INTRODUCTION

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Pulmonary endothelial dysfunction is a major event in acute lung injury, and impaired endothelial production of nitric oxide (NO) has been described in the early stages of vascular disorders (1). Recently, evidence has emerged suggesting that the capacity of pulmonary endothelial cells to produce endogenous NO is impaired in acute lung injury (1). Constitutive NO synthase (cNOS) and inducible NOS (iNOS) have been localized within lung tissue (2), but the regulation of NOS expression in physiologic and pathologic states in the lung remains poorly understood.

The activity of cNOS and iNOS is modulated by the cofactor tetrahydrobiopterin (BH₄) (5, 6). De novo biosynthesis of BH₄ from GTP is highly regulated (7, 8). Endogenous BH₄ is insufficient for full iNOS activity in rat smooth muscle cells (9, 10) and exogenous BH₄ regulates iNOS expression by stabilization of its mRNA (11). Exogenous BH₄ induces relaxation of isolated rat and canine arteries (12, 13). Reconstitution of endothelium-dependent vasodilation after reperfusion injury by BH₄ has been demonstrated in a pig model (14). In man, BH₄ intra-arterially infused in relatively high concentrations has marked local vasodilating properties in healthy subjects but does not affect systemic blood pressure or heart rate (15). Moreover, BH₄ intraarterially infused in a relatively low concentration selectively and locally restores NO-dependent vasodilation in patients with endothelial dysfunction due to hypercholesterolemia, while it has no effect in healthy control subjects (16). These results suggest that impaired endogenous BH₄ synthesis contributes to disturbed NO production in the vascular system.

Supplementation of the impaired endogenous NO production by inhalational NO has been shown to be beneficial for treatment of patients with acute or chronic pulmonary diseases (17, 18). However, questions remain concerning the side effects as well as the practical aspects of inhalational NO, and strict guidelines must be followed for its safe administration (17). Thus, it is of interest to search for additional pharmacological approaches aimed at restoring NO dependent vasodilation in the lung. Inhalation of the NO synthase cofactor BH₄ might be such a therapeutic modality.

Whether BH₄ could affect NO dependent lung dysfunction is still unknown. Therefore, the purpose of this preliminary report is to investigate whether BH₄ can be administered by inhalation in humans, whether inhaled BH₄ is locally resorbed and whether inhaled BH₄ exerts any adverse effect on pulmonary function or systemic hemodynamic parameters in healthy individuals.

	METHODS

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Study Population

The pilot test with BH₄ dissolved in N-acetylcysteine was performed on one subject. Inhalation experiments with buffered BH₄ were performed on four subjects. All subjects (four male, one female; age 30- 46 yr) were in good health and had no history of lung disease. The study was performed according to the standards of the local ethics committee. Blood samples were drawn from the A. radialis and V. brachialis before and immediately after completing the inhalation in heparinized tubes (Vacutainer; Becton Dickinson, Rutherford, NJ). Plasma was separated and kept frozen at 20° C. Midstream urine was collected in sterile tubes and immediately frozen at 20° C.

Determination of BH₄ Stability

Three hundred mg of BH₄-dihydrochloride were dissolved in 3 ml sterile water. The clear and colorless solution was titrated with NaHCO₃ to a pH of 3.0, 3.8, 4.5, and 5.0, respectively, and strongly stirred at room temperature in air atmosphere and dim light. After 30 min, the maximal time needed for inhalation, the color was noted and pterins were quantified by HPLC.

Administration of BH₄

For the pilot experiment, (6R)-5,6,7,8-tetrahydro-L-biopterin-dihydrochloride (Dr. B. Schircks Laboratories, Jona, Switzerland) was dissolved in N-acetylcysteine 10% (Inpharzame, Cadempino, Switzerland). N-acetylcysteine is used for galenic stabilization of BH₄ tablets (Milupa, Friedrichsdorf, Germany) for long-term storage at room temperature. Inhaled N-acetylcysteine has no known effect on lung function per se, it has, however, a bad taste. Therefore, in all other experiments, 500 mg BH₄ were dissolved, directly prior to inhalation in dim light, in 3 ml sterile water containing 150 mg NaHCO₃, resulting in a buffered solution with a pH of 4.5-5. For inhalation, the hand-activated DeVilbiss technique was used (19). The DeVilbiss 646 nebulizer was run with compressed dry medical air at a flow rate of 8 L/min. This technique results in an aerosol mass median diameter of 4.4 µm (range 3.7-5.8 µm) and in 57% of respirable droplets (< 5 µm) (20). The mouthpiece of the nebulizer was placed between the teeth of the subject, who was directed to exhale to functional residual capacity, inhale slowly over 1-2 s toward total lung capacity, and then hold his or her breath for 2-3 s. Throughout the inhalation, the subject activated the nebulizer by placing a finger over the activator valve. This procedure was repeated until all of the solution was nebulized. The subjects completed the inhalation within 20 to 30 min.

