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IntroductionAsthma is a heterogeneous disease with poorly defined phenotypes. Patients with severe asthma often receive multiple treatments including oral corticosteroids (OCS). Treatment may modify the observed metabotype, rendering it challenging to investigate underlying disease mechanisms. Here, we aimed to identify dysregulated metabolic processes in relation to asthma severity and medication.MethodsBaseline urine was collected prospectively from healthy participants (n=100), patients with mild-to-moderate asthma (n=87) and patients with severe asthma (n=418) in the cross-sectional U-BIOPRED cohort; 12–18-month longitudinal samples were collected from patients with severe asthma (n=305). Metabolomics data were acquired using high-resolution mass spectrometry and analysed using univariate and multivariate methods.ResultsA total of 90 metabolites were identified, with 40 significantly altered (p−20), OCS-treated asthmatic patients differed significantly from non-treated patients (p=9.5×10−4), and longitudinal metabotypes demonstrated temporal stability. Carnitine levels evidenced the strongest OCS-independent decrease in severe asthma. Reduced carnitine levels were associated with mitochondrial dysfunction via decreases in pathway enrichment scores of fatty acid metabolism and reduced expression of the carnitine transporter SLC22A5 in sputum and bronchial brushings.ConclusionsThis is the first large-scale study to delineate disease- and OCS-associated metabolic differences in asthma. The widespread associations with different therapies upon the observed metabotypes demonstrate the need to evaluate potential modulating effects on a treatment- and metabolite-specific basis. Altered carnitine metabolism is a potentially actionable therapeutic target that is independent of OCS treatment, highlighting the role of mitochondrial dysfunction in severe asthma.

The rat single-pass intestinal perfusion (SPIP) model is commonly used to investigate gastrointestinal physiology and membrane drug transport. The SPIP model can be used with the intestinal segment inside or outside the abdomen. The rats can also be treated with parecoxib, a selective cycloxygenase-2 inhibitor that has been shown to affect some intestinal functions following abdominal surgery, such as motility, epithelial permeability, fluid flux and ion transport. However, the impact of extra-abdominal placement of the intestinal segment in combination with parecoxib on intestinal drug transport has not been investigated. There is also uncertainty how well intestinal permeability determinations based on luminal drug disappearance and plasma appearance correlate in the rat SPIP model. The main objective of this rat in vivo study was to investigate the effect of intra- vs. extra abdominal SPIP, with and without, pretreatment with parecoxib. The effect was evaluated by determining the difference in blood-to-lumen Cr-51-EDTA clearance, lumen-to-blood permeability of a cassette-dose of four model compounds (atenolol, enalaprilat, ketoprofen, and metoprolol), and water flux. The second objective was to compare the jejunal permeability values of the model drugs when determined based on luminal disappearance or plasma appearance. The study showed that the placement of the perfused jejunal segment, or the treatment with parecoxib, had minimal effects on membrane permeability and water flux. It was also shown that intestinal permeability of low permeability compounds should be determined on the basis of data from plasma appearance rather than lumina] disappearance. If permeability is calculated on the basis of luminal disappearance, it should preferably include negative values to increase the accuracy in the determinations.

While bronchodilators and inhaled corticosteroids are the mainstay of asthma treatment, up to 50% of asthmatics remain uncontrolled. Many studies show that the cysteinyl leukotriene cascade remains highly activated in some asthmatics, even those on high-dose inhaled or oral corticosteroids. Hence, inhibition of the leukotriene C4 synthase (LTC4S) enzyme could provide a new and differentiated core treatment for patients with a highly activated cysteinyl leukotriene cascade. Starting from a screening hit (3), a program to discover oral inhibitors of LTC4S led to (1S,2S)-2-({5-[(5-chloro-2,4-difluorophenyl)(2-fluoro-2-methylpropyl)amino]-3-methoxypyrazin-2-yl}carbonyl)cyclopropanecarboxylic acid (AZD9898) (36), a picomolar LTC4S inhibitor (IC50 = 0.28 nM) with high lipophilic ligand efficiency (LLE = 8.5), which displays nanomolar potency in cells (peripheral blood mononuclear cell, IC50,free = 6.2 nM) and good in vivo pharmacodynamics in a calcium ionophore-stimulated rat model after oral dosing (in vivo, IC50,free = 34 nM). Compound 36 mitigates the GABA binding, hepatic toxicity signal, and in vivo toxicology findings of an early lead compound 7 with a human dose predicted to be 30 mg once daily.

Background and purpose Human pharmacokinetic studies of lung-targeted drugs are typically limited to measurements of systemic plasma concentrations, which provide no direct information on lung target-site concentrations. We aimed to evaluate lung pharmacokinetics of commonly prescribed drugs by sampling different lung compartments after inhalation and oral administration. Experimental approach Healthy volunteers received single, sequential doses of either inhaled salbutamol, salmeterol, and fluticasone propionate (n=12), or oral salbutamol and propranolol (n=6). Each participant underwent bronchoscopies and gave breath samples for analysis of particles in exhaled air at two time points after drug administration (1 and 6, 2 and 9, 3 and 12, or 4 and 18 hours). Lung samples were taken via bronchosorption, bronchial brush, mucosal biopsy, and bronchoalveolar lavage during each bronchoscopy. Blood samples were taken during the 24 hours after administration. Pharmacokinetic profiles were generated by combining data from multiple individuals, covering all sample timings. Key results Pharmacokinetic profiles were obtained for each drug in lung epithelial lining fluid, lung tissue, and plasma. Inhalation of salbutamol resulted in approximately 100-fold higher concentrations in lung versus plasma. Salmeterol and fluticasone concentration ratios in lung versus plasma were higher still. Bronchosorption- and bronchoalveolar-lavage-generated profiles of inhaled drugs in epithelial lining fluid were comparable, whereas, for oral drugs, epithelial-lining-fluid concentrations appeared to be overestimated in bronchoalveolar-lavage-generated profiles. Conclusion and implications Combining pharmacokinetic data derived from multiple individuals and techniques sampling different lung compartments enabled generation of pharmacokinetic profiles for evaluation of lung targeting after inhaled and oral drug delivery.

