Compression of Vectors for Small Interfering RNAs Delivery: Toward Oral Administration of siRNA Lipoplexes in Tablet Forms.

Currently, most nonviral nucleic acid vectors are in the form of colloidal suspensions administered primarily parenterally. This type of formulation and the mode of administration impose strong constraints such as the size of the administered vectors or the production of sterile preparations. The tablet form provides access to easy oral administration, well accepted by patients; As regards nucleic acid vectors, a dry form represents an advance in terms of stability. Using an optimized lipid-based small interfering RNA-delivery system, we studied the tabletability of a liquid suspension of these vectors. We optimized the conditions of freeze-drying by choosing excipients and process, allowing for the conservation of both the gene-silencing efficacy of the formulated siRNAs and the supramolecular structure of the lipid particulate system. Gene-silencing efficacy was assayed on luciferase-expressing cells and the structure of the siRNA vector in freeze-dried and tablet forms was examined using small-angle X-ray scattering (SAXS) synchrotron radiation. The freeze-dried powders were then mixed with excipients necessary for the good progress of the compression by allowing for a regular supply of the matrix and the reduction of friction. The compression was carried out using a rotary press simulator that allows for complete monitoring of the compression conditions. After compression, formulated siRNAs retained more than 60% of their gene-silencing efficacy. Within the tablets, a specific SAXS signal was detectable and the lamellar and cubic phases of the initial liquid suspension were restored after resuspension of siRNA vectors by disintegration of the tablets. These results show that the bilayer lipid structures of the particles were preserved despite the mechanical constraints imposed by the compression. If such a result could be expected after the freeze-drying step, it was never shown, to our knowledge, that siRNA-delivery systems could retain their efficacy and structure after mechanical stress such as compression. This opens promising perspectives to oral administration of siRNA as an alternative to parenteral administration.

An overview on dosage forms and formulation strategies for vaccines and antibodies oral delivery.

Most of biopharmaceuticals in clinical use today are available in a solution or suspension form and delivered by invasive routes (i.e. injection). However, several attempts have been made in order to develop effective oral formulations of 'biomolecules' characterized by a fragile structure and a low bioavailability. To achieve an efficient delivery of such molecules by non-parenteral route, in particular, via the oral route, novel concepts are needed not only to overcome significant enzymatic and diffusion barriers but also to ensure stability and biological activity. Vaccines and antibodies have a special interest as biomolecules because of their high therapeutic efficacy both in prevention and treatment of several chronic diseases. In this review, we would like to highlight the trends made in the development of pharmaceutical forms to deliver these molecules by the oral route. Hence, we will focus on the description of the different forms (solutions, suspensions, powders, tablets, micro and nanocarriers …) available today or under research study, in which product stability and efficacy are maintained. A special attention will be paid to the formulation strategies that may include the addition of several functional excipients and/or adjuvants, aiming to protect, to functionalize or to modulate their release in the body.

Tableting behavior of freeze and spray-dried excipients in pharmaceutical formulations.

Most of biopharmaceuticals, in their liquid form, are prone to instabilities during storage. In order to improve their stability, lyophilization is the most commonly used drying technique in the pharmaceutical industry. In addition, certain applications of biopharmaceutical products can be considered by oral administration and tablets are the most frequent solid pharmaceutical dosage form used for oral route. Thus, the tableting properties of freeze-dried products used as cryo and lyoprotectant could be a key element for future pharmaceutical developments and applications. In this study, we investigated the properties that might play a particular role in the specific compaction behavior of freeze-dried excipients. The tableting properties of freeze-dried trehalose, lactose and mannitol were investigated and compared to other forms of these excipients (spray-dried, commercial crystalline and commercial crystalline milled powders). The obtained results showed a specific behavior in terms of compressibility, tabletability and brittleness for the amorphous powders obtained after freeze-drying. The comparison with the other powders showed that this specific tableting behavior is linked to both the specific texture and the physical state (amorphization) of these freeze-dried powders.

