Engineered phage with antibacterial CRISPR-Cas selectively reduce E. coli burden in mice.

Antibiotic treatments have detrimental effects on the microbiome and lead to antibiotic resistance. To develop a phage therapy against a diverse range of clinically relevant Escherichia coli, we screened a library of 162 wild-type (WT) phages, identifying eight phages with broad coverage of E. coli, complementary binding to bacterial surface receptors, and the capability to stably carry inserted cargo. Selected phages were engineered with tail fibers and CRISPR-Cas machinery to specifically target E. coli. We show that engineered phages target bacteria in biofilms, reduce the emergence of phage-tolerant E. coli and out-compete their ancestral WT phages in coculture experiments. A combination of the four most complementary bacteriophages, called SNIPR001, is well tolerated in both mouse models and minipigs and reduces E. coli load in the mouse gut better than its constituent components separately. SNIPR001 is in clinical development to selectively kill E. coli, which may cause fatal infections in hematological cancer patients.

A novel welfare and scientific approach to conducting dog metabolism studies allowing dogs to be pair housed.

Metabolism cages are designed to conduct absorption, distribution, metabolism and excretion (ADME) studies, enabling an 'excretion balance' scientific objective to be met. Historically, the design of dog metabolism cages has involved single housing. This type of housing has limitations for normal social behaviours and has been largely unchanged for 25-30 years. Improving animal welfare is a focus area for the authorities as well as the industry throughout the European Union. A collaboration was developed between Novo Nordisk and Covance to enhance the design of metabolism cages, allowing dogs to be pair housed. The purpose of the study was to compare excretion balance data from pair-housed and singly housed dogs in order to demonstrate that conducting excretion balance studies with a pair-housing design improves animal welfare without compromising the scientific integrity of the study. A radiolabelled test compound, [14C]-Quetiapine, was selected for this investigation based on its excretion profile. The assessment of the dogs' stress levels was investigated by measuring the levels of serum cortisol as an indicative biomarker. Results were inconclusive due to large variations in cortisol levels. However, dogs appeared calmer in the pair-housing setting. The overall mean recovery (±standard deviation) for pair-housed animals (94.0 ± 0.66% of the dose) was equivalent to that from singly housed dogs (93.0 ± 2.29%). Based on these data, we conclude that pair housing of dogs for future metabolism ADME studies does not compromise the scientific integrity, and therefore is a major progression in the design of these studies, enhancing welfare.

Synthesis and evaluation of zirconium-89 labelled and long-lived GLP-1 receptor agonists for PET imaging.

INTRODUCTION: Lately, zirconium-89 has shown great promise as a radionuclide for PET applications of long circulating biomolecules. Here, the design and synthesis of protracted and long-lived GLP-1 receptor agonists conjugated to desferrioxamine and labelled with zirconium-89 is presented with the purpose of studying their in vivo distribution by PET imaging. The labelled conjugates were evaluated and compared to a non-labelled GLP-1 receptor agonist in both in vitro and in vivo assays to certify that the modification did not significantly alter the peptides' structure or function. Finally, the zirconium-89 labelled peptides were employed in PET imaging, providing visual verification of their in vivo biodistribution. METHODS: The evaluation of the radiolabelled peptides and comparison to their non-labelled parent peptide was performed by in vitro assays measuring binding and agonistic potency to the GLP-1 receptor, physicochemical studies aiming at elucidating change in peptide structure upon bioconjugation and labelling as well as an in vivo food in-take study illustrating the compounds' pharmacodynamic properties. The biodistribution of the labelled GLP-1 analogues was determined by ex vivo biodistribution and in vivo PET imaging. RESULTS: The results indicate that it is surprisingly feasible to design and synthesize a protracted, zirconium-89 labelled GLP-1 receptor agonist without losing in vitro potency or affinity as compared to a non-labelled parent peptide. Physicochemical properties as well as pharmacodynamic properties are also maintained. The biodistribution in rats shows high accumulation of radiolabelled peptide in well-perfused organs such as the liver, kidney, heart and lungs. The PET imaging study confirmed the findings from the biodistribution study with a significant high uptake in kidneys and presence of activity in liver, heart and larger blood vessels. CONCLUSIONS AND ADVANCES IN KNOWLEDGE: This initial study indicates the potential to monitor the in vivo distribution of long-circulating incretin hormones using zirconium-89 based PET.

[Miliary tuberculosis in a patient with morbus Bechterew treated with tumour necrosis factor-alpha].

Tumour necrosis factor (TNF)-α inhibitors are registered for treatment of several severe dermatologic, rheumatologic and gastrointestinal diseases. Due to TNF-α's important role in the immune system patients have increased risk of serious infections during treatment with TNF-α inhibitor. We here present a case of miliary tuberculosis complicated by pericardial effusion in a Danish man treated with TNF-α inhibitor that emphasizes the need for rapid examination of new symptoms in this patient category.

[Boerhaave's syndrome can be a rare cause of tension pneumothorax]. / Boerhaaves syndrom kan være en sjælden årsag til trykpneumothorax.

We report a case of tension pneumothorax as an initial symptom caused by spontaneous rupture of the distal oesophagus (Boerhaave's syndrome). Prompt recognition of this potentially lethal syndrome is essential. The best way to diagnose the syndrome is by performing an oesophagram.

Bioavailability of seocalcitol III. Administration of lipid-based formulations to minipigs in the fasted and fed state.

