How wound environments trigger the release from Rifampicin-loaded liposomes.

BACKGROUND: Chronic wounds often contain high levels of proinflammatory cytokines that prolong the wound-healing process. Patients suffering from these conditions are likely to benefit from topical rifampicin therapy. Although recent research indicates considerable anti-inflammatory properties of the antibiotic, currently, there are no commercial topical wound healing products available. To address this medical need, a liposomal drug delivery system was developed. A mechanistic investigation outlined major influences of wound environments that affect the release kinetics and, as a consequence, local bioavailability. METHODS: Liposomes were prepared using the thin-film hydration method and subsequently freeze-dried at the pilot scale to improve their stability. We investigated the influence of oxidation, plasma proteins, and lipolysis on the in vitro release of rifampicin and its two main degradation products using the Dispersion Releaser technology. A novel simulated wound fluid provided a standardized environment to study critical influences on the release. It reflects the pathophysiological environment regarding pH, buffer capacity, and protein content. RESULTS: During storage, the liposomes efficiently protect rifampicin from degradation. After the dispersion of the vesicles in simulated wound fluid, despite the significant albumin binding (>70%), proteins have no considerable effect on the release. Also, the presence of lipase at pathophysiologically elevated concentrations did not trigger the liberation of rifampicin. Surprisingly, the oxidative environment of the wound bed represents the strongest accelerating influence and triggers the release. CONCLUSION: A stable topical delivery system of rifampicin has been developed. Once the formulation comes in contact with simulated wound fluid, drug oxidation accelerates the release. The influence of lipases that are assumed to trigger the liberation from liposomes depends on the drug-to-lipid ratio. Considering that inflamed tissues exhibit elevated levels of oxidative stress, the trigger mechanism identified for rifampicin contributes to targeted drug delivery.

Predicting drug release and degradation kinetics of long-acting microsphere formulations of tacrolimus for subcutaneous injection.

Today, tacrolimus represents a cornerstone of immunosuppressive therapy for liver and kidney transplants and remains subject of preclinical and clinical investigations, aiming at the development of long-acting depot formulations for subcutaneous injection. One major challenge arises from establishing in vitro-in vivo correlations due to the absence of meaningful in vitro methods predictive for the in vivo situation, together with a strong impact of multiple kinetic processes on the plasma concentration-time profile. In the present approach, two microsphere formulations were compared with regards to their in vitro release and degradation characteristics. A novel biorelevant medium provided the physiological ion and protein background. Release was measured using the dispersion releaser technology under accelerated conditions. A release of 100% of the drug from the carrier was achieved within 7 days. The capability of the in vitro performance assay was verified by the level A in vitro-in vivo correlation analysis. The contributions of in vitro drug release, drug degradation, diffusion rate and lymphatic transport to the absorption process were quantitatively investigated by means of a mechanistic modelling approach. The degradation rate, together with release and diffusion characteristics provides an estimate of the bioavailability and therefore can be a guide to future formulation development.

Predicting human pharmacokinetics of liposomal temoporfin using a hybrid in silico model.

Over the years, the performance of the liposomal formulations of temoporfin, Foslip® and Fospeg®, was investigated in a broad array of cell-based assays and preclinical animal models. So far, little attention has been paid to the influence of drug release and liposomal stability on the plasma concentration-time profile. The drug release is a key attribute which impacts product quality and the in vivo efficacy of nanocarrier formulations. In the present approach, the in vitro drug release and the drug-protein transfer of Foslip® and Fospeg® was determined using the dispersion releaser technology. To analyze the stability of both formulations in physiological fluids, nanoparticle tracking analysis was applied. A comparable drug release behavior and a high physical stability with a vesicle size of approximately 92 ± 2 nm for Foslip® and at 111 ± 5 nm for Fospeg® were measured. The development of a novel hybrid in silico model resulted in an optimal representation of the in vivo data. Based on the information available for previous formulations, the model enabled a prediction of the performance of Foslip® in humans. To verify the simulations, plasma concentration-time profiles of a phase I clinical trial were used. An absolute average fold error of 1.4 was achieved. Moreover, a deconvolution of the pharmacokinetic profile into different fractions relevant for the in vivo efficacy and safety was achieved. While the total plasma concentration reached a cmax of 2298 ng/mL after 0.72 h, the monomolecular drug accounted for a small fraction of the photosensitizer with a cmax of 321 ng/mL only.

Advanced in silico modeling explains pharmacokinetics and biodistribution of temoporfin nanocrystals in humans.

