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.

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.

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 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.

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.

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.

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.

Relationship between Age and the Ability to Break Scored Tablets.

BACKGROUND: Practical problems with the use of medicines, such as difficulties with breaking tablets, are an often overlooked cause for non-adherence. Tablets frequently break in uneven parts and loss of product can occur due to crumbling and powdering. Health characteristics, such as the presence of peripheral neuropathy, decreased grip strength and manual dexterity, can affect a patient's ability to break tablets. As these impairments are associated with aging and age-related diseases, such as Parkinson's disease and arthritis, difficulties with breaking tablets could be more prevalent among older adults. The objective of this study was to investigate the relationship between age and the ability to break scored tablets. METHODS: A comparative study design was chosen. Thirty-six older adults and 36 young adults were systematically observed with breaking scored tablets. Twelve different tablets were included. All participants were asked to break each tablet by three techniques: in between the fingers with the use of nails, in between the fingers without the use of nails and pushing the tablet downward with one finger on a solid surface. It was established whether a tablet was broken or not, and if broken, whether the tablet was broken accurately or not. RESULTS: The older adults experienced more difficulties to break tablets compared to the young adults. On average, the older persons broke 38.1% of the tablets, of which 71.0% was broken accurately. The young adults broke 78.2% of the tablets, of which 77.4% was broken accurately. Further analysis by mixed effects logistic regression revealed that age was associated with the ability to break tablets, but not with the accuracy of breaking. CONCLUSIONS: Breaking scored tablets by hand is less successful in an elderly population compared to a group of young adults. Health care providers should be aware that tablet breaking is not appropriate for all patients and for all drugs. In case tablet breaking is unavoidable, a patient's ability to break tablets should be assessed by health care providers and instructions on the appropriate method of breaking should be provided.

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.

Safe and effective pharmacotherapy in infants and preschool children: importance of formulation aspects.

Safe and effective paediatric pharmacotherapy requires careful evaluation of the type of drug substance, the necessary dose and the age-appropriateness of the formulation. Generally, the younger the child, the more the attention that is required. For decades, there has been a general lack of (authorised) formulations that children are able to and willing to take. Moreover, little was known on the impact of pharmaceutical aspects on the age-appropriateness of a paediatric medicine. As a result of legislative incentives, such knowledge is increasingly becoming available. It has become evident that rapidly dissolving tablets with a diameter of 2 mm (mini-tablets) can be used in preterm neonates and non-rapidly dissolving 2 mm mini-tablets in infants from 6 months of age. In addition, uncoated 4 mm mini-tablets can be used in infants from the age of 1 year. Also, there is some evidence that children prefer mini-tablets over a powder, suspension or syrup. Other novel types of age-appropriate oral formulations such as orodispersible films may further add to the treatment possibilities. This review provides an overview of the current knowledge on oral formulations for infants and preschool children, the advantages and disadvantages of the different types of dosage forms and the age groups by which these can likely be used.

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.

Quantitative LC-MS determination of liposomal encapsulated prednisolone phosphate and non-encapsulated prednisolone concentrations in murine whole blood and liver tissue.

The underlying pharmacokinetic profile of liposomal drug delivery systems is not yet fully known. This is primarily due to a lack of suitable quantitative bioanalytical methodology to simultaneously determine separate liposomal-encapsulated and non-encapsulated drug tissue concentrations in complex biological samples. Here, an LC-MS method was developed which enables the simultaneous quantification of separate liposomal-encapsulated prednisolone phosphate and non-encapsulated prednisolone concentrations in whole blood and liver tissue. Liquid chromatography, negative electrospray ionization and Orbitrap-MS analysis allowed highly accurate and sensitive detection of prednisolone phosphate (PP) and prednisolone (P) in complex matrix. Using dexamethasone phosphate and dexamethasone as internal standards, the quantitative LC-MS method was optimized and validated for high selectivity, sensitivity and quantitative accuracy of PP and P from liposomes. The lower limits of quantitation were 0.99µmol/L blood and 0.53nmol/g liver for PP, and 229nmol/L blood and 0.514nmol/g liver for P. Quantitative accuracies of 84-118% were observed. The intra-run precision was ≤11%. Application of this new LC-MS method will yield the first liposomal pharmacokinetic profile showing accurate encapsulated and non-encapsulated drug tissue concentrations separately. This is also the first quantitative LC-MS method for the simultaneous quantification of the prodrug PP and its parent drug P in whole blood and liver tissue samples.

