Vecuronium bromide

Study objectives: To determine the selective vasodilatory effects of two inhaled "NONOate" aerosols in a closed chest pig model of acute pulmonary hypertension (APH).

Methods: APH was induced by IV infusion of the prostaglandin [H.sub.2]/thromboxane [A.sub.2] receptor agonist (U46619). Aerosolized diethylenetriamine nitric oxide (NO) adduct (DETA/NO, n = 4), dipropylenetriamine NO adduct (DPTA/NO, n = 4) [60 [micro]mol each], or placebo (n = 4) was delivered via the trachea. Hemodynamic parameters and blood samples were measured before and after inhalation therapy.

Results: Compared to control animals, pulmonary vascular resistance and pulmonary arterial pressure were significantly reduced from 10 to 105 min after DETA/NO administration and from 10 to 45 min after DPTA/NO aerosol administration (p < 0.05). Both aerosols had no significant effect on systemic vascular resistance or systemic BP. Serum nitrite significantly increased after the inhalation of both NONOates (p < 0.01). There was a tendency for reduced intrapulmonary shunting, particularly after treatment with DETA/NO.

Conclusion: Both DETA/NO and DPTA/NO administered as aerosols selectively reduced pulmonary hypertension induced by U46619.

Key words: ARDS; NONOates; pulmonary hypertension; selective pulmonary vasodilators

Abbreviations: ANOVA = analysis of variance; APH = acute pulmonary hypertension; CI = cardiac index; DETA/ NO = diethylenetriamine nitric oxide adduct; DPTA/NO = dipropylenetriamine nitric oxide adduct; MPAP = mean pulmonary artery pressure; NO = nitric oxide; N[O.sub.2] = nitrite; PAP = pulmonary artery pressure; PCWP = pulmonary capillary wedge pressure; PVRI = pulmonary vascular resistance index; SBP = systemic BP; SPV = selective pulmonary vasodilator; V/Q = ventilation/perfusion.

**********

Selective pulmonary vasodilators (SPVs) dilate the pulmonary vasculature without causing systemic hypotension and without worsening ventilation/perfusion (V/Q) matching. (1) Established SPVs, such as inhaled nitric oxide (NO) and inhaled aerosolized prostacyclin, are used in the treatment of hypoxemia and pulmonary hypertension in conditions such as ARDS. (2) SPVs reduce pulmonary arterial pressure (PAP), improve right-heart function, and improve oxygenation in patients with ARDS. (3) To date, inhaled NO is the most widely used SPV. It has minimal effects on the systemic circulation, improves V/Q matching, and thereby improves oxygenation. (l,4) However, inhaled NO has some major disadvantages: (1) since it has a very short half-life and is rapidly inactivated, it has to be delivered continuously to be effective (5); (2) abrupt discontinuation of therapy can lead to acute rebound pulmonary hypertension with significant morbidity (6); and (3) high concentrations of NO and its oxidant products (such as nitrogen dioxide [N[O.sub.2.sup.-]]) are toxic and require complex delivery and environmental monitoring systems to protect patients and medical attendants from unwanted exposure. (5,7)

"NONOates," or diazeniumdiolates, are chemical compounds that carry the [[N(O)NO].sup.-] functional group. (8) When dissolved in physiologic solutions or buffers, they spontaneously release 2 mol of NO per mole of parent compound. The half-lives of NO generation from NONOates vary from 1 min to 1 day under normal physiologic conditions. (8,9) NONOates may prove to be superior to gaseous NO as SPVs in clinical therapeutics in that: (1) the longer decomposition half-lives may allow intermittent therapy, (2) theoretically rebound pulmonary hypertension is less likely because of the gradual reduction in tissue levels of NO, and (3) NONOates are stable in solid form and highly water soluble. In addition, they can be delivered to the lung using a small-volume nebulizer and do not require safety monitoring systems.

Diethylenetriamine NO adduct (DETA/NO) and dipropylenetriamine NO adduct (DPTA/NO) are NONOates with NO-generating half-lives of approximately 20 h and 3 h, respectively, at 37[degrees]C. (8,9) Compared to the extremely short biological half-life of NO (< 5 s), the slow and continuous release of NO from these novel compounds potentially makes them very useful agents for the treatment of pulmonary hypertension and hypoxemia in such conditions as ARDS. The aim of this study was to investigate the selective pulmonary vasodilatory effects of DETA/NO and DPTA/NO in an acute pulmonary hypertension (APH) model.

