A novel inhaled phosphodiesterase 4 inhibitor (CHF6001) reduces the allergen challenge response in asthmatic patients.

CHF6001 is an inhaled phosphodiesterase 4 (PDE4) inhibitor in development for the treatment of obstructive lung diseases. The efficacy and safety of CHF6001 were investigated in a double blind, placebo controlled, 3-way cross-over study using the allergen challenge model. Thirty-six atopic asthmatics who were not taking inhaled corticosteroids and who demonstrated a late asthmatic response (LAR) to inhaled allergen at screening were randomised to receive CHF6001 400 µg or 1200 µg or placebo administered once a day using a dry powder inhaler. The three treatment periods were 9 days; allergen challenges were performed on day 9 and induced sputum was obtained after 10 h from challenge. Washout periods between treatments were up to 5 weeks. Both CHF6001 doses significantly attenuated the LAR; the primary endpoint analysis showed that CHF6001 400 µg and 1200 µg caused reductions of 19.7% (p = 0.015) and 28.2% (p < 0.001) respectively of the weighted FEV1 AUC4-10h compared with placebo. The difference between the CHF6001 doses was not statistically significant (p = 0.223). Compared with placebo, CHF6001 caused greater reduction in sputum eosinophil counts, although these changes were not statistically significant. CHF6001 was well tolerated, with similar numbers of adverse events in each treatment period. This inhaled PDE4 inhibitor has the potential to provide clinical benefits in patients with atopic asthma.

Bronchoabsorption; a novel bronchoscopic technique to improve biomarker sampling of the airway.

BACKGROUND: Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely. METHODS: Eight healthy smokers aged 40-65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1ß), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis. RESULTS: A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL. CONCLUSIONS: The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression. TRIAL REGISTRATION: NHS Health Research Authority (13/LO/0256).

The effects of the novel SHIP1 activator AQX-1125 on allergen-induced responses in mild-to-moderate asthma.

BACKGROUND: SH2-containing inositol-5'-phosphatase 1 (SHIP1) is an endogenous inhibitor of the phosphoinositide-3-kinase pathway that is involved in the activation and chemotaxis of inflammatory cells. AQX-1125 is a first-in-class, oral SHIP1 activator with a novel anti-inflammatory mode of action. OBJECTIVE: To evaluate the effects of AQX-1125 on airway responses to allergen challenge in mild-to-moderate asthmatic patients. METHODS: A randomized, double-blind, placebo-controlled, two-way crossover study was performed in 22 steroid-naïve mild-to-moderate asthmatics with a documented late-phase response to inhaled allergen (LAR). AQX-1125 (450 mg daily) or placebo was administered orally for 7 days. Allergen challenge was performed on day 6 (2 h postdose), followed by methacholine challenge (day 7), and induced sputum collection and fractional exhaled nitric oxide (FeNO). RESULTS: AQX-1125 significantly attenuated the late-phase response compared with placebo (FEV1 4-10 h: mean difference 150 mL, 20%; P = 0.027) and significantly increased the minimum FEV1 during LAR (mean difference 180 mL; P = 0.014). AQX-1125 had no effect on the early-phase response. AQX-1125 showed a trend in reduction of sputum eosinophils, neutrophils and macrophages although this did not achieve significance as there were only 11 paired samples for analysis. There was no effect on methacholine responsiveness or FeNO. Pharmacokinetic data showed AQX-1125 was rapidly absorbed with geometric mean Cmax and AUC0-24 h values of 1417 ng/mL and 16 727 h ng/mL, respectively. AQX-1125 was well tolerated, but mild GI side-effects (dyspepsia, nausea and abdominal pain) were described in 4/22 subjects on active treatment. These side-effects were mild self-limiting, required no further treatment and did not lead to discontinuation of therapy. CONCLUSION AND CLINICAL RELEVANCE: AQX-1125, a novel oral SHIP1 activator, significantly reduces the late response to allergen challenge, with a trend to reduce airway inflammation. AQX-1125 was safe and well tolerated and merits further investigation in inflammatory disorders.