Drug release from an oromucosal paste for the selective decontamination of the oropharynx (in ICU patients and healthy volunteers).

Selective oropharyngeal decontamination (SOD) is used in many ICUs in the Netherlands and some other European countries. While its clinical effect has been studied intensively, no studies have been done to assess the biopharmaceutical aspects of the paste, i.e. it is not known which local concentrations exist. For this study, five healthy volunteers were subjected to 400mg of the generally used paste. Ten ICU patients were treated according to the normal standard in the ICU of the University Medical Center Utrecht. Salivary levels of the various substances were measured over time using two separate analytical methods. Also the microbial burden of the oropharynx was assessed. The results show significant variation in release, both ICU patients and healthy volunteers. The antimicrobials tobramycin and colistin showed a relatively fast release, while nystatin exhibited a controlled release-like pattern. Amphotericin B is hardly released from the formulation. The concentration of the antimicrobial agents drop to sub-MIC levels relatively fast. From a biopharmaceutical perspective, amphotericin B should be replaced by nystatin. The application of the mouth paste is subject to massive variation in daily practice; each nurse applies a different amount, in a different way. In addition, the formulation is hard to apply and unpleasant with regards to the taste and feel for the conscious patients. This is not a clinical study, but a study that aimed to give a biopharmaceutical justification for SOD Both the clinical practice and the clinically determined levels of drugs enable critical evaluation of the outcome of clinical studies performed until now.

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.

The role of formulation on the pharmacokinetics of antiretroviral drugs.

INTRODUCTION: A multitude of antiretroviral drug formulations are now available for HIV-infected adults and children. These formulations include individual and co-formulated drugs, many of which are also supplied in generic versions. Many antiretroviral drugs have a low aqueous solubility and poor bioavailability. Drug formulation can significantly affect bioavailability, and given the increasing number of new formulations and drug combinations, it is important to be aware that formulation can influence the pharmacokinetics of antiretroviral drugs. AREAS COVERED: This review provides an overview of studies assessing the pharmacokinetics of different antiretroviral drug formulations in adults and children, including fixed-dose combinations. For some antiretroviral drugs, differences in pharmacokinetics have been described, with largest differences in exposure when a liquid formulation is compared to a tablet or capsule formulation. Biopharmaceutical properties of antiretroviral drugs relevant to bioavailability are discussed. EXPERT OPINION: Antiretroviral drug formulations and their excipients can significantly impact drug exposure. However, this is not yet fully recognized. It is important to realize that children use different formulations than adults. Effort should be made to ensure that adequate drug exposures are achieved to treat HIV-infected children. In addition, manipulation of drug formulations may lead to differences in pharmacokinetics.

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.

The development of a method to quantify encapsulated and free prednisolone phosphate in liposomal formulations.

This paper presents the development of a new method for the simple and reliable quantification of the free drug amount in liposomal preparations of prednisolone phosphate (PP). In this method the free drug is distinguished from the encapsulated drug by means of hydrolysis of the free PP into prednisolone (P) by alkaline phosphatase (AP). During method development reaction progress curves were recorded to determine the required AP concentration and the corresponding incubation time to achieve hydrolysis of all free PP. Reaction progress curves also showed that small changes in the amount of weighted AP and the incubation periods used do not cause a change in outcome. Further, several organic solvents were tested as precipitation solvent and the use of tetrahydrofuran (THF) yielded clean chromatograms, rapid AP deactivation and complete liposome rupture avoiding under- and overestimations of the encapsulated and free drug concentrations. Method accuracy was evaluated during a cross-validation involving dialysis. Intra- and interday precision were evaluated by determining the standard deviation (SD) and relative standard deviation (RSD) after applying the new method on one day (n=4) and on different days (n=3). The accuracy of the developed method is comparable to the accuracy determined by dialysis, while clearly the method using AP is more precise. In conclusion, comprehensive method development yielded an accurate and precise method, which can replace traditional methods like dialysis and solid phase extraction (SPE). With little effort the method can be upgraded and become part of the liposome certification prior to human use. The overall principle behind the method offers possibilities for many drug carrier systems.