Sustained-release hot melt extrudates of the weak acid TMP-001: A case study using PBB modelling.

Over the last 30 years, hot melt extrusion has become a leading technology in the manufacture of amorphous drug delivery systems. Mostly applied as an 'enabling formulation' for poorly soluble compounds, application in the design of sustained-release formulations increasingly attracts the attention of the pharmaceutical industry. The drug candidate TMP-001 is currently under evaluation for the early treatment of Multiple Sclerosis. Although this weak acid falls into class II of the Biopharmaceutics Classification System, the compound exhibits high solubility in the upper intestine resulting in high peroral bioavailability. In the present studies, four different formulation prototypes varying in their sustained-release behavior were developed, using L-arginine as a pore-forming agent in concentrations ranging between 0 and 20%. Initially, biorelevant release testing was applied to assess the dissolution behavior of the prototypes. For these formulations, a total drug release of 44.7%, 64.6%, 75%, and 90.5% was achieved in FaSSIF-v2 after 24 h. Two candidates were selected for further characterization considering the crystal structure and the physical stability of the amorphous state of TMP-001 in the formulations together with the release behavior in Level II biorelevant media. Our findings indicate L-arginine as a valuable excipient in the formulation of hot melt extrudates, as its presence led to a considerable stabilization of the amorphous state and favorably impacted the milling process and release behavior of TMP-001. To properly evaluate the proposed formulations and the importance of colonic dissolution and absorption on the overall bioavailability, a physiologically-based biopharmaceutics model was used.

Predicting the environmental emissions arising from conventional and nanotechnology-related pharmaceutical drug products.

Today, environmental pollution with pharmaceutical drugs and their metabolites poses a major threat to the aquatic ecosystems. Active substances such as fenofibrate, are processed to pharmaceutical drug formulations before they are degraded by the human body and released into the wastewater. Compared to the conventional product Lipidil® 200, the pharmaceutical product Lipidil 145 One® and Ecocaps take advantage of nanotechnology to improve uptake and bioavailability of the drug in humans. In the present approach, a combination of in vitro drug release studies and physiologically-based biopharmaceutics modeling was applied to calculate the emission of three formulations of fenofibrate (Lipidil® 200, Lipidil 145 One®, Ecocaps) into the environment. Special attention was paid to the metabolized and non-metabolized fractions and their individual toxicity, as well as to the emission of nanomaterials. The fish embryo toxicity test revealed a lower aquatic toxicity for the metabolite fenofibric acid and therefore an improved toxicity profile. When using the microparticle formulation Lipidil® 200, an amount of 126 mg of non-metabolized fenofibrate was emitted to the environment. Less than 0.05% of the particles were in the lower nanosize range. For the nanotechnology-related product Lipidil 145 One®, the total drug emission was reduced by 27.5% with a nanomaterial fraction of approximately 0.5%. In comparison, the formulation prototype Ecocaps reduced the emission of fenofibrate by 42.5% without any nanomaterials entering the environment. In a streamlined life cycle assessment, the lowered dose in combination with a lowered drug-to-metabolite ratio observed for Ecocaps led to a reduction of the full life cycle impacts of fenofibrate with a reduction of 18% reduction in the global warming potential, 61% in ecotoxicity, and 15% in human toxicity. The integrated environmental assessment framework highlights the outstanding potential of advanced modeling technologies to determine environmental impacts of pharmaceuticals during early drug development using preclinical in vitro data.

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.

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.

A comparison of two biorelevant in vitro drug release methods for nanotherapeutics based on advanced physiologically-based pharmacokinetic modelling.

As a growing number of nanotherapeutics enters the market, improved analytical techniques for measuring the drug release are required. Biorelevant release tests have become a standard in the prediction of in vivo pharmacokinetics but also in quality control of novel dosage forms. In the present study, two methods for testing the drug release from nanocarriers, namely the filtration technique and the dispersion releaser technology, have been investigated. Initially, the in vitro release rates were determined using two different biorelevant media. Additionally, the effect of each method on a simulated in vivo pharmacokinetic profile was studied using advanced PBPK modelling. The two methods resulted in slightly different release profiles. Applying the filtration method, an early plateau of 91.0 ±â€¯5.3% was reached at the first sampling time point. In comparison, the release rate steadily increased to a maximum of 100.9 ±â€¯4.1% when the dispersion releaser technology was used. Sensitivity analysis revealed how these differences translated into the PBPK-based simulation. A change in the total dissolution rate of 10% resulted in cmax values of +1.6% and -11.0%, respectively, when using input data obtained with the dispersion releaser. Data obtained by filtration translated into cmax values of ±1.8%.

The γH2AX assay for genotoxic and nongenotoxic agents: comparison of H2AX phosphorylation with cell death response.

DNA double-strand breaks (DSBs) and blocked replication forks resulting from bulky adducts and inhibitors of replication activate the DNA damage response (DDR), a signaling pathway marked by phosphorylation of histone 2AX (H2AX). The phosphorylated form, γH2AX, accumulates at the site of the damage and can be visualized as foci by immunocytochemistry. The objective of this study was to assess if γH2AX is a reliable biomarker for genotoxic exposures. To this end, we selected 14 well-known genotoxic compounds and compared them with 10 nongenotoxic chemicals, using CHO-9 cells because they are well characterized as to DNA repair and DDR. We quantified γH2AX foci manually and automatically. In addition, total γH2AX activation was determined by flow cytometry. For all chemicals the cytotoxic dose response was assayed by a metabolic cytotoxicity assay. We show that (1) all genotoxic agents induced γH2AX dose-dependently whereas nongenotoxic agents do not; (2) γH2AX was observed for genotoxicants in the cytotoxic dose range, revealing a correlation between cytotoxicity and γH2AX for genotoxic agents; for nongenotoxic agents cytotoxicity was not related to γH2AX; (3) manual scoring of γH2AX and automated scoring provided comparable results, the automated scoring was faster and investigator independent; (4) data obtained by foci counting and flow cytometry showed a high correlation, suggesting that γH2AX scoring by flow cytometry has the potential for high-throughput analysis. However, the microscopic evaluation can provide additional information as to foci size, distribution, colocalization and background staining; (5) γH2AX foci were colocalized with 53BP1 and Rad51, supporting the notion that they represent true DSBs. Collectively, the automated analysis of γH2AX foci allows for rapid determination of genetic damage in mammalian cells. The data revealed that the induction of γH2AX by genotoxicants is related to loss of viability and support γH2AX as a reliable bio-indicator for pretoxic DNA damage.