Setipiprant, a selective CRTH2 antagonist, reduces allergen-induced airway responses in allergic asthmatics.

BACKGROUND: CRTH2 is a G-protein-coupled receptor on T helper2 cells that mediates pro-inflammatory effects of prostaglandin D2 in allergic responses. OBJECTIVE: To investigate the tolerability and pharmacokinetics of setipiprant (ACT-129968), a selective orally active CRTH2 antagonist, in allergic asthmatics and to assess the protective effects of multiple doses of this drug against allergen-induced airway responses. METHODS: In this 3-centre, double-blinded, placebo-controlled, cross-over study, 18 allergic asthmatic males were randomized to setipiprant 1000 mg or matching placebo b.i.d. for 5 consecutive days. Study periods were separated by a washout of ≥ 3 weeks. On study day 4, subjects underwent a standardized allergen challenge and airway response was recorded by FEV1 until 10 h post-allergen. Airway responsiveness to methacholine and exhaled nitric oxide (eNO) were measured pre- and post-dosing. The effects of both treatments on the allergen-induced airway responses were compared by a paired Student's t-test. RESULTS: Fifteen subjects completed the study per-protocol and were included in the analysis. Overall, setipiprant was well tolerated and no clinically relevant adverse events occurred. Trough plasma concentrations showed a high inter-subject variability. Compared with placebo, setipiprant significantly reduced the allergen-induced late asthmatic response (LAR), inhibiting the area under the response vs. time curve (AUC(3-10 h) ) by on average 25.6% (P = 0.006) and significantly protected against the allergen-induced airway hyperresponsiveness (AHR) to methacholine (P = 0.0029). There was no difference in the early asthmatic response (EAR) or in allergen-induced changes in eNO between treatments. CONCLUSION AND CLINICAL RELEVANCE: Setipiprant at multiple oral doses was well tolerated and reduced both the allergen-induced LAR and the associated AHR in allergic asthmatics. Our findings confirm that CRTH2 may be a promising target for the treatment of allergic disorders.

Pharmacological modulation of gut mucosal and large vessel blood flow.

BACKGROUND: Constipation, diminished gut blood flow, ischaemic colitis and drug therapy may be associated. AIM: To study the effect of constipating medication on, and the regulation of, gut blood flow. METHODS: 24 healthy females (mean age 30) received, in a double-blind, three-way crossover study: (i) placebo, (ii) ipratropium 40 microg by inhalation (positive control known to reduce rectal mucosal blood flow) and (iii) oral loperamide 4 mg. Mucosal blood flow was measured at the splenic flexure and rectum using laser Doppler flowmetry. Blood flow in the superior and inferior mesenteric arteries was measured by trans-abdominal Doppler ultrasound. RESULTS: Ipratropium decreased rectal mucosal blood flow by 16% (P=0.009) and splenic flexure mucosal blood flow by 8% (P=0.075). Loperamide caused no change in rectal (P=0.40) or splenic flexure mucosal blood flow (P=0.73). Neither treatment changed superior or inferior mesenteric artery blood flow. Splenic flexure mucosal blood flow showed a positive correlation with rectal mucosal blood flow (r=0.69; P<0.0001). CONCLUSIONS: Vasoactive agents may reduce gut mucosal blood flow in the absence of reduced large vessel flow. Constipating drugs do not necessarily reduce gut blood flow. Rectal mucosal blood flow correlates with splenic flexure mucosal flow, and potentially may be used as a more convenient surrogate for studying splenic flexure blood flow.

The nasal mucosal late allergic reaction to grass pollen involves type 2 inflammation (IL-5 and IL-13), the inflammasome (IL-1ß), and complement.

