Dose-response effects of TPI ASM8 in asthmatics after allergen.

BACKGROUND: TPI ASM8 contains two modified antisense oligonucleotides (AON) targeting the beta subunit (ß(c) ) of the IL-3, IL-5, GM-CSF receptors and the chemokine receptor CCR3. A previous study suggested that TPI ASM8 had broader effects than just inhibition of eosinophils in asthmatics. OBJECTIVE: We assessed whether TPI ASM8 caused a dose-dependent attenuation in the inflammatory and physiological changes after inhaled allergen challenge (AIC). METHODS: This single-center, open-label, stepwise-ascending dose study was conducted in fourteen stable, mild allergic asthmatics. Following placebo AIC, subjects underwent AIC after 4 days treatment with 1, 2, and 4 mg BID and finally 8 mg once daily (OD) of TPI ASM8, inhaled via the I-Neb™ nebuliser. Treatments were separated by 2-3-week washout periods. RESULTS: TPI ASM8 was safe and well tolerated at all doses. TPI ASM8 8 mg OD reduced eosinophils in sputum after AIC (by 60.9% at 7 h and 68.4% at 24 h post-AIC, P=0.016 and P=0.007, respectively). Additionally, TPI ASM8 8 mg OD significantly attenuated the early and late airway responses as shown by the reduction in the area under the curve by 45% (P=0.016) and 59%, (P=0.0015), respectively, the increase in eosinophil cationic protein (ECP) by up to 57% (P=0.021), and airway responsiveness to methacholine by more than 1 doubling dose (P=0.012). A dose-response relationship was noted, and efficacy was maintained with once per day administration. CONCLUSIONS: TPI ASM8 attenuated a broad range of inflammatory and physiological changes after AIC, suggesting that CCR3, IL-3, and GM-CSF also are important targets for the management of asthma.

Protection against methacholine-induced bronchospasm: salbutamol pMDI versus Clickhaler DPI.

Passive dry-powder inhalers (DPIs) have been developed as an alternative to pressurised metered-dose inhalers (pMDIs) to improve aerosol delivery on inhalation and eliminate the need for propellants. However, new DPI formulations of generic drugs must be rigorously compared with conventional pMDI therapy. This randomised, double-blind, double-dummy, placebo-controlled, seven-way crossover study evaluated bronchoprotection from methacholine challenge in order to compare a novel salbutamol DPI (Clickhaler) with a reference salbutamol pMDI (Ventolin). Adult asthma patients with airway hyperresponsiveness to methacholine (provocative concentration of methacholine causing a 20% fall in the forced expiratory volume in one second (PC20) <4 mg x mL(-1)) were treated on separate days with 0, 100, 200 or 400 microg of salbutamol via the DPI or pMDI. Methacholine challenge was performed before and after salbutamol treatment and the PC20 ratios analysed by Finney's bioassay to test for therapeutic equivalence of the inhalers. Eighteen patients completed the study and showed significant dose-related responses to salbutamol. The relative potency of DPI:pMDI was 1.29 (90% confidence interval 1.04-1.63). There were no treatment differences in safety (cardiac frequency, blood pressure, adverse events). Methacholine-challenge methodology provides a sensitive bioassay and has demonstrated therapeutic equivalence of the salbutamol Clickhaler dry-powder inhaler with the conventional salbutamol pressurised metered-dose inhaler.

Reproducibility of bronchodilator response for a reservoir dry powder inhaler following routine clinical use.

The performance of dry powder inhaler (DPI) devices, particularly reservoir DPIs, may be influenced by environmental conditions. This study compared the bronchodilator efficacy and in vitro aerosol characteristics of salbutamol, delivered via a novel reservoir DPI (Clickhaler) and a conventional pressurized metered-dose inhaler (MDI) before and after use of the DPI in clinical practice. Following a screening visit, patients received cumulative doses of salbutamol (100, 200, and 400 microg) via DPI or MDI on separate days in a double-blind, crossover design before and after a 4-week period, during which the DPI was used as the patients' first-line bronchodilator. Lung function responses (forced expiratory volume in 1 sec [FEV1], forced vital capacity [FVC], and peak expiratory flow [PEF]) to salbutamol delivered by DPI and MDI and in vitro aerosol characteristics were not significantly different before and after the period of DPI patient use. DPI performance, assessed in vivo and in vitro, is maintained following an extended period of patient use.

