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A selective inhibitor of inducible nitric oxide synthase inhibits exhaled breath nitric oxide in healthy volunteers and asthmatics¹

TREVOR T. HANSEL^*,2, SERGEI A. KHARITONOV, LOUISE E. DONNELLY, EDWARD M. ERIN^*, MARK G. CURRIE^,3, WILLIAM M. MOORE^,3, PAMELA T. MANNING, DAVID P. RECKER^,4 and PETER J. BARNES

^* Clinical Studies Unit and
Department of Thoracic Medicine, National Heart and Lung Institute (NHLI), Imperial College, London, UK; and
Pharmacia Corporation, Skokie, Illinois, and St. Louis, Missouri, USA

²Correspondence: NHLI Clinical Studies Unit, Royal Brompton Hospital, London, SW3 6HP, UK. E-mail: t.hansel{at}ic.ac.uk

SPECIFIC AIMS

The inducible isoenzyme of nitric oxide synthase (iNOS) generates nitric oxide (NO) in inflammatory diseases such as asthma. In the present study we tested the ability of the active metabolite L-N⁶-(1-iminoethyl)lysine (L-NIL) to inhibit iNOS activity in human airway epithelial cells. We performed a randomized double-blind, placebo-controlled crossover trial using the prodrug L-N⁶-(1-iminoethyl)lysine 5-tetrazole amide (SC-51) to assess tolerability in healthy volunteers and mild asthmatic patients and the effects on levels of exhaled breath NO.

PRINCIPAL FINDINGS

1. L-NIL inhibits iNOS activity but not expression in human airway epithelial cells
Bronchial epithelial cells in brushings derived from normal healthy individuals were shown by immunocytochemistry to express iNOS; the protein is evenly distributed throughout the cytoplasm in basal cells but located beneath the cilia in the columnar, ciliated cells. Treatment of human primary airway epithelial cells with 50 ng/mL of IL-1ß, TNF-, and IFN- (cytomix) for 24 h caused an induction of iNOS protein and an increase in the accumulation of nitrite in cell culture media from 2.20 ± 0.2 µM in the absence of cytomix to 3.5 ± 0.4 µM in the presence of cytomix. This increase in nitrite in the media was inhibited in a dose-dependent manner by addition of L-NIL to the cell media (IC₅₀ 5.7 µM). This decrease in nitrite production is not mediated by an inhibition of iNOS protein expression since the inclusion of L-NIL did not affect iNOS expression as detected by immunoblotting. Instead, as expected, it appears to result from inhibition of enzymatic activity.

2. L-NIL inhibits exhaled NO in healthy volunteers and patients with asthma
The levels of exhaled NO in normal subjects and asthmatic patients (Table 1 )after administration of placebo and doses of either 20 or 200 mg of SC-51 are shown in Fig. 1. The AUC of change in exhaled NO was not significantly different from zero for placebo in any of the four groups of subjects. After administration of both doses of SC-51, there was a rapid reduction in exhaled NO that was significantly different in the healthy volunteers and the asthmatics throughout the 72 h assessment. The difference in AUC of change in NO for the subjects receiving 200 mg of SC-51 was higher in both healthy volunteers (P<0.001) and the asthmatics (P=<0.01) than in subjects receiving 20 mg of SC-51 (P=0.004). The timing, duration, and magnitude in the reduction of exhaled NO after the 20 and 200 mg doses of SC-51 was consistent with the pharmacokinetic profiles observed. Both doses were rapidly absorbed, exhibiting a time to maximal plasma concentration (Tmax) of 0.5 h and a terminal elimination half-life (T_1/2) of 40–53 h. The exposure (AUC and Cmax) was roughly dose proportional between the two doses.

View larger version (23K): [in this window] [in a new window]   Figure 1. Change in exhaled breath nitric oxide (NO) levels after oral administration of A20 mg and B) 200 mg SC-51 in patients with mild to moderate asthma (filled triangle) compared with placebo (open triangles) and in healthy subjects (filled circles) compared with placebo (open circles). Vertical arrows represent times when dosing with SC-51 or placebo was performed. Mean values are shown (n=12).

3. L-NIL was well tolerated
SC-51 was well tolerated with no apparent effects on heart rate, blood pressure, or FEV₁. There was no effect on hematology or blood biochemistry.

CONCLUSIONS AND SIGNIFICANCE

The selective iNOS inhibitor SC-51 caused a rapid reduction in exhaled breath NO that lasted for >72 h in healthy volunteers and asthmatics. The rapid reduction in exhaled NO correlated closely with the maximal concentration (Cmax) of compound in the plasma, and the duration of the effect was consistent with the relatively long terminal elimination half-life of SC-51 (40–54 h). Even though exhaled NO levels were reduced dramatically after treatment with SC-51, no change was seen in FEV₁ in either asthma patients or healthy volunteers. This suggests that NO derived from iNOS does not have an endogenous bronchodilator role in airways.