Non-invasive mucosal sampling (nasosorption and nasal curettage) was used following nasal allergen challenge with grass pollen in subjects with allergic rhinitis, in order to define the molecular basis of the late allergic reaction (LAR). It was found that the nasal LAR to grass pollen involves parallel changes in pathways of type 2 inflammation (IL-4, IL-5 and IL-13), inflammasome-related (IL-1ß), and complement and circadian-associated genes. A grass pollen nasal spray was given to subjects with hay fever followed by serial sampling, in which cytokines and chemokines were measured in absorbed nasal mucosal lining fluid, and global gene expression (transcriptomics) assessed in nasal mucosal curettage samples. Twelve of 19 subjects responded with elevations in interleukin (IL)-5, IL-13, IL-1ß and MIP-1ß/CCL4 protein levels in the late phase. In addition, in these individuals whole-genome expression profiling showed upregulation of type 2 inflammation involving eosinophils and IL-4, IL-5 and IL-13; neutrophil recruitment with IL-1α and IL-1ß; the alternative pathway of complement (factor P and C5aR); and prominent effects on circadian-associated transcription regulators. Baseline IL-33 mRNA strongly correlated with these late-phase responses, whereas a single oral dose of prednisone dose-dependently reversed most nasal allergen challenge-induced cytokine and transcript responses. This study shows that the LAR to grass pollen involves a range of inflammatory pathways and suggests potential new biomarkers and therapeutic targets. Furthermore, the marked variation in mucosal inflammatory events between different patients suggests that in the future precision mucosal sampling may enable rational specific therapy.

A novel flow cytometric-based method to measure kinase inhibition in sputum from COPD subjects.

INTRODUCTION: Janus kinases (JAKs) regulate inflammatory gene expression through phosphorylation of signal transducer and activator of transcription (STAT) proteins. Expression of STAT proteins is increased in chronic obstructive pulmonary disease (COPD), and may be involved in driving chronic inflammation. Oral JAK inhibitors are effective as anti-inflammatory therapy but exhibit dose-limiting adverse effects. Development of inhaled compounds would be enhanced by robust biomarkers that directly reflect the anti-inflammatory and pharmacological activity in the lung. METHODS: A novel flow cytometry assay was developed to measure STAT1 phosphorylation in sputum inflammatory cells. The standard sputum processing method was refined to improve sputum cell viability. The flow cytometric assay was used to assess the reproducibility of the measurement of STAT1 phosphorylation and the in vitro activity of a pan JAK-inhibitor on three separate visits in patients with COPD. RESULTS: Upregulation of STAT1 phosphorylation was measured following in vitro IFNγ stimulation of sputum macrophages (stimulated/unstimulated ratio 1.57; p<0.00001). Upregulation was inhibited following in vitro preincubation with a pan JAK-inhibitor (inhibited+stimulated/unstimulated ratio 0.97). STAT1 phosphorylation activity could only be measured in macrophages. CONCLUSIONS: Sputum from patients with COPD can be used to reproducibly measure phospho-STAT expression in sputum macrophages. The flow cytometry-based method can be used to evaluate kinase inhibitors in vitro and subsequently in ex vivo studies. The assay is particularly useful for the assessment of inhaled compounds where whole blood assays may not be relevant.

Development of novel cell surface CD34-targeted recombinant adenoassociated virus vectors for gene therapy.

Recombinant adenoassociated virus (rAAV) type 2 vectors have been used to transduce a wide variety of cell types, including hematopoietic progenitor cells. For in vivo gene transfer, it is desirable to have an rAAV vector that specifically transduces selected target cells. As a first step toward generating an rAAV vector capable of targeting delivery in vivo, we have engineered a chimeric protein combining the AAV capsid protein and the variable region of a single-chain antibody against human CD34 molecules, a cell surface marker for hematopoietic stem/progenitor cells. Inclusion of the chimeric CD34 single-chain antibody-AAV capsid proteins within an rAAV virion significantly increased the preferential infectivity of rAAV for the CD34+ human myoleukemia cell line KG-1, which is normally refractory to rAAV transduction. Antibodies against the single-chain antibody and the CD34 protein blocked this transduction. This chimeric vector represents a significant improvement in the host range of rAAV and the first step toward specific gene delivery by rAAV vectors to cells of choice, in this case, hematopoietic progenitor cells, for the treatment of human disease.

