Cranberry extract inhibits in vitro adhesion of F4 and F18+Escherichia coli to pig intestinal epithelium and reduces in vivo excretion of pigs orally challenged with F18+ verotoxigenic E. coli.

F4+E. coli and F18+E. coli infections are an important threat for pig industry worldwide. Antibiotics are commonly used to treat infected piglets, but the emerging development of resistance against antibiotics raises major concerns. Hence, alternative therapies to prevent pigs from F4+E. coli and F18+E. coli infections need to be developed. Since cranberry previously showed anti-adhesive activity against uropathogenic E. coli, we aimed to investigate whether cranberry extract could also inhibit binding of F4+E. coli and F18+E. coli to pig intestinal epithelium. Using the in vitro villus adhesion assay, we found that low concentrations of cranberry extract (20µg or 100µg/ml) have strong inhibitory activity on F4+E. coli (75.3%, S.D.=9.31 or 95.8%, S.D.=2.56, respectively) and F18+E. coli adherence (100% inhibition). This effect was not due to antimicrobial activity. Moreover, cranberry extract (10mg or 100mg) could also abolish in vivo binding of F4 and F18 fimbriae to the pig intestinal epithelium in ligated loop experiments. Finally, two challenge experiments with F18+E. coli were performed to address the efficacy of in-feed or water supplemented cranberry extract. No effect could be observed in piglets that received cranberry extract only in feed (1g/kg or 10g/kg). However, supplementation of feed (10g/kg) and drinking water (1g/L) significantly decreased excretion and diarrhea. The decreased infection resulted in a decreased serum antibody response indicating reduced exposure to F18+E. coli.

Genipin-crosslinked gelatin microspheres as a strategy to prevent postsurgical peritoneal adhesions: In vitro and in vivo characterization.

BACKGROUND: Peritoneal adhesions are a common complication after abdominal surgery. They cause small bowel obstruction, female infertility and chronic abdominal pain. Peritoneal adhesions also hamper uniform drug distribution in the peritoneal cavity, thereby reducing the efficacy of intraperitoneal chemotherapy after cytoreductive surgery. AIM: The goal of this study was to develop a formulation that prevents peritoneal adhesions, evenly distributes in the abdominal cavity, and simultaneously extends residence time and improves local drug concentration. This report describes the formulation and characterization of genipin-crosslinked gelatin microspheres (GP-MS). METHODS AND RESULTS: Spheroid gelatin microspheres were prepared by an emulsification solvent extraction method. A higher degree of crosslinking was obtained by increasing genipin concentration and crosslinking time. The degree of crosslinking allowed to tailor the degradation rate of GP-MS, hence their residence time. GP-MS did not affect cell viability. In vivo experiments showed excellent GP-MS biocompatibility and degradation characteristics. GP-MS were distributed evenly throughout the abdominal cavity. Adhesions were induced in Balb/c mice by application of an abraded peritoneal wall-cecum model. GP-MS-treated mice developed significantly less postsurgical adhesions compared to saline and Hyalobarrier(®) group. Histopathological examination showed a decrease of peritoneal inflammation over time in GP-MS-treated mice with complete recovery of peritoneal wounds post-operative day 14. CONCLUSION: GP-MS are a promising strategy to prevent postoperative peritoneal adhesions and improve efficacy of postoperative intraperitoneal chemotherapy.

Analysis of drug use in institutionalized individuals with intellectual disability and tube feeding.

