iNClusive: a database collecting useful information on non-canonical amino acids and their incorporation into proteins for easier genetic code expansion implementation.

The incorporation of non-canonical amino acids (ncAAs) into proteins is a powerful technique used in various research fields. Genetic code expansion (GCE) is the most common way to achieve this: a specific codon is selected to be decoded by a dedicated tRNA orthogonal to the endogenous ones. In the past 30 years, great progress has been made to obtain novel tRNA synthetases (aaRSs) accepting a variety of ncAAs with distinct physicochemical properties, to develop robust in vitro assays or approaches for codon reassignment. This sparked the use of the technique, leading to the accumulation of publications, from which gathering all relevant information can appear daunting. Here we present iNClusive (https://non-canonical-aas.biologie.uni-freiburg.de/), a manually curated, extensive repository using standardized nomenclature that provides organized information on ncAAs successfully incorporated into target proteins as verified by mass spectrometry. Since we focused on tRNA synthetase-based tRNA loading, we provide the sequence of the tRNA and aaRS used for the incorporation. Derived from more than 687 peer-reviewed publications, it currently contains 2432 entries about 466 ncAAs, 569 protein targets, 500 aaRSs and 144 tRNAs. We foresee iNClusive will encourage more researchers to experiment with ncAA incorporation thus contributing to the further development of this exciting technique.

Bioavailability enhancement of itraconazole-based solid dispersions produced by hot melt extrusion in the framework of the Three Rs rule.

Solid dispersion formulations made of itraconazole (ITZ) and Soluplus® (polyethylene glycol, polyvinyl acetate and polyvinylcaprolactame-based graft copolymer abbreviated SOL) were produced using hot melt extrusion. Since ITZ possesses a water solubility of less than 1ng/mL, the aim of this work was to enhance the aqueous solubility of ITZ, and thereby improve its bioavailability. The three formulations consisted of a simple SOL/ITZ amorphous solid dispersion (ASD), an optimized SOL/ITZ/AcDiSol® (super-disintegrant) ASD and an equimolar inclusion complex of ITZ in hydroxypropyl-ß-cyclodextrin (substitution degree=0.63, CD) with SOL. The three formulations were compared in vitro and in vivo to the marketed product Sporanox®. The in vitro enhancement of dissolution rate was evaluated using a biphasic dissolution test. In vitro dissolution results showed that all three formulations had a higher percentage of ITZ released than Sporanox® with the following ranking: SOL/ITZ/CD>SOL/ITZ/AcDiSol®>SOL/ITZ>Sporanox®. The bioavailability of these four formulations was evaluated in rats. The bioanalytical method was optimized so that only 10µL of blood was withdrawn from the rats using specific volumetric absorptive microsampling devices. This enabled to keep the same rats during the whole study, which was in accordance with the Three Rs rules (reduction, refinement and replacement). Furthermore, this technique allowed the suppression of inter-individual variability. Higher Cmax and AUC were obtained after the administration of all three formulations compared to the levels after the use of Sporanox® as follows: SOL/ITZ/AcDiSol®>SOL/ITZ/CD>SOL/ITZ>Sporanox®. The inversion in the ranking between SOL/ITZ/CD and SOL/ITZ/AcDiSol® made impossible the establishment of an in vitro-vivo correlation. Indeed, very different release rates were obtained in vitro and in vivo for the two optimized formulations. These results suggest that ITZ would be protected inside the core of the SOL micelles even during the absorption step at the intestine, while some agents present in the intestinal fluids could displace ITZ from the hydrophobic cavity of CD by competition.

Hot-melt extrusion as a continuous manufacturing process to form ternary cyclodextrin inclusion complexes.