Image analysis quantification of sticking and picking events of pharmaceutical powders compressed on a rotary tablet press simulator.

PURPOSE: The aim of this work was to develop a quantification method based on image analysis, able to characterize sticking during pharmaceutical tableting. Relationship between image analysis features and relevant mechanical parameters recorded on an instrumented tablet press simulator were investigated. METHODS: Image analysis, based on gray levels co-occurrence matrices (GLCM), generated textural features of the tablet surface. The tableting simulator (Stylcam® 200R, Medelpharm), instrumented with force and displacement transducers, provided accurate records. The tablet defects and compaction process parameters were studied using three pharmaceutical powders (Fast-Flo® lactose, anhydrous Emcompress® and Avicel® PH200 microcrystalline cellulose), five compression pressures (60 to 250 MPa), five lubricating levels, and three types of punches (standard steel, amorphous hard carbon and anti-sticking punches). RESULTS: Texture parameters made it possible to quantify with precision tablets' aspect. The selected parameter IC2 (Information on Correlation 2) plotted versus the ratio between the ejection shear stress (Esh) and the compression pressure (Cp) let appear a relevant knowledge space where it was possible to identify a normal and a degraded tableting mode. A positive link between those two parameters was shown. CONCLUSION: Since the Esh/Cp ratio was related to image analysis results, it proved to be an interesting defect tag.

Tableting properties of freeze-dried trehalose: Physico-chemical and mechanical investigation.

Freeze-drying of biopharmaceutical products is the method of choice in order to improve their stability and storage conditions. Such freeze-dried products are usually intended for parenteral route administration. However, many biopharmaceutical materials administered by parenteral route are used to treat local diseases particularly in the gastro-intestinal tract. Therefore, many studies concentrate nowadays their effort on developing alternative dosage forms to deliver biopharmaceutical molecules by the oral route. Tablets are the most popular solid pharmaceutical dosage form used for oral administration since they present many advantages, but poor informations are available on the possibility of tableting freeze-dried powders. In this study, we evaluate the compaction behavior of freeze-dried trehalose powder since trehalose is one of the most used cryo and lyoprotectant for the lyophilisation of biopharmaceutical entities. Results show that freeze-dried trehalose powder can be tableted while remaining amorphous and the obtained compacts present very specific properties in terms of compressibility, tabletability, brittleness and viscoelasticity compared to the crystalline trehalose and compared to classical pharmaceutical excipients.

Changes in the specific surface area of tablets composed of pharmaceutical materials with various deformation behaviors.

OBJECTIVE: The aim of this work is to study the effect of compaction on the specific surface area of tablets composed of various pharmaceutical materials (microcrystalline cellulose, lactose, and anhydrous calcium phosphate) compacted under seven degrees of compaction pressure. METHODS: In a first part, the influence of the deformation behavior of the compacted materials on the evolution of the specific surface area is observed. In a second part, the brittle and ductile abilities of the materials are calculated using the specific surface area values. The experimental results are used to calculate the number and the force of interparticulate bonds inside the tablet. RESULTS AND DISCUSSION: Tablets made of microcrystalline cellulose, which deform plastically, have specific surface areas that fall under pressure. In the case of lactose, the tablet specific surface area first increases to reach a maximum value at a pressure of 150 MPa. At higher pressure, however, the specific surface area decreases. The specific surface area of tablets composed of anhydrous calcium phosphate consistently increases, whatever the compaction pressure applied. Moreover, the evolution of the specific surface area is correlated with the tensile strength of the corresponding tablets. The number and the force of interparticulate bonds make it possible to classify the materials according to their deformation behavior and to quantify their ability to form cohesive tablets.

Polymorphic transformation of anhydrous caffeine under compression and grinding: a re-evaluation.