Foscan®, a formulation comprising temoporfin dissolved in a mixture of ethanol and propylene glycol, has been approved in Europe for palliative photodynamic therapy of squamous cell carcinoma of the head and neck. During clinical and preclinical studies it was observed that considering the administration route, the drug presents a rather atypical plasma profile as plasma concentration peaks delayed. Possible explanations, as for example the formation of a drug depot or aggregation after intravenous administration, are discussed in current literature. In the present study an advanced in silico model was developed and evaluated for the detailed description of Foscan® pharmacokinetics. Therefore, in vitro release data obtained from experiments with the dispersion releaser technology investigating dissolution pressures of various release media on the drug as well as in vivo data obtained from a clinical study were included into the in silico models. Furthermore, precipitation experiments were performed in presence of biorelevant media and precipitates were analyzed by nanoparticle tracking analysis. Size analysis and particle fraction were also incorporated in this model and a sensitivity analysis was performed. An optimal description of the in vivo situation based on in vitro release and particle characterization data was achieved, as demonstrated by an absolute average fold error of 1.21. This in vitro-in vivo correlation provides an explanation for the pharmacokinetics of Foscan® in humans.

Nanomedicines - Tiny particles and big challenges.

After decades of research, nanotechnology has been used in a broad array of biomedical products including medical devices, drug products, drug substances, and pharmaceutical-grade excipients. But like many great achievements in science, there is a fine balance between the risks and opportunities of this new technology. Some materials and surface structures in the nanosize range can exert unexpected toxicities and merit a more detailed safety assessment. Regulatory agencies such as the United States Food and Drug Administration or the European Medicines Agency have started dealing with the potential risks posed by nanomaterials. Considering that a thorough characterization is one of the key aspects of controlling such risks this review presents the regulatory background of nanosafety assessment and provides some practical advice on how to characterize nanomaterials and drug formulations. Further, the challenges of how to maintain and monitor pharmaceutical quality through a highly complex production processes will be discussed.

Stimulating brain recovery after stroke using theranostic albumin nanocarriers loaded with nerve growth factor in combination therapy.

For many years, delivering drug molecules across the blood brain barrier has been a major challenge. The neuropeptide nerve growth factor is involved in the regulation of growth and differentiation of cholinergic neurons and holds great potential in the treatment of stroke. However, as with many other compounds, the biomolecule is not able to enter the central nervous system. In the present study, nerve growth factor and ultra-small particles of iron oxide were co-encapsulated into a chemically crosslinked albumin nanocarrier matrix which was modified on the surface with apolipoprotein E. These biodegradable nanoparticles with a size of 212 ±â€¯1 nm exhibited monodisperse size distribution and low toxicity. They delivered NGF through an artificial blood brain barrier and were able to induce neurite outgrowth in PC12 cells in vitro. In an animal model of stroke, the infarct size was significantly reduced compared to the vehicle control. The combination therapy of NGF and the small-molecular MEK inhibitor U0126 showed a slight but not significant difference compared to U0126 alone. However, further in vivo evidence suggests that successful delivery of the neuropeptide is possible as well as the synergism between those two treatments.

Predictive PBPK modeling as a tool in the formulation of the drug candidate TMP-001.

Since many drugs in the therapy scheme of multiple sclerosis (MS) are applied parenterally with significant side effects, oral treatment is the most accepted therapy option for chronic diseases like MS. The drug candidate TMP-001, which has disease-modifying properties, can be applied orally. Beside other symptoms, swallowing disorders have a major impact not only on the health status and quality of life of MS patients, but also impede reliable drug therapy. Consequently, the development of an easy-to-swallow liquid oral dosage form supported by a combined PBPK-IVIVC model was approached. In this context, the impact of formulation parameters was studied. Biorelevant in vitro drug release studies resulted in an almost complete release of 96.91% ±â€¯1.00% in the intestine which was translated to rapidly increasing in silico plasma profiles. The predictions were compared to the outcome of a phase I clinical trial. A partial parameter sensitivity analysis of the in silico model deepened our understanding of the physiological processes underlying human pharmacokinetics.

Nanopharmaceuticals for wound healing - Lost in translation?

Today, many of the newly developed pharmaceuticals and medical devices take advantage of nanotechnology and with a rising incidence of chronic diseases such as diabetes and cardiovascular disease, the number of patients afflicted globally with non-healing wounds is growing. This has created a requirement for improved therapies and wound care. However, converting the strategies applied in early research into new products is still challenging. Many of them fail to comply with the market requirements. This review discusses the legal and scientific challenges in the design of nanomedicines for wound healing. Are they lost in translation or is there a new generation of therapeutics in the pipeline?

Tip-enhanced Raman scattering for tracking of invasomes in the stratum corneum.