Pulmonary Function Measurements

Spirometry, lung volumes, resistance, and diffusion capacity for CO were measured using the body plethysmograph Sensor Medics 66200 Autobox^® (Yorba Linda, CA), which satisfies the American Thoracic Society performance criteria (21). Criteria for acceptability, reproducibility and predicted values were according to the European Community for Steel and Coal (22, 23).

Hemodynamic Parameters

The heart rate and systolic, diastolic and mean arterial blood pressure were measured every 2.5 min with a Colin BP-306 blood pressure monitor (Carbamed, Liebefeld, Switzerland). Data were collected from 15 min before to 15 min after the inhalation procedure.

Pterin Measurements

Pterins in plasma and urine were quantified with fluorimetric detection by HPLC after acidic or differential oxidation as previously described (5, 24). For stability studies, BH₄ solution was analyzed directly without prior oxidation to determine spontaneous oxidation in solution.

	RESULTS

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Pilot Experiment

This experiment was performed on one volunteer. Since BH₄ is administered orally in a dose up to 20 mg/kg body weight (25), we considered it safe to start inhalation with 200 mg BH₄ dissolved in 3 ml N-acetylcysteine 10%. Immediately after inhalation, venous plasma levels of BH₄, detected as biopterin by HPLC, increased from 22.4 to 130.4 nmol/L. In a second step, we applied 800 mg BH₄ dissolved in 5 ml N-acetylcysteine 10% within the same experiment. Venous plasma BH₄ accordingly increased further to 599.8 nmol/L. A similar increase of BH₄ was observed in the arterial blood sample, where biopterin concentrations increased from 72.1 to 548.9 nmol/L. Midstream urine was collected prior to experimentation, immediately after and 4 h after the end of the second step. Urinary BH₄ excretion increased from 0.49 to 1.14 and further to 8.37 mmol/ mol creatinine. Respiratory functions, measured as spirometry, lung volumes, pulmonary resistance and diffusing capacity for CO were normal and were not altered by inhaled BH₄ (data not shown). However, BH₄ dissolved in N-acetylcysteine has an unpleasant odor and sour taste. Therefore, we decided to use buffered BH₄ solution for further inhalation studies.

Stability of Buffered BH₄

Testing of buffered BH₄ for stability was done as described in the METHODS section. Irrespective of the pH, titrated with NaHCO₃ to 3.0, 3.8, 4.5, or 5.0, respectively, more than 95% of the initially dissolved BH₄ was detected as unoxidized BH₄ by HPLC after vigorous stirring for 30 min in air. As the solution was stirred, its color changed from colorless to slight yellow but remained clear and odorless.

Inhalation of Buffered BH₄

The next series of experiments was performed on four further volunteers. Just before inhalation, BH₄ solution buffered to a pH of 4.5-5 was freshly prepared in dim light in a total volume of 3 ml. This solution was color- and odorless and had a slight sour taste. Directly after the inhalation period (20 to 30 min), biopterin levels were increased 13-160-fold in arterial as well as in venous plasma (Figure 1A). Similar results were observed in urine, where biopterin increased 5-25 fold (Figure 1B). The renally excreted degradation products of BH₄, pterin and isoxanthopterin (see Figure 3), rose accordingly (Figure 1C). However, differential oxidation of the urinary samples revealed that the percentage of BH₄ from total excreted biopterin remained in the physiologic range (60-80% tetrahydro form), indicating that a major part of circulating BH₄ after inhalation remained in the reduced form. There were no significant effects of inhaled BH₄ on respiratory function (Table 1), and buffered BH₄ was well tolerated by all subjects. The stability of buffered BH₄ solution was verified by HPLC determination of BH₄ concentrations in samples taken from the nebulizer shortly before the end of inhalation and was greater than 95% throughout.

View larger version (17K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   View larger version (23K): [in this window] [in a new window]   Figure 1.   Alteration of plasma (A) and urinary (B, C ) pterin concentrations by inhalation of a buffered BH4 solution. Five hundred mg BH4 were dissolved in 3 ml sterile water containing 150 mg NaHCO3 just before inhalation. Blood samples were drawn before and immediately after the inhalation. Midstream urine was collected before, immediately after and 4 h after the end of inhalation. Results of experiments, performed on four individuals, are presented on a logarithmic scale.

