Inhibition of allergen-induced pulmonary responses by the selective tryptase inhibitor 1,5-bis-[4-[(3-carbamimidoyl-benzenesulfonylamino)-methyl]-phenoxy]-pen tane (AMG-126737).

Emerging evidence suggests that mast cell tryptase is a therapeutic target for the treatment of asthma. The effects of this serine protease are associated with both pathophysiologic pulmonary responses and pathologic changes of the asthmatic airway. In this study, the tryptase inhibitor 1,5-bis-[4-[(3-carbamimidoyl-benzenesulfonylamino)-methyl]-p henoxy]-pentane (AMG-126737) was evaluated for its pharmacologic effects against allergen-induced airway responses. AMG-126737 is a potent inhibitor of human lung mast cell tryptase (Ki = 90 nM), with greater than 10- to 200-fold selectivity versus other serine proteases. Intratracheal administration of AMG-126737 inhibited the development of airway hyperresponsiveness in allergen-challenged guinea pigs with an ED50 of 0.015 mg/kg. In addition, the compound exhibited oral activity in the guinea pig model. The in vivo activity of AMG-126737 was confirmed in a sheep model of allergen-induced airway responses, where the compound inhibited early and late phase bronchoconstriction responses and the development of airway hyperresponsiveness. These results support the proposed role of tryptase in the pathology of asthma and suggest that AMG-126737 has potential therapeutic utility in this pulmonary disorder.

Secretory leukocyte protease inhibitor prevents allergen-induced pulmonary responses in animal models of asthma.

Secretory leukocyte protease inhibitor (SLPI) is a naturally occurring protein of human airways that exhibits broad spectrum inhibitory activity against mast cell and leukocyte serine proteases implicated in asthma pathology. To assess the potential therapeutic utility of SLPI in this disorder, its effects on antigen-induced pulmonary responses were evaluated. In Ascaris-sensitized sheep, SLPI (3 mg) administered by aerosol daily for 4 days, with the final dose 0.5 h before antigen challenge, reduced the areas under the curve for early- and late-phase bronchoconstriction (73 and 95%, respectively; p <.05 versus control responses). SLPI also inhibited the development of airway hyperresponsiveness to carbachol (84%, p <. 05 versus control response) measured 24 h after antigen challenge. In ovalbumin-sensitized guinea pigs, intratracheal administration of SLPI daily for 3 days, with the final dose 1 h before antigen challenge, inhibited the development of airway hyperresponsiveness to histamine with an ED50 of <0.05 mg/kg. Prolonged pharmacodynamic activity of SLPI was observed in both species. In a murine model of atopic asthma, SLPI inhibited leukocyte influx into the airways after chronic allergen challenge. SLPI administered to sheep by the predosing protocol described above also prevented the antigen-induced decrease of tracheal mucus velocity (p <.05). In addition, a single aerosol administration of SLPI (30 mg) to sheep 1 h after antigen challenge inhibited the subsequent late-phase bronchoconstriction and development of hyperresponsiveness and reversed the stimulated decrease in tracheal mucus velocity. These results suggest that SLPI may provide therapeutic intervention against the pathophysiology of asthma and its underlying pathology.

Intravenous keratinocyte growth factor protects against experimental pulmonary injury.

Keratinocyte growth factor (KGF) administered by intratracheal instillation is well documented to stimulate the proliferation of alveolar and bronchial cells. In the present study, intravenous KGF was also shown to stimulate the proliferation of alveolar and bronchial cells in mice and rats, although to a lesser degree than intratracheal KGF. Despite the decreased potency of intravenous KGF on pulmonary cell 5-bromo-2'-deoxyuridine incorporation compared with intratracheal KGF, intravenous KGF was very effective in preventing experimental bleomycin-induced pulmonary dysfunction, weight loss, and mortality in either mice or rats and experimental hyperoxia-induced mortality in mice. The effectiveness of intravenous administration of KGF in preventing lung injury suggests that the mechanisms of the protective effect of KGF may involve more than pulmonary cell proliferation and also suggests the potential use of systemic KGF for clinical trials in settings of pulmonary injury.

Keratinocyte growth factor protects mice from chemotherapy and radiation-induced gastrointestinal injury and mortality.

