Butyrate Prevents Induction of CXCL10 and Non-Canonical IRF9 Expression by Activated Human Intestinal Epithelial Cells via HDAC Inhibition.

Non-communicable diseases are increasing and have an underlying low-grade inflammation in common, which may affect gut health. To maintain intestinal homeostasis, unwanted epithelial activation needs to be avoided. This study compared the efficacy of butyrate, propionate and acetate to suppress IFN-γ+/-TNF-α induced intestinal epithelial activation in association with their HDAC inhibitory capacity, while studying the canonical and non-canonical STAT1 pathway. HT-29 were activated with IFN-γ+/-TNF-α and treated with short chain fatty acids (SCFAs) or histone deacetylase (HDAC) inhibitors. CXCL10 release and protein and mRNA expression of proteins involved in the STAT1 pathway were determined. All SCFAs dose-dependently inhibited CXCL10 release of the cells after activation with IFN-γ or IFN-γ+TNF-α. Butyrate was the most effective, completely preventing CXCL10 induction. Butyrate did not affect phosphorylated STAT1, nor phosphorylated NFκB p65, but inhibited IRF9 and phosphorylated JAK2 protein expression in activated cells. Additionally, butyrate inhibited CXCL10, SOCS1, JAK2 and IRF9 mRNA in activated cells. The effect of butyrate was mimicked by class I HDAC inhibitors and a general HDAC inhibitor Trichostatin A. Butyrate is the most potent inhibitor of CXCL10 release compared to other SCFAs and acts via HDAC inhibition. This causes downregulation of CXCL10, JAK2 and IRF9 genes, resulting in a decreased IRF9 protein expression which inhibits the non-canonical pathway and CXCL10 transcription.

The availability of drug by liposomal drug delivery : Individual kinetics and tissue distribution of encapsulated and released drug in mice after administration of PEGylated liposomal prednisolone phosphate.

Lately, the usefulness of liposomal drug delivery systems has been debated. To better understand the underlying pharmacokinetics of the targeted drug delivery by liposomes, individual encapsulated and non-encapsulated drug concentrations in blood, tumor, liver, spleen and kidneys were quantified after i.v. administration of liposomal prednisolone phosphate in mice. Kinetic analysis shows that the tumor influx of encapsulated drug is not dominant compared to the uptake by the other tissues. Further, from a quantitative point of view, the availability of non-encapsulated drug in the tumor tissue after liposomal delivery is not pronounced as compared to the other tissues studied. However, drug release in the tumor seems more extended than in the other tissues and the non-encapsulated drug concentration decreases more slowly in the tumor than in the liver and spleen. The spleen shows a high affinity for the uptake of encapsulated drug as well as the release of drug from the liposomes. Subsequently, released drug in the spleen, and possibly also in other tissues, is probably quickly redistributed towards the blood and other tissues. This also impairs the drug delivery effect of the liposomes. In contrast to the released drug in the central circulation, liver and spleen, the released drug concentration in the tumor remains at a fairly constant level likely due to the extended release kinetics from the liposomes. These extended release characteristics in the tumor most probably contribute to the beneficial effect. Nevertheless, it should be noted that larger released drug concentrations are formed in healthy tissues.

Scale up of Semisolid Dosage Forms Manufacturing Based on Process Understanding: from Lab to Industrial Scale.

The scale up of production processes is a major challenge in pharmaceutical industry. Using a quality by design approach, upscaling can be based on the design space, which can be assessed on a small scale. In a previous study, the critical process parameters were identified by a definitive screening design on cetomacrogol ointment. In the current study, this lab scale (0.5 kg) study was scaled up to industrial scale (2000 kg, filling 100g tubes at 75 tubes/min). A similar trend for the influence of filling temperature on ointment yield stress was found for lab and industrial scale production. Furthermore, a process window for ointment filling viscosities was established. It was shown that between 26 and 170 Pa.s ointment could be filled into tubes with a low weight variation (< 0.5% RSD) resulting in a product with a yield stress that meets the pre-set criteria. This approach was subsequently verified using several creams and ointments and showed general applicability.

Elucidation of the variability in consistency of pharmacopoeia quality petrolatum.

