A High-Throughput Chemical Screen in DJ-1ß Mutant Flies Identifies Zaprinast as a Potential Parkinson's Disease Treatment.

Dopamine replacement represents the standard therapy for Parkinson's disease (PD), a common, chronic, and incurable neurological disorder; however, this approach only treats the symptoms of this devastating disease. In the search for novel disease-modifying therapies that target other relevant molecular and cellular mechanisms, Drosophila has emerged as a valuable tool to study neurodegenerative diseases due to the presence of a complex central nervous system, the blood-brain barrier, and a similar neurotransmitter profile to humans. Human PD-related genes also display conservation in flies; DJ-1ß is the fly ortholog of DJ-1, a gene for which mutations prompt early-onset recessive PD. Interestingly, flies mutant for DJ-1ß exhibit PD-related phenotypes, including motor defects, high oxidative stress (OS) levels and metabolic alterations. To identify novel therapies for PD, we performed an in vivo high-throughput screening assay using DJ-1ß mutant flies and compounds from the Prestwick® chemical library. Drugs that improved motor performance in DJ-1ß mutant flies were validated in DJ-1-deficient human neural-like cells, revealing that zaprinast displayed the most significant ability to suppress OS-induced cell death. Zaprinast inhibits phosphodiesterases and activates GPR35, an orphan G-protein-coupled receptor not previously associated with PD. We found that zaprinast exerts its beneficial effect in both fly and human PD models through several disease-modifying mechanisms, including reduced OS levels, attenuated apoptosis, increased mitochondrial viability, and enhanced glycolysis. Therefore, our results support zaprinast as a potential therapeutic for PD in future clinical trials.

GPR35 regulates osteogenesis via the Wnt/GSK3ß/ß-catenin signaling pathway.

It is well known that osteoporosis is a significant chronic disease with the increase of the aging population. Here, we report that expression of G protein-coupled receptor 35 (GPR35) in bone marrow mesenchymal stem cells (BMSCs) is suppressed in diagnosed osteoporosis patients and osteoporotic mice. The expression of GPR35 on BMSCs is enhanced during osteogenic differentiation. GPR35 knockout suppresses the proliferation and osteogenesis of BMSCs and deteriorates bone mass in both sham-treated and ovariectomized mice. Moreover, GPR35 deficiency reduces ß-catenin activity in BMSCs. In contrast, the overexpression of GPR35 contributes to these processes in BMSCs. Finally, using zaprinast, a synthetic GPR35 agonist, we show that zaprinast rescues OVX-induced bone loss and promotes bone generation in mice. Thus, GPR35 may as a new target and its agonist zaprinast may serve as a novel treatment for osteoporosis.

Zaprinast diminished pain and enhanced opioid analgesia in a rat neuropathic pain model.

The mechanism of neuropathic pain is complex and unclear. Based on our results, we postulate that an intensification of the kynurenine pathway occurs as a consequence of nerve injury. The G protein-coupled receptor 35 (GPR35) is important for kynurenine pathway activation. Cyclic GMP-specific phosphodiesterase inhibitors have also been shown to have beneficial effects on neuropathic pain. Therefore, the aims of our research were to elucidate how a substance that acts as both an agonist of GPR35 and an inhibitor of phosphodiesterase influences neuropathic pain in a rat model. Here, we demonstrated that preemptive and repeated intrathecal (i.t.) administration (16 h and 1 h before injury and then after nerve ligation daily for 7 days) of zaprinast (1 µg/5 µl) significantly attenuated mechanical (von Frey test) and thermal (cold plate test) hypersensitivity measured on day 7 after chronic constriction injury, and the effect of even a single injection lasted up to 24 h. Our data indicate that zaprinast diminished the number of IBA1-positive cells and consequently attenuated the levels of IL-1beta, IL-6, IL-18, and NOS2 in the lumbar spinal cord and/or dorsal root ganglia. Our results also demonstrated that zaprinast potentiated the analgesic properties of morphine and buprenorphine. In summary, in a neuropathic pain model, zaprinast significantly reduced pain symptoms and enhanced the effectiveness of opioids. Our data provide new evidence that modulation of both GPR35 and phosphodiesterase could be an important strategy for innovative pharmacological treatments designed to decrease hypersensitivity evoked by nerve injury.

Antidepressant-like effects of translocator protein (18 kDa) ligand ZBD-2 in mouse models of postpartum depression.