Background: The lower respiratory tract of the landrace pig has close anatomical and physiological similarities with that of the human, and hence, for inhalation studies this species is well suited for biopharmaceutical research. Methods: The objective of this study was to evaluate pharmacokinetics in pigs following one dose of Diskus™ Seretide™ forte device, labeled 500/50 fluticasone propionate (FP) and salmeterol xinafoate (SX), respectively. The PreciseInhale™ (PI) instrument was used to actuate the inhaler for in vitro testing and aerosol dosing to pigs. In vitro, the aerosol was characterized with a cascade impactor with respect to mass median aerodynamic diameter, geometric standard deviation, and fine particle dose. In vivo, dry powder inhalation exposure was delivered as a short bolus dose, to anesthetized and mechanically ventilated landrace pigs. In addition to plasma PK, PK assessment of airway epithelial lining fluid (ELF) was used in this study. ELF of the depth of three to fourth airway generation of the right lung was accessed using standard bronchoscopy and a synthetic absorptive matrix. Results and Conclusions: Dry powder inhalation exposures with good consistency and well characterized aerosols to the pig lung were achieved by the use of the PreciseInhale™ instrument. Drug concentrations of ELF for both FP and SX were demonstrated to be four to five orders of magnitude higher than its corresponding systemic plasma drug concentrations. Clinical PK following inhalation of the same dose was used as benchmark, and the clinical study did demonstrate similar plasma PK profiles and drug exposures of both FP and SX as the current pig study. Two factors explain the close similarity of PK (1) similiar physiology between species and (2) the consistency of dosing to animals. To conclude, our study demonstrated the utility and translational potential of conducting PK studies in pigs in the development of inhaled pharmaceuticals.

The relevance of the rat single-pass intestinal perfusion model for investigating in vivo time-dependent effects of absorption-modifying excipients (AMEs) is not fully established. Therefore, the dynamic effect and recovery of the intestinal mucosa was evaluated based on the lumen-to-blood flux (Jabs) of six model compounds, and the blood-to-lumen clearance of 51Cr-EDTA (CLCr), during and after 15- and 60-min mucosal exposure of the AMEs, sodium dodecyl sulfate (SDS) and chitosan, in separate experiments. The contribution of enteric neurons on the effect of SDS and chitosan was also evaluated by luminal coadministration of the nicotinic receptor antagonist, mecamylamine. The increases in Jabs and CLCr (maximum and total) during the perfusion experiments were dependent on exposure time (15 and 60 min), and the concentration of SDS, but not chitosan. The increases in Jabs and CLCr following the 15-min intestinal exposure of both SDS and chitosan were greater than those reported from an in vivo rat intraintestinal bolus model. However, the effect in the bolus model could be predicted from the increase of Jabs at the end of the 15-min exposure period, where a six-fold increase in Jabs was required for a corresponding effect in the in vivo bolus model. This illustrates that a rapid and robust effect of the AME is crucial to increase the in vivo intestinal absorption rate before the yet unabsorbed drug in lumen has been transported distally in the intestine. Further, the recovery of the intestinal mucosa was complete following 15-min exposures of SDS and chitosan, but it only recovered 50% after the 60-min intestinal exposures. Our study also showed that the luminal exposure of AMEs affected the absorptive model drug transport more than the excretion of 51Cr-EDTA, as Jabs for the drugs was more sensitive than CLCr at detecting dynamic mucosal AME effects, such as response rate and recovery. Finally, there appears to be no nicotinergic neural contribution to the absorption-enhancing effect of SDS and chitosan, as luminal administration of 0.1 mM mecamylamine had no effect.

Background Antibiotic treatment is a well-known risk factor for healthcare facility-associated Clostridioides (Clostridium) difficile infection (HCF-CDI). Antibiotic stewardship programmes (ASPs) targeting high-risk antibiotics have been shown to decrease HCF-CDI incidence. HCF-CDI incidence is high in Nordic countries despite relatively low antibiotic use in hospital. Objectives To determine if HCF-CDI incidence was modified by a hospital ASP that restricted cephalosporin use. Methods The effects of an ASP on HCF-CDI incidence were evaluated in a two-centre setting using a retrospective design. We exploited a strategy of both individual case ascertainment based on chart reviews and aggregated data from the hospitals. Cases were attributed to the antibiotics given prior to disease onset, in proportion to the number of DDDs used. Three periods were studied: 2007 (before the ASP), 2012 and 2015. Results At the ASP hospital, cephalosporin use decreased by 87% and the number of HCF-CDI/1000 hospital admissions decreased significantly from 2.25 (2007) to 1.16 (2015) (P = 0.0014). The corresponding results at the non-ASP hospital showed a non-significant increase from 2.09 to 2.38. A high number of cases could be attributed to cephalosporins at both hospitals. The increased use of other broad-spectrum antibiotics, e.g. piperacillin/tazobactam, at the ASP hospital was not associated with offsetting increases in attributable HCF-CDI cases. Conclusions Decreased use of cephalosporins is an effective strategy to decrease HCF-CDI incidence over time in a setting with high incidence and low antibiotic use.

Based on expert group work, Swedish recommendations for the management of community-acquired pneumonia in adults are here updated. The management of sepsis-induced hypotension is addressed in detail, including monitoring and parenteral therapy. The importance of respiratory support in cases of acute respiratory failure is emphasized. Treatment with high-flow oxygen and non-invasive ventilation is recommended. The use of statins or steroids in general therapy is not found to be fully supported by evidence. In the management of pleural infection, new data show favourable effects of tissue plasminogen activator and deoxyribonuclease installation. Detailed recommendations for the vaccination of risk groups are afforded.