Stratum corneum is the primary skin barrier to percutaneous absorption. Since 1980, topical liposomal formulations have been proposed and successfully employed for increasing the drug penetration through the skin. There is no clear consensus on the drug penetration mechanism from topically applied liposomes, despite a vast amount of research. One of the reasons for the ambiguity is that the interactions between the stratum corneum and liposomes are in nanoscale, which makes them difficult to probe. In this study, we employed tip-enhanced Raman scattering (TERS) to gain a better understanding of the interactions between the human stratum corneum and topically applied liposomal system called invasomes. TERS is capable of imaging at nanometer spatial resolution and can provide structural information at the nanometer scale. A sample preparation technique was developed and calibrated to enable TERS on complex stratum corneum samples. Invasomes prepared from a head deuterated phospholipid were employed to aid identification of topically applied liposomal phospholipid in the stratum corneum. Results presented in this study give for the first time a strong spectroscopic evidence along with high-resolution images to show intact invasome vesicles deep in the stratum corneum upon topical application.

Micro-spherical cochleate composites: method development for monodispersed cochleate system.

Cochleates have been of increasing interest in pharmaceutical research due to their extraordinary stability. However the existing techniques used in the production of cochleates still need significant improvements to achieve sufficiently monodispersed formulations. In this study, we report a simple method for the production of spherical composite microparticles (3-5 µm in diameter) made up of nanocochleates from phosphatidylserine and calcium (as binding agent). Formulations obtained from the proposed method were evaluated using electron microscopy and small angle X-ray scattering and were compared with conventional cochleate preparation techniques. In this new method, an ethanolic lipid solution and aqueous solution of a binding agent is subjected to rapid and uniform mixing with a microfluidic device. The presence of high concentration of organic solvent promotes the formation of composite microparticles made of nanocochleates. This simple methodology eliminates elaborate preparation methods, while providing a monodisperse cochleate system with analogous quality.

Transdermal delivery from liposomal formulations - Evolution of the technology over the last three decades.

Strong barrier properties of stratum corneum often limits the efficiency of drug delivery through skin. Several strategies were tried to improve permeation of drug through skin for local as well as systemic drug delivery. Incorporation of the drug within flexible liposomal vesicles has been one of the popular and well-studied approaches for delivering drug to deeper layers of the skin or even systemic circulation. Flexible/deformable/elastic liposomal systems such as invasomes, Transfersomes®, ethosomes, niosomes, etc. have demonstrated encouraging results in delivering small molecules and large proteins to the skin. It is necessary to recognize the promising concepts and analyze their potential, since a clear understanding of the drawbacks and advantages of these approaches will lead towards future development. In the current review we have attempted to give an overview of different liposomal drug carriers for transdermal drug delivery and their efficiency as drug delivery system through different in vivo and in vitro studies. Also, an overview of the studies which investigated the interactions between skin and vesicles, which have lead us to our current understanding of the skin penetration mechanisms of liposomal formulations is presented.

Optimizing novel implant formulations for the prolonged release of biopharmaceuticals using in vitro and in vivo imaging techniques.

As a rapidly growing class of therapeutics, biopharmaceuticals have conquered the global market. Despite the great potential from a therapeutic perspective, such formulations often require frequent injections due to their short half-life. Aiming to establish a parenteral dosage form with prolonged release properties, a biodegradable implant was developed, based on a combination of nanoencapsulation of protein-heparin complexes, creation of a slow release matrix by freeze-drying, and compression using hyaluronan and methylcellulose. In order to investigate this novel delivery system, formulations containing IFN-ß-1a and trypsinogen as model proteins were developed. No degradation of the proteins was observed at any stage of the formulation processing. The potential of the delivery system was evaluated in vivo and in vitro after fluorescence-labeling of the biopharmaceuticals. An optimized agarose gel was utilized as in vitro release medium to simulate the subcutaneous environment in a biorelevant manner. In addition, the formulations were administered to female SJL mice and release was innovatively tracked by fluorescence imaging, setting up an in vitro-in vivo correlation. A prolonged time of residence of approximately 12days was observed for the selected formulation design.

Understanding cochleate formation: insights into structural development.

Understanding the structure and the self-assembly process of cochleates has become increasingly necessary considering the advances of this drug delivery system towards the pharmaceutical industry. It is well known that the addition of cations like calcium to a dispersion of anionic lipids such as phosphatidylserines results in stable, multilamellar cochleates through a spontaneous assembly. In the current investigation we have studied the intermediate structures generated during this self-assembly of cochleates. To achieve this, we have varied the process temperature for altering the rate of cochleate formation. Our findings from electron microscopy studies showed the formation of ribbonlike structures, which with proceeding interaction associate to form lipid stacks, networks and eventually cochleates. We also observed that the variation in lipid acyl chains did not make a remarkable difference to the type of structure evolved during the formation of cochleates. More generally, our observations provide a new insight into the self-assembly process of cochleates based on which we have proposed a pathway for cochleate formation from phosphatidylserine and calcium. This knowledge could be employed in using cochleates for a variety of possible biomedical applications in the future.