The Pharmacopeia monograph for petrolatum poorly defines the material's physical properties. Indeed, differences between petrolatum grades can be substantial; yield stress varies between 65 and 280 Pa which can be compared with the consistency of respectively thin cream or thick ointment. This variation is not only due to differences in composition or refining process but also as a result of different processing; for example, thermal history influences petrolatum structure considerably. Slow cooling of petrolatum resulted in a yield stress of 26 Pa and fast cooling in 79 Pa. X-ray showed that crystallinity was 0.7% for the first cooling case and 1.5% for the second one. Crystallite size was estimated to be 20-50 nm. To investigate if this relatively small difference in crystallinity may induce the difference in consistency, 15 nm SiO2 particles were added to petrolatum. Indeed, a small increase in SiO2 concentration led to a major increase in yield stress. This was argued to be due to the small size of the particles, resulting in a large increase in absolute number of particles. The Pharmacopeia does not unambiguously define the pharmaceutical excipient petrolatum. As a consequence, the formulator has to take care of selecting the appropriate grade as well as to carefully control the processing of the material in order to achieve a consistent pharmaceutical product.

Plasma, blood and liver tissue sample preparation methods for the separate quantification of liposomal-encapsulated prednisolone phosphate and non-encapsulated prednisolone.

Besides the development of sample preparation methods for the determination of separate liposomal-encapsulated prednisolone phosphate and non-encapsulated prednisolone concentrations in murine plasma and blood, this article also presents the first description of an accurate sample preparation method for the determination of such separate concentrations in the murine liver. The quantitative differentiation is based on the immediate hydrolysis of prednisolone phosphate (PP) into prednisolone (P) after its release from the liposomes in vivo: PP represents the encapsulated drug, while P represents the non-encapsulated drug. The use of 10 ml methanol/g tissue during homogenization of liver tissue ensures complete liposome rupture, prevention of the dephosphorylation of PP released during homogenization, sufficient clean supernatants, excellent extraction of P and sufficient extraction of PP and excellent accuracies and precision complying with the internal guidelines for pre-clinical studies (80-120% and maximal 20%, respectively). Similarly, the matching sample preparation methods for plasma and blood involve protein precipitation with four equivalents of methanol also ensuring accuracies and precision complying with the internal guidelines for pre-clinical studies. Application of these sample preparation methods is going to generate the first pharmacokinetic (PK) profile of a liposomal preparation, in which the encapsulated and non-encapsulated drug concentrations in a tissue are measured separately. Such separated concentration profiles can gain important insights into the PKs of liposomal PP and probably also with regard to liposomal formulations in general, like the quantification of the in vivo drug release from the liposomes.

In vitro confirmation of the quantitative differentiation of liposomal encapsulated and non-encapsulated prednisolone (phosphate) tissue concentrations by murine phosphatases.

The quantitative differentiation of liposomal encapsulated and non-encapsulated drug tissue concentrations is desirable, since the efficacy and toxicity are only related to the level of non-encapsulated drug. However, such separate concentration profiles in tissues have still not been reported due to lacking analytical methodology. The encapsulation of prodrugs like prednisolone phosphate (PP) in liposomes offers new, analytical opportunities. Instantaneous dephosphorylation of PP into prednisolone (P) by phosphatases after its release from the liposome in vivo makes it possible to differentiate between the encapsulated and the non-encapsulated drug for such preparations of liposomal PP: PP represents the encapsulated drug, while P represents the non-encapsulated drug. In the here described study, the instantaneous dephosphorylation of PP by murine liver and kidney phosphatases has been verified by incubation of PP in liver and kidney homogenates followed by estimation of the dephosphorylation rate constants k and the dephosphorylation time of the expected maximal in vivo non-encapsulated drug concentrations. In vitro PP has been rapidly converted into P in the presence of homogenate from the excretory organs. The calculated values for k have shown that the liver contains more active sites per gram of tissue than the kidneys. However, the dephosphorylation of PP by these active sites is slower compared with the kidneys. Compared with other pharmacokinetic processes of P, the estimated dephosphorylation times of the expected maximal in vivo non-encapsulated drug concentrations in the liver and the kidneys are considered to be instantaneous. This enables the separate determination of the encapsulated and non-encapsulated drug concentrations in the excretory organs after administration of liposomal PP in mice generating the first pharmacokinetic profile of a liposomal preparation, in which the in vivo encapsulated and free drug tissues concentrations are measured separately. This can also gain important insights into the pharmacokinetics of liposomal formulations in general.

Quantitative characterization of agglomerate abrasion in a tumbling blender by using the Stokes number approach.