Polymorphic transformations that may occur during mechanical treatment are of great interest for the production of pharmaceutical solids. Anhydrous caffeine is a well-known example of such transformations but a careful reading of the already existing literature shows that published results are contradictory. In this study, both forms of caffeine, form I and form II, respectively metastable and stable at ambient pressure and temperature, were submitted to compression in an instrumented alternative press and to grinding, and were studied before and after treatment by X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC). Compression experiments showed no changes of form II during compression, whereas form I was partially transformed into form II. Nevertheless, this transformation did not happen immediately. Form II appeared after a kinetically slow transformation and was clearly detectable only after a few days, fact that was never mentioned by previous authors. Same phenomenon was observed after the grinding of form I but also after an extensive grinding of form II. DSC and XRPD measurements indicated that polymorphic transformation did not happen directly, but that a new intermediate form was obtained after mechanical treatment, which slowly turned into form II within a few days.

Fate of Tableted Freeze-Dried siRNA Lipoplexes in Gastrointestinal Environment.

The incorporation of siRNA into nanocarriers is mandatory to facilitate its intracellular delivery, as siRNA itself cannot enter cells. However, the incorporation of these nanocarriers into oral, solid dosage forms and their fate in the gastrointestinal environment is yet to be explored. In the present work, the fate of, (i) naked siRNA, (ii) freshly prepared siRNA lipoplexes, and (iii) tableted siRNA lipoplexes, in simulated gastric and intestinal fluids was studied. The siRNA, either released from or protected within the lipoplexes, was quantified by gel electrophoresis and siRNA efficacy was assessed in cell transfection. The freshly prepared lipoplexes kept their siRNA load and transfection efficiency totally preserved during 1 h of incubation in simulated gastric fluid at 37 °C. However, in simulated intestinal fluid, despite no release of siRNA from lipoplexes after 6 h of incubation, gene silencing efficacy was dramatically decreased even after 1 h of exposure. The lipoplexes obtained from tablets efficiently protected siRNA in simulated gastric fluid, thus preserving the gene silencing efficacy, whereas their incubation in simulated intestinal fluid resulted in a marked siRNA release and decreased gene silencing efficacy. These results provided a detailed explanation for understanding the fate of siRNA in gastrointestinal conditions, when simply loaded in lipoplexes or formulated in the form of tablets.

Anisotropic porous structure of pharmaceutical compacts evaluated by PGSTE-NMR in relation to mechanical property anisotropy.

PURPOSE: The pore space anisotropy of pharmaceutical compacts was evaluated in relation to the mechanical property anisotropy. METHODS: The topology and the pore space anisotropy were characterized by PGSTE-NMR measurements. Parallelepipedical compacts of anhydrous calcium phosphate (aCP) and microcrystalline cellulose (MCC) were tested on top, bottom and side faces. A microindentation and three-point single beam tests were used to measure Brinell hardness, tensile strength and Young's modulus. All the data were submitted to a statistical analysis to test for significance. RESULTS: The porous structure of MCC compacts was anisotropic, contrary to those of aCP. The analysis of the pore space by PGSTE-NMR method showed that its structural anisotropy was controlled by the behaviour under compaction of the excipients. At the same time, the Young's modulus and the tensile strength were the same whatever the direction of testing. For the aCP compacts, all the faces had the same Brinell hardness. With MCC compacts, only the bottom face showed a lower Brinell hardness. CONCLUSIONS: Except for Brinell hardness measured on MCC compacts, the tested samples were characterized by anisotropic mechanical properties when its porous structures were sometimes anisotropic. Then, there is not a straight link between porosity anisotropy and mechanical properties.

Application of PGSTE-NMR technique to characterize the porous structure of pharmaceutical tablets.