OBJECTIVES: Little is known about the medication used by people with intellectual disabilities (ID) and enteral feeding tube (EFT). However, in light of the complexity associated with drug administration through EFT, data on medication use in this population may be helpful in the development of practical guidelines and staff training initiatives. METHODS: A cross-sectional, observational study was conducted in six Belgian residential care facilities (RCFs) for individuals with ID. Anonymized medication records of all residents receiving chronic medication through EFT were collected (n = 156). All chronic drugs were categorized according to the ATC classification, and medication records were checked for potential major drug-drug interactions (DDI). RESULTS: The 156 residents used a total of 1029 chronic drugs via EFT, with a median of six drugs per resident (range 1-14). A total of 148 different drug molecules were identified, belonging to 38 main ATC therapeutic groups (ATC level 2). Antiepileptics, drugs for constipation and drugs for acid-related disorders were the most frequently used groups. Seventy-four of the 156 screened medication records (47%) contained at least one potential DDI; in total, 116 potential interactions were identified, which represent 38 different interacting drug pairs. CONCLUSION: This study describes medication use through EFT among people with ID in Belgian RCFs, with antiepileptics being the most frequently used group. Our study also demonstrated that a high number of drugs is administered through EFT, and that the number of potential DDIs is high. These observations warrant an increased attention for drug administration through the EFT in individuals with ID.

Co-extrusion as a processing technique to manufacture a dual sustained release fixed-dose combination product.

OBJECTIVES: This study aimed to design a fixed-dose combination dosage form which provides a sustained release profile for both the freely water-soluble metformin HCl and the poorly soluble gliclazide, two antidiabetic compounds used to treat diabetes mellitus. METHODS: Hot-melt co-extrusion was used as an innovative manufacturing technique for a pharmaceutical fixed-dose combination product. In this way, a matrix formulation that sustained metformin release could be developed, despite the high drug load in the formulation and the freely soluble nature of the drug. KEY FINDINGS: It was clear that co-extrusion was perfectly suited to produce a fixed-dose combination product with adequate properties for each of the incorporated APIs. A coat layer, containing at least 30% CAPA(®) 6506 as a hydrophobic polymer, was necessary to adequately sustain the release of the highly dosed freely soluble drug from the 70% metformin HCl-loaded CAPA(®) 6506 core of the co-extrudate. To obtain a complete gliclazide release over 24-h solubilization in Kollidon(®) VA, added as a second polymer to the CAPA(®) 6506 in the coat, was needed. CONCLUSIONS: Both active pharmaceutical ingredients (APIs), which have different physicochemical characteristics, were formulated in a single dosage form, using co-extrusion.

Tumor-environment biomimetics delay peritoneal metastasis formation by deceiving and redirecting disseminated cancer cells.

Peritoneal metastasis is life threatening and is the result of an extensive communication between disseminated cancer cells, mesothelial cells and cancer-associated fibroblasts (CAF). CAFs secrete extracellular matrix (ECM) proteins creating a receptive environment for peritoneal implantation. Considering cancer as an ecosystem may provide opportunities to exploit CAFs to create biomimetic traps to deceive and redirect cancer cells. We have designed microparticles (MP) containing a CAF-derived ECM-surface that is intended to compete with natural niches. CAFs were encapsulated in alginate/gelatine beads (500-750 µm in diameter) functionalised with a polyelectrolyte coating (MP[CAF]). The encapsulated CAFs remain viable and metabolically active (≥35 days), when permanently encapsulated. CAF-derived ECM proteins are retained by the non-biodegradable coating. Adhesion experiments mimicking the environment of the peritoneal cavity show the selective capture of floating cancer cells from different tumor origins by MP[CAF] compared to control MP. MP[CAF] are distributed throughout the abdominal cavity without attachment to intestinal organs and without signs of inflammatory reaction. Intraperitoneal delivery of MP[CAF] and sequential removal redirects cancer cell adhesion from the surgical wound to the MP[CAF], delays peritoneal metastasis formation and prolongs animal survival. Our experiments suggest the use of a biomimetic trap based on tumor-environment interactions to delay peritoneal metastasis.

Understanding medication adherence among patients of Turkish descent with type 2 diabetes: a qualitative study.