Keratinocyte growth factor (KGF) stimulates the proliferation and differentiation of epithelial cells including those of the gastrointestinal tract. Although chemotherapeutics and radiation exposure kill rapidly proliferating tumor cells, rapidly dividing normal cells of the host's gastrointestinal tract are also frequently damaged, leading to the clinical condition broadly termed "mucositis." In this report, recombinant human KGF used as a pretreatment in several mouse models of chemotherapy and/or radiation-induced gastrointestinal injury significantly improved mouse survival. Using multiple-dose 5-fluorouracil, methotrexate, and radiation in combination and total body radiation alone models, KGF increased survival by 55% or greater. In the models that used chemotherapy with or without radiation, KGF significantly ameliorated weight loss after injury and accelerated weight gain during recovery. The basis of these systemic benefits appears to be due in part to the trophic effects of the growth factor on the intestinal epithelium because KGF pretreatment caused an increase in measures of mucosal thickness (villus height and crypt depth) that persisted during the course of 5-fluorouracil chemotherapy. Treatment with KGF also afforded a 3.5-fold improvement in crypt survival in the small intestine, suggesting that KGF also has a direct effect on the crypt stem cells. These data indicate that KGF may be therapeutically useful to lessen the intestinal side effects of current cancer therapy regimens.

Tryptase mediates hyperresponsiveness in isolated guinea pig bronchi.

Hyperresponsiveness of airway smooth muscle to allergens and environmental factors has long been associated with the pathophysiology of asthma. Tryptase, a serine protease of lung mast cells, has been implicated as one of the mediators involved in the induction of hyperresponsiveness. As a consequence, tryptase inhibitors have become the subject of study as potential novel therapeutic agents for asthma. Secretory leukocyte protease inhibitor (SLPI) is a naturally occurring protein of human airways which exhibits anti-tryptase activity. To assess the potential therapeutic utility of SLPI in asthma, its effects were evaluated using in vitro and ex vivo models of airway hyperresponsiveness and compared with the effects of the small molecule tryptase inhibitor APC-366. Our results demonstrate that SLPI inhibits tryptase-mediated hyperresponsiveness in vitro and attenuates the hyperresponsiveness observed in airway smooth muscle from antigen-sensitized animals subjected to antigen exposure. The small molecule tryptase inhibitor APC-366 has a similar inhibitory effect. Thus, tryptase appears to be a significant contributor to the development of hyperresponsiveness in these models. To the extent that tryptase contributes to the development and progression of asthma, SLPI may possess therapeutic potential in this disease setting.

Prevention of bleomycin-induced lung injury in rats by keratinocyte growth factor.

Intratracheal instillation of bleomycin produces pulmonary fibrosis in rats. Alveolar type II cell proliferation is thought to minimize the fibrotic response after lung injury. Because keratinocyte growth factor (KGF) stimulates type II cell proliferation in the rat, we designed experiments to evaluate whether intratracheal KGF before or after intratracheal bleomycin would prevent pulmonary fibrosis. Intratracheal bleomycin without KGF resulted in moderate to severe lung injury and subsequent fibrosis. Conversely, intratracheal KGF pretreatment at 48 or 72 hr before bleomycin resulted in minimal to no visible lung injury. Rats pretreated with phosphate buffered saline before bleomycin had significantly more neutrophils and protein in bronchoalveolar lavage fluid at 4 and 6 days and higher hydroxyproline levels after bleomycin as compared to KGF-pretreated rats. Pretreatment with KGF at 48 hr protected against bleomycin-induced alterations in pulmonary physiology and increased surfactant protein C-positive (SP-C)-positive cells and SP-A, SP-B, SP-C, and SP-D mRNA levels after bleomycin instillation when compared to saline pretreated rats on day 1 or day 7. KGF posttreatment protocols did not prevent bleomycin lung injury and fibrosis. We conclude that KGF pretreatment attenuates bleomycin lung injury and increases type II cell proliferation and surfactant protein gene expression after bleomycin instillation in the rat.

Keratinocyte growth factor increases surfactant pool sizes in premature rabbits.

Keratinocyte growth factor (KGF) is a recently described 19-kD glycoprotein that is a mitogen for epithelial-derived tissue and is expressed during embryonic tissue differentiation. We investigated whether human recombinant KGF (rKGF) could increase alveolar and lung-tissue saturated phosphatidyl-choline (Sat PC) in preterm rabbits delivered at 28.5 d gestation. Preterm rabbits were given 1, 5, or 10 mg/kg rKGF by tracheal injection at 30 min of age. Lung tissue and total lung (alveolar plus lung tissue) Sat PC increased by 48 h (5 and 10 mg/kg) (p < 0.01) and 72 h (1 and 10 mg/kg) (p < 0.05). A single intravascular or intramuscular dose of 10 mg/kg rKGF also increased long tissue and total-lung Sat PC by 48 h (p < 0.01). A single dose of rKGF given shortly after birth to preterm rabbits increased lung Sat PC in the preterm rabbits.