Clickhaler (a novel dry powder inhaler) provides similar bronchodilation to pressurized metered-dose inhaler, even at low flow rates.

STUDY OBJECTIVE: Comparison of the bronchodilator response to an albuterol novel dry powder inhaler (DPI) (Clickhaler [CH]; ML Laboratories PLC; St. Albans, UK) activated at various inspiratory flow rates and to an albuterol pressurized metered-dose inhaler (pMDI) by patients with moderate to moderately severe stable asthma. DESIGN: Randomized, double-blind, placebo-controlled comparison of the bronchodilator response to albuterol DPI (200 microg) at inspiratory flow rates of approximately 15, 30, and 60 L/min in patients with stable asthma with demonstrated reversibility to albuterol. Active (albuterol via pMDI inhaled at 30 L/min) and placebo controls were included. SETTING: Single center study at the chest/allergy unit of a teaching hospital in Canada. PATIENTS: Sixteen patients with moderate to moderately severe stable asthma. MEASUREMENTS AND RESULTS: Efficacy end points were FEV1, FVC, FEV1/FVC, maximum expiratory flow, and forced expiratory flow between 25% and 75% of vital capacity. Safety end points included heart rate, BP, and tremor. There was no significant difference between the bronchodilator response to albuterol via the CH at 15, 30, and 60 L/min inspiratory flow rate and, at all flow rates, no significant difference was found comparing albuterol CH with the pMDI. All of the techniques for delivering albuterol provided significantly better bronchodilatation than placebo. Adverse events were minimal and did not differ between CH and pMDI or between the various flow rates inhaled through the CH. CONCLUSION: A novel passive albuterol DPI (CH) provides a similar bronchodilator response at 15, 30, and 60 L/min inspiratory flow rates compared with a pMDI used optimally.

Design, development and performance of a novel multidose dry-powder inhaler.

The authors describe the design and development of a breath-actuated multidose dry-powder inhaler and summarize the in vitro and in vivo data demonstrating its robustness and performance in the laboratory and during clinical use. Drugs for the treatment of asthma--including budesonide, beclomethasone dipropionate and salbutamol--when formulated with lactose powder as a carrier and dispensed via this device, have exhibited clinical efficacy and safety profiles comparable with standard pressurized metered-dose inhalers and dry-powder formulations.

Furosemide decreases the sensitivity of glucose transport to insulin in skeletal muscle in vitro.

The effects of the diuretic furosemide on the sensitivity of glucose disposal to insulin were investigated in rat soleus muscle in vitro. At basal levels of insulin, the rates of 3-O-methylglucose transport, 2-deoxyglucose phosphorylation and lactate formation were not affected significantly by furosemide (0.5 mmol/l). However, furosemide significantly decreased these rates at physiological and maximal levels of insulin. The contents of 2-deoxyglucose and glucose 6-phosphate in the presence of furosemide were not significantly different from those in control muscles at all levels of insulin studied. It is concluded that furosemide decreases the sensitivity of glucose utilization to insulin in skeletal muscle by directly inhibiting the glucose transport process.

Use of whole-body plethysmography to compare bronchodilator inhaler efficacy.

Whole-body plethysmography is not included in guidelines from regulatory authorities for the development of treatments or delivery devices for lung disease, despite its potential advantages compared to spirometry. Two separate studies were undertaken to assess the use of specific airway conductance (sGaw) as a pharmacodynamic endpoint for the comparison of two bronchodilator delivery systems (a novel dry powder inhaler and a standard metered dose inhaler). The first pilot study involved delivery of a single dose of salbutamol (200 micrograms) to 12 healthy volunteers and determination of sGaw up to 120 min after treatment. The second study involved delivery of cumulative doses of salbutamol (100, 200 and 400 micrograms) to 19 healthy volunteers with demonstrated reversibility of sGaw to the bronchodilator and measurement of sGaw up to 240 min after treatment. In both studies, increases in sGaw after treatment were significant compared to placebo and larger than the recorded increases in FEV1. Increases in sGaw were similar for both delivery devices and support the therapeutic equivalence of the two products. Power calculations indicated that the second study had appropriate statistical power to discriminate between treatments. It is concluded that the assessment of sGaw in healthy volunteers may be a useful and sensitive pharmacodynamic endpoint for use in the development of bronchodilators and their delivery devices.