The 18 kDa translocator protein (TSPO) is primarily localized in the outer mitochondrial membrane of steroid-synthesizing cells in the central and peripheral nervous systems. One of the protein's main functions is transporting substrate cholesterol into the mitochondria in a prerequisite process for steroid synthesis. Clinical trials have indicated that TSPO ligands might be valuable in treating some neuropathies and psychopathies. However, limited information is known about the role of TSPO in postpartum depression (PPD). The TSPO ligand ZBD-2, a derivative of XBD173, was synthesized in our laboratory. Behavioral tests, enzyme linked immunosorbent assay, and Western blot were employed to evaluate ZBD-2's efficacy against PPD and to elucidate the potential underlying molecular mechanism. The TSPO levels significantly decreased in the basolateral amygdala of PPD models. After treatment for 2 weeks, ZBD-2 alleviated depression-like behaviors and enhanced the TSPO level in a PPD animal model. The underlying mechanisms of ZBD-2 were related to regulate the hypothalamic-pituitary-adrenal axis, enhance 5-HT and BDNF secretion, and maintain the excitatory and inhibitory synaptic protein expression to normal levels. Our results directly confirm that ZBD-2 exerts a therapeutic effect on PPD, which provides a new target for anti-PPD drug development.

Antidepressant-Like and Anxiolytic-Like Effects of ZBD-2, a Novel Ligand for the Translocator Protein (18 kDa).

Activation of translocator protein (18 kDa) (TSPO) plays an important role to mediate rapid anxiolytic efficacy in stress response and stress-related disorders by the production of neurosteroids. However, little is known about the ligand of TSPO on the anxiety-like and depressive behaviors and the underlying mechanisms in chronic unpredictable mild stress (UCMS) mice. In the present study, a novel ligand of TSPO, ZBD-2 [N-benzyl-N-ethyl-2-(7,8-dihydro-7-benzyl-8-oxo-2-phenyl-9H-purin-9-yl) acetamide] synthesized by our laboratory, was used to evaluate the anxiolytic and antidepressant efficacy and to elucidate the underlying mechanisms. ZBD-2 (3 mg/kg) significantly attenuated anxiety-like and depressive behaviors in the UCMS mice, which was blocked by TSPO antagonist PK11195 (3 mg/kg). Treatment of ZBD-2 reversed the decrease in biogenic amines (norepinephrine, dopamine, and serotonin) in the brain region of hippocampus in the UCMS mice. The decreases in TSPO, GluN2B-containing N-methyl-D-aspartate (NMDA) receptors, GluA1, p-GluA1-Ser831, p-GluA1-Ser845, PSD-95, and GABAA-a2 were integrated with the increases of CaMKII and iNOS levels in the hippocampus of the UCMS mice. ZBD-2 significantly reversed the changes of above proteins. However, ZBD-2 or PK11195 treatment did not affect the levels of GluN2A-containing NMDA receptors and the total levels of GAD67. Our study provides strong evidences that ZBD-2 has a therapeutic effect on chronic stress-related disorders such as depression and anxiety through regulating the biogenic amine levels and the synaptic proteins in the hippocampus.

A novel translocator protein 18 kDa ligand, ZBD-2, exerts neuroprotective effects against acute spinal cord injury.

Traumatic spinal cord injury (SCI) happens accidently and often leads to motor dysfunction due to a series of biochemical and pathological events and damage, either temporarily or permanently. Translocator protein 18 (TSPO) has been found to be involved in the synthesis of endogenous neurosteroids which have multiple effects on neurons, but the internal mechanisms are not clear. N-benzyl-N-ethyl-2-(7,8-oxo-2-phenyl-9H-purin-9-yl) acetamide (ZBD-2), a newly reported ligand of TSPO, shows some neuroprotective effect against focal cerebral ischemia in vivo and NMDA-induced neurotoxicity in vitro. The present study aims to examine the role of ZBD-2 in SCI mice and elucidate the underlying molecular mechanisms. The SCI model was established by crushing spinal cord. ZBD-2 (10 mg/kg) significantly enhanced the hindlimb locomotor functions after SCI and decreased the tissue damage and conserved the white matter of the spinal cord. High-dose ZBD-2 alleviated the oxidative stress induced by SCI and regulated the imbalance between NR2B-containing NMDA and GABA receptors by increasing the levels of GAD67 in the spinal cord of SCI mice. Additionally, ZBD-2 (10 mg/kg) increased phosphorylated Akt (p-Akt) and decreased the ratio of Bax/Bcl-2. These results demonstrate that ZBD-2 performs neuroprotection against SCI through regulating the synaptic transmission and the PI3K/AKT signaling pathway.