Induction by aerosol allergen of sustained and nonspecific IgE-mediated airway hyperreactivity in the guinea pig.

Described is a guinea pig model of allergic asthma, in which animals are sensitized by intraperitoneal injections of ovalbumin complexed with aluminum hydroxide to elicit an IgE response. Aerosol exposure to ovalbumin induces acute bronchoconstriction and nonspecific airways hyperreactivity, evaluated by intravenous challenges of histamine or acetylcholine. This model mimics the hallmark features of human asthma, is induced by respired allergen and is pharmacologically modulated by prophylactic drugs (methyl prednisolone, ketotifen, theophylline) and the competitive PAF receptor antagonist SDZ 264-412.

Prevention of non-specific airway hyperreactivity after allergen challenge in guinea-pigs by the PAF receptor antagonist SDZ 64-412.

1. Allergen challenge by aerosol in sensitized guinea-pigs elicited non-specific airway hyperreactivity assessed by reactivity to i.v. histamine or acetylcholine. Airway hyperreactivity to histamine persisted for at least 48 h and was accompanied by pulmonary eosinophilia as determined by bronchoalveolar lavage cell analysis. 2. Airway hyperreactivity was independent of vagal reflex mechanisms since it was not abrogated by bilateral vagotomy. 3. The novel platelet-activating factor (PAF) receptor antagonist SDZ 64-412 inhibited the development of airway hyperreactivity, as measured 24 h after aerosol allergen challenge, when given as a single treatment orally 2 h before allergen challenge. The PAF receptor antagonist WEB 2086 as well as methylprednisolone and ketotifen also showed efficacy in preventing development of airway hyperreactivity. 4. Neither the two PAF antagonists nor ketotifen had any effect on bronchoalveolar lavage (BAL) eosinophil numbers. Methylprednisolone was the only substance which readily prevented eosinophil recruitment in addition to airway hyperreactivity. 5. We conclude that allergen-induced airway hyperreactivity in guinea-pigs is inhibited by prophylactic anti-asthma drugs and specific PAF receptor antagonists, thus demonstrating a pivotal role of PAF in this response. There was a lack of correlation between airway hyperreactivity and the presence of BAL eosinophils.

Resolution of impaired pulmonary function and pulmonary hypertension after phorbol ester administration in rabbits.

To assess metabolic functions of the pulmonary circulation during lung injury and subsequent recovery from injury, we measured angiotensin-converting enzyme (ACE) activity by means of benzoyl-phenylalanyl-alanyl-proline (BPAP) hydrolysis and 5-hydroxytryptamine (5-HT) removal in vivo in three groups of anesthetized rabbits. One group was treated with 30 micrograms/kg/day phorbol myristate acetate (PMA) intravenously 10 times over 14 days (PMA group). A second group received the same PMA treatment but was not studied until 14 days after the last treatment (PMA/recovery group). A third group was treated with vehicle alone. At the end of PMA treatment, rabbits had an elevated pulmonary artery pressure and depressed ACE activity, expressed as the ratio Vmax/Km. Decreased Vmax/Km for ACE was due to a significant reduction in apparent Vmax for BPAP (control = 235 +/- 37, PMA = 139 +/- 12 nmol/s). Km was unchanged (control = 25 +/- 4, PMA = 31 +/- 7 microM). Uptake of 5-HT was unaffected by PMA treatment. After 2 wk of recovery (PMA/recovery group), pulmonary hypertension had resolved. In this group, Vmax for BPAP hydrolysis was not significantly different from control (280 +/- 18 nmol/s), but Km was significantly increased (48 +/- 5 microM). We conclude that repeated exposure of rabbits to PMA results in lung injury manifested as depressed pulmonary ACE activity and pulmonary artery hypertension. Although much of these alterations were reversible within 2 wk after discontinuing PMA, an increase in apparent Km of ACE may be a more persistent alteration in vascular endothelial cell dysfunction.

Pulmonary angiotensin-converting enzyme kinetics after acute lung injury in the rabbit.