Background and Purpose Hyperglycaemia increases glucose concentrations in airway surface liquid and increases the risk of pulmonary Pseudomonas aeruginosa infection. We determined whether reduction of blood and airway glucose concentrations by the anti-diabetic drug dapagliflozin could reduce P. aeruginosa growth/survival in the lungs of diabetic mice. Experimental Approach The effect of dapagliflozin on blood and airway glucose concentration, the inflammatory response and infection were investigated in C57BL/6J (wild type, WT) or leptin receptor-deficient (db/db) mice, treated orally with dapagliflozin prior to intranasal dosing with LPS or inoculation with P. aeruginosa. Pulmonary glucose transport and fluid absorption were investigated in Wistar rats using the perfused fluid-filled lung technique. Key Results Fasting blood, airway glucose and lactate concentrations were elevated in the db/db mouse lung. LPS challenge increased inflammatory cells in bronchoalveolar lavage fluid from WT and db/db mice with and without dapagliflozin treatment. P. aeruginosa colony-forming units (CFU) were increased in db/db lungs. Pretreatment with dapagliflozin reduced blood and bronchoalveolar lavage glucose concentrations and P. aeruginosa CFU in db/db mice towards those seen in WT. Dapagliflozin had no adverse effects on the inflammatory response in the mouse or pulmonary glucose transport or fluid absorption in the rat lung. Conclusion and Implications Pharmacological lowering of blood glucose with dapagliflozin effectively reduced P. aeruginosa infection in the lungs of diabetic mice and had no adverse pulmonary effects in the rat. Dapagliflozin has potential to reduce the use, or augment the effect, of antimicrobials in the prevention or treatment of pulmonary infection.

After inhaled drug administration, pharmacokinetic (PK) data is typically limited to plasma concentrations and provides no direct information on lung target site levels. This study aims to increase understanding of regional lung PK of selected drugs by taking samples from various lung compartments using innovative sampling methods. Eighteen healthy volunteers were enrolled to either receive single doses of orally inhaled salbutamol (200µg), salmeterol (50µg) and fluticasone propionate (500µg) (n=12) or single oral doses of salbutamol (4 mg) and propranolol (40 mg) (n=6). Two bronchoscopies were performed in each subject. During each bronchoscopy 2 samples of bronchosorption, bronchial brush, mucosal biopsy and one bronchoalveolar lavage (BAL) were performed at different pre-specified times (1, 2, 3, 4, 6, 9, 12, 18 hours) after the drug administration. PK profiles of the model drugs were obtained for concentrations in human lung epithelial lining fluid and lung tissue, enabling insight into the spatial lung distribution of drugs after inhaled and oral administration (Fig. 1). Innovative study design with direct sampling from different compartments in lung provided high quality PK profiles in human lung and demonstrated extent of lung targeting following inhaled drug delivery.

The Swedish Association of General Practice has published a set of guidelines for general practitioner's (GPs') learning in general practice/family medicine. This article is a summary of principles, guidelines and applications regarding Swedish GPs' continuing professional development (CPD). We argue that reflection on own practice, reflection with colleagues in small groups and use of written learning plans and portfolios for courses and lectures are important parts of CPD and should be increasingly used and supported by employers. Collection of CME credits for certification purposes does not ensure that educational measures have been effective. Statements of recurring CPD should be incorporated in contracts between health care authorities and health care units.

[Image: see text] The further optimization of a recently disclosed series of inverse agonists of the nuclear receptor RORC2 is described. Investigations into the left-hand side of compound 1, guided by X-ray crystal structures, led to the substitution of the 4-aryl-thiophenyl residue with the hexafluoro-2-phenyl-propan-2-ol moiety. This change resulted in to compound 28, which combined improved drug-like properties with good cell potency and a significantly lower dose, using an early dose to man prediction. Target engagement in vivo was demonstrated in the thymus of mice by a reduction in the number of double positive T cells after oral dosing.

Modulating and optimizing the local pharmacokinetics of inhaled drugs by chemical design or formulation is challenged by the lack of predictive in vitro systems and in vivo techniques providing a detailed description of drug location in the lung. The present study investigated whether a new experimental setup of freshly prepared agarose-filled lung slices can be used to estimate lung retention in vitro, by comparing with in vivo lung retention after intratracheal instillation. Slices preloaded with inhaled β-adrenergic compounds (salbutamol, formoterol, salmeterol, indacaterol or AZD3199) were incubated in a large volume of buffer (w/wo monensin to assess the role of lysosomal trapping), and the amount remaining in slices at different time points was determined with liquid chromatography-tandem mass spectrometry. The in vitro lung retention closely matched the in vivo lung retention (half-lives within 3-fold for 4/5 compounds), and monensin shortened the half-lives for all compounds. The results suggest that freshly prepared rat lungs slices can be used to predict lung retention and that slow kinetics of lysosomal trapping is a key mechanism by which retention in the lung and the effect duration of inhaled β-adrenergic bronchodilators are prolonged.

The development of oral modified-release (MR) dosage forms requires an active pharmaceutical ingredient (API) with a sufficiently high absorption rate in both the small and large intestine. Dogs are commonly used in preclinical evaluation of regional intestinal absorption and in the development of novel MR dosage forms. This study determined regional intestinal effective permeability (Peff) in dogs with the aim to improve regional Peff prediction in humans. Four model drugs, atenolol, enalaprilat, metoprolol, and ketoprofen, were intravenously and regionally dosed twice as a solution into the proximal small intestine (P-SI) and large intestine (LI) of three dogs with intestinal stomas. Based on plasma data from two separate study occasions for each dog, regional Peff values were calculated using a validated intestinal deconvolution method. The determined mean Peff values were 0.62, 0.14, 1.06, and 3.66 × 10(-4) cm/s in the P-SI, and 0.13, 0.02, 1.03, and 2.20 × 10(-4) cm/s in the LI, for atenolol, enalaprilat, metoprolol, and ketoprofen, respectively. The determined P-SI Peff values in dog were highly correlated (R(2) = 0.98) to the historically directly determined human jejunal Peff after a single-pass perfusion. The determined dog P-SI Peff values were also successfully implemented in GI-Sim to predict the risk for overestimation of LI absorption of low permeability drugs. We conclude that the dog intestinal stoma model is a useful preclinical tool for determination of regional intestinal permeability. Still, further studies are recommended to evaluate additional APIs, sources of variability, and formulation types, for more accurate determination of the dog model in the drug development process.