Studies with intravenous administration of L-NMMA, a nonselective inhibitor of NOS, caused hypertension in animals and humans. The description of hypertension in mutant mice lacking the gene for eNOS suggests that L-NMMA is causing hypertension through inhibition of eNOS. Therefore, the excellent tolerability of SC-51 suggests that at the doses tested, this compound is not appreciably inhibiting eNOS. The apparent lack of inhibition of eNOS (and of nNOS) is supported by the finding that the maximal plasma concentration reached after the 200 mg dose of SC-51 (3000 ng/mL or 15 µM) was much lower than the IC₅₀ concentrations for these enzymes. eNOS selectivity can reasonably be inferred from the absence of any effect on blood pressure or heart rate at the high (200 mg) dose and the selectivity ratio in vitro (30-fold); no such indicator helps with assessing whether nNOS has been inhibited. With only a 13-fold in vitro selectivity, it is possible that nNOS is being inhibited in addition to iNOS, especially at the 200 mg dose, despite the pharmacokinetic data mentioned above. Therefore, it should be acknowledged that some of the exhaled NO could originate from nNOS rather than solely iNOS.

The long duration of effects in terms of lowering exhaled NO after a single dose suggests that the dosing regimen for this compound could be adjusted to permit single daily dosing. The effects on NO were demonstrated to be of rapid onset and dose related. The higher 200 mg dose may be a supramaximal dose lying on the plateau of the dose response curve. The 200 mg dose caused a 95% inhibition of exhaled NO in asthma, which is greater than the 70% inhibition obtained with high doses of nonspecific inhibitors such as L-NAME and L-NMMA, as well as with the more selective aminoguanidine. Assuming that complete inhibition of iNOS has been selectively obtained, the residual exhaled NO of <1 ppb in healthy volunteers and asthmatics may be produced by constitutive nNOS and eNOS as well as exogenous atmospheric sources.

The patients with asthma included in our study had very mild disease, and in many cases were nonsymptomatic with only mildly impaired lung function, having been selected on the basis of elevated exhaled breath NO (>15 ppb). Hence, although no convincing effects were noted on lung function or symptoms of asthma, this is not a suitable population of asthmatics to identify potential clinical efficacy. However, the measurement of exhaled breath NO in asthmatics should be a useful method to assess the pharmacodynamic properties of iNOS inhibitors

An expanding body of research supports an important role for NO in human asthma. Selective iNOS inhibitors have been shown to suppress eosinophil infiltration to the lung in rodent models of allergic airway inflammation with associated decreased lung chemokine expression and allergic airway inflammation inhibited in mice deficient in iNOS. Gaseous NO is detectable in elevated amounts in exhaled breath from asthmatic patients, and peroxynitrite is increased in bronchial biopsies from asthmatics. Nitrotyrosine is increased in exhaled breath condensate and in the lung parenchyma and airways of patients who have died from asthma. Recently, S-nitrosothiols have been shown to signal the ventilatory response to hypoxia, S-nitrosoglutathione being formed when NOS is activated in neuronal and other tissue.

Selective inhibition of iNOS has considerable therapeutic potential in a population of severe asthmatics receiving oral steroids with elevated exhaled breath NO, since iNOS inhibitors may be effective where steroids have failed. The action of iNOS inhibitors in asthma may relate to vasomotor properties, as decreased airway wall thickness could result from decreased vasodilation and blood flow. There is potential for reduction in proinflammatory effects and decreased airway hyperreactivity (AHR). Studies are needed to assess the clinical efficacy of iNOS inhibitors in terms of effects on the clinical features of asthma, blood, and sputum eosinophils, and AHR to bronchoconstrictor agents.

There is a considerable therapeutic rationale for using selective iNOS therapy in chronic obstructive pulmonary disease (COPD). Use of exhaled NO as a practical marker for COPD remains controversial, although there is elevated nitrotyrosine and iNOS in sputum cells. Patients with severe stable COPD have reduced levels of exhaled NO, possibly due to NO being consumed by reactive oxidant species.

In conclusion, this report demonstrates that a selective inhibitor of iNOS has rapid onset and causes marked, long-term suppression of exhaled breath NO levels both in healthy control subjects and in patients with mild asthma. It is encouraging that this therapy is well tolerated in terms of preservation of vasomotor and bronchial smooth muscle tone. Inhibition of iNOS has therapeutic potential for asthma in addition to a range of inflammatory diseases involving other organ systems.

View larger version (37K): [in this window] [in a new window]   Figure 2. Schematic diagram. Summary of a clinical study with the selective inducible nitric oxide synthase (iNOS) inhibitor SC-51. The pro-drug SC-51 is rapidly hydrolyzed to the active metabolie L-NIL.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0633fje; doi: 10.1096/fj.02-0633fje

³ Present address: Microbia Inc., Cambridge, MA 02141, USA.

⁴ Present address: Takeda Pharmaceuticals, Lincolnshire, IL 60069, USA

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