The effects of insulin on transport and metabolism of glucose in skeletal muscle from hyperthyroid and hypothyroid rats.

The effects of insulin on the rates of glucose disposal were studied in soleus muscles isolated from hyper- or hypothyroid rats. Treatment with triiodothyronine for 5 or 10 days decreased the sensitivity of glycogen synthesis but increased the sensitivity of lactate formation to insulin. The sensitivity of 3-O methylglucose to insulin was increased only after 10 days of treatment and was accompanied by an increase in the sensitivity of 2-deoxyglucose phosphorylation; however, 2-deoxyglucose and glucose 6-phosphate in response to insulin remained unaltered. In hypothyroidism, insulin-stimulated rates of 3-O-methylglucose transport and 2-deoxyglucose phosphorylation were decreased; however, at basal levels of insulin, 3-O-methylglucose transport was increased, while 2-deoxyglucose phosphorylation was normal. In these muscles, the sensitivity of lactate formation to insulin was decreased; this defect was improved after incubation of the muscles with prostaglandin E2. The results suggest: (a) in hyperthyroidism, insulin-stimulated rates of glucose utilization in muscle to form lactate are increased mainly because of a decrease in glycogen synthesis; when hyperthyroidism progresses in severity, increases in the sensitivity of glucose transport to insulin and in the activity of hexokinase may also be involved; (b) in hypothyroidism, the decrease in insulin-stimulated rates of glucose utilization is caused by decreased rates of glycolysis; (c) prostaglandins may be involved in the changes in sensitivity of glucose utilization to insulin observed in muscle in altered thyroid states.

Relative bioavailability of salbutamol to the lung following inhalation via a novel dry powder inhaler and a standard metered dose inhaler.

AIMS: The number of dry powder inhaler (DPI) devices could increase because they are easier to use than a metered dose inhaler (MDI). Using urinary excretion, the relative bioavailability of salbutamol to the lungs and the body for a prototype DPI has been compared with an MDI. METHODS: A randomized, double-blind, two way crossover study compared the amount of salbutamol in the urine 30 min following inhalation of 2 x 100 micrograms salbutamol from a prototype DPI (Innovata Biomed Ltd, UK) and a Ventolin (Allen and Hanburys Ltd, UK) MDI in 10 volunteers. The amount of salbutamol and its metabolite, the ester sulphate conjugate, renally excreted up to 24 h post inhalation was also determined to evaluate the relative bioavailability of salbutamol to the body. RESULTS: The mean (s.d.) 30 min post-treatment urinary excretion for the prototype DPI and MDI was 8.4 (2.6) and 5.0 (1.9) micrograms, respectively (P < 0.001). The total amount of salbutamol and its ester metabolite excreted in the urine over the 24 h period after inhalation was 187.9 (77.6) and 137.6 (40.0) micrograms (P < 0.05). CONCLUSIONS: The prototype DPI delivered more salbutamol to the body and the lungs than a conventional MDI. This finding supports further development of the prototype DPI. The urinary salbutamol method is able to discriminate between two different inhalation systems.

Effects of glucocorticoid excess on the sensitivity of glucose transport and metabolism to insulin in rat skeletal muscle.

GENBANK/dy examines the mechanisms of glucocorticoid-induced insulin resistance in rat soleus muscle. Glucocorticoid excess was induced by administration of dexamethasone to rats for 5 days. Dexamethasone decreased the sensitivity of 3-O-methylglucose transport, 2-deoxyglucose phosphorylation, glycogen synthesis and glucose oxidation to insulin. The total content of GLUT4 glucose transporters was not decreased by dexamethasone; however, the increase in these transporters in the plasma membrane in response to insulin (100 m-units/litre) was lessened. In contrast, the sensitivity of lactate formation to insulin was normal. The content of 2-deoxyglucose in the dexamethasone-treated muscle was decreased at 100 m-units/litre insulin, while the contents of glucose 6-phosphate and fructose 2,6-bisphosphate were normal at all concentrations of insulin studied. The maximal activity of hexokinase in the soleus muscle was not affected by dexamethasone; however, inhibition of this enzyme by glucose 6-phosphate was decreased. These results suggest the following. (1) Glucocorticoid excess causes insulin resistance in skeletal muscle by directly inhibiting the translocation of the GLUT4 glucose transporters to the plasma membrane in response to insulin; since the activity of hexokinase is not affected, the changes in the sensitivity of glucose phosphorylation to insulin seen under these conditions are secondary to those in glucose transport. (2) The sensitivity of glycogen synthesis and glucose oxidation to insulin is decreased, but that of glycolysis is not affected: a redistribution of glucose away from the pathway of glycogen synthesis and glucose oxidation could maintain a normal rate of lactate formation although the rate of glucose transport is decreased.