The aim of this study was to evaluate hot-melt extrusion (HME) as a continuous process to form cyclodextrin (CD) inclusion complexes in order to increase the solubility and dissolution rate of itraconazole (ITZ), a class II model drug molecule of the Biopharmaceutics Classification System. Different CD derivatives were tested in a 1:1 (CD:ITZ) molar ratio to obtain CD ternary inclusion complexes in the presence of a polymer, namely Soluplus® (SOL). The CD used in this series of experiments were ß-cyclodextrin (ßCD), hydroxypropyl-ß-cyclodextrin (HPßCD) with degrees of substitution of 0.63 and 0.87, randomly methylated ß-cyclodextrin (Rameb®), sulfobutylether-ß-cyclodextrin (Captisol®) and methyl-ß-cyclodextrin (Crysmeb®). Rheology testing and mini extrusion using a conical twin screw mini extruder were performed to test the processability of the different CD mixtures since CD are not thermoplastic. This allowed Captisol® and Crysmeb® to be discarded from the study due to their high impact on the viscosity of the SOL/ITZ mixture. The remaining CD were processed by HME in an 18mm twin screw extruder. Saturation concentration measurements confirmed the enhancement of solubility of ITZ for the four CD formulations. Biphasic dissolution tests indicated that all four formulations had faster release profiles compared to the SOL/ITZ solid dispersion. Formulations of HPßCD 0.63 and Rameb® even reached 95% of ITZ released in both phases after 1h. The formulations were characterized using thermal differential scanning calorimetry and attenuated total reflectance infra-red analysis. These analyses confirmed that the increased release profile was due to the formation of ternary inclusion complexes.

Selection of a discriminant and biorelevant in vitro dissolution test for the development of fenofibrate self-emulsifying lipid-based formulations.

Fenofibrate, a BCS class II compound, has a low bioavailability especially when taken orally on an empty stomach. The challenge to find a new formulation for providing bioavailability, independent of food, is still ongoing. If the development of a suitable oral delivery formulation of BCS class II compounds is a frequent and great challenge to formulation scientists, the in vitro evaluation of these new formulations is also a great challenge. The purpose of this study was therefore to select an in vitro dissolution test that would be useful and as biorelevant as possible for the development of fenofibrate self-emulsifying lipid-based formulations. In this context, three different fenofibrate formulations, for which in vivo data are available in the literature, were tested using different dissolution tests until we found the one that was the most suitable. As part of this approach, we started with the simplest in vitro dissolution tests and progressed to tests that were increasingly more complex. The first tests were different single phase dissolution tests: a test under sink conditions based on the USP monograph, and different tests under non-sink conditions in non-biorelevant and biorelevant media. Given the inconclusive results obtained with these tests, biphasic dissolution systems were then tested: one with USP apparatus type II alone and another which combined USP apparatus types II and IV. This last combined test seemed the most suitable in vitro dissolution test for the development of the future fenofibrate lipid-based formulations we intend to develop in our own laboratory.

Investigation of a suitable in vitro dissolution test for itraconazole-based solid dispersions.

The difficulty to find a relevant in vitro dissolution test to evaluate poorly soluble drugs is a well-known issue. One way to enhance their aqueous solubility is to formulate them as amorphous solid dispersions. In this study, three formulations containing itraconazole (ITZ), a model drug, were tested in seven different conditions (different USP apparatuses and different media). Two of the formulations were amorphous solid dispersions namely Sporanox®, the marketed product, and extrudates composed of Soluplus® and ITZ produced by hot melt extrusion; and the last one was pure crystalline ITZ capsules. After each test, a ranking of the formulations was established. Surprisingly, the two amorphous solid dispersions exhibited very different behavior depending primarily on the dissolution media. Indeed, the extrudates showed a better release profile than Sporanox® in non-sink and in biphasic conditions, whilst Sporanox® showed a higher release profile than the extrudates in sink and fasted simulated gastric conditions. The disintegration, dynamic light scattering and nuclear magnetic resonance results highlighted the presence of interaction between the surfactants and Soluplus®, which slowed down the erosion of the polymer matrix. Indeed, the negative charge of sodium dodecyl sulfate (SDS) and bile salts interacted with the surface of the extrudates that formed a barrier through which the water hardly diffused. Moreover, Soluplus® and SDS formed mixed micelles in solution in which ITZ interacts with SDS, but no longer with Soluplus®. Regarding the biphasic dissolution test, the interactions between the octanol dissolved in the aqueous media disrupted the polymer--ITZ system leading to a reduced release of ITZ from Sporanox®, whilst it had no influence on the extrudates. All together these results pointed out the difficulty of finding a suitable in vitro dissolution test due to interactions between the excipients that complicates the prediction of the behavior of these solid dispersions in vivo.

Continuous production of itraconazole-based solid dispersions by hot melt extrusion: Preformulation, optimization and design space determination.