The challenge of defining the concentration of unbound drug at the lung target site after inhalation limits the possibility to optimize target exposure by compound design. In this study, a novel rat lung slice methodology has been developed and applied to study drug uptake in lung tissue, and the mechanisms by which this occurs. Freshly prepared lung slices (500 μm) from drug-naive rats were incubated with drugs followed by determination of the unbound drug volume of distribution in lung (Vu,lung), as the total concentration of drug in slices divided by the buffer (unbound) concentration. Vu,lung determined for a set of inhaled drug compounds ranged from 2.21 mL/g for salbutamol to 2970 mL/g for dibasic compound A. Co-incubation with monensin, a modulator of lysosomal pH, resulted in inhibition of tissue uptake of basic propranolol to 13%, indicating extensive lysosomal trapping. Partitioning into cells was particularly high for the cation MPP+ and the dibasic compound A, likely because of the carrier-mediated transport and lysosomal trapping. The results show that different factors are important for tissue uptake and the presented method can be used for profiling of inhaled compounds, leading to a greater understanding of distribution and exposure of drug in the lung.

The small intestine mucosal barrier is physiologically regulated by the luminal conditions, where intestinal factors, such as diet and luminal tonicity, can affect mucosal permeability. The intestinal barrier may also be affected by absorption-modifying excipients (AME) in oral drug delivery systems. Currently, there is a gap in the understanding of how AMEs interact with the physiological regulation of intestinal electrolyte transport and fluid flux, and epithelial permeability. Therefore, the objective of this single-pass perfusion study in rat was to investigate the effect of three AMEs on the intestinal mucosal permeability at different luminal tonicities (100, 170, and 290 mOsm). The effect was also evaluated following luminal administration of a nicotinic receptor antagonist, mecamylamine, and after intravenous administration of a COX-2 inhibitor, parecoxib, both of which affect the enteric neural activity involved in physiological regulation of intestinal functions. The effect was evaluated by changes in intestinal lumen-to-blood transport of six model compounds, and blood-to-lumen clearance of

Pharmaceutical excipients that may affect gastrointestinal (GI) drug absorption are called critical pharmaceutical excipients, or absorption-modifying excipients (AMEs) if they act by altering the integrity of the intestinal epithelial cell membrane. Some of these excipients increase intestinal permeability, and subsequently the absorption and bioavailability of the drug. This could have implications for both the assessment of bioequivalence and the efficacy of the absorption-enhancing drug delivery system. The absorption-enhancing effects of AMEs with different mechanisms (chitosan, sodium caprate, sodium dodecyl sulfate (SDS)) have previously been evaluated in the rat single-pass intestinal perfusion (SPIP) model. However, it remains unclear whether these SPIP data are predictive in a more in vivo like model. The same excipients were in this study evaluated in rat and dog intraintestinal bolus models. SDS and chitosan did exert an absorption-enhancing effect in both bolus models, but the effect was substantially lower than those observed in the rat SPIP model. This illustrates the complexity of the AME effects, and indicates that additional GI physiological factors need to be considered in their evaluation. We therefore recommend that AME evaluations obtained in transit-independent, preclinical permeability models (e.g. Ussing, SPIP) should be verified in animal models better able to predict in vivo relevant GI effects, at multiple excipient concentrations.

Rational: AZD9898 is a small molecule inhibitor of leukotriene C4 synthase (LTC4S) in development for oral treatment of leukotriene driven asthma. AZD9898 is expected to inhibit the downstream production of CysLTs but, unlike the 5-LO inhibitor Zileuton, spare the production of LTB4 and the pro-resolving lipoxins. Hence, AZD9898 has the potential to give additional benefits in asthma compared to leukotriene modifiers on the market. Here we describe the pharmacological properties of AZD9898. Methods: The PK/PD properties and potency of the LTC4S inhibitor AZD9898 was tested in vivo in rat and in vitro in recombinant enzyme assays, human PBMCs, eosinophils and mast cells upon stimulation. Ex vivo stimulation of human whole blood and nasal polyp tissue was used for additional proof of mechanism in human tissue and for investigation of a differentiated profile of AZD9898 compared to CysLT inhibitors currently on the market. Results: The potency of AZD9898 was in the nanomolar range in enzyme assays, rat airways and human PBMCs. AZD9898 completely inhibited LTC4 production in eosinophils and mast cells and showed a differentiated profile compared to current leukotriene modifiers in explant assays of human whole blood and nasal polyps, by inhibiting CysLTs while sparing LTB4 and the pro-resolving lipoxins. There was a direct relationship between plasma concentrations of AZD9898 and CysLT inhibition in the airways of rats. Conclusions: AZD9898 effectively inhibits CysLT production in vivo and in vitro and shows favorable pharmacological properties. LTC4 synthase may be the preferred point at to which inhibit cysteinyl leukotrienes in asthma patients and will be taken forward in clinical studies.