MATERIALS AND METHODS

Twelve white, Landrace-cross, female piglets weighing 19 to 25 kg were obtained from the Animal Resources Center, Murdoch, Western Australia. The study was approved by the Animal Experimentation Ethics Committee of the University of Western Australia.

The piglets were anesthetized with 3% halothane in oxygen-enriched air (fraction of inspired oxygen, 0.5). Anesthesia was maintained with halothane, 0.5 to 1%, in air/oxygen (fraction of inspired oxygen, 0.5). They received mechanical ventilation (Siemens Servo 900B; Siemens; Elema, Sweden) throughout the study period via a 6-mm endotracheal tube in the assist-control ventilation mode. Tidal volumes were [less than or equal to] 10 mL/kg, and minute ventilation was adjusted to achieve normocapnia. Vecuronium bromide was infused IV at 2 to 6 mg/h to facilitate mechanical ventilation. Hydration was maintained by infusing isotonic saline solution via an ear vein at a rate of 5 mL/kg/h.

A single-lumen catheter was inserted into the femoral artery and connected to a monitor (Sirecust 404; Siemens) to continuously monitor the systemic BP (SBP). A five-lumen 7.5F gauge, balloon-tipped, flow-directed pulmonary artery catheter (Arrow Model AH-05050-H; Arrow International; Reading, PA) was placed via the femoral vein. The catheter was advanced into the inferior vena cava, and the tip of catheter was ultimately placed in the right or left pulmonary artery, verified by typical pressure-wave forms. The proximal end of the five-lumen catheter measured the pressure at the level of the superior vena cava (central venous pressure), while the distal end of the catheter monitored the mean PAP (MPAP). Pulmonary capillary wedge pressure (PCWP) was measured following inflation of the distal catheter balloon with not more than 1.5 mL of air. PCWP was measured at the end of expiration and immediately before measuring the cardiac output in each instance. A cardiac output measuring set (CritikitTM-SP4500; Becton Dickinson, Critical Care Systems; Singapore) was connected to the infusion port of the pulmonary artery catheter. Cardiac output was measured using a thermodilution technique, by injection of a 5 mL bolus of cold sterile 5% dextrose water into the right atrium. Cardiac output was calculated from the detected change in temperature at the thermistor in the pulmonary artery by the internal algorithm of the monitor (utilizing the Stewart-Hamilton equation). (10) Values of cardiac output were divided by body mass (in kilograms) to obtain a cardiac index (CI), expressed in units of liters per minute per kilogram. Blood samples for gas analysis were drawn from the femoral arterial line (arterial blood gas) and the distal port of the pulmonary artery catheter (mixed venous blood gas). These blood samples were analyzed by a blood gas analyzer (ABL 520; Radiometer; Copenhagen, Denmark) within 10 min of being drawn. The intrapulmonary shunt (percentage) was calculated from the blood gas measurements using the standard equation. Arterial blood was also collected for analysis of total serum nitrite (nitrate was reduced to N[O.sub.2.sup.-] before assay) concentrations using the Griess reaction as described by Giovannoni et al. (11)

Baseline measurements of hemodynamic parameters and blood samples for N[O.sub.2.sup.-] quantification were obtained after the anesthetized animals had been stabilized for at least 20 min. Once all the baseline data had been collected, APH was induced by IV infusion of a prostaglandin [H.sub.2]/thromboxane [A.sub.2] receptor agonist (U46619; Cayman Chemical; Ann Arbor, MI). U46619 (10 mg) was dissolved in 10 mL of isotonic phosphate buffer (WA Hospital Central Pharmaceutical Manufacturing Facility; Perth, Australia) [pH 7.4] and diluted to 50 mL in normal saline solution to a final concentration of 0.2 mg/mL. U46619 was then infused continuously via the femoral vein at an initial rate of 0.01 [micro]g/kg/min, and increased every 6 min in the following steps: 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, and 10 [micro]g/kg/min. (12) Hemodynamic parameters were measured every 6 min, and the infusion rate was increased step-wise until the target pulmonary vascular resistance index (PVRI) of [greater than or equal to] 1.5 times above the baseline value was achieved. PVRI was calculated as 79.9 x (MPAP - PCWP)/CI, expressed in dynes per second per centimeter (5) per kilogram. U46619 was then infused continuously, throughout the study at the same rate used to achieve the target PVRI. The cumulative dose of U46619 for each animal from the achievement of target PVRI to the end of the study was noted.