Depression of lung endothelial cell metabolic function may be an early and sensitive indicator of lung damage. When such functions are measured in vivo, substrates injected usually must be limited to "trace" doses due to the significant hemodynamic effects of high doses of substrate. Under first-order conditions (i.e., trace doses) the enzyme or transport system rate constant Vmax/Km may be calculated, but independent estimates of each variable (Vmax and Km) are not available. We therefore used multiple indicator-dilution methods and higher substrate concentrations to apply a mathematical model, based on saturable kinetics that yield independent estimates of the apparent kinetic parameters Vmax and Km for pulmonary angiotensin-converting enzyme (ACE). We used the ACE substrate, [3H]benzoyl-phenylalanyl-alanyl-proline ([3H]BPAP) and made these measurements and also estimates of serotonin [5-hydroxytryptamine (5-HT)] removal, before and after acute lung injury induced by intratracheal administration of phorbol myristate acetate (PMA). PMA significantly depressed the percent 5-HT removal (62 +/- 3 to 44 +/- 4%) and BPAP percent metabolism (74 +/- 2 to 66 +/- 2), when trace amounts of either compound were injected as a bolus.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of nafazatrom and indomethacin on pulmonary removal of prostaglandin E1 after endotoxin in rabbits.

1 We compared the effects of endotoxin on pulmonary prostaglandin E1 (PGE1) removal in groups of rabbits pretreated with the cyclo-oxygenase inhibitor, indomethacin, or nafazatrom (Bay g 6575), which has been shown to increase plasma prostacyclin concentrations. 2 In untreated animals, endotoxin transiently decreased pulmonary removal of [3H]-PGE1, caused pulmonary hypertension, systemic hypotension and increased plasma concentrations of PGE2 and 6-keto-PGF1 alpha. 3 Indomethacin pretreatment prevented the transient decrease in pulmonary removal of [3H]-PGE1 in response to endotoxin, prevented the haemodynamic effects and inhibited prostaglandin synthesis. Pretreatment with nafazatrom did not affect the decreased pulmonary removal of [3H]-PGE1, exacerbated the haemodynamic response, reduced survival and potentiated the increase in circulating 6-keto-PGF1 alpha. 4 We conclude that indomethacin acts to prevent the depression of pulmonary [3H]-PGE1 removal by eliminating surface area changes associated with endotoxin-induced pulmonary vasoconstriction. 5 These data suggest that nafazatrom treatment results in exacerbation of the endotoxin-induced systemic hypotension presumably due to its effect on increased plasma prostacyclin during the later phase of endotoxaemia.

Resolution of increased permeability pulmonary edema in rats.

The rate and sequence of interstitial and alveolar fluid removal from the lung after the occurrence of pulmonary edema were examined. Rats were given intraperitoneal injections of 20 mg/kg alpha-naphthylthiourea (ANTU), resulting in an increased permeability edema with alveolar flooding. Animals were killed at intervals between 2 and 48 hours after ANTU for the gravimetric determination of extravascular lung water (Qwl/dQl) and histologic study of the lung. Interstitial fluid volume was quantified by a morphometric technique. The assumptions were made that edema fluid equaled the experimental Qwl/dQl minus the normal Qwl/dQl, and that the edema fluid volume equaled the sum of interstitial and alveolar fluid volume. It was found that between 2 and 4 hours after the induction of pulmonary edema, fluid was removed from the alveolar space faster than it was removed from the interstitial space. Between 4 and 48 hours after ANTU, the fluid removal rate from both compartments was much slower, and interstitial fluid was removed at a faster rate than alveolar fluid. It is hypothesized that the later phase of fluid removal from the lung is dependent on the removal of protein.

Role of interstitium in clearance of alveolar fluid in normal and injured lungs.

We investigated the contribution of the pulmonary interstitial space to the removal of alveolar fluid and solute. We prepared anesthetized sheep for the collection of lung lymph. A balloon-tipped catheter was advanced into a lower lung lobe, and 20 ml Ringer lactate solution (RL) were instilled in one group. Other groups received 20 ml RL with 4 mg/ml Evans blue dye (EB) or 10 micrograms/kg phorbol myristate acetate (PMA) or both. Instillation of 20 ml RL and EB resulted in an increase in lymph flow over RL alone, presumably by an osmotic mechanism. After 4 h, small perivascular fluid cuffs, which contained little EB, were present, and 1.9% of the instilled EB was removed by the lymphatics. An average of 9.2 ml of excess water remained in the lung. Instillation of RL, EB, and PMA resulted in an increase in lymph flow and large perivascular fluid cuffs, which contained large amounts of EB. Lymphatic removal of the instilled EB accounted for 1.2% of the total amount instilled. An average of 19.1 ml water was present in the lung after 4 h. We conclude that alveolar instillation of PMA results in epithelial and endothelial membrane injury and that when lung injury is present interstitial fluid reservoirs may be important sites of alveolar fluid accumulation and important routes of fluid removal from the air space.