There is a renewed interest from the pharmaceutical field to develop oral formulations of compounds, such as peptides, oligonucleotides, and polar drugs. However, these often suffer from insufficient absorption across the intestinal mucosal barrier. One approach to circumvent this problem is the use of absorption modifying excipient(s) (AME). This study determined the absorption enhancing effect of four AMEs (sodium dodecyl sulfate, caprate, chitosan, N-acetylcysteine) on five model compounds in a rat jejunal perfusion model. The aim was to correlate the model compound absorption to the blood-to-lumen clearance of the mucosal marker for barrier integrity

The aim of the present study was to evaluate and interpret the pharmacokinetic profiles of two compounds after subcutaneous (s.c.) administration. The compounds have similar physicochemical properties, but are a base (BA99) and an acid (AC88), respectively. The compounds were administered as nano- (5 and 500 mu mol/kg) and microsuspensions (5 mu mol/kg) s.c. to Sprague-Dawley rats. At the low dose, the exposure was higher for both compounds administered as nanocrystals compared to microparticles. The high dose of the compounds resulted in even higher exposure, but not in a dose-linear manner. The differences in exposure between nano- and microparticles were mainly ascribed to higher dissolution rate and improved solubility for smaller particles. In addition to differences in exposure, there were also differences in the elimination pattern. After s.c. injection of 5 mu mol/kg of BA99 as nano- and microsuspensions, the elimination profile was similar as observed earlier after oral administration. However, after injection of the higher dose of BA99 and all formulations of AC88, an extended elimination profile was observed, forming a maintained plateau under the investigated time-period. Essentially, constant plasma levels were caused by a balanced equilibrium between total body clearance of the drug and supply rate of drug from the formulations.

Currently there are only a limited number of determinations of human P-eff in the distal small intestine and none in the large intestine. This has hindered the validation of preclinical models with regard to absorption in the distal parts of the intestinal tract, which can be substantial for BCS class II-IV drugs, and drugs formulated into modified-release (MR) dosage forms. To meet this demand, three model drugs (atenolol, metoprolol, and ketoprofen) were dosed in solution intravenously, and into the jejunum, ileum, and colon of 14 healthy volunteers. The P-eff of each model drug was then calculated using a validated deconvolution method. The median P-eff of atenolol in the jejunum, ileum, and colon was 0.45, 0.15, and 0.013 X 10(-4) cm/s, respectively. The corresponding values for metoprolol were 1.72, 0.72, and 1.30 X 10(-4) cm/s, and for ketoprofen 8.85, 6.53, and 3.37 X 10(-4) cm/s, respectively. This is the first study where the human Peff of model drugs has been determined in all parts of the human intestinal tract in the same subjects. The jejunal values were similar to directly determined values using intestinal single-pass perfusion, indicating that the deconvolution method is a valid approach for determining regional P-eff. The values from this study will be highly useful in the validation of preclinical regional absorption models and in silico tools.

Pulmonary drug disposition after inhalation is complex involving mechanisms, such as regional drug deposition, dissolution, and mucociliary clearance. This study aimed to develop a systems pharmacology approach to mechanistically describe lung disposition in rats and thereby provide an integrated understanding of the system. When drug- and formulation-specific properties for the poorly soluble drug fluticasone propionate were fed into the model, it proved predictive of the pharmacokinetics and receptor occupancy after intravenous administration and nose-only inhalation. As the model clearly distinguishes among drug-specific, formulation-specific, and system-specific properties, it was possible to identify key determinants of pulmonary selectivity of receptor occupancy of inhaled drugs: slow particle dissolution and slow drug-receptor dissociation. Hence, it enables assessment of factors for lung targeting, including molecular properties, formulation, as well as the physiology of the animal species, thereby providing a general framework for rational drug design and facilitated translation of lung targeting from animal to man.\ud

In Sigfridsson et al. (2011, Drug Dev Ind Pharm, 37: 243-251), there was no difference in plasma concentration of BA99 when administering the drug as nanosuspension or microsuspension and analyzing the blood samples by liquid chromatography-mass spectrometry. This was related to the dissolved amount of drug in the gastric tract, which was high enough to support fast absorption when the drug reached the small intestine. One single physicochemical property (pK(a), about 3 for BA99) abolished the benefit of small particles. These results were further confirmed in the present study, where a proton pump inhibitor, AZD0865, was used to elevate the gastric pH and then drastically decreased the gastric solubility. In this way, BA99 could be considered as a model compound for a neutral substance. By increasing the gastric pH to 5-6 and 8-9, respectively, in rats, the plasma concentrations of BA99, after administering nanosuspensions, were unchanged compared with untreated (i.e. no AZD0865) animals. For microsuspensions of the test compound, on the other hand, the exposure of BA99 was 2- to 3-fold lower than for nanosuspensions at both pHs. Moreover, the blood concentrations of BA99 administered as microsuspension were also 2- to 3-fold lower compared with untreated (no AZD0865) individuals receiving both nanoparticles and microparticles of BA99. Obviously, for neutral compounds, with similar physicochemical properties as the present compound, size reduction will be crucial for increased plasma exposure. For basic compounds, with similar physicochemical properties as the present compound, the crucial step for absorption is the dissolution and solubility in the gastric tract.

The aim of the present study was to find out if nanosuspensions were a better choice compared with microsuspensions, for the present substances with water solubility in the order of 2-3 μM (pH 6.8, small intestinal pH) and no permeability limitations. The ambition was also to understand what the higher solubility in the stomach for BA99 means in terms of absorption properties of the substance.The pharmacokinetic parameters of a poorly soluble acid (AC88) and a poorly soluble base (BA99) administered orally as nanosuspensions have been compared with those from microsuspensions using rat as in vivo species.A significant difference was observed between the two suspensions for AC88 already at the lowest dose, 5 μmol/kg (the particle size of the nanosuspensions and the microsuspensions was about 200 nm and 14 μm, respectively). These results were further confirmed at a high dose (500 μmol/kg). However, for BA99, there were no significant differences between the two formulations at any dose investigated (the particle size of the nanosuspensions and the microsuspensions was about 280 nm and 12 μm, respectively).The study demonstrated a clear correlation between particle size and in vivo exposures for an acidic compound, the nanosuspensions providing the highest exposure. For a basic compound, on the other hand, with the present properties and doses, a microsuspension was sufficient. In the latter case, the higher solubility at gastric pH, because of the basic pK(a), limits the need for particle reduction.