Direct compaction of pharmaceutical tablets is a complex process that results in a heterogeneous density distribution inside the compact. In the present study, we have used a non-invasive and non-destructive technique: the pulsed-gradient stimulated-echo (PGSTE) NMR method to access to topological information (connectivity, tortuosity) about the porous structure of the tablets obtained with three different pharmaceutical excipients: the microcrystalline cellulose, the lactose and the anhydrous calcium phosphate. These materials were chosen since their mechanical properties under pressure are highly differentiated. To probe the pore space with the PGSTE-NMR technique, the tablets were initially impregnated with silicone oil that is NMR sensitive (1H NMR). The time-dependent apparent self-diffusion coefficient was measured over a suitable range of diffusion time in the directions perpendicular and parallel to the compression axis, from which the tortuosity factor and the anisotropy of the porous structure can be studied. These results show that the porous structure varies with pressure and depends on the excipient behaviour under pressure. Then, this work demonstrates that PGSTE-NMR could be an alternative and a very interesting technique to obtain useful information on the structural properties of such compacted materials.

Application of percolation model to the tensile strength and the reduced modulus of elasticity of three compacted pharmaceutical excipients.

Percolation theory has been applied to several mechanical properties of pharmaceutical tablets. This power law describes the change of tablet's properties with the relative density. It defines critical tablet densities from which the mechanical properties start to change. The exponent in the law is expected to be universal for a mechanical property and numerical values are proposed in the literature. In this work, the percolation model was applied to the tensile strength and the reduced modulus of elasticity (obtained from surface indentation test) of three compacted pharmaceutical excipients (a microcrystalline cellulose, a lactose and an anhydrous calcium phosphate). Two approaches were proposed. First, the exponent was kept constant and equal to the values used in the literature (2.7 for the tensile strength and 3.9 for the reduced modulus of elasticity). Secondly, the critical tablet density (i.e. the percolation threshold) and the exponent were determined from the model. In the first approach, the percolation thresholds were higher than the relative tapped density. Using the second approach, the experimentally determined exponents were not close to the values of the literature and the critical relative densities were higher than the relative tapped density or equal to zero. Then, this study showed that the exponent seems not universal and that the model must be used carefully.

Effect of the Curvature of the Punches on the Shape of the Interface and the Delamination Tendency of Bilayer Tablets.

Bilayer tablets are of special interest in the pharmaceutical industry. The main problem during their manufacturing is the occurrence of delamination during or after the ejection from the die. This work studies the influence of using punches with a curvature on the interfacial strength and thus on the delamination tendency of bilayer tablets. Bilayer tablets were produced with a compaction simulator using different flat and concave punches with different radii of curvature. The main compaction pressure was kept constant but the tamping force was varied. Two bilayer model systems were studied. The interfacial strength was determined using a previously described indentation test. The factors studied were analyzed for statistical significance with respect to the responses. The curvature of the interface was found to be higher when the curvature of the punch and the tamping force increased. Breaking tests then demonstrated that, for bilayer tablets obtained using the same compression parameters, the interfacial strength was lower when the curvature of the interface increased. As a consequence, when producing bilayer tablets with concave punches, it is important to choose properly the tableting parameters in order to have an interface between the layers as flat as possible to avoid delamination issues.

Quantitative measurements of localized density variations in cylindrical tablets using X-ray microtomography.

Direct compaction is a complex process that results in a density distribution inside the tablets which is often heterogeneous. Therefore, the density variations may affect the compact properties. A quantitative analysis of this phenomenon is still lacking. Recently, X-ray microtomography has been successfully used in pharmaceutical development to study qualitatively the impact of tablet shape and break-line in the density of pharmaceutical tablets. In this study, we evaluate the density profile in microcrystalline cellulose (Vivapur 12) compacts obtained at different mean porosity (ranging from 7.7% to 33.5%) using X-ray tomography technique. First, the validity of the Beer-Lambert law is studied. Then, density calibration is performed and density maps of cylindrical tablets are obtained and visualized using a process with colour-scale calibration plot which is explained. As expected, important heterogeneity in density is observed and quantified. The higher densities in peripheral region were particularly investigated and appraised in regard to the lower densities observed in the middle of the tablet. The results also underlined that in the case of pharmaceutical tablets, it is important to differentiate the mechanical properties representative of the total volume tablet and the mechanical properties that only characterize the tablet surface like the Brinell hardness measurements.