Removal of microcrystalline cellulose agglomerates in a dry-mixing system (lactose, 100 M) predominantly occurs via abrasion. The agglomerate abrasion rate potential is estimated by the Stokes abrasion (StAbr) number of the system. The StAbr number equals the ratio between the kinetic energy density of the moving powder bed and the work of fracture of the agglomerate. Basically, the StAbr number concept describes the blending condition of the dry-mixing system. The concept has been applied to investigate the relevance of process parameters on agglomerate abrasion in tumbling blenders. Here, process parameters such as blender rotational speed and relative fill volumes were investigated. In this study, the StAbr approach revealed a transition point between abrasion rate behaviors. Below this transition point, a blending condition exists where agglomerate abrasion is dominated by the kinetic energy density of the powder blend. Above this transition point, a blending condition exists where agglomerates show (undesirable) slow abrasion rates. In this situation, the blending condition is mainly determined by the high fill volume of the filler.

Butyrate Protects Barrier Integrity and Suppresses Immune Activation in a Caco-2/PBMC Co-Culture Model While HDAC Inhibition Mimics Butyrate in Restoring Cytokine-Induced Barrier Disruption.

Low-grade inflammation and barrier disruption are increasingly acknowledged for their association with non-communicable diseases (NCDs). Short chain fatty acids (SCFAs), especially butyrate, could be a potential treatment because of their combined anti-inflammatory and barrier- protective capacities, but more insight into their mechanism of action is needed. In the present study, non-activated, lipopolysaccharide-activated and αCD3/CD28-activated peripheral blood mononuclear cells (PBMCs) with and without intestinal epithelial cells (IEC) Caco-2 were used to study the effect of butyrate on barrier function, cytokine release and immune cell phenotype. A Caco-2 model was used to compare the capacities of butyrate, propionate and acetate and study their mechanism of action, while investigating the contribution of lipoxygenase (LOX), cyclooxygenase (COX) and histone deacetylase (HDAC) inhibition. Butyrate protected against inflammatory-induced barrier disruption while modulating inflammatory cytokine release by activated PBMCs (interleukin-1 beta↑, tumor necrosis factor alpha↓, interleukin-17a↓, interferon gamma↓, interleukin-10↓) and immune cell phenotype (regulatory T-cells↓, T helper 17 cells↓, T helper 1 cells↓) in the PBMC/Caco-2 co-culture model. Similar suppression of immune activation was shown in absence of IEC. Butyrate, propionate and acetate reduced inflammatory cytokine-induced IEC activation and, in particular, butyrate was capable of fully protecting against cytokine-induced epithelial permeability for a prolonged period. Different HDAC inhibitors could mimic this barrier-protective effect, showing HDAC might be involved in the mechanism of action of butyrate, whereas LOX and COX did not show involvement. These results show the importance of sufficient butyrate levels to maintain intestinal homeostasis.

The Stokes number approach to support scale-up and technology transfer of a mixing process.

Transferring processes between different scales and types of mixers is a common operation in industry. Challenges within this operation include the existence of considerable differences in blending conditions between mixer scales and types. Obtaining the correct blending conditions is crucial for the ability to break up agglomerates in order to achieve the desired blend uniformity. Agglomerate break up is often an abrasion process. In this study, the abrasion rate potential of agglomerates is described by the Stokes abrasion (St(Abr)) number of the system. The St(Abr) number equals the ratio between the kinetic energy density of the moving powder bed and the work of fracture of the agglomerate. In this study, the St(Abr) approach demonstrates to be a useful tool to predict the abrasion of agglomerates during blending when technology is transferred between mixer scales/types. Applying the St(Abr) approach revealed a transition point between parameters that determined agglomerate abrasion. This study gave evidence that (1) below this transition point, agglomerate abrasion is determined by a combination of impeller effects and by the kinetic energy density of the powder blend, whereas (2) above this transition point, agglomerate abrasion is mainly determined by the kinetic energy density of the powder blend.

Influence of commonly used excipients on the chemical degradation of enalapril maleate in its solid state: The role of condensed water.

The physicochemical stability of enalapril maleate was investigated in the presence of fourteen different excipients divided into four different classes. The extent of a drug-excipient interaction was investigated by following the chemical stability using HPLC. It was found that there is a certain order in the stability of enalapril maleate. Enalapril maleate remained most stable in the presence of: disaccharides > celluloses > starches > superdisintegrants. The amount of degradation can be related to the excipient characteristics. A material with a higher water sorption capacity and lower crystallinity presents a more reactive particle surface. It was revealed that the condensation layer deposited on the surface of the excipient is responsible for the degradation of enalapril maleate. A confirmation was found by changing the surface of the excipient and influencing the environmental humidity that allowed a variable build-up of the condensation layer. For this particle-particle interaction, the microenvironmental pH only presents a minor effect as it was found to not be a determining factor for degradation. Moreover, there appears to be a firm relationship between the degradation of enalapril maleate and the water sorption-activity of excipients.