LeciPlex, invasomes, and liposomes: A skin penetration study.

The present study compares three vesicular systems, cationic LeciPlex, invasomes, and conventional liposomes for their ability to deliver drugs deep into the skin. Skin penetration ability of the three vesicular systems was studied for two drugs namely idebenone (antioxidant/anticancer) and azelaic acid (antiacne). All systems showed sizes in nanometer range with small polydispersity indices. Vesicular systems were characterized by CryoTEM studies to understand the differences in morphology of the vesicular systems. Ex vivo human skin penetration studies suggested a pattern in penetration of drugs in different layers of the skin: LeciPlex showed higher penetration for idebenone whereas invasomes showed higher penetration of azelaic acid. Ex vivo study using a fluorescent dye (DiI) was performed to understand the differences in the penetration behavior of the three vesicular systems on excised human skin. In vitro cytotoxicity studies on B16F10 melanoma cell lines revealed, when loaded with idebenone, LeciPlex formulations had the superior activity followed by invasomes and liposomes. In vitro antimicrobial study of azelaic acid loaded systems on Propionibacterium acne revealed high antimicrobial activity for DDAB leciplex followed by almost equal activity for invasomes and CTAB LeciPlex followed by liposomes. Whereas antiacne efficacy study in rats for azelaic acid loaded systems, invasomes exhibited the best antiacne efficacy followed by liposomes and LeciPlex.

Electron microscopy and theoretical modeling of cochleates.

Cochleates are self-assembled cylindrical condensates that consist of large rolled-up lipid bilayer sheets and represent a novel platform for oral and systemic delivery of therapeutically active medicinal agents. With few preceding investigations, the physical basis of cochleate formation has remained largely unexplored. We address the structure and stability of cochleates in a combined experimental/theoretical approach. Employing different electron microscopy methods, we provide evidence for cochleates consisting of phosphatidylserine and calcium to be hollow tubelike structures with a well-defined constant lamellar repeat distance and statistically varying inner and outer radii. To rationalize the relation between inner and outer radii, we propose a theoretical model. Based on the minimization of a phenomenological free energy expression containing a bending, adhesion, and frustration contribution, we predict the optimal tube dimensions of a cochleate and estimate ratios of material constants for cochleates consisting of phosphatidylserines with varied hydrocarbon chain structures. Knowing and understanding these ratios will ultimately benefit the successful formulation of cochleates for drug delivery applications.

Non-invasive depth profile imaging of the stratum corneum using confocal Raman microscopy: first insights into the method.

The stratum corneum is a strong barrier that must be overcome to achieve successful transdermal delivery of a pharmaceutical agent. Many strategies have been developed to enhance the permeation through this barrier. Traditionally, drug penetration through the stratum corneum is evaluated by employing tape-stripping protocols and measuring the content of the analyte. Although effective, this method cannot provide a detailed information regarding the penetration pathways. To address this issue various microscopic techniques have been employed. Raman microscopy offers the advantage of label free imaging and provides spectral information regarding the chemical integrity of the drug as well as the tissue. In this paper we present a relatively simple method to obtain XZ-Raman profiles of human stratum corneum using confocal Raman microscopy on intact full thickness skin biopsies. The spectral datasets were analysed using a spectral unmixing algorithm. The spectral information obtained, highlights the different components of the tissue and the presence of drug. We present Raman images of untreated skin and diffusion patterns for deuterated water and beta-carotene after Franz-cell diffusion experiment.

Predicting the effect of fed-state intestinal contents on drug dissolution.

PURPOSE: There are several endogenous and exogenous species in the gastrointestinal (GI) tract which can act as solubilizing agents and thereby affect drug dissolution. The purpose of this study is to understand food effects on drug dissolution and provide insight into their anticipated overall effect on absorption and bioavailability. METHODS: Dissolution kinetics of 15 drugs of variable logP, charge, and molecular weight were tested in simulated intestinal environment. The ability of a film-equilibrium-based model to predict the influence of a simulated intestinal environment on drug dissolution was examined. RESULTS: The most significant improvement in dissolution kinetics and solubility (up to 6-fold) was evident with highly hydrophobic compounds (logP > 4). Improvement in solubility did not always constitute improvement in dissolution kinetics on a relevant time scale. Comparison of simulation and experimental results indicates that a model considering micelle partitioning as a pseudo-equilibrium process can predict trends in the influence of food-related solubilizing agents on drug dissolution kinetics. CONCLUSIONS: The significance of food-related solubilizing agents to drug dissolution is not always obvious, as it depends on multiple physicochemical parameters; however, simple modeling may provide insight into food effects on dissolution and, ultimately, overall absorption and bioavailability of compounds considered for oral formulation.