The purpose of this work was to increase the solubility and the dissolution rate of itraconazole, which was chosen as the model drug, by obtaining an amorphous solid dispersion by hot melt extrusion. Therefore, an initial preformulation study was conducted using differential scanning calorimetry, thermogravimetric analysis and Hansen's solubility parameters in order to find polymers which would have the ability to form amorphous solid dispersions with itraconazole. Afterwards, the four polymers namely Kollidon® VA64, Kollidon® 12PF, Affinisol® HPMC and Soluplus®, that met the set criteria were used in hot melt extrusion along with 25wt.% of itraconazole. Differential scanning confirmed that all four polymers were able to amorphize itraconazole. A stability study was then conducted in order to see which polymer would keep itraconazole amorphous as long as possible. Soluplus® was chosen and, the formulation was fine-tuned by adding some excipients (AcDiSol®, sodium bicarbonate and poloxamer) during the hot melt extrusion process in order to increase the release rate of itraconazole. In parallel, the range limits of the hot melt extrusion process parameters were determined. A design of experiment was performed within the previously defined ranges in order to optimize simultaneously the formulation and the process parameters. The optimal formulation was the one containing 2.5wt.% of AcDiSol® produced at 155°C and 100rpm. When tested with a biphasic dissolution test, more than 80% of itraconazole was released in the organic phase after 8h. Moreover, this formulation showed the desired thermoformability value. From these results, the design space around the optimum was determined. It corresponds to the limits within which the process would give the optimized product. It was observed that a temperature between 155 and 170°C allowed a high flexibility on the screw speed, from about 75 to 130rpm.

Understanding the impact of microcrystalline cellulose physicochemical properties on tabletability.

The quality by design (QbD) initiative is promoting a better understanding of excipient performance and the identification of critical material attributes (CMAs). Despite microcrystalline cellulose (MCC) being one of the most popular direct compression binders, only a few studies attempted identifying its CMAs. These studies were based either on a limited number of samples or on MCC produced on a small scale and/or in conditions that deviate from those normally encountered in production. The present work utilizes multivariate analyses first to describe a large database of MCCs produced on a commercial scale, including an overview of their physicochemical properties, and secondly to correlate the most significant material attributes with tabletability. Particle size and moisture content are often considered as the most common if not the sole CMAs with regard to MCC performance in direct compression. The evaluation of more than 80 neat MCCs and the performance of selected samples in a model formulation revealed the importance of other potential critical attributes such as tapped density. Drug product developers and excipient suppliers should work together to identify these CMAs, which may not always be captured by the certificate of analysis.

Optimization of a PGSS (particles from gas saturated solutions) process for a fenofibrate lipid-based solid dispersion formulation.

The aim of this study was to develop a formulation containing fenofibrate and Gelucire(®) 50/13 (Gattefossé, France) in order to improve the oral bioavailability of the drug. Particles from gas saturated solutions (PGSS) process was chosen for investigation as a manufacturing process for producing a solid dispersion. The PGSS process was optimized according to the in vitro drug dissolution profile obtained using a biphasic dissolution test. Using a design of experiments approach, the effects of nine experimental parameters were investigated using a PGSS apparatus provided by Separex(®) (Champigneulles, France). Within the chosen experimental conditions, the screening results showed that the drug loading level, the autoclave temperature and pressure, the connection temperature and the nozzle diameter had a significant influence on the dissolution profile of fenofibrate. During the optimization step, the three most relevant parameters were optimized using a central composite design, while other factors remained fixed. In this way, we were able to identify the optimal production conditions that would deliver the highest level of fenofibrate in the organic phase at the end of the dissolution test. The closeness between the measured and the predicted optimal dissolution profiles in the organic phase demonstrated the validity of the statistical analyses.

Microcrystalline cellulose, a direct compression binder in a quality by design environment--a review.

The ICH quality vision introduced the concept of quality by design (QbD), which requires a greater understanding of the raw material attributes, of process parameters, of their variability and their interactions. Microcrystalline cellulose (MCC) is one of the most important tableting excipients thanks to its outstanding dry binding properties, enabling the manufacture of tablets by direct compression (DC). DC remains the most economical technique to produce large batches of tablets, however its efficacy is directly impacted by the raw material attributes. Therefore excipients' variability and their impact on drug product performance need to be thoroughly understood. To help with this process, this review article gathers prior knowledge on MCC, focuses on its use in DC and lists some of its potential critical material attributes (CMAs).

Towards a real time release approach for manufacturing tablets using NIR spectroscopy.