OBJECTIVES: To explore perspectives of Turkish migrants with type 2 diabetes mellitus (T2DM) on adherence to oral hypoglycaemic agents (OHA). DESIGN: In-depth interviews with 21 T2DM patients of Turkish descent recruited from primary care and community sources in Ghent, Belgium, using a theoretical sampling procedure. Analysis was guided by a grounded theory approach, using Nvivo 8. RESULTS: Respondents reported a multitude of barriers and facilitators of adherence to OHA. Some of these barriers are distinctive for T2DM patients of Turkish descent. Respondents' causal beliefs about stress and the Belgian climate often led to non-adherence during less stressful periods, like summer holidays in Turkey. Some respondents adjusted their medication use to food intake or during Ramadan fasting. Concerns about OHA were the main reason for the widespread use of herbal medicine in this sample. The religious framework used to interpret diabetes led, in combination with feelings of depression, to non-adherence in some respondents while it facilitated medication adherence in others. A potential gender effect with respect to the self-management of OHA was observed. Non-distinctive factors include: beliefs about OHA, polypharmacy, beliefs about the course of diabetes, forgetfulness, the perception of the doctor's medical expertise, feelings of depression and social support. CONCLUSION: Health care providers should explore in detail and regularly patients' perspectives on illness beliefs, medication beliefs and their trust in doctors' medical expertise as this will provide useful starting points for promoting medication adherence. Whenever possible health care workers should engage with their patients in therapeutic alliances.

Experimental investigation of granule size and shape dynamics in twin-screw granulation.

A twin-screw granulator (TSG), a promising equipment for continuous high shear wet granulation (HSWG), achieves the desired level of mixing by a combination of the appropriate screw configuration and a suitable set of process settings (e.g. feed rate, screw speed, etc.), thus producing a certain granule size and shape distribution (GSSD). However, the primary sizing and shaping mechanism behind the resulting distribution is not well understood due to the opacity of the multiphase system in the granulator. This study experimentally characterised the GSSD dynamics along the TSG barrel length in order to understand the function of individual screw modules and process settings, as well as their interaction. Particle size analysis of granules collected at the outlet of the TSG suggested significant interaction between the process and screw configuration parameters influencing the heterogeneity in the GSSD. By characterising the samples collected along the screw length, a variable influence of the screw modules at different process conditions was observed. At low liquid-to-solid ratio (L/S), the first kneading module seemed to play a significant role in mixing, whereas the second kneading module was found to be more involved in reshaping the granules. At high L/S and high throughput, aggregation mainly took place in the second kneading module changing the GSSD. The results obtained from this study will be further used for the calibration and validation of a mechanistic model and, hence, support future development of a more detailed understanding of the HSWG process in a TSG.

Nanoporous polyelectrolyte vaccine microcarriers. A formulation platform for enhancing humoral and cellular immune responses.

In this paper we report on the design, characterization and immuno-biological evaluation of nanoporous polyelectrolyte microparticles as vaccine carrier. Relative to soluble antigen, formulation of antigen as a sub-10 µm particle can strongly enhance antigen-specific cellular immune responses. The latter is crucial to confer protective immunity against intracellular pathogens and for anti-cancer vaccines. However, a major bottleneck in microparticulate vaccine formulation is the development of generic strategies that afford antigen encapsulation under benign and scalable conditions. Our strategy is based on spray drying of a dilute aqueous solution of antigen, oppositely charged polyelectrolytes and mannitol as a pore-forming component. The obtained solid microparticles can be redispersed in aqueous medium, leading to leaching out of the mannitol, thereby creating a highly porous internal structure. This porous structure enhances enzymatic processing of encapsulated proteins. After optimizing the conditions to process these microparticles we demonstrate that they strongly enhance cross-presentation in vitro by dendritic cells to CD8 T cells. In vivo experiments in mice confirm that this vaccine formulation technology is capable of enhancing cellular immune responses.

Flatworm models in pharmacological research: the importance of compound stability testing.

Flatworms possess adult pluripotent stem cells, which make them extraordinary experimental model organisms to assess in vivo the undesirable effects of substances on stem cells. Currently, quality practices, implying evaluation of the stability of the test compound under the proposed experimental conditions, are uncommon in this research field. Nevertheless, performing a stability study during the rational design of in vivo assay protocols will result in more reliable assay results. To illustrate the influence of the stability of the test substance on the final experimental outcome, we performed a short-term International Conference on Harmonization (ICH)-based stability study of cyclophosphamide in the culture medium, to which a marine flatworm model Macrostomum lignano is exposed. Using a validated U(H)PLC method, it was demonstrated that the cyclophosphamide concentration in the culture medium at 20°C is lowered to 80% of the initial concentration after 21days. The multiwell plates, flatworms and diatoms, as well as light exposure, did not influence significantly the cyclophosphamide concentration in the medium. The results of the stability study have practical implications on the experimental set-up of the carcinogenicity assay like the frequency of medium renewal. This case study demonstrates the benefits of applying appropriate quality guidelines already during fundamental research increasing the credibility of the results.