Kynurenic acid and zaprinast induce analgesia by modulating HCN channels through GPR35 activation.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have a key role in the control of cellular excitability. HCN2, a subgroup of the HCN family channels, are heavily expressed in small dorsal root ganglia (DRG) neurons and their activation seems to be important in the determination of pain intensity. Intracellular elevation of cAMP levels activates HCN-mediated current (Ih) and small DRG neurons excitability. GPR35, a Gi/o coupled receptor, is highly expressed in small DRG neurons, and we hypothesized that its activation, mediated by endogenous or exogenous ligands, could lead to pain control trough a reduction of Ih current. Patch clamp recordings were carried out in primary cultures of rat DRG neurons and the effects of GPR35 activation on Ih current and neuronal excitability were studied in control conditions and after adenylate cyclase activation with either forskolin or prostaglandin E2 (PGE2). We found that both kynurenic acid (KYNA) and zaprinast, the endogenous and synthetic GPR35 agonist respectively, were able to antagonize the forskolin-induced depolarization of resting membrane potential by reducing Ih-mediated depolarization. Similar results were obtained when PGE2 was used to activate adenylate cyclase and to increase Ih current and the overall neuronal excitability. Finally, we tested the analgesic effect of both GPR35 agonists in an in vivo model of PGE2-induced thermal hyperalgesia. In accord with the hypothesis, both KYNA and zaprinast showed a dose dependent analgesic effect. In conclusion, GPR35 activation leads to a reduced excitability of small DRG neurons in vitro and causes a dose-dependent analgesia in vivo. GPR35 agonists, by reducing adenylate cyclase activity and inhibiting Ih in DRG neurons may represent a promising new group of analgesic drugs.

Effect of the phosphodiesterase-5 inhibitor zaprinast on ischemia-reperfusion injury in rats.

PURPOSE: The cardiac and renal protective effects of phosphodiesterase-5 (PDE-5) inhibitors against ischemia-reperfusion injury have recently been demonstrated in animal studies. We evaluated the effect of pretreatment with the PDE-5 inhibitor zaprinast on warm renal ischemia in a rat model. METHODS: Female Sprague-Dawley rats underwent concomitant right nephrectomy and left renal hilar occlusion for 30 minutes. Twelve animals were equally divided into three groups: Group 1 received no pharmacologic pretreatment, group 2 was pretreated with zaprinast 10 mg/kg, and group 3 was pretreated with zaprinast 20 mg/kg. Zaprinast was dissolved in 25% dimethyl sulfoxide and given as a single intraperitoneal injection 30 minutes before surgery. Serum blood urea nitrogen (BUN) and creatinine levels, histopathology, and TUNEL staining for apoptosis were assessed 24 hours postoperatively. RESULTS: The mean creatinine level for groups 1, 2, and 3 was 0.73 mg/dL, 0.55 mg/dL, and 0.38 mg/dL, respectively. These values were not statistically different (P=0.099). The mean BUN levels of 35.8 mg/dL for group 1, 27.3 mg/dL for group 2, and 23.3 mg/dL for group 3 were also statistically similar (P=0.278). There were no objective differences in histopathologic evaluation or TUNEL staining between the groups. CONCLUSION: This study did not demonstrate a beneficial effect of zaprinast pretreatment on renal parameters after warm ischemic injury.

Phosphodiesterase inhibitors for persistent pulmonary hypertension of the newborn: a review.

Persistent pulmonary hypertension of the newborn (PPHN) is a complex syndrome with multiple causes, with an incidence of 0.43-6.8/1,000 live births and a mortality of 10-20%. Survivors have high morbidity in the forms of neurodevelopmental and audiological impairment, cognitive delays, hearing loss, and a high rate of rehospitalization. The optimal approach to the management of PPHN remains controversial. Inhaled nitric oxide (iNO) is currently regarded as the gold standard therapy, but with as many as 30% of cases failing to respond, has not proven to be the single magic bullet. Given the complex pathophysiology of the disease, any such magic bullet is unlikely. A number of recent studies have suggested a role for specific phosphodiesterase (PDE) inhibitors in the management of PPHN. Sildenafil, a specific PDE5 inhibitor, appears the most promising of such agents. We aim to review the current status and limitations of iNO and the potential of PDE inhibitors in the management of PPHN. The reasons why caution is warranted before specific PDE5 inhibitors like sildenafil are labelled as potential magic bullets for PPHN will be discussed. The need for randomized-controlled trials to determine the safety, efficacy, and long-term outcome following treatment with sildenafil in PPHN is emphasized.