After target pulmonary hypertension had been achieved and stabilized for at least 20 min, hemodynamic measurements were made and blood samples were obtained for N[O.sub.2.sup.-] measurement. Animals were then treated with placebo (n = 4), DETA/NO (n=4), or DPTA/NO (n=4) aerosol. DETA/NO and DPTA/NO (60 [micro]mol; Sigma Chemical; St. Louis, MO) solutions (12 mM) were freshly prepared by dissolving each drug in 5 mL of phosphate buffer (WA Hospital Central Pharmaceutical Manufacturing Facility) [pH 7.4] immediately before inhalation therapy. The dose used in this study was derived from the data of Cornfield et al, (13) adjusted for body weight. Normal saline, DETA/NO, or DPTA/NO solutions were aerosolized into the respiratory tract via a Sidestream Jet Nebuliser (Medic-Aid; West Sussex, UK) driven by 100% oxygen at a flow rate of 2 to 3 L/min. Aerosols were delivered into the lungs over 20 min by connecting the nebulizer to the inspiratory limb of the breathing circuit. Hemodynamic measurements were repeated at 5, 10, 15, 20, 25, 30, 45, 60, 75, 90, 105, 120, 180, and 240 min after aerosol therapy was commenced. Further blood samples for N[O.sub.2.sup.-] measurement were collected at 15, 30, 45, 60, 75, 90, 180, and 240 min. Piglets were killed with IV pentobarbitone at the end of study.

Selective pulmonary vasodilation was defined as a reduction in pulmonary vascular resistance (with a lack of effect on SBP). Reduction in intrapulmonary shunting was a secondary end point, as this was not a hypoxemic animal model primarily.

Data were analyzed using SigmaStat Scientific Software (Version 2.0; Jandel Corporation; San Rafael, CA). All data were first tested for normality and equivalence of variance. Nonnormal distributed variables were log transformed. All the data were analyzed by two-way repeated-measures analysis of variance (ANOVA). All data are presented as mean [+ or -] SEM.

RESULTS

APH was successfully induced in four piglets in the control group, three piglets in the DETA/NO group, and four piglets in the DPTA/NO group. Due to failure in the placement of the pulmonary artery catheter, pulmonary hypertension was not induced in one of the piglets in the DETA/NO group. However, all piglets received inhaled therapy (placebo, DETA/NO, or DPTA/NO), and serum N[O.sub.2.sup.-] concentrations were measured in all 12 animals.

Induction of APH

The mean doses of U46619 infused to achieve target PVRI in the control, DETA/NO-treated, and DPTA/NO-treated groups were 73 [+ or -] 7.9 [micro]g, 78 [+ or -] 15 [micro]g, and 79 [+ or -] 17 [micro]g, respectively. The cumulative doses of U46619 at the end of the study were 880 [+ or -] 65 [micro]g, 827 [+ or -] 100 [micro]g, and 906 [+ or -] 75 [micro]g, respectively, for control, DETA/NO-treated, and DPTA/NO-treated groups. There were no significant differences in the doses of U46619 used in each group.

The mean baseline values of PVBI for the control, DETA/NO-treated, and DPTA/NO-treated animals were 8.6 [+ or -] 0.6 dyne * [s.sup.-1] * [cm.sup.-5] * [kg.sup.-1], 8.6 [+ or -] 0.2 dyne * [s.sup.-1] * [cm.sup.-5] * [kg.sup.-1], and 7.9 [+ or -] 0.4 dyne * [s.sup.-1] * [cm.sup.-5] * [kg.sup.-1], respectively. Immediately before aerosol administration (zero time), PVRI had risen to 19 [+ or -] 0.6 dyne * [s.sup.-1] * [cm.sup.-5] * [kg.sup.-1], 25 [+ or -] 2.3 dyne * [s.sup.-1] * [cm.sup.-5] * [kg.sup.-1], and 20 [+ or -] 2.7 dyne * [s.sup.-1] * [cm.sup.-5] * [kg.sup.-1] in the control, DETA/NO-treated, and DPTA/NO-treated groups, respectively. There were no significant differences in PVRI between either of the NONOate groups and the control group at these two time points (baseline and zero time).