Study on the Mechanism Responsible for the Incompatibility of Enalapril Maleate with Sodium Starch Glycolate.

Enalapril maleate (EM) is known to suffer from incompatibilities in the solid state. This study investigates the destabilizing effect of sodium starch glycolate (SSG) on EM. This was done by varying the mixing ratio and moisture content of binary mixtures. Differential scanning calorimetry and microscopy show a loss of crystallinity of EM at the contact surface with SSG. It is shown that this is followed by decomposition of E to diketopiperazine (DKP). These phenomena are modulated by moisture. The environmental pH turned out to be crucial; when the zwitterion is formed at the appropriate pH, ring closure into DKP is promoted.

The consequences of granulate heterogeneity towards breakage and attrition upon fluid-bed drying.

High-shear granulated lactose granulates were dried in a fluid-bed dryer at various conditions. Granules were characterized by water content and size analysis. It is shown that the drying process is very dynamic in terms of growth and breakage phenomena. Granular size heterogeneity, composition and water content determine the granule behavior upon drying. Large granules consist of small primary particles and contain more water than small granules that consist of large primary particles. This differentiates the drying rate and extent of size reduction of the different granule size classes. The results enable a critical evaluation of process control and process monitoring. Understanding of granule behavior and continuous monitoring of the fluid-bed drying process enables process and product optimization.

Using the internal stress concept to assess the importance of moisture sorption-induced swelling on the moisture transport through the glassy HPMC films.

The purpose of this research was to elucidate the significance of the changes in the mechanical and the volumetric properties on the moisture diffusivity through the polymer films. The internal stress concept was adapted and applied to estimate the relative impact of these property changes on the total stress experienced by a polymer film during storage. Hydroxypropyl Methylcellulose free films were used as a model material prepared at various conditions and stored at different relative humidities. The changes in the internal stress of these films due to the moisture sorption were studied. It was demonstrated that the stress-relaxation of the films increases at increasing moisture content. At the point when there is a definite loss of stress in the film, which is at moisture content higher than 6%, was shown to correlate with the significant increase of the moisture diffusivity. Further investigations revealed that the loss of stress is especially due to the swelling of the polymer rather than the changes in the inherent strain (the quotient between the tensile strength and the modulus of elasticity) of the HPMC films. This implies that the impact of the moisture sorption on the diffusivity is predominantly via volume addition rather than via altering the mechanical properties. Additionally, the approach presented here also brings up a new application of the internal stress concept, which in essence suggests the possibility to estimate the diffusion coefficient from the sorption isotherm and the mechanical analysis data.

Thermal stability study of crystalline and novel spray-dried amorphous nilotinib hydrochloride.

The thermal characteristics and the thermal degradation of crystalline and amorphous nilotinib hydrochloride (NH) were studied. The spray drying technique was successfully utilized for the amorphization of NH and was evaluated by spectroscopic techniques and differential scanning calorimetry (DSC). The ethanolic spray drying process yielded amorphous NH with a glass transition temperature (Tg) of 147°C. Thermal characterization of the amorphous phase was performed by heat capacity measurements using modulated DSC (mDSC). Thermal degradation was studied by thermogravimetric analysis (TGA). The derived thermodynamic properties of the amorphous NH indicate fragile behaviour and a low crystallization tendency. NH was found to be molecularly stable up to 193°C. After which, the thermal degradation displayed two phases. The values of the thermal degradation parameters were estimated using the Ozawa-Flynn-Wall and Friedman non-isothermal, model-free, isoconversional methods The results indicate the two phases to be single-step reactions. The examination of the physical stability of amorphous NH during storage and at elevated temperatures showed stability at 180°C for at least 5h and at 20-25°C/60% RH for at least 6 months. During these periods, no crystallization was observed. This study is the first to report the thermal characteristics of NH. Additionally, it is also the first to describe the full thermal analysis of a spray-dried amorphous drug. The thermal data may be used in the projection of future production processes and storage conditions of amorphous NH.

Granule breakage during drying processes.