View larger version (12K): [in this window] [in a new window]   Figure 3.   Metabolic breakdown of BH4. BH4 has a half-life of approximately 7 h in serum and over 80% of the total amount circulates in the fully reduced form (27). If degraded, it is either chemically cleaved to pterin or further enzymatically metabolized by xanthine dehydrogenase (XDH) to isoxanthopterin in the liver. Both of the degradation products, pterin and isoxanthopterin, are renally excreted and can be analyzed in urine.

Hemodynamic Parameters

As shown in Figure 2A-C, the systemic hemodynamic parameters were unaffected during and after inhalation of 500 mg BH₄. None of the volunteers experienced orthostatic hypotension during or after the experiment.

View larger version (18K): [in this window] [in a new window]   View larger version (12K): [in this window] [in a new window]   View larger version (12K): [in this window] [in a new window]   Figure 2.   Hemodynamic parameters before, during and after BH4 inhalation. Systolic and diastolic blood pressure (A), mean arterial pressure (B) and heart rate (C ) were measured every 2.5 min before (squares), during (circles), and after (triangles) inhalation of 500 mg BH4. Data were collected from 15 min before to 15 min after the inhalation procedure. The results from four different subjects of one representative experiment are presented. SBP = systolic blood pressure; DBP = diastolic blood pressure; MAP = mean arterial pressure; HR = heart rate; bpm = beats per minute.

	DISCUSSION

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This preliminary study demonstrates: (1) the feasibility of BH₄ inhalation; (2) the local resorption of BH₄ in the lung; and (3) the tolerability of inhaled BH₄. There is no acute effect, and in particular no adverse effect, on pulmonary function parameters. BH₄ inhalation therefore represents a novel modality of local BH₄ administration. Our studies indicate that freshly prepared, sodium bicarbonate buffered, BH₄ solutions are stable for 30 min (the maximal time needed for inhalation of 500 mg BH₄) at a pH ranging from 3.0 to 5.0. Therefore, the pH chosen for the solution used for inhalation in this study is 4.5-5. This value is comparable to the pH of the N-acetylcysteine 10% solution routinely used for inhalation for many years. Therefore, we believe that inhalation of a solution buffered to a pH of 4.5-5 is likely to be safe, even for long-term applications. Furthermore, our experience with orally administered BH₄, used for life-long daily therapy of inborn BH₄ deficiencies for over 15 years in doses comparable to that used for inhalation, reveals no adverse side-effects or toxicity.

The vasodilating properties of BH₄ have been shown in vitro and in vivo (12). One great advantage of inhaled NO in the treatment of pulmonary diseases is its selective pulmonary vasodilation without major influence on the systemic circulation, explained by the inactivation of NO by rapid combination with hemoglobin in the pulmonary circulation (26). Our data show that systemic hemodynamics are not affected by inhalation of 500 mg BH₄ despite elevated systemic arterial and venous BH₄ levels. Elevated urinary excretion of pterin and isoxanthopterin, the breakdown products of BH₄ exclusively, but not of more highly oxidized biopterins (see Figure 3), indicate that the circulating substance was in the fully reduced form. Infusion of high doses of BH₄ (8-32 mg/min) in the brachial artery in man led to a marked local vasodilation in the perfused limb. Blood flow in the nonperfused limb, systemic blood pressure and heart rate remained unchanged despite elevated circulating BH₄ concentrations (15). These results suggest a requirement of high local BH₄ concentrations for vasodilating effects of exogenous BH₄ in healthy subjects. Most interestingly, a relatively low dose of BH₄ (500 µg/min) locally restored the disturbed NO-dependent vasodilation of patients with endothelial dysfunction due to hypercholesterolemia, while the same concentration of BH₄ had no effect in control subjects (16). Thus, local administration of lower doses of BH₄ might be a therapeutic approach for selective restoration of impaired endothelial NO production leading to vasodilation, and local administration of BH₄ by inhalation might therefore be advantageous to intravenous BH₄. Furthermore, inhaled BH₄ should be distributed predominantly to well ventilated alveoli, thereby improving the matching of ventilation to perfusion, resulting in improved arterial oxygenation.

In summary, our preliminary study in five healthy subjects shows that BH₄ inhalation is a feasible modality for local application of this regulatory NO synthase cofactor in the lung. It remains to be established whether BH₄ restores the impaired pulmonary endothelial cell NO production in patients with NO dependent pulmonary diseases and might thereby offer an alternative therapeutic approach aimed at restoration of NO dependent vasodilation in the lung.