Effects on the Pulmonary Vasculature

Compared to control animals, PVRIs were significantly reduced at 10, 15, 20, 25, 30, 45, and 60 min after aerosol delivery in the DETA/NO-treated group (p < 0.05; Fig 1). PVRIs were also significantly reduced at 10, 15, 20, 25, 30, and 45 min after aerosol delivery in the DPTA/NO-treated group (p < 0.05; Fig 1).

[FIGURE 1 OMITTED]

There were no significant differences in MPAP between either of the NONOate-treated groups and the control group at baseline and zero time. MPAP was significantly reduced compared to control animals from 10 to 105 min after treatment in the DETA/NO-treated group and from 10 to 45 min after treatment in the DPTA/NO-treated group (p < 0.05; Fig 2). PVRI and MPAP were not significantly reduced compared to their values at zero time in the control group. However, MPAP and PVRI were significantly lower compared to zero time values in the DETA/NO-treated group from 10 to 60 min after aerosol delivery (p < 0.05). Compared to zero time values, MPAP was also significantly reduced from 10 to 30 min after aerosol delivery in the DPTA/NO-treated group (p < 0.05), but none of the reductions in PVRI after DPTA/NO administration in this group reached statistical significance (Table 1).

[FIGURE 2 OMITTED]

Other Hemodynamic Effects

No significant differences were found in PCWP or CI at any time point between the three treated groups or at any time period within any group compared to respective zero time values. There were no significant differences in the systemic vascular resistance index or SBP between the three groups. The major hemodynamic effects of inhaled DETA/NO and DPTA/NO are summarized in Table 1.

Effects on Gas Exchange

Intrapulmonary shunting after DETA/NO and DPTA/NO administration tended to be reduced (Fig 3). Compared to the control animals, the reduction was more marked in the DETA/NO-treated group (up to 120 min) than that in the DPTA/NO-treated group, but the differences did not reach statistical significance (p = 0.07). The alveolar-arterial oxygen partial pressure gradient also tended toward an improvement; again, this was not statistically significant (data not shown).

[FIGURE 3 OMITTED]

Serum NO Metabolites

There was an increase in the total serum N[O.sub.2.sup.-] concentrations after aerosol administration in both DETA/NO-treated and DPTA/NO-treated groups compared to control animals and to baseline (p < 0.01; Fig 4). The levels increased significantly from 15 min after aerosol administration and remained above control levels throughout the remainder of the experiment.

[FIGURE 4 OMITTED]

DISCUSSION

In this study, we have shown that both DETA/NO and DPTA/NO delivered by inhalation significantly reduced pulmonary vascular resistance without a detectable effect on the systemic circulation. The vasodilatory effect of both NONOates lasted from 45 min, up to 120 min. Total serum N[O.sub.2.sup.-] concentrations were significantly increased for the duration of the experiment, indicating continuous release and absorption of NO from the lung. Both NONOates also tended to reduce intrapulmonary shunting, but this effect was not statistically significant.

NONOates are a unique class of NO donors that release NO from physiologic solutions at predictable rates. The vasodilatory effect of NONOates is derived from the NO molecules released from the parent compound. (14) When inhaled, NONOates not only exhibit the advantages of gaseous NO as a selective pulmonary vasodilator, but also avoid some of the disadvantages of NO delivery and monitoring. The NO-release half-life of DPTA/NO is approximately 3 h, while that for DETA/NO is approximately 20 h. (8)

The pulmonary vasodilation effect of inhaled DETA/NO was first demonstrated by Hampl et al (15) in rodents, where chronic pulmonary hypertension had been induced by monocrotaline. DETA/NO (50 [micro]mol) significantly reduced the PAP and total pulmonary resistance index without affecting the systemic circulation. The same group of researchers further investigated the acute vasodilatory effect of DETA/NO in fetal lambs. (13) Pulmonary arterial blood flow increased in a dose-dependent manner after 0.1 mg, 0.4 mg, and 1.0 mg of nebulized DETA/NO administration into the fetal lungs. Inhaled DETA/NO had no effect on aortic BP or heart rate.