Zaprinast, a phosphodiesterase 5 inhibitor, overcomes sexual dysfunction produced by fluoxetine, a selective serotonin reuptake inhibitor in hamsters.

A high incidence of sexual dysfunction among women is reported in the clinical literature. Little experimental investigation has been initiated on the ability of phosphodiesterase (PDE) inhibitors to overcome deficits in sexual functioning because of selective serotonin reuptake inhibitors (SSRIs). The effects of fluoxetine, an SSRI, and zaprinast, a PDE-5 inhibitor, on the lateral displacement response (used as a measure of sensitivity to reproductively relevant stimuli) of hamsters in behavioral estrus were investigated. In Experiment 1, hamsters that were maximally sensitive to reproductively relevant stimuli because they were at the peak of behavioral estrus were administered fluoxetine (10 mg/kg, i.p.); they had significantly decreased lateral displacement responses compared to vehicle-administered hamsters. In Experiment 2, hamsters that were relatively less sensitive to sexual stimuli because they were at the termination of behavioral estrus were administered zaprinast (3 mg/kg; i.p.); they had significantly enhanced lateral displacement responses compared to responses seen following vehicle administration. In Experiment 3, fluoxetine-induced deficits in the lateral displacement of hamsters at the peak of behavioral estrus were overcome by the coadministration of zaprinast. These data confirm previous findings that sexual dysfunction can be induced by SSRIs and extend the current knowledge to suggest that administration of a PDE-5 inhibitor can override SSRI-induced deficits in sexual functioning.

Pharmacology of phosphodiesterase-5 inhibitors.

The clinical properties (efficacy and safety profile) of a medicine are related not only to its mode of action, but also to its selectivity for its target (usually a receptor or enzyme) and are also influenced by its pharmacokinetic properties (absorption, distribution, metabolism and elimination). The growing number of phosphodiesterase inhibitors that are selective for phosphodiesterase-5 (PDE5) represent a promising new class of compounds that are useful for the treatment of erectile dysfunction and perhaps other disorders. Some of the basic pharmacodynamic and pharmacokinetic parameters that describe drug action are discussed with regard to the new PDE5 inhibitors. Central topics reviewed are the concentration that produces a given in vitro response, or potency (IC50), maximum plasma concentration (Cmax), time to Cmax (Tmax), half-life (t 1/2), area under the curve (AUC), bioavailability, onset and duration of action, and the balance to achieve optimum safety and efficacy. To illustrate these concepts, a group of inhibitors with varying selectivities and potencies for PDE5 (theophylline, IBMX, zaprinast, sildenafil, tadalafil and vardenafil) are discussed. Each drug has its own set of unique pharmacological characteristics based on its specific molecular structure, enzyme inhibition profile and pharmacokinetic properties. Each PDE5 inhibitor has a distinct selectivity that contributes to its safety profile. As with all new drugs, and especially those in a new class, careful evaluation will be necessary to ensure the optimal use of the PDE5 inhibitors.

Cessation of platelet-mediated cyclic canine coronary occlusion after thrombolysis by combining nitric oxide inhalation with phosphodiesterase-5 inhibition.