The exposure of UG558 was not good enough using traditional microsuspensions.The aim of this study was to find out whether nanosuspensions were a better choice compared with a microsuspension, for an acidic substance with a water solubility in the order of 2 microM (pH 6.8, small intestinal pH) and no permeability limitations.UG558 was ground by a planetary ball mill. The particle size was measured by laser diffraction and the stability of the particle sizes was followed. The pharmacokinetic parameters of UG558 administered as nanosuspension have been compared with those from microsuspension using rat as in vivo specie. Both formulations were administered orally. The nanosuspension was also administered intravenously.The particle size of the nanosuspensions was about 190 nm and about 12 microm for the microsuspensions. At the administered doses, solutions were no alternative (e.g. due to limited solubility). Already at the lowest dose, 5 micromol/kg (5 ml/kg), a significant difference was observed between the two suspensions. These results were further confirmed at a high dose (500 micromol/kg, 5 mL/kg). Thus, the study demonstrated a clear correlation between particle size and in vivo exposures, where the nanosuspensions provided the highest exposure. Furthermore, no adverse events were observed for the substance nor the nanosuspension formulations (i.e., the particles) in spite of the higher exposures obtained with the nanoparticles. To make it possible to calculate the bioavailability, 5 micromol/kg doses of the nanosuspensions (5 ml/kg) were also administered intravenously. No adverse events were observed.The nanoparticles have a larger surface, resulting in faster in vivo dissolution rate, faster absorption, and increased bioavailability, compared to microparticles. The lower overall bioavailability observed at the high dose, compared with the low dose, was due to a combination of low dissolution rate, low solubility, and a narrow intestinal absorption window for UG558.

Asymptotic distances between probability distributions appearing in πps sampling theory are studied. The distributions are Poisson, Conditional Poisson (CP), Sampford, Pareto, Adjusted CP and Adjusted Pareto sampling. We start with the Kullback-Leibler divergence and the Hellinger distance and derive a simpler distance measure using a Taylor expansion of order two. This measure is evaluated first theoretically and then numerically, using small populations. The numerical examples are also illustrated using a multidimensional scaling technique called principal coordinate analysis (PCO). It turns out that Adjusted CP, Sampford, and adjusted Pareto are quite close to each other. Pareto is a bit further away from these, then comes CP and finally Poisson which is rather far from all the others.

Pareto sampling was introduced by Rosen in the late 1990s. It is a simple method to get a fixed size πps sample though with inclusion probabilities only approximately as desired. Sampford sampling, ...

The hepatic cytochrome P450 reductase null (HRN) mouse, which has no functional hepatic Cyp P450s, may represent a useful model for examining extra-hepatic P450-related oxidative metabolism. Here the pharmacokinetics and metabolic fate of midazolam, a drug known to undergo such extra-hepatic metabolism, have been investigated in the HRN mouse and compared with a phenotypically normal strain (C57BL/6J). In addition, the effects of co-administration of the pan-P450 inhibitor 1′-aminobenzotriazole (ABT) on the metabolic profile have been compared in both strains. Significant pharmacokinetic differences for midazolam were observed between the two strains of mice with the HRN mice showing lower circulating concentrations of 1′-hydroxymidazolam but higher concentrations of 1′-hydroxymidazolam- O -glucuronide. A significant increase in midazolam exposure was seen upon ABT exposure for both strains of mice, but no differences in the area under the concentration time curves (AUC) for the monitored metabolites were observed. Although oxidative metabolism of midazolam was not abolished, significant decreases in 1′-hydroxymidazolam formation ratios were observed for both strains of mice exposed to ABT. Metabolite profiling of blood and bile showed a number of qualitative and quantitative differences between HRN and normal mice. These differences in midazolam metabolism between the two strains of mice clearly demonstrate the role that liver P450 enzymes play in the murine metabolism of midazolam. The fate of the compound in the HRN mice shows the importance of extrahepatic metabolism and also showed that these mice appear to be more capable of forming circulating phase II glucuronides than the normal strain.

A method to study single-molecule reactions confined in a biomimetic container is described. The technique combines rapid vesicle preparation, optical trapping and fluorescence confocal microscopy for performing simultaneous single-vesicle trapping and single-molecule detection experiments. The collisional environment between a single enzyme and substrate inside a vesicle is characterized by a Brownian dynamics Monte Carlo simulation. q 1999 Elsevier Science B.V. All rights reserved.

The clinical relevance of parvovirus B19 DNA persistence in bone marrow was examined in 10 immunocompetent individuals undergoing examinations for unexplained fever, arthralgia or chronic leukopenia. Common causes of these symptoms had been ruled out and bone marrow aspiration was indicated at this stage of investigation. In addition to morphological analysis of the bone marrow, a test for B19 DNA was performed with 2 nested PCRs. Five of these 10 selected patients had detectable B19 DNA in their bone marrow, whereas no viraemia was observed. Additional bone marrow samples were collected at least 6 months after the first sample from the B19 DNA-positive patients, of whom 3 were found to be still positive. Indeed, 2 of the patients have been positive for more than 5 y of follow-up. Sera from all patients with persistent B19 DNA in bone marrow could neutralize the virus. One patient responded to treatment with immunoglobulin but later relapsed. No other cause of the symptoms was found, despite extensive investigations, and at least some of the prolonged disease manifestations may be due to parvovirus B19.

Secretory vesicles obtained from the atrial gland of the gastropod mollusk Aplysia californica were chemically analyzed individually with a combination of optical trapping, capillary electrophoresis separation, and a laser-induced fluorescence detection. With the use of optical trapping, a single vesicle that had attoliters (10 −18 liters) of volume was introduced into the tapered inlet of a separation capillary. Once the vesicle was injected, it was lysed, and its components were fluorescently labeled with naphthalene-2,3-dicarboxaldehyde before separation. The resultant electropherograms indicated distinct variations in the contents of single vesicles.