Growth hormone suppression and glutamine flux associated with cardiac surgery.

Pharmacological doses of growth hormone (GH) in humans and rats increase plasma and muscle glutamine values. As major surgery results in a physiological rise in serum GH concentration, we investigated whether this physiological increase in GH altered glutamine metabolism. Eighteen patients undergoing coronary artery bypass graft (CABG) surgery were randomly assigned to receive somatostatin, 100 micrograms subcutaneously at induction of anaesthesia and 8 hourly for 48 h, or placebo. Somatostatin effectively blocked the physiological surge of GH following injury but did not affect plasma or muscle glutamine concentrations, which fell significantly in both groups. Plasma glutamine decreased by 31% (P < 0.01) and 28% (P < 0.01) in the control and somatostatin groups respectively. Muscle glutamine was reduced 45% (P < 0.001) in the control group and 50% (P < 0.001) in the somatostatin group. There was no difference in muscle or circulating glutamate, alanine or branched chain amino acid concentrations or in metabolite values between the somatostatin-treated patients and the control group. There was no relationship between the GH response to surgery and glutamine metabolism following major surgery.

Studies on the effects of growth hormone administration in vivo on the rates of glucose transport and utilization in rat skeletal muscle.

The effects of growth hormone (GH) administration to rats in vivo on the sensitivity of the rate of glucose utilization to insulin were studied in soleus muscles isolated from these rats. A single injection of GH did not increase the rate of glucose transport within 1-2 h. However, 12 h after, the rate of glucose transport was increased at 10 mU insulin l-1 and was accompanied by a similar increase in the rate of lactate formation but no change in the rate of glycogen synthesis. Prolonged treatment with GH decreased the rate of glucose transport and glycogen synthesis and increased the content of glucose 6-phosphate at physiological levels of insulin but did not affect the rate of lactate formation. These results suggest that: (a) GH does not increase the rate of glucose transport acutely; however, after several hours, the sensitivity of glucose transport and glycolysis to insulin are increased; (b) prolonged elevations of the level of GH in plasma decrease the sensitivity of the rate of glucose transport and glycogen synthesis to insulin. However, redirection of glucose residues away from the pathway of glycogen synthesis towards that of glycolysis and a possible increase in the rate of glycogenolysis maintain a normal rate of lactate formation, although the rate of glucose transport is decreased.

The effects of growth hormone administration in vivo on skeletal muscle glutamine metabolism of the rat.

Human growth hormone administration to the rat for 3 or 10 days increased the concentrations of glutamine in both skeletal muscle and plasma and the rate of glutamine release was increased from muscle isolated from rats treated with growth hormone for 3 days. Growth hormone may therefore play an important role in the control of glutamine metabolism in muscle.

The effects of growth hormone and insulin-like growth factors I and II on glutamine metabolism by skeletal muscle of the rat in vitro.

Soleus muscle preparations of the rat were incubated in the presence of growth hormone, IGF-I or IGF-II. Growth hormone (10 and 100 ng/ml) increased, but IGF-I and IGF-II were without effect on the concentration of glutamine in soleus muscle. Growth hormone was without effect on the rate of release of glutamine from muscle. In contrast, the rate of release of glutamine was decreased in the presence of IGF-I (750 ng/ml) and increased in the presence of IGF-II (250 and 750 ng/ml). Growth hormone and the IGFs may therefore play an important role in the control of glutamine metabolism by skeletal muscle.

Effects of glucocorticoids on lymphocyte metabolism.