A model based analysis of IPEC dosing of paclitaxel in rats.

PURPOSE: A strong pharmacokinetic rational exists for the use of (Hyperthermic) Intraperitoneal Perioperative Chemotherapy in peritoneal carcinomatosis. However, controversy remains regarding the optimal treatment strategies. Paclitaxel is believed to be a good compound for IPEC treatment because of its favourable pharmacokinetic properties. METHODS: Rat experiments were set up to gain insight in PTX's pharmacokinetics and pharmacodynamics after IPEC treatment with Taxol®. Afterwards a Pharmacokinetic-Pharmacodynamic model was developed, that concurrently describes plasma and tumour exposure post IPEC dosing. Moreover, the developed model adequately describes the time-course of tumour apoptosis as well as the treatment effect on tumour volume. RESULTS: We show that the complex absorption processes underlying PTX absorption from the peritoneal cavity post IPEC dosing, give rise to a markedly non-linear dose response relationship. Furthermore, we show that, in order to optimize treatment efficiency whilst concurrently minimizing the possibility of systemic toxicities, lowering the dose and extending exposure to the cytotoxic solution is the way forward. CONCLUSIONS: Based on the close resemblance between tumour exposure in our animal model and tumour exposure in patients treated under similar conditions, we hypothesise that, according to our findings in the rat, in the treatment of PC using IPEC administration of PTX, less is truly more.

Influence of raw material properties upon critical quality attributes of continuously produced granules and tablets.

Continuous manufacturing gains more and more interest within the pharmaceutical industry. The International Conference of Harmonisation (ICH) states in its Q8 'Pharmaceutical Development' guideline that the manufacturer of pharmaceuticals should have an enhanced knowledge of the product performance over a range of raw material attributes, manufacturing process options and process parameters. This fits further into the Process Analytical Technology (PAT) and Quality by Design (QbD) framework. The present study evaluates the effect of variation in critical raw material properties on the critical quality attributes of granules and tablets, produced by a continuous from-powder-to-tablet wet granulation line. The granulation process parameters were kept constant to examine the differences in the end product quality caused by the variability of the raw materials properties only. Theophylline-Lactose-PVP (30-67.5-2.5%) was used as model formulation. Seven different grades of theophylline were granulated. Afterward, the obtained granules were tableted. Both the characteristics of granules and tablets were determined. The results show that differences in raw material properties both affect their processability and several critical quality attributes of the resulting granules and tablets.

Effectiveness of pharmaceutical care for patients with chronic obstructive pulmonary disease (PHARMACOP): a randomized controlled trial.

AIMS: Few well-designed randomized controlled trials have been conducted regarding the impact of community pharmacist interventions on pharmacotherapeutic monitoring of patients with chronic obstructive pulmonary disease (COPD). We assessed the effectiveness of a pharmaceutical care programme for patients with COPD. METHODS: The pharmaceutical care for patients with COPD (PHARMACOP) trial is a single-blind 3 month randomized controlled trial, conducted in 170 community pharmacies in Belgium, enrolling patients prescribed daily COPD medication, aged ≥ 50 years and with a smoking history of ≥ 10 pack-years. A computer-generated randomization sequence allocated patients to an intervention group (n = 371), receiving protocol-defined pharmacist care, or a control group (n = 363), receiving usual pharmacist care (1:1 ratio, stratified by centre). Interventions focusing on inhalation technique and adherence to maintenance therapy were carried out at start of the trial and at 1 month follow-up. Primary outcomes were inhalation technique and medication adherence. Secondary outcomes were exacerbation rate, dyspnoea, COPD-specific and generic health status and smoking behaviour. RESULTS: From December 2010 to April 2011, 734 patients were enrolled. Forty-two patients (5.7%) were lost to follow-up. At the end of the trial, inhalation score [mean estimated difference (Δ),13.5%; 95% confidence interval (CI), 10.8-16.1; P < 0.0001] and medication adherence (Δ, 8.51%; 95% CI, 4.63-12.4; P < 0.0001) were significantly higher in the intervention group compared with the control group. In the intervention group, a significantly lower hospitalization rate was observed (9 vs. 35; rate ratio, 0.28; 95% CI, 0.12-0.64; P = 0.003). No other significant between-group differences were observed. CONCLUSIONS: Pragmatic pharmacist care programmes improve the pharmacotherapeutic regimen in patients with COPD and could reduce hospitalization rates.