In the present paper, BA99 and AC88 were used as model compounds for intraperitoneal (i.p.) administration to Sprague-Dawley rats. A major problem for the compounds, like many others newly developed pharmaceutical drugs, is the poor solubility in water. To solve solubility related problems, development of nanosuspensions is an attractive alternative. Both compounds are suitable for nanosuspensions, using the milling approach. After 2 weeks in freezer, the nanoparticles aggregated to form particles in the 400-2000 nm interval. However, following a 20 s ultrasonication step, the original particle sizes (about 200 nm) were obtained. Adding 5% mannitol before the samples were frozen abolished aggregation. It is also possible to freeze-dry the nanosuspension in the presence of 5% mannitol and re-disperse the formulation in water. Nanosuspensions of both compounds were injected i.p. to rats at 5 and 500 mu moL/kg. At the low dose, also a microsuspension was administered. I. p. administration resulted in overall improved C-max for both AC88 and BA99 compared to s. c. and oral administration. I. p. is the preferred route of administration of tolerable drugs when a fast onset of action is desired and when a significant first passage metabolism occurs. The net charge of the active molecule appeared to affect the absorption kinetics. In the present work, the neutral molecule was favored over the negatively charged one.

The quantification of P-glycoprotein [P-gp, ABCB1, multidrug resistance 1 (MDR1)] protein in biological matrices is considered a key factor missing for useful translation of in vitro functional data to the in vivo situation and for comparison of transporter data among different in vitro models. In the present study a liquid chromatography (LC)-mass spectrometry method was developed to quantify P-gp membrane protein levels in different biological matrices. The amount of P-gp transporter protein was measured in Caco-2 cell monolayers and in inside-out human embryonic kidney (HEK)-MDR1 vesicles. From both in vitro systems, two preparations with different functionality were used. Transporter function was determined as digoxin efflux in Caco-2 cell monolayers and N-methylquinidine (NMQ) uptake in membrane vesicles, and, in addition, mRNA expression in the Caco-2 monolayers was measured. The results showed an excellent relationship between NMQ uptake functionality in inside-out HEK-MDR1 vesicles and protein contents. Similar concordance between the digoxin efflux and P-gp content in different Caco-2 cell cultures was observed, whereas mRNA levels are indicative of increased P-gp content and activity in older Caco-2 cultures, however, not yielding the same quantitative relationship. The results from both Caco-2 and HEK-MDR1 membrane vesicles confirm that the protein content is directly related to the level of activity in the respective system. The method presented here to quantify P-gp protein by LC-multiple reaction monitoring will facilitate the development of future methodologies to bridge between expression systems and cell/tissue models and to scale from in vitro models to whole organs.

This paper describes a case study in the use of a network gatekeeper we developed to enable a telecommunications Services-Oriented Architecture for the purposes of exposing a service provider network to third parties. Service provider revenue growth will inevitably be software-centric, with the move to software-based systems in the Next Generation Network. Web Services and SOA are quickly becoming a critical component of software middleware to address this need. Initially proven in the IT enterprise space, service providers are adopting SOA and Web Services. For example, at the edge of their networks, service providers are adopting Telecom Web Services for third party access. In the core of their networks, they are adopting IMS Application Server-hosted services. In this paper, we discuss the specific requirements and architectural aspects for both scenarios and point to the applicable trends that impact this domain. We illustrate how Web Services and SOA serve as the foundation for reliable services that can be built more efficiently than with traditional approaches without compromising reliability and manageability.

Summary The aim of this study was to characterize the proinflammatory and T helper (Th)1/Th2 cytokine responses during acute parvovirus B19 (B19) infection and determine whether an imbalance of the Th1/Th2 cytokine pattern is related to persistent B19 infection. Cytokines were quantified by multiplex beads immunoassay in serum from B19-infected patients and controls. The cytokine responses were correlated with B19 serology, quantitative B19 DNA levels and clinical symptoms. In addition to a proinflammatory response, elevated levels of the Th1 type of cytokines interleukin (IL)-2, IL-12 and IL-15 were evident at time of the initial peak of B19 viral load in a few patients during acute infection. This pattern was seen in the absence of an interferon (IFN)-γ response. During follow-up (20–130 weeks post-acute infection) some of these patients had a sustained Th1 cytokine response. The Th1 cytokine response correlated with the previously identified sustained CD8+ T cell response and viraemia. A cross-sectional study on patients with persistent B19 infection showed no apparent imbalance of their cytokine pattern, except for an elevated level of IFN-γ response. No general immunodeficiency was diagnosed as an explanation for the viral persistence in this later group. Neither the acutely infected nor the persistently infected patients demonstrated a Th2 cytokine response. In conclusion, the acutely infected patients demonstrated a sustained Th1 cytokine response whereas the persistently infected patients did not exhibit an apparent imbalance of their cytokine pattern except for an elevated IFN-γ response.

Udgivelsesdato: 2006-Jan A subset of parvovirus B19 (B19) infected patients retains the infection for years, as defined by detection of B19 DNA in bone marrow. Thus far, analysis of B19-specific humoral immune responses and viral genome variations has not revealed a mechanism for the absent viral clearance. In this study, ex-vivo cellular immune responses were assessed by enzyme linked immunospot assay mounted against the majority of the translated viral genome. Compared to seropositive healthy individuals, individuals with B19 persistence (2-8 years) showed larger number of responses to the structural proteins (P = 0.0022), whereas responses to the non-structural protein were of lower magnitude (P = 0.012). These observations provide the first findings of immunological discrepancies between individuals with B19 persistence and healthy individuals, findings that may reflect both failed immunity and antigenic exhaustion.