OBJECTIVES: We sought to evaluate the ability of type 5 phosphodiesterase (PDE5) inhibitors to augment the antithrombotic effects of inhaled nitric oxide (NO) in a canine model of platelet-mediated coronary thrombosis after thrombolysis. BACKGROUND: Type 5 phosphodiesterase inhibitors potentiate the ability of NO to inhibit platelet aggregation in vitro by preventing platelet cyclic guanosine monophosphate catabolism. We previously reported that breathing low concentrations of NO gas attenuated, but did not prevent, cyclic flow reductions (CFRs) in a canine model of coronary thrombosis after thrombolysis. METHODS: Cyclic flow reductions were induced after creation of a left anterior descending coronary artery stenosis, endothelial injury, thrombus formation and thrombolysis. Dogs were either untreated or treated with inhaled NO (20 ppm by volume), intravenous zaprinast, intravenous dipyridamole or the combination of inhaled NO with either PDE5 inhibitor (n = 4 per group). RESULTS: Cyclic flow reductions ceased, and complete coronary patency was achieved in all dogs after they breathed NO combined with zaprinast (by 12.0+/-4.7 min [mean +/- SEM]) or dipyridamole (by 9.8+/-4.7 min). The frequency of CFRs was unaffected by NO, dipyridamole or zaprinast alone. Systemic arterial blood pressure and bleeding time were unchanged with any treatment. Ex vivo thrombin-induced platelet aggregation in dogs breathing NO and receiving dipyridamole was reduced by 75+/-7% (p < 0.05). CONCLUSIONS: The PDE5 inhibitors potentiated the antithrombotic properties of inhaled NO in a canine model of platelet-mediated coronary artery thrombosis after thrombolysis, without prolonging the bleeding time or causing systemic hypotension.

[Endothelin receptor antagonist, phosphodiesterase inhibitor, thromboxane inhibitor].

According to the advanced comprehension of pathophysiology of primary pulmonary hypertension (PPH), a therapeutical approach to PPH has changed recently. One of the breakthrough to the treatment of PPH is application of prostacyclin. It has been revealed that intravenous administration of prostacyclin has improved the prognosis and patient's quality of life. Another development of endothelin receptor antagonists and phosphodiesterase inhibitors have provided a novel pulmonary-specific effect. An endothelin receptor antagonist has a great inhibitory effect against pulmonary vasculature remodeling. In this regard, this regard, this receptor antagonist has superior effect to other medicines. Furthermore, a phosphodiesterase inhibitor shows a great decreasing effect on pulmonary hypertension with less effect on systemic blood pressure. These drugs will provide a great potential to the treatment of pulmonary hypertension.

Combined therapy with zaprinast and inhaled nitric oxide abolishes hypoxic pulmonary hypertension.

OBJECTIVE: To determine whether the combination of the phosphodiesterase 5 inhibitor zaprinast and inhaled nitric oxide (NO) decreases hypoxic pulmonary hypertension in the rat. DESIGN: Prospective, experimental study. SETTING: Animal laboratory of a university medical center. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Anesthetized rats were mechanically ventilated and instrumented for measurement of mean systemic arterial pressure, pulmonary arterial pressure, and cardiac output. In group 1, four acute hypoxic challenges (FIO2 = 0.17 for 5 mins) were performed: initial, during 40 ppm inhaled NO, immediately after discontinuation of 5 mins of inhaled NO, and final. In group 2 rats, an initial hypoxic challenge was performed and rats then received zaprinast (3 mg/kg bolus followed by 0.3 mg/kg/min infusion). Four hypoxic challenges analogous to group 1 were then performed during zaprinast administration. MEASUREMENTS AND MAIN RESULTS: Initial hypoxic challenge produced similar increases in pulmonary arterial pressure in both groups. In group 1, inhaled NO either only before or only during hypoxia decreased the pulmonary hypertensive response to hypoxia. In group 2, zaprinast administration did not alter hemodynamics. Zaprinast alone decreased the pulmonary hypertensive response to hypoxia. The combination of zaprinast and inhaled NO (either before or during hypoxia) abolished the pulmonary hypertensive response to hypoxia. CONCLUSIONS: Treatment with inhaled NO for 5 mins before but not during hypoxia is as effective as inhaled NO during hypoxia. Inhaled NO and zaprinast both decrease the pulmonary hypertensive response to hypoxia, and the combination abolishes the response. The combination of a phosphodiesterase 5 inhibitor and inhaled NO may have clinical applicability in the treatment of pulmonary hypertension.

Effects of intravenous Zaprinast and inhaled nitric oxide on pulmonary hemodynamics and gas exchange in an ovine model of acute respiratory distress syndrome.