Comparison of breaking tests for the characterization of the interfacial strength of bilayer tablets.

The bilayer tableting technology is gaining more acceptance in the drug industry, due to its ability to improve the drug delivery strategies. It is currently assessed by the European Pharmacopoeia, that the mechanical strength of tablets can be evaluated using a diametral breaking tester. This device applies a force diametrically, and records the tablet breaking point. This approach has been used to measure the structural integrity of single layer tablets as well as bilayer (and multi-layer) tablets. The latter ones, however, have a much complex structure. Therefore, testing a bilayer tablet with the currently used breaking test methodology might not be appropriate. The aim of this work was to compare results from several tests that have been proposed to quantify the interfacial strength of bilayer tablets. The obtained results would provide an indication on which tests are appropriate to evaluate the robustness of a bilayer tablet. Bilayer tablets were fabricated using a model formulation: Microcrystalline Cellulose (MCC) for the first layer, and spray dried lactose (SDLac) as second layer. Each set of tablets were tested using the following tests: Diametral Test, Shear Test and Indentation Test. The tablets were examined before and after the breaking test using Scanning Electron Microscopy (SEM). When a bilayer tablet was subjected to shearing or indentation, it showed signs of clear delamination. Differently, using the diametral test system, the tablets showed no clear difference, before and after the testing. However, when examining each layer via SEM, it was clear that a fracture occurred in the layer made of SDLac. Thus, the diametral test is a measure of the strength of one of the two layers and therefore it is not suited to test the mechanical strength of bilayer tablets.

Evolution of the Die-Wall Pressure during the Compression of Biconvex Tablets: Experimental Results and Comparison with FEM Simulation.

Capping is a classical manufacturing problem for tablets, which is known to affect more biconvex tablets than flat-faced ones. One reason could be the development of a higher residual die-wall pressure during unloading. Unfortunately, contradictory results were published on the subject. In this work, the evolution of the die-wall pressure during the compaction of biconvex tablets was studied experimentally and using finite element method (FEM) modeling. It was compared with the case of flat-faced tablets. Experimental and numerical results showed that during the compression of biconvex tablet, a lower maximum die-wall pressure and a higher residual die-wall pressure were obtained compared with the case of flat-faced tablet. Moreover, both approaches showed, for biconvex tablets, a temporary increase of the die-wall pressure at the end of the unloading phase. FEM demonstrated that this phenomenon was due to a gradual loss of contact between the punch and the tablet from the side to the center. This complex unloading behavior causes the temporary increase of the die-wall pressure and the development of a shear stress between the convex part and the land of the tablet. This could explain the capping tendency of biconvex tablets.

Investigating the effect of tablet thickness and punch curvature on density distribution using finite elements method.

Finite elements method was used to study the influence of tablet thickness and punch curvature on the density distribution inside convex faced (CF) tablets. The modeling of the process was conducted on 2 pharmaceutical excipients (anhydrous calcium phosphate and microcrystalline cellulose) by using Drucker-Prager Cap model in Abaqus(®) software. The parameters of the model were obtained from experimental tests. Several punch shapes based on industrial standards were used. A flat-faced (FF) punch and 3 convex faced (CF) punches (8R11, 8R8 and 8R6) with a diameter of 8mm were chosen. Different tablet thicknesses were studied at a constant compression force. The simulation of the compaction of CF tablets with increasing thicknesses showed an important change on the density distribution inside the tablet. For smaller thicknesses, low density zones are located toward the center. The density is not uniform inside CF tablets and the center of the 2 faces appears with low density whereas the distribution inside FF tablets is almost independent of the tablet thickness. These results showed that FF and CF tablets, even obtained at the same compression force, do not have the same density at the center of the compact. As a consequence differences in tensile strength, as measured by diametral compression, are expected. This was confirmed by experimental tests.