The aim of this study was to use the near-infrared spectroscopy (NIRS) as a process analytical tool to evaluate the conformity of paracetamol tablets in terms of four Pharmacopoeia tests (content uniformity, hardness, disintegration time, friability) and to control in-line blend uniformity. Tablets were manufactured by direct compression. Three different active pharmaceutical ingredient (API) concentrations were manufactured and three different compaction pressures were used. Intact tablets were analysed by transmission mode with NIRS prior to European Pharmacopoeia tests that were used as reference methods. Partial least square (PLS) regression was selected to build the prediction NIR models for content uniformity, tablet hardness and disintegration time. The prediction of NIR content uniformity and tablet hardness methods were validated using the accuracy profile approach. The values of the root mean squared error of calibration (RMSEC) and the root mean squared error of prediction (RMSEP) for the disintegration time indicated the robustness and the global accuracy of the NIR model. Regarding the tablet friability test, the classification was based on K-nearest neighbours (KNN). Then tablet NIR analyses successfully allowed the prediction of their conformity. Compared to the time consuming Pharmacopoeia reference methods, the benefit of this nondestructive method is significant, especially for reducing batch release time.

PAT tools for the control of co-extrusion implants manufacturing process.

Hot melt extrusion is a novel pharmaceutical manufacturing process technique. In this study, we identified four Critical Quality Attributes (CQAs) of the implant manufacturing process by hot melt extrusion: the implant diameter, the quantity of the Active Pharmaceutical Ingredient (API), the homogeneity distribution of API and the thickness of the membrane. We controlled the implant diameter and the quantity of API in-line with a laser measurement, NIR and Raman spectroscopy, respectively. These two different spectroscopic techniques provided comparable results. In fact, the RMSEC and RMSECV were very close in each PAT technique but NIR spectroscopy was easier to use and less sensitive to external changes. For the control of the homogeneity of API distribution and the thickness of the membrane, we used successfully Raman spectroscopy imaging. These PAT tools help reducing analysis time.

Design space approach in the optimization of the spray-drying process.

From a quality by design perspective, the aim of the present study was to demonstrate the applicability of a Bayesian statistical methodology to identify the Design Space (DS) of a spray-drying process. Following the ICH Q8 guideline, the DS is defined as the "multidimensional combination and interaction of input variables (e.g., materials attributes) and process parameters that have been demonstrated to provide assurance of quality." Thus, a predictive risk-based approach was set up in order to account for the uncertainties and correlations found in the process and in the derived critical quality attributes such as the yield, the moisture content, the inhalable fraction of powder, the compressibility index, and the Hausner ratio. This allowed quantifying the guarantees and the risks to observe whether the process shall run according to specifications. These specifications describe satisfactory quality outputs and were defined a priori given safety, efficiency, and economical reasons. Within the identified DS, validation of the optimal condition was effectuated. The optimized process was shown to perform as expected, providing a product for which the quality is built in by the design and controlled setup of the equipment, regarding identified critical process parameters: the inlet temperature, the feed rate, and the spray flow rate.

Optimisation and validation of a fast HPLC method for the quantification of sulindac and its related impurities.

The European Pharmacopoeia describes a liquid chromatography (LC) method for the quantification of sulindac, using a quaternary mobile phase including chloroform and with a rather long run time. In the present study, a new method using a short sub-2 µm column, which can be used on a classical HPLC system, was developed. The new LC conditions (without chloroform) were optimised by means of a new methodology based on design of experiments in order to obtain an optimal separation. Four factors were studied: the duration of the initial isocratic step, the percentage of organic modifier at the beginning of the gradient, the percentage of organic modifier at the end of the gradient and the gradient time. The optimal condition allows the separation of sulindac and of its 3 related impurities in 6 min instead of 18 min. Finally, the method was successfully validated using an accuracy profile approach in order to demonstrate its ability to accurately quantify these compounds.

In vivo biocompatibility of three potential intraperitoneal implants.

The intraperitoneal biocompatibility of PDMS, polyHEMA and pEVA was investigated in rats, rabbits and rhesus monkeys. No inflammation was evidenced by hematological analyses and measurement of inflammatory markers throughout the experiment and by post-mortem examination of the pelvic cavity. After 3 or 6 months, histological analysis revealed fibrous tissue encapsulating PDMS and PEVA implants in all species and polyHEMA implants in rabbits and monkeys. Calcium deposits were observed inside polyHEMA implants. The intraperitoneal biocompatibility of all 3 polymers makes them suitable for the design of drug delivery systems, which may be of great interest for pathologies confined to the pelvic cavity.