Our study showed that following U-46619--induced pulmonary hypertension, both DETA/NO and DPTA/NO delivered as single 60 [micro]mol dose aerosols could significantly reduce PVRI and PAP. In keeping with their respective release half-lives, DETA/NO had a greater and more prolonged effect than DPTA/NO. Neither drug caused any systemic vasodilation, presumably because the released NO is rapidly inactivated by hemoglobin once it enters the circulation, (16) and also due to the very short biological half-life of NO. (17) The expected small but not significant rise in SBP in response to U46619 infusion was seen in all groups (Table 1). These changes are in keeping with previous studies. (5,18)

There was also a trend toward reduced intrapulmonary shunting with both DETA/NO and DPTA/ NO. The effect was more noticeable with DETA/NO but was not statistically significant for either compound. The failure to detect a significant difference in this measurement may be due to the relatively small sample sizes. Alternatively, lack of a significant effect may arise because the model used in this study was not designed primarily to cause an initial high level of intrapulmonary shunting (ie, this was not a hypoxemia model). In ARDS, increased intrapulmonary shunting is a common finding. (1,19) This is in effect a physiologic right to left shunt and may contribute to hypoxemia. A true selective pulmonary vasodilator should not worsen V/Q mismatching. (1,20)

The release of NO from inhaled DETA/NO and DPTA/NO can be inferred from our measurements of total serum N[O.sub.2.sup.-]. Serum N[O.sub.2.sup.1] concentrations were threefold to fivefold higher in both treatment groups and persisted for the 4 h of the experiment. Baseline concentrations of N[O.sub.2.sup.-] (mean of 45 [+ or -] 2.7 [micro]mol/L and 29 [+ or -] 7 [micro]mol/L for DETA/NO and DPTA/NO, respectively) were significantly lower than those reported by Jacobs et al (21) before DMAEP/NO inhalation in piglets (255 [micro]mol/L), but were similar to those from several other studies (range, 20 to 45 [micro]mol/L) using similar analytical methodology. (11,22-24) The baseline concentrations in the study by Jacobs et al (21) were obtained after the induction of acute lung injury by the infusion of oleic acid; in this study, the levels were obtained before the induction of APH. Mean concentrations of total N[O.sub.2.sup.-] in serum during the period of the experiment were 194 [+ or -] 23 [micro]mol/L and 165 [+ or -] 22 [micro]mol/L for a 60 [micro]mol single aerosolized dose of DETA/NO and DPTA/NO, respectively, which is similar to the means of 261 [+ or -] 45 [micro]mol/L and 242 [+ or -] 28 [micro]mol/L reported 30 min and 60 min after nebulized DMAEP/NO (340 [micro]mol). (21)

CONCLUSION

We have shown that both inhaled DETA/NO and DPTA/NO are selective pulmonary vasodilators in a closed-chest animal model. They not only reduced the pulmonary vascular resistance, with no detectable effect on systemic circulation, but they also tended to reduce intrapulmonary shunting. DETA/NO was more effective, presumably as a result of its longer NO-generating half-life. The potential therapeutic benefit of these agents has been demonstrated in a patient with ARDS. (25)

ACKNOWLEDGMENT: The authors thank Ms. Brigit Roberts and Mr. Denham Heliams in the animal laboratory.

REFERENCES

(1) Thebaud B, Arnal JF, Mercier JC, et al. Inhaled and exhaled nitric oxide. Cell Mol Life Sci 1999; 55:1103-1112

(2) Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med 2000; 342:1334-1349

(3) Hart CM. Nitric oxide in adult lung disease. Chest 1999; 115:1407-1417

(4) Frostell C, Fratacci MD, Wain JC, et al. Inhaled nitric oxide: a selective pulmonary vasodilator reversing hypoxic pulmonary vasoconstriction. Circulation 1991; 83:2038-2047

(5) Brilli RJ, Krafte-Jacobs B, Smith DJ, et al. Aerosolization of novel nitric oxide donors selectively reduce pulmonary hypertension. Crit Care Med 1998; 26:1390-1396

(6) Christenson J, Lavoie A, O'Connor M, et al. The incidence and pathogenesis of cardiopulmonary deterioration after abrupt withdrawal of inhaled nitric oxide. Am J Respir Crit Care Med 2000; 161:1443-1449

(7) Cuthbertson BH, Dellinger P, Dyar O, et al. UK guidelines for the use of inhaled nitric oxide therapy in adult ICUs. Intensive Care Med 1997; 23:1212-1218

(8) Keefer L, Nims RW, Davies KM, et al. NONOates (1-substituted Diazen-1-ium-1,2-diolates) as nitric oxide donors: convenient nitric oxide dosage forms. Methods Enzymol 1996; 268:281-293