Compaction of crystallographic forms of pharmaceutical granular lactoses. II. Compacts mechanical properties.

It is well known that the choice of the crystal form affects the physicochemical properties such as compaction behaviour. In this work, the mechanical properties of compacts obtained from compaction of lactoses by using a micropress prototype are calculated. Tensile strength, Young's modulus, toughness and Brinell hardness were measured and used to compare the various crystalline forms: alpha-lactose monohydrate (LalphaM), anhydrous beta-lactose (LbetaA), anhydrous alpha-lactose (LalphaA) and partly amorphous lactose (FF). With all the mechanical properties measured, the lactoses could be differentiated. Then, the specific energy of failure G*(IC) was obtained from the toughness and the Young's modulus for each lactose. LalphaM showed small specific energy of failure due to its low toughness which is not balanced by its Young's modulus. The highest values were obtained with the two anhydrous forms, LalphaA and LbetaA. Finally, these mechanical properties were linked with general compaction behaviour and cohesive energy density which is a characterization at a molecular level.

Development of a new test for the easy characterization of the adhesion at the interface of bilayer tablets: proof-of-concept study by experimental design.

Although, adhesion at the interface of bilayer tablets is critical for their design it is difficult to characterize this adhesion between layers. In view of this, a new test with an easy implementation was proposed for the characterization of the interface of bilayer tablets. This work is presented as a proof-of-concept study to investigate the reliability of this new test with regard to the effects of some critical process parameters (e.g., compaction pressure applied on each layer) and material attributes (e.g., elasticity of the layered materials) on the interfacial adhesion of bilayer tablets. This was investigated using a design of experiment approach and the results obtained were in good accordance with those obtained with other tests and thus, confirms the potential of such a method for the measurement of the interfacial adhesion of bilayer tablets.

Study of the validity of the three-point bending test for pharmaceutical round tablets using finite element method modeling.

Mechanical strength is an important quality attribute of the tablets produced in the pharmaceutical industry. The three-point bending test is one of the methods described by the United States (US) pharmacopeia to test this property. In this work, finite element method modeling was perform to study the stress distribution in a round, flat tablet submitted to this test and to verify whether the equation given by US pharmacopeia to calculate the tensile strength could be used without restrictions. For this test, the center of the lower face of the tablet was submitted to the highest tensile stress and, at this point, the stress state was nearly uniaxial. This test is thus well suited to measure the tensile strength of pharmaceutical tablets. Moreover, simulations were performed with a large range of geometrical dimensions using the dimensionless parameters D/L and h/D (where D is the tablet diameter, h is the tablet thickness, and L is the distance between the supports). In order to obtain the value of the tensile strength with a good precision when using the equation given by the US pharmacopeia, the measurements should only be performed in a restricted area of the domain defined by D/L and h/D.

Comparison of different failure tests for pharmaceutical tablets: applicability of the Drucker-Prager failure criterion.

Several tests can be used to study the strength of pharmaceutical tablets. Equations exist in the literature to transform the failure force measured into a failure stress which can be considered as a characteristic of the strength of the material. For each failure test, the stress state at failure is different, and as a consequence, the failure stresses obtained are also different. It would thus be interesting to find a failure criterion to unify the different results. In this study four different tests were performed on pharmaceutical compacts of various densities: diametral compression, three-point flexure, biaxial flexure and uniaxial compressive tests. The Drucker-Prager criterion was tested as a possible fracture envelope. The results showed that this criterion is well suited to explain the failures obtained by diametral compression, three-point flexure and biaxial flexure. Nevertheless, for the uniaxial compressive test, the use of this criterion led to a significative underestimation of the experimental value of the failure stress. As a consequence, the Drucker-Prager criterion must be used with caution and is not able to explain all the failures that can occur in a pharmaceutical compact.