Udgivelsesdato: 2005-May BACKGROUND: Human parvovirus B19 (B19) polymerase chain reaction (PCR) is now a routine analysis and serves as a diagnostic marker as well as a complement or alternative to B19 serology. The clinical significance of a positive B19 DNA finding is however dependent on the type of tissue or body fluid analysed and of the immune status of the patient. OBJECTIVES: To analyse the clinical significance of B19 DNA positivity in bone marrow samples from rheumatic patients. STUDY DESIGN: Parvovirus B19 DNA was analysed in paired bone marrow and serum samples by nested PCR technique. Serum was also analysed for B19-specific IgG and IgM antibodies and the results were compared with clinical and epidemiological data. RESULTS AND CONCLUSIONS: B19 IgG was found in 41 of 50 patients (82%) whereas none was B19 IgM positive. The serologic evaluation showed that none of the patients had acute B19 infection. However, B19 DNA was detected by PCR in 13 of 50 (26%) bone marrow samples from these patients indicating a high frequency of persistent infection compared with previous reports of patient groups and healthy controls. In the study, 22 patients had rheumatoid arthritis (RA) and 7 of these RA patients were B19 DNA positive in bone marrow. Rheumatoid factor was positive in 4 of the 7 B19 DNA positive RA patients as compared with Rheumatoid factor positivity in all of the 15 B19 DNA negative RA patients. Erosive arthritis in X-ray was less common in the B19 DNA positive group than in the B19 DNA negative group. A high frequency of parvovirus B19 DNA was thus detected in bone marrow samples in rheumatic patients. The clinical data does not support a direct association between B19 PCR positivity and rheumatic disease manifestation. Therefore, the clinical significance of B19 DNA positivity in bone marrow samples from rheumatic patients must be interpreted with caution.

Invasive group A streptococcal infection is an uncommon but severe disease. Bloodcultures usually yield growth of Streptococcus pyogenes and the bacteria may also be isolated from sterile sites e.g. muscle or pleura. The most serious manifestation is Streptococcal Toxic Shock Syndrome (STSS) with or without necrotizing fasciitis (NF). This condition may occur in about a fifth of the cases with a very high case-fatality rate. Preclinical data and case reports suggest that the fatality rate can be reduced by treatment of high-dose intravenous immunoglobulin. A European placebo-controlled clinical trial with immunoglobulin has been conducted but was prematurely terminated due to difficulties in patient recruitment. In our clinic we have during the last 3.5 years seen 41 patients with invasive group A streptococcal infection and 11 of these have received high-dose immunoglobulin. The indication for that treatment has been hypotension and multiorgan failure. All 11 patients but one fullfilled the criteria for STSS and 5 had NF. Most of the patients were treated with a single dose intravenous infusion of 50 g immunoglobulin. All patients were treated with both benzylpenicillin and clindamycin. Ten patients were fully recovered--case-fatality rate 9 percent. We suggest that patients with invasive group A streptococcal infections who develop STSS with or without NF should be treated with high-dose intravenous immunoglobulin.

We explore possibilities to construct nanoscale analytical devices based on lipid membrane technology. As a step toward this goal, we present nanotube-vesicle networks with fluidic control, where the nanotube segments reside at, or very close (

Laser-induced fluorescence detection of labeled amino acids in a flowing stream at femtomolar (10(-15)M) concentrations was achieved by using a fused-silica capillary flow-cell comprising a constricted thin-walled detection region with inner diameters (IDs) ranging from 2 to 8 microm. The diameter of the constricted region was made to match a diffraction-limited focus of a uniphase transverse electromagnetic mode (TEM(00)) laser beam. Optimization of capillary dimensions and geometries (i.e., curvature, wall thickness, and outer-inner diameter ratio were performed in order to minimize cylindrical lensing of the focused laser beam. The fluorescence was collected in a confocal optical setup using a 1.3 numerical aperture (NA), 100x oil-immersion objective and a single-photon-counting avalanche diode (SPAD). Under conditions of fluid flow, the constriction in the capillary forces all analytes to traverse across the laser probe volume, resulting in a high sampling efficiency. Fluorescein isothiocyanate-labeled glutamate (FITC-Glu) was electrophoretically separated and detected in capillaries having an ID of 2 microm at the constricted region with detection limits of 250 fM (signal-to-noise ratio (S/N) = 3) in the injected solution.

A simple and low-cost pulling device for fused-silica capillaries was developed. By using a tantalum heating filament and the self-tension in a bent capillary, tips and constricted regions with outer diameters of approximately 1 microm and inner diameters of a few hundred nanometers could be reproducibly pulled from 50-microm-i.d., 375-microm-o.d. capillaries. The tips can be used in different applications such as microinjection, micromanipulation, and single-channel patch-clamp, injection ends for CE or as electrospray tips. Constricted capillaries with optimized dimensions to minimize cylindrical lensing effects and to match the size of a diffraction-limited laser focus can be used as optical detection windows in CE and micro-HPLC. Fused silica has several advantages over other glasses such as high melting temperature and superior optical and mechanical properties.

Telia and Ericsson have executed a joint project to evaluate the Ericsson WCDMA experimental system in Stockholm, Sweden. This system has been an important tool for gaining valuable hands-on WCDMA experience and to support the development of the commercial 3G WCDMA system. Some results from the ongoing tests are presented in this paper.

Patients with haematological disorders (n = 100) were examined for prevalence of parvovirus B19 DNA in the bone marrow and serum, irrespective of B19-related symptoms. B19 DNA was studied using 2 nested PCRs and the serum samples were further analysed with B19-specific IgG, IgM and avidity as well as seroreactivity against linear and conformational epitopes of the B19 VP2 antigen. The latter assays specify whether the IgG antibody response represents acute or past B19 infection. B19 DNA was detected in 4 of the 100 bone marrow samples, whereas all the serum samples were B19 DNA negative. None of the 4 B19 DNA positive patients had symptoms typical of B19 infection and serology showed past infection. Furthermore, 2 were still B19 DNA positive in bone marrow more than 1 y after the first sample indicating virus persistence. The seroprevalence for B19 IgG was 59% and 2 patients were B19 IgM positive. Thus, presence of B19 DNA in bone marrow from patients with haematological disorders is not a general finding in seropositive patients. B19 DNA can persist in bone marrow, but in our material this finding showed no clear correlation with symptomatic B19 infection.

Generalized herpes simplex virus infections constitute a severe threat to mother and child during pregnancy. Before antiviral treatments were available, both mother and foetus had a high mortality rate. The following case illustrates a successful outcome after long-term antiviral treatment. It is of great importance to suspect, diagnose and treat patients with HSV 2 early, especially during pregnancy.