(9) Fitzhugh AL, Keefer LK. Diazeniumdiolates: pro- and antioxidant applications of the "NONOates." Free Radic Biol Med 2000; 28:1463-1469

(10) Maxted KJ. Haemodynamic monitoring. In: Bain WH, Taylor KM, eds. Handbook of intensive care. London, UK: Wright PSG, 1983; 25-47

(11) Giovannoni G, Land JM, Keir G, et al. Adaptation of the nitric reductase and Griess reaction methods for the measurement of serum nitrate plus nitrite levels. Ann Clin Biochem 1997; 34:193-198

(12) Snapper JR, Thabes JS, Lefferts PL, et al. Role of endothelin in endotoxin-induced sustained pulmonary hypertension in sheep. Am J Respir Crit Care Med 1998; 157:81-88

(13) Cornfield DN, Martin EB, Hampl V, et al. Aerosol delivery of diethylenetriamine/nitric oxide, a nitric oxide adduct, causes selective pulmonary vasodilation in perinatal lambs. J Lab Clin Med 1999; 134:419-425

(14) Morley D, Keefer LK. Nitric oxide/nucleophile complexes: a unique class of nitric oxide-based vasodilators. J Cardiovasc Pharmacol 1993; 22:S3-S9

(15) Hampl V, Tristani-Firouzi M, Hutsell TC, et al. Nebulized nitric oxide/nucleophile adduct reduces chronic pulmonary hypertension. Cardiovasc Res 1996; 31:55-62

(16) Rimar S, Gillis CN. Selective pulmonary vasodilation by inhaled nitric oxide is due to hemoglobin inactivation. Circulation 1993; 88:2884-2887

(17) Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991; 43:109-142

(18) Adrie C, Ichinose F, Holzmann A, et al. Pulmonary vasodilation by nitric oxide gas and prodrug aerosols in acute pulmonary hypertension. J Appl Physiol 1998; 84:435-441

(19) West JB. Ventilation, blood flow, and gas exchange. In: Murray J, Nadel JA, eds. Respiratory medicine. 2nd ed. Sydney, Australia: W.B. Saunders Company, 1994; 51-89

(20) Barbera JA, Roger N, Roca J, et al. Worsening of pulmonary gas exchange with nitric oxide inhalation in chronic obstructive pulmonary disease. Lancet 1996; 347:436-440

(21) Jacobs BR, Brilli RJ, Ballard ET, et al. Aerosolized soluble nitric oxide donor improves oxygenation and pulmonary hypertension in acute lung injury. Am J Respir Crit Care Med 1998; 158:1536-1542

(22) Fike CD, Kaplowitz MR, Thomas CJ, et al. Chronic hypoxia decreases nitric oxide production and endothelial nitric oxide synthase in newborn pig lungs. Am J Physiol 1998; 274:L517-L526

(23) Godfrey M, Majid DSA. Renal handling of circulating nitrates in anesthetized dogs. Am J Physiol 1998; 275:F68-F73

(24) Kumura E, Kosaka H, Shiga T, et al. Elevation of plasma nitric oxide end products during focal cerebral ischemia and reperfusion in the rat. J Cerebral Blood Flow Metab 1994; 14:487-491

(25) Lam CF, van Heerden PV, Sviri S, et al. The effects of inhalation of a novel nitric oxide donor, DETA/NO in a patient with severe hypoxaemia due to the acute respiratory distress syndrome. Anaesth Intensive Care 2002; 30:472-476

* From the Department of Pharmacology (Drs. Lam and Ilett) University of Western Australia, Crawley; Department of Intensive Care (Dr. van Heerden), Sir Charles Gairdner Hospital, Perth; and Department of Anatomical Pathology (Dr. Caterina and Mr. Filion), The Western Australia Center for Pathology and Medical Research, Nedlands, Australia.

This study was performed at the University of Western Australia, Crawley, Australia.

This study was supported by the Sir Charles Gairdner Hospital Research Fund.

Manuscript received February 14, 2002; revision accepted September 4, 2002.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: permissions@chestnet.org).

Correspondence to: Chen F. Lam, MD, Department of Pharmacology, University of Western Australia, Crawley, WA 6009, Australia; e-mail: cflam@cyllene.uwa.edu.au

COPYRIGHT 2003 American College of Chest Physicians
COPYRIGHT 2003 Gale Group