Distribution of binder in granules produced by means of twin screw granulation.

According to the quality by design principle processes may not remain black-boxes and full process understanding is required. The granule size distribution of granules produced via twin screw granulation is often found to be bimodal. The aim of this study was to gain a better understanding of binder distribution within granules produced via twin screw granulation in order to investigate if an inhomogeneous spread of binder is causing this bimodal size distribution. Theophylline-lactose-polyvinylpyrrolidone K30 (PVP) (30-67.5-2.5%, w/w) was used as a model formulation. The intra-granular distribution of PVP was evaluated by means of hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy. For the evaluated formulation, no PVP rich zones were detected when applying a lateral spatial resolution of 0.5 µm, indicating that PVP is homogenously distributed within the granules.

Hydrogen bonded polymeric multilayer films assembled below and above the cloud point temperature.

Polymeric multilayer films assembled via hydrogen-bonding are witnessing increased interest from the scientific community. Here we report on hydrogen bonded multilayers of tannic acid and neutral poly(2-oxazoline)s. Importantly we demonstrate, to the best of our knowledge, for the first time that a temperature responsive polymer, in this case poly(2-(n-propyl)-2-oxazline), can be assembled below and above its TCP with distinctly different growth mechanisms.

Vaginal distribution and retention of tablets comprising starch-based multiparticulates: evaluation by colposcopy.

We have developed fast-disintegrating tablets comprising starch-based pellets and excipient granules for intravaginal drug delivery. The purpose of this study was to evaluate the intravaginal disintegration, distribution and retention behavior of these tablets in sheep and women using colposcopy as visualization technique. One tablet was administered to each study subject (n = 6) and repeated colposcopy examination was performed over a 48 h and 24 h period in sheep and women, respectively. Colposcopy in sheep indicated that in vivo tablet disintegration was initiated within 30 min of vaginal administration and that due to disintegration of the pellets themselves, the formulation was transformed into a gel-like mass which distributed throughout the entire vaginal cavity within 2-4 h. In vivo tablet disintegration after intravaginal administration to women was complete within 4 h, whereby the formulation gradually spread throughout the vaginal cavity as complete covering was observed after 12 and 24 h. The persistent retention (up to 24 and 48 h in women and sheep, respectively) confirmed the long retention time of this vaginal formulation.

One-step spray-dried polyelectrolyte microparticles enhance the antigen cross-presentation capacity of porcine dendritic cells.

Vaccination is regarded as the most efficient and cost-effective way to prevent infectious diseases. Vaccine design nowadays focuses on the implementation of safer recombinant subunit vaccines. However, these recombinant subunit antigens are often poor immunogens and several strategies are currently under investigation to enhance their immunogenicity. The encapsulation of antigens in biodegradable microparticulate delivery systems seems a promising strategy to boost their immunogenicity. Here, we evaluate the capacity of polyelectrolyte complex microparticles (PECMs), fabricated by single step spray-drying, to deliver antigens to porcine dendritic cells and how these particles affect the functional maturation of dendritic cells (DCs). As clinically relevant model antigen F4 fimbriae, a bacterial adhesin purified from a porcine-specific enterotoxigenic Escherichia coli strain was chosen. The resulting antigen-loaded PECMs are efficiently internalised by porcine monocyte-derived DCs. F4 fimbriae-loaded PECMs (F4-PECMs) enhanced CD40 and CD25 surface expression by DCs and this phenotypical maturation correlated with an increased secretion of IL-6 and IL-1ß. More importantly, F4-PECMs enhance both the T cell stimulatory and antigen presentation capacity of DCs. Moreover, PECMs efficiently promoted the CD8(+) T cell stimulatory capacity of dendritic cells, indicating an enhanced ability to cross-present the encapsulated antigens. These results could accelerate the development of veterinary and human subunit vaccines based on polyelectrolyte complex microparticles to induce protective immunity against a variety of extra- and intracellular pathogens.