BACKGROUND: Inhaled nitric oxide (No) selectively dilates the pulmonary vasculature and improves gas exchange in acute respiratory distress syndrome. Because of the very short half-life of NO, inhaled NO is administered continuously. Intravenous Zaprinast (2-o-propoxyphenyl-8-azapurin-6-one), a cyclic guanosine monophosphate phosphodiesterase inhibitor, increases the efficacy and prolongs the duration of action of inhaled NO in models of acute pulmonary hypertension. Its efficacy in lung injury models is uncertain. The authors hypothesized that the use of intravenous Zaprinast would have similar beneficial effects when used in combination with inhaled NO to improve oxygenation and dilate the pulmonary vasculature in a diffuse model of acute lung injury. METHODS: The authors studied two groups of sheep with lung injury produced by saline lavage. In the first group, 0, 5, 10, and 20 ppm of inhaled NO were administered in a random order before and after an intravenous Zaprinast infusion (2 mg/kg bolus followed by 0.1 mg. kg-1. min-1). In the second group, inhaled NO was administered at the same concentrations before and after an intravenous infusion of Zaprinast solvent (0.05 m NaOH). RESULTS: After lavage, inhaled NO decreased pulmonary arterial pressure and resistance with no systemic hemodynamic effects, increased arterial oxygen partial pressure, and decreased venous admixture (all P < 0.05). The intravenous administration of Zaprinast alone decreased pulmonary artery pressure but worsened gas exchange (P < 0.05). Zaprinast infusion abolished the beneficial ability of inhaled NO to improve pulmonary gas exchange and reduce pulmonary artery pressure (P < 0. 05 vs. control). CONCLUSIONS: This study suggests that nonselective vasodilation induced by intravenously administered Zaprinast at the dose used in our study not only worsens gas exchange, but also abolishes the beneficial effects of inhaled NO.

Site-specific effect of guanosine 3',5'-cyclic monophosphate phosphodiesterase inhibition in isolated lamb lungs.

OBJECTIVE: To determine the effect of combining inhaled nitric oxide (NO) with an inhibitor of guanosine 3',5'-cyclic monophosphate-specific phosphodiesterase on total and segmental lung resistances. STUDY DESIGN: A controlled laboratory study in isolated blood-perfused lungs prepared from lambs. SETTING: Animal research facility affiliated with a university teaching hospital. SUBJECTS: Five newborn lambs at <48 hrs of life. INTERVENTIONS: Isolated blood-perfused lungs were prepared and treated with indomethacin (40 microg/mL) to inhibit prostaglandin synthesis. After a baseline period of normoxia (28% oxygen), pulmonary hypertension was induced with the thromboxane mimetic U46619 (0.1-0.4 microg/kg/min). During pulmonary hypertension, lungs were studied with inhaled NO only, with infusion of zaprinast only (0.25 mg/kg bolus and 0.05 mg/kg/min infusion), and with a combination of the two. For each study condition, the total pressure decrease across the lung was measured, and the inflow-outflow occlusion technique was used to partition the total pressure gradient measured at constant flow (100 mL/kg/min) into gradients across relatively noncompliant large arteries and veins and more compliant small arteries and veins. MEASUREMENTS AND MAIN RESULTS: U46619 infusion produced significant pulmonary vasoconstriction. The combination of inhaled NO and zaprinast decreased the total pressure decrease across the lung significantly more than NO alone. This effect was primarily attributable to a significantly greater decrease in gradient across the small artery segment after inhaled NO and zaprinast compared with NO alone. CONCLUSIONS: Guanosine 3',5'-cyclic monophosphate phosphodiesterase inhibition with zaprinast enhances the effect of inhaled NO, particularly in conditions in which small arteries represent the site of resistance. Phosphodiesterase inhibition may be a promising adjunct to inhaled NO for the treatment of persistent pulmonary hypertension.

Reduced airway hyperresponsiveness by phosphodiesterase 3 and 4 inhibitors in guinea-pigs.

The aim of the present study was to compare the effects of selective phosphodiesterase (PDE) 3, 4 and 5 inhibitors on antigen-induced airway hyperresponsiveness in sensitized guinea-pigs. When the sensitized guinea-pigs were orally pre-treated with the selective PDE4 inhibitor, Ro 20-1724 (30 mg/kg), and studied 48h after OA, a significant reduction (P<0.01) of the leftward shift of the dose-response curve to ACh was noted, whereas it was ineffective at the lower dose (10 mg/kg). Administration of the selective PDE3 inhibitor, milrinone (30 mg/kg) also elicited a significant reduction (P<0.01) of the airway hyperresponsiveness, whereas the PDE5 inhibitor zaprinast (30 mg/kg) was ineffective. These results show that both PDE3 and PDE4 inhibitors are able to inhibit the antigen-induced airway hyperresponsiveness in sensitized guinea-pigs and support the potential utility of selective PDE inhibitors in the treatment of asthma.