Analysis of antibody reactivity in the sera of 42 patients with paraproteinaemia.

The clinical expression of disease in patients with conditions in which autoimmunity is thought to contribute to the pathogenesis of disease is the result of an unfortunate combination of predisposing and environmental factors. The presence of autoantibodies showing a variety of antigen specificities in sera from many of these patients has been closely correlated with particular spectra of organ involvement or tissue destruction. Their precise role in the disease process is as yet unclear. Sera from patients with paraproteinaemia also often contain autoantibodies to a variety of cell components, although symptoms of autoimmune disease are rarely found in this group of individuals. In this study of 42 sera from patients with paraproteinaemia we have confirmed the presence of autoantibodies in 33% (13/42) of samples. Amongst the autoantibodies detected were those to human neutrophils (3), U1RNP (8) and cardiolipin (4). In five sera, the immunoglobulin class of autoantibody did not correlate with that of the monoclonal band. This study extends previous reports of the repertoire of autoantibodies present in sera from patients with paraproteinaemia.

Loin pain and haematuria syndrome: a somatoform disorder.

Fifteen patients with the loin pain and haematuria syndrome (LPH) were compared with 10 patients with complicated renal stone disease referred to the same tertiary centre and matched for age, sex and duration of illness. LPH patients had a history of three times more medically unexplained somatic symptoms other than loin pain (p < 0.01) and a higher proportion took analgesics regularly (p < 0.01). The onset of pain was associated with an adverse psychologically important life-event in eight of the LPH patients but in none of the controls (p < 0.02). LPH patients more frequently recalled serious parental illness and disability in childhood (p < 0.001) than controls, and a higher proportion felt responsible for causing or alleviating parental illness or distress (p < 0.05). LPH subjects scored higher in the 'paternal care' dimension of the Parental Bonding Instrument (p < 0.05). No difference was found between LPH patients and controls in terms of current depression and anxiety but both groups exhibited high rates of lifetime depression. LPH patients expressed lower levels of anger and hostility (p < 0.002) than did controls. Our observations suggest that psychological factors are of major importance in the aetiology of LPH, which may represent a type of somatoform pain disorder.

Recombinant human erythropoietin shortens the uraemic bleeding time without causing intravascular haemostatic activation.

Blood rheology and haemostasis have been investigated in 8 haemodialysis patients during treatment with recombinant human erythropoietin (rHuEPO). The aim was to elucidate the mechanism by which rHuEPO improves haemostasis, and to determine whether rHuEPO promotes intravascular coagulation. Investigations were performed before, and after 3 months of treatment. Haemoglobin and haematocrit rose significantly after rHuEPO (p less than 0.001) and there was a concurrent shortening of the bleeding time. No significant changes were observed in platelet aggregation, thromboxane generation, or platelet nucleotide content during the treatment period. Whole blood viscosity increased following rHuEPO (p less than 0.01), but plasma viscosity and red cell deformability were unchanged, as were markers of intravascular platelet activation and plasma levels of cross-linked fibrin derivatives. No patient suffered from thrombosis during the study period, and elevation of the haematocrit in uraemic patients up to 0.35 with rHuEPO did not appear to lead to intravascular coagulation. Shortening of the prolonged bleeding time in haemodialyzed patients following rHuEPO appeared to be due to the increase in circulating red cells, rather than to changes in platelet reactivity.

Dynamic multi-planar EPI of the urinary bladder during voiding with simultaneous detrusor pressure measurement.

Magnetic resonance imaging gives high quality images of the urinary bladder with excellent contrast. We report here the first application of dynamic, multi-slice, echo planar imaging to a study of urinary bladder emptying. Changes in urinary bladder volumes and rates of urine expulsion from the bladder have been measured simultaneously with bladder pressure. The method shows promise for clinical applications involving compromised bladder function, for reappraising bladder contraction strength-volume relationships, and for investigating the rate of change of length, three-dimensional shape, and wall tension in different parts of the bladder during micturition.