The immunosuppressive effect of glucocorticoids has been widely reported; however, the mechanism of action of these hormones on the immune system has not been fully established. In the present study, the effect of glucocorticoids on glucose, glutamine, and pyruvate metabolism in lymph node lymphocytes was investigated. Addition of dexamethasone to the incubation medium did not alter glucose and glutamine metabolism but inhibited pyruvate utilization by 40%. This latter effect took 1 h to occur and remained for up to 6 h, even after removal of dexamethasone from the culture medium. Measurements of the activity of pyruvate dehydrogenase in lymphocytes and the rate of [1-14C]-pyruvate conversion into 14CO2 in incubated lymphocyte mitochondria demonstrated that glucocorticoids decrease pyruvate utilization by inhibiting the activity of this key regulatory enzyme. The effect of such an inhibition of pyruvate utilization on the function of cells of the immune system remains to be clarified.

Plasma amino acid concentrations in the overtraining syndrome: possible effects on the immune system.

Overtraining and long-term exercise are associated with an impairment of immune function. We provide evidence in support of the hypothesis that the supply of glutamine, a key fuel for cells of the immune system, is impaired in these conditions and that this may contribute to immunosuppression. Plasma glutamine concentration was decreased in overtrained athletes and after long-term exercise (marathon race) and was increased after short-term, high intensity exercise (sprinting). Branched chain amino acid supplementation during long-term exercise was shown to prevent this decrease in the plasma glutamine level. Overtraining was without effect on the rate of T-lymphocyte proliferation in vitro or on the plasma levels of interleukin-1 and -6, suggesting that immune function is not impaired in this condition. Given the proposed importance of glutamine for cells of the immune system, it is concluded that the decrease in plasma glutamine concentration in overtraining and following long-term exercise, and not an intrinsic defect in T lymphocyte function, may contribute to the immune deficiency reported in these conditions.

Acute ethanol administration and the metabolism of glutamine by skeletal muscle of the rat: implications for ethanol-induced reductions in protein synthesis.

The effect of acute ethanol administration (75 mmol/kg) on the metabolism of glutamine by skeletal muscle of the rat was studied in order to investigate the hypothesis that the concentration of this amino acid in muscle controls the rate of protein synthesis. Ethanol administration was without effect on the concentration of glutamine in EDL (extensor digitorum longus) and plantaris muscles (Type II fibre-rich muscles), but increased the concentration in soleus muscle (Type I fibre-rich muscle). The rate of release of glutamine was increased from EDL muscle, but unchanged from soleus muscle of treated animals. Ethanol administration was without effect on the maximal activity of glutamine synthetase in both soleus and EDL muscles. It is concluded that changes in the concentration of glutamine in muscle cannot explain the ethanol-induced changes in the rate of protein synthesis. Nevertheless, the increase in the concentration of glutamine in soleus muscle following ethanol administration is of interest and may be mediated via modulation of the glutamine transporter and/or the activity of glutamine synthetase in vivo.

Effects of major and minor surgery on plasma glutamine and cytokine levels.

The plasma levels of glutamine and cytokines have been measured frequently in patients before, during, and after elective abdominal aortic aneurysm surgery ("major surgery") or inguinal hernia repair ("minor surgery"). The plasma glutamine level declined rapidly following major surgery and remained markedly below preoperative levels until at least 7 days after surgery. This response of the plasma glutamine levels was significantly correlated with the production of interleukin 6 but not with that of interleukin 1, tumor necrosis factor, or interferon gamma. In contrast, following minor surgery, the plasma glutamine level was unchanged and the elaboration of interleukin 6 was attenuated. The decrease in the plasma glutamine level following major surgery may contribute to the state of immunosuppression, which follows major surgery, and the relationship between amino acid and cytokine metabolism is worthy of further study.

The effect of insulin-like growth factor II on glucose uptake and metabolism in rat skeletal muscle in vitro.

The effect of insulin-like growth factor II (IGF II) on the rates of lactate formation, glycogen synthesis and glucose transport in the presence of a range of concentrations of insulin were investigated using an isolated preparation of rat skeletal muscle. IGF II, at a concentration of 65 ng/ml, caused a small but significant increase in the rates of these processes at a basal physiological insulin concentration (10 muunits/ml), but was without effect in the presence of 1, 100, 1000 or 10,000 muunits of insulin/ml. Hence IGF II increased the insulin sensitivity of this tissue. This effect was removed if the incubation medium was supplemented with an equimolar concentration of IGF binding protein 1 (BP1). It is suggested that changes in the concentration of IGF II and/or BP1 may regulate glucose uptake and metabolism in skeletal muscle and have physiological significance in the control of blood glucose level.