The drying of wet granules often involves an unwanted and uncontrolled size reduction. Current FDA PAT guidance stresses importance of process control and understanding. The aim of this study is to determine and understand the breakage phenomena during drying processes in order to control these processes. High shear granulated lactose granules with water as binding liquid were dried during variable periods. Subsequently the (partially) dried granules were exposed to agitation by the impeller and chopper in the granulator. Granule characterization revealed that the change in granule size of (partially) dried granules is dependent on water content and follows a three phase system characterized by a growth, plateau and breakage phase. The derived yield stress of the granules is a function of velocity. From this it is concluded that in the plateau phase above minimum water content, stress behavior of granules can be described with Rumpfs' dynamic granule strength, whereas below minimum water content (breakage phase) granule strength is determined by the solid bridges. The extent and velocity of stress and water content of the granules during the process determine the size reduction phenomena.

Learning from patients: Identifying design features of medicines that cause medication use problems.

Usability is a key factor in ensuring safe and efficacious use of medicines. However, several studies showed that people experience a variety of problems using their medicines. The purpose of this study was to identify design features of oral medicines that cause use problems among older patients in daily practice. A qualitative study with semi-structured interviews on the experiences of older people with the use of their medicines was performed (n=59). Information on practical problems, strategies to overcome these problems and the medicines' design features that caused these problems were collected. The practical problems and management strategies were categorised into 'use difficulties' and 'use errors'. A total of 158 use problems were identified, of which 45 were categorized as use difficulties and 113 as use error. Design features that contributed the most to the occurrence of use difficulties were the dimensions and surface texture of the dosage form (29.6% and 18.5%, respectively). Design features that contributed the most to the occurrence of use errors were the push-through force of blisters (22.1%) and tamper evident packaging (12.1%). These findings will help developers of medicinal products to proactively address potential usability issues with their medicines.

The effect of crystal imperfections on particle fracture behaviour.

Micronisation of active pharmaceutical ingredients is a process which is sometimes difficult to control. The main purpose of this study was to assess the effect of the pre-existing flaws in the material to be milled. The rate of breakage of four samples of a model compound (sodium chloride), originating from different sources, was determined in a jet mill. It appeared that each type of sodium chloride has a distinct particle rate of breakage and breakage pattern. The numbers of flaws in the different types of sodium chloride have been determined by immersing the sodium chloride particles in a liquid with the same refractive index. This makes the cracks better visible. Microphotographs were made and flaws were counted manually. The study shows that the flaw density has an impact on the fracture behaviour of particles. The degree of fracture tends to increase with increasing flaw density. The paper shows however that the mechanical properties of the material as well as the starting particle size dominate the significance of the impact of flaws on fracture behaviour.

Pharmacokinetics in Elderly Women of Benzyl Alcohol From an Oil Depot.

Pharmaceutical oil depots are meant to release active substances at a sustained rate. Most of these depots contain benzyl alcohol (BOH) to facilitate the production and administration. Because BOH changes the solubility of components in both the body fluid and the oil formulation, it is relevant to know the change in the BOH concentration in the oil over time. In this study, volunteers were subcutaneously injected with an oil depot that contained 10% BOH, nandrolone decanoate, and cholecalciferol. The aim of this study was to determine the pharmacokinetic profiles of BOH and its metabolites benzoic acid and hippuric acid simultaneously in serum to estimate the BOH release out of the depot. For this, an HPLC bioassay was developed and adequately validated. Hereafter, the bioassay was applied to serum samples obtained at several time points between 0 and 35 days. BOH appeared immediately in serum after injection. The pharmacokinetic profile revealed that all BOH was depleted from the depot within 52 h after injection. Thus, the partition coefficient of active substances between the oil formulation and the body tissue changes rapidly in the first days after injection but will remain constant hereafter.

Fundamental understanding of drug absorption from a parenteral oil depot.

Oil depots are parenteral drug formulations meant for sustained release of lipophilic compounds. Until now, a comprehensive understanding of the mechanism of drug absorption from oil depots is lacking. The aim of this paper was to fill this gap. A clinical study with healthy volunteers was conducted. An oil depot with nandrolone decanoate and benzyl alcohol was subcutaneously administered in the upper arm of female volunteers. Pharmacokinetic profiles of both substances were related to each other and to literature data. Benzyl alcohol absorbs much more rapidly than nandrolone. In detail, it appears that benzyl alcohol enters the central compartment directly, while nandrolone decanoate is recovered in serum after a lag time. This lag time is also seen in literature data, although not reported explicitly. The absorption of nandrolone is enhanced by the presence of benzyl alcohol. This is most likely an effect of altered oil viscosity and partition coefficient between the oil and aqueous phase. The absorption rate constant of compounds is found to be related to the logP of the solubilized prodrug. The absorption rate is however not only determined by the physico-chemical properties of the formulation but also by the tissue properties. Here, it is argued that lymphatic flow must be considered as a relevant parameter.