 5-fold deformation/elongation. (Lipid) Bilayer vesicles are normally more adaptable than the comparably large (lipid coated) fluid droplets. One of the reasons for this, and an essential condition for achieving a high bilayer adaptability and pore penetration, is a high bilayer membrane elasticity. The other reason is the relaxation of changing colloid's volume-to-surface constraint during pore penetration; it stands to reason that such relaxation requires a concurrent, but only transient and local, bilayer permeabilisation. Both these phenomena are reflected in bilayer composition sensitivity, which implies non-linear pressure dependency of the apparent barrier penetrability, for example. Amphipats that acceptably weaken a membrane (surfactants, (co)solvents, such as certain alcohols, etc.) consequently facilitate controlled, local bilayer destabilisation and increase lipid bilayer flexibility. When used in the right quantity, such additives thus lower the energetic expense for elastic bilayer deformation, associated with pore penetration. Another prerequisite for aggregate transport through the skin is the colloid-induced opening of the originally very narrow ( approximately 0.4 nm) gaps between cells in the barrier to pores with diameter above 30 nm. Colloids incapable of enforcing such widening-and simultaneously of self-adapting to the size of 20-30 nm without destruction-are confined to the skin surface. All relatively compact colloids seem to fall in this latter category. This includes mixed lipid micelles, solid (nano)particles, nano-droplets, biphasic vesicles, etc. Such colloids, therefore, merely enter the skin through the rare wide gaps between groups of skin cells near the organ surface. Transdermal drug delivery systems based on corresponding drug formulations, therefore, rely on simple drug diffusion through the skin; the colloid then, at best, can modulate drug transport through the barrier. In contrast, the adaptability-and stability-optimised mixed lipid vesicles (Transfersomes, a trademark of IDEA AG) can trespass much narrower pathways between most cells in the skin; such highly adaptable colloids thus mediate drug transport through the skin. Sufficiently stable ultra-adaptable carriers, therefore, can ensure targeted drug delivery deep below the application site. This has already been shown in numerous preclinical tests and several phase I and phase II clinical studies. Drug delivery by means of highly adaptable drug carriers, moreover, allows highly efficient and well-tolerated drug targeting into the skin proper. Sustained drug release through the skin into systemic blood circulation is another field of ultradeformable drug carrier application.