Development of a nanocrystalline Paclitaxel formulation for HIPEC treatment.

PURPOSE: To develop a nanocrystalline paclitaxel formulation with a high paclitaxel-to-stabilizer ratio which can be used for hyperthermic intraperitoneal chemotherapy (HIPEC). METHODS: Paclitaxel (PTX) nanocrystals were prepared via wet milling using Pluronic F127(®) as stabilizer. The suitability of paclitaxel nanosuspensions for HIPEC treatment was evaluated by analyzing the cytotoxicity of both stabilizer and formulation, and by determining the maximum tolerated dose (MTD) and bioavailability. The effect on tumor growth was evaluated by magnetic resonance imaging (MRI) at day 7 and 14 after HIPEC treatment in rats with peritoneal carcinomatosis of ovarian origin. RESULTS: Monodisperse nanosuspensions (±400 nm) were developed using Pluronic F127(®) as single additive. The cytotoxicity and MTD of this nanocrystalline formulation was similar compared to Taxol(®), while its bioavailability was higher. MRI data after HIPEC treatment with a PTX nanocrystalline suspension showed a significant reduction of tumor volume compared to the non-treated group. Although no significant differences on tumor volume were observed between Taxol(®) and the nanosuspension, the rats treated with the nanosuspension recovered faster following the HIPEC procedure. CONCLUSION: Nanosuspensions with a high paclitaxel-to-stabilizer ratio are of interest for the treatment of peritoneal carcinomatosis of ovarian origin via HIPEC.

Engineered 3D microporous gelatin scaffolds to study cell migration.

Here we present a facile method to fabricate microporous hydrogel scaffolds that can be functionalized with a chemokine gradient. These scaffolds allow studying cellular responses in a 3D environment.

Surface-engineered polyelectrolyte multilayer capsules: synthetic vaccines mimicking microbial structure and function.

Immunizing: to evoke highly potent immune responses against recombinant antigens, hollow capsules consisting of layers of dextran sulphate and poly-L-arginine that encapsulate the antigen ovalbumin (orange circles) were coated with immune-activating CpG-containing oligonucleotides (green). These capsules were readily internalized by dendritic cells and showed activity in further immunization experiments.

Polymeric multilayer capsule-mediated vaccination induces protective immunity against cancer and viral infection.

Recombinant antigens hold high potential to develop vaccines against lethal intracellular pathogens and cancer. However, they are poorly immunogenic and fail to induce potent cellular immunity. In this paper, we demonstrate that polymeric multilayer capsules (PMLC) strongly increase antigen delivery toward professional antigen-presenting cells in vivo, including dendritic cells (DCs), macrophages, and B cells, thereby enforcing antigen presentation and stimulating T cell proliferation. A thorough analysis of the T cell response demonstrated their capacity to induce IFN-γ secreting CD4 and CD8 T cells, in addition to follicular T-helper cells, a recently identified CD4 T cell subset supporting antibody responses. On the B cell level, PMLC-mediated antigen delivery promoted the formation of germinal centers, resulting in increased numbers of antibody-secreting plasma cells and elevated antibody titers. The functional relevance of the induced immune responses was validated in murine models of influenza and melanoma. On a mechanistic level, we have demonstrated the capacity of PMLC to activate the NALP3 inflammasome and trigger the release of the potent pro-inflammatory cytokine IL-1ß. Finally, using DC-depleted mice, we have identified DCs as the key mediators of the immunogenic properties of PMLC.