The bioavailability of seocalcitol from two lipid-based formulations and a propylene glycol (PG) solution was studied in minipigs in the fasted and fed state. The lipid-based formulations were a medium chain triglyceride (MCT) solution and a self-microemulsifying drug delivery system (MC-SMEDDS) having a composition of 25% MCT, 48% cremophor RH 40, 27% akoline MCM. An IV solution was administered in order to determine the absolute bioavailability. In the fasted state the absolute bioavailability of seocalcitol was 15, 21 and 28% for the PG, MCT and MC-SMEDDS, respectively. The bioavailability from the PG solution was affected by the presence of food (29%), whereas the bioavailability from the lipid-based formulations was less affected by the presence of food; MCT (22%) and MC-SMEDDS (33%). The increased bioavailability from the PG solution in the fed state is believed to be due to the presence of lipids in the food. The present study illustrates an often mentioned beneficial effect of dosing lipid-based formulations; the reduced food effect on bioavailability. Previously published solubility data in simulated intestinal media relates very well to the present in vivo findings as the solubility studies showed that addition of lipids to the formulation could reduce/eliminate the difference in solubility between the fasted and fed state. Previously the same formulations were dosed to rats, resulting in a lower bioavailability from the MC-SMEDDS compared to the MCT. This illustrates that the animal model used should be carefully considered when studying formulations that are dependent on the dynamic processes in the GIT.

Bioavailability of seocalcitol IV: evaluation of lymphatic transport in conscious rats.

PURPOSE: To study the use of long chain triglycerides (LCT) as a lymphotropic carrier of (3)H-seocalcitol by comparing the lymphatic transport and the portal absorption of (3)H-seocalcitol when dissolved in a (1) LCT solution or a (2) reference solution without lipid containing propylene glycol (PG). MATERIALS AND METHODS: A lymph cannulated conscious rat model was dosed orally with (3)H-seocalcitol dissolved in either LCT or PG. Lymph was collected continuously, and blood was sampled over 9 h. (3)H-seocalcitol in blood and lymph and triglycerides in lymph were analysed. RESULTS: A statistically significantly (p < 0.05) higher recovery of the dosed (3)H-seocalcitol was found in the intestinal lymph upon administration of the LCT solution (1.3 +/- 0.6%) compared to the PG solution (0.5 +/- 0.4%). The portal absorption of (3)H-seocalcitol was significantly (p < 0.05) higher from the LCT solution (16.2 +/- 2.2%) than from the PG solution (10.8 +/- 0.8%). CONCLUSIONS: The LCT solution resulted in a statistical significantly higher level of lymphatic and portal transport of (3)H-seocalcitol compared with the PG solution. However, even though LCT facilitates the formation of chylomicrons, (3)H-seocalcitol favours absorption directly to the portal blood probably due to the moderate lipophilicity of the molecule.

Bioavailability of seocalcitol II: development and characterisation of self-microemulsifying drug delivery systems (SMEDDS) for oral administration containing medium and long chain triglycerides.

By constructing ternary phase diagrams it was possible to identify two self-microemulsifying drug delivery systems (SMEDDS) containing either medium chain triglycerides (MC-SMEDDS) or long chain triglycerides (LC-SMEDDS), with the same ratio between lipid, surfactant and co-surfactant. The SMEDDS ended up having a composition of 25% lipid, 48% surfactant and 27% co-surfactant, MC-SMEDDS: viscoleo, cremophor RH40, akoline MCM and LC-SMEDDS: sesame oil, cremophor RH40, peceol. Upon dilution with water both SMEDDS resulted in clear to bluish transparent microemulsions with a narrow droplet size of 30nm. The industrial usefulness of the developed SMEDDS was evaluated with regard to bioavailability and chemical stability using the vitamin D analogue, seocalcitol, as model compound. The absorption and bioavailability of seocalcitol in rats were approximately 45% and 18%, respectively, from both the MC-SMEDDS and LC-SMEDDS indicating similar in vivo behavior of the two formulations, despite the difference in nature of lipid component. There was no improvement in bioavailability by the use of SMEDDS, compared to the bioavailability achieved from simple MCT and LCT solutions (22-24%) (Grove, M., Pedersen, G.P., Nielsen, J.L., Mullertz, A., 2005. Bioavailability of seocalcitol. I. Relating solubility in biorelevant media with oral bioavailability in rats-effect of medium and long chain triglycerides. J. Pharm. Sci. 94, 1830-1838.). After 3 months' storage at accelerated conditions (40 degrees C/75% RH), a decrease in concentration of seocalcitol of 10-11% was found in MC-SMEDDS and LC-SMEDDS compared with a degradation of less than 3% for the simple lipid solutions of MCT and LCT. In this study the simple lipid solutions seem to be a better choice compared with the developed SMEDDS due to a slightly higher bioavailability and a better chemical stability of seocalcitol.

Bioavailability of seocalcitol I: Relating solubility in biorelevant media with oral bioavailability in rats--effect of medium and long chain triglycerides.

Simulated intestinal media (SIM) containing bile salt (BS) and phospholipids (PL) with and without medium chain lipolytic products (MC-LP) or long chain lipolytic products (LC-LP) were developed to study the solubility of seocalcitol. Both MC-LP and LC-LP were studied in order to investigate the influence of fatty acid chain length on the in vitro solubility of seocalcitol. The same solubility of seocalcitol was found in media containing either MC-LP or LC-LP. The bioavailability after oral administration of seocalcitol dissolved in medium chain triglyceride (MCT), long chain triglyceride (LCT), and a reference formulation containing propylene glycol (PG) was studied in vivo in rats. The lipid formulations showed a twofold increase in bioavailability compared with the reference formulation, indicating positive effects of lipids on the bioavailability reflecting a better solubility in the intestine and protection against precipitation of seocalcitol in the gastro intestinal tract. There was no difference in the in vivo bioavailability of seocalcitol between the MCT and the LCT solutions, which correlates with the identical in vitro solubility of seocalcitol in SIM containing MC-LP or LC-LP.