A possible involvement of oxidative lung injury in endotoxin-induced bronchial hyperresponsiveness to substance P in guinea pigs.

Reactive oxygen-derived free radical species have been implicated in the pathogenesis and pathophysiology of inflammatory lung diseases. In a guinea pig model of aerosolized endotoxin-induced bronchial hyperresponsiveness to substance P, a possible involvement of oxidative lung injury was assessed by measuring the changes in membrane-bound neutral endopeptidase activity in the airway tissues and the level of lipid peroxides in the plasma. Vehicle-treated animals developed a neutrophilic airway inflammation, bronchial hyperresponsiveness to substance P associated with neutral endopeptidase hypoactivity, and elevation of lipid peroxides at 18 to 24 h after an exposure to endotoxin (75 microgram/ml, 40 min). A nonselective phosphodiesterase inhibitor, aminophylline, and selective phosphodiesterase isoenzyme inhibitors, SDZ-ISQ-844 (type III/IV) and SDZ-MKS-492 (type III), attenuated the neutrophilic airway inflammation induced by endotoxin. Aminophylline, SDZ-MKS-492, and a superoxide anion-generating NADPH-oxidase inhibitor apocynin inhibited bronchial hyperresponsiveness to substance P with attenuation of neutral endopeptidase inactivation induced by endotoxin. SDZ-ISQ-844, SDZ-MKS-492, and apocynin attenuated the elevation of lipid peroxides. The generation of hypochlorite (OCl-) from whole blood leukocytes was attenuated by aminophylline, SDZ-ISQ-844, SDZ-MKS-492, and apocynin at 1 to 2 h after exposure. These results suggest that reactive oxygen-derived free radical species-mediated oxidative lung injury may play an important role in endotoxin-induced bronchial hyperresponsiveness to substance P, and that phosphodiesterase isoenzyme inhibitors may be potentially useful as anti-inflammatory drugs.

Effect of selective phosphodiesterase 3 inhibition on the early and late asthmatic responses to inhaled allergen.

AIMS: Phosphodiesterase isoenzymes may play an important role in the regulation of airway calibre and bronchial smooth muscle function. Immunomodulatory actions may also be important in allergic airway inflammation. We have examined the effect of a phosphodiesterase (PDE) 3 inhibitor (MKS492) on the early and late responses to inhaled allergen in 18 atopic asthmatic subjects. METHODS: On three separate occasions, 2-4 weeks apart, patients were administered either placebo, 20 mg or 40 mg of oral MKS492 as a single dose. After 90 min they inhaled a dose of allergen (grass pollen or D. pteronyssinus) that had been demonstrated to produce a 20% decrease in FEV1. Measurements of FEV1 were repeated at 30 min intervals over the subsequent 7.5 h. RESULTS: After placebo, allergen inhalation produced bronchoconstriction with a maximum mean minimum FEV1 of 75.9% (12.7) (mean [s.d.]) of post-saline baseline values and a late response developed in 13 subjects (decrease in FEV1 > or = 15%). MKS492 (40 mg) increased the mean minimum % post-saline FEV1 at 30 min from 81.6% (14.0) for placebo to 93.1% (13.8), difference 11.5, 95% CI (2.6, 20.4). The mean minimum % post-saline FEV1 in the early response (0-2 h) was also increased by the 40 mg dose to 86.5% (11.8) vs 77.9% (13.8) for placebo, but not by the 20 mg dose. Significant bronchodilatation was noted 90 min after dosing (pre-allergen) in the MKS492 40 mg group. Late phase responses as assessed by 4 h % post-saline FEV1 was significantly improved by MKS 492 40 mg to 90.0% (6.3) vs 83.0% (12.4) for placebo-difference 7.0, 95% CI (0.1, 13.9), but again not by the 20 mg dose. Analysis of the area under the FEV1 response-time course curves showed non-significant reduction in the late response after the 40 mg dose (placebo 159.0, 134.8) vs 128.1, [81.5] for 40 mg). CONCLUSIONS: A novel PDE 3 inhibitor significantly decreases the early bronchoconstrictor response in asthma and attenuates the late response; effects that may be more marked at higher doses.