Triamterene-induced crystalluria and cylinduria: clinical and experimental studies.

We examined the occurrence of crystals and casts in the urine of healthy subjects after administration of triamterene and the site of crystal formation in experimental animals. Twenty out of twenty healthy subjects had abundant triamterene crystals and casts in acid urine after receiving a single 100 mg dose. Casts were present in the urine from 2-11 hours after administration of the diuretic. Cast formation occurred in acidic urine and was prevented by alkalinization of the urine with potassium citrate. Animal studies showed that crystallization and cast formation occurred in the medullary and papillary collecting ducts of the rat kidney. These findings provide a possible explanation for the reported nephrotoxicity of triamterene, particularly when given to patients who are receiving non-steroidal anti-inflammatory agents.

Rapid improvement in severe lupus glomerular lesions following intensive plasma exchange associated with immunosuppression.

We report our experience of intensive plasma exchange (PE) in the treatment of 12 females with severe diffuse proliferative lupus nephritis. All had active disease with crescentic lesions demonstrated on biopsy immediately before PE. Nine patients were also treated with high dose steroids, three patients with low dose steroids and most patients also received cytotoxic therapy. Eight of the 12 patients were biopsied immediately after a course of PE. All but one patient (low dose steroid group) showed considerable diminution of histologic activity with resolution of crescentic lesions. Plasma exchange may accelerate such resolution over conventional therapy, prevent subsequent sclerosis and preserve functional renal tissue.

Accuracy of reagent strip testing for urinary tract infection in the elderly.

The accuracy of reagent strip testing for urinary tract infection (UTI) was assessed in 100 elderly patients (50 acute patients admitted to hospital and 50 attending the day hospital). Reagent strip sensitivities were: acute patients-urinary nitrite 83%, blood 67%, protein 72% and leucocytes 72%, and day hospital patients-urinary nitrite 90%, blood 65%, protein 30% and leucocytes 60%. Urinary nitrite specificities were 100% for both groups of patients. Only 28% of patients with a UTI had specific symptoms of the infection; pyrexia and a raised WBC also proved poor indicators. Urinary nitrite was thus the most accurate immediate indicator of UTI.

Effects of a reversible beta-tryptase and trypsin inhibitor (RWJ-58643) on nasal allergic responses.

BACKGROUND: beta-Tryptase is a multifunctional mast cell serine protease released during mast cell degranulation and tryptase/trypsin inhibitors are a novel potential therapeutic approach for allergic inflammatory diseases. OBJECTIVES: This study was performed to assess the effects of RWJ-58643 on nasal symptoms, eosinophil influx, and cytokine and chemokine release following nasal allergen challenge (NAC). METHODS: Male patients with grass pollen allergic rhinitis (n=16) out of season received single doses of RWJ-58643 (100, 300, 600 microg) or matched placebo given 30 min before NAC in a double-blind, randomized crossover design. A single dose of 200 microg budesonide was studied in an open-label extension phase. NAC was performed with Timothy grass pollen (ALK) via a nasal device, and nasal lavage was performed at times 0 (pre-drug, pre-allergen), 0.5 (30 min post-drug, pre-NAC) 1.5, 2.5, 4.5, 6.5, 8.5, and 24 h after drug administration. Nasal lavage mediators were analysed using a sensitive multiplexed bead immunoassay system. RESULTS: Low-dose RWJ-58643 (100 microg) and budesonide (200 microg) significantly reduced symptoms, eosinophils and levels of IL-5 following NAC. However, higher doses of RWJ-58643 (300 and 600 microg) caused a late eosinophilia and preceding increases in IL-5 compared with placebo. CONCLUSIONS: This study suggests that combined beta-tryptase and trypsin inhibition has therapeutic potential in allergic inflammation, however, this property is dose responsive and higher doses are ineffective and may cause eosinophilia.