Role of intrapulmonary release of eicosanoids and superoxide anion as mediators of pulmonary dysfunction and endothelial injury in sheep with intermittent complement activation.

In 30 anesthetized sheep, we show that repeated bolus injections of autologous zymosan-activated plasma produce pulmonary hypertension, hypoxemia, intrapulmonary thromboxane release, pulmonary leukostasis, and sustained increases in lung lymph flow and protein clearance. Studies with platelet-rich plasma demonstrated that addition of zymosan-activated plasma does not induce platelet aggregation or thromboxane release. We studied the role of cyclooxygenase products as mediators of these pathophysiological responses by pretreating sheep with either meclofenamate (4 mg/kg) or ibuprofen (12.5 mg/kg). Both drugs inhibited thromboxane release and hypoxemia. Ibuprofen, but not meclofenamate, reproducibly attenuated the hypertensive responses and the increases in lymph flow and protein clearance. Neither drug prevented pulmonary leukostasis. These results demonstrate that cyclooxygenase products mediate the development of complement-induced hypoxemia but are not sole mediators of pulmonary hypertension or increases in vascular permeability. Furthermore, ibuprofen has anti-inflammatory actions, not shared by meclofenamate, which enhance the effectiveness of this drug. Since activated leukocytes release reactive oxygen metabolites, we treated sheep with superoxide dismutase (2800 U/kg per hour) to determine the role of superoxide anions in these responses. This treatment significantly attenuated the increases in lung lymph flow and protein clearance. The results suggest that multiple mediators, which may originate from activated leukocytes sequestered in the pulmonary circulation, contribute to the pathophysiological changes seen with intermittent complement activation. Cyclooxygenase products of arachidonic acid contribute to the hypertension and are solely responsible for the hypoxemia. Reactive oxygen metabolites are important mediators of the complement-induced increases in lung vascular permeability.

alpha-Naphthylthiourea produces dose-dependent lung vascular injury in sheep.

We tested the effects of alpha-naphthylthiourea (ANTU) on lung fluid balance and prostanoid concentrations in anesthetized sheep acutely prepared for collection of pulmonary lymph. Sheep were given 20, 50, 75, or 100 mg/kg ANTU or the vehicle dimethylsulfoxide (DMSO) intravenously. The first phase of the response consisted of transient increases in pulmonary artery pressure and plasma and lymph thromboxane B2 concentrations. Lymph flow increased with no change in the lymph-to-plasma protein concentration ratio (L/P). These changes occurred in sheep given DMSO alone or DMSO with ANTU; they were not dependent on the dose of ANTU given. Two to 4 h after drug administration, pulmonary artery pressure and thromboxane concentrations were normal or near normal. Lymph flow rate reached steady-state levels averaging 1.5 (DMSO alone) to 5.3 (100 mg/kg ANTU) times base line with L/P ratios unchanged from base line. ANTU/DMSO produces transient, severe pulmonary hypertension that may be prostaglandin mediated. The sustained response consists of increased flow rate of protein-rich lymph.

Relationships among lung extravascular fluid compartments with alveolar flooding.

We studied the relationship between lung perivascular cuff fluid and alveolar fluid in dogs with alveolar flooding. In one group of dogs, we produced edema with alloxan. We waited 1 h and injected Evans blue dye intravenously. We then caused additional alveolar flooding by a rapid fluid load. The concentration of dye in perivascular fluid cuffs averaged 40% of that in airway edema fluid. We conclude that alveolar fluid derives from microvascular filtrate in the perimicrovascular compartment rather than from fluid in the loose connective tissue spaces. In a second group of dogs, we produced alveolar flooding by mechanically increasing left atrial pressure with a simultaneous fluid load. After flooding stopped and pressures stabilized, we injected Evans blue dye. Morphological examination of lung sections showed no dye present in alveolar fluid, although dye was present in alveolar septa and perivascular cuffs. Flooding occurs during a transient period consistent with an apparent increase in alveolar epithelial permeability, after which an isolated alveolar reservoir is created.