WO2002041924A1

WO2002041924A1 - Nitric oxide donor compounds and methods of treatment using same

Info

Publication number: WO2002041924A1
Application number: PCT/AU2001/001525
Authority: WO
Inventors: Peter Vernon Van Heerden; Kenneth Ilett; Chen Fuh Lam
Original assignee: Emphatec Pty Ltd
Priority date: 2000-11-23
Filing date: 2001-11-23
Publication date: 2002-05-30
Also published as: AU2002223283A1; AUPR164400A0

Abstract

The use of nitric oxide donor compounds in the preparation of a medicament for the inhibition of growth factors in the pulmonary vasculature of a mammal, improving effort tolerance in humans suffering smoking induced lung injury and heart disease processes resulting in pulmonary hypertension, the prevention and/or treatment of remodelling of the pulmonary vasculature and/or right heart of a mammal. A method for the treatment of pulmonary hypertension, acute hypoxaemia, smoking induced lung injury, remodelling of the pulmonary vasculature and/or right heart, and/or chronic hypoxaemia, the method comprising the administration of a pharmacologically effective amount of a nitric oxide donor compound to a patient in need thereof by way of a nebuliser or metered dose inhaler.

Description

"Nitric Oxide Donor Compounds and Methods of Treatment Using Same"

Field of the Invention

The present invention relates to nitric oxide donor compounds and methods of treatment using same.

More particularly, the methods of treatment of the present invention include use of nitric oxide donor compounds in the inhibition of growth factors in the pulmonary vasculature, the prevention and treatment of remodelling of the pulmonary vasculature and the right heart, and improving effort tolerance in patients suffering from smoking-ihduced lung injury and other lung disease and heart disease processes resulting in pulmonary hypertension.

Background Art

Pulmonary hypertension is a serious medical condition, often resulting from, inter alia, adult respiratory distress syndrome, neonatal respiratory distress syndrome, pneumonia, asthma, bronchiolitis, chronic obstructive pulmonary disease, restrictive lung disease, near drowning, cardiopulmonary arrest, cardiopulmonary bypass, emphysema, sepsis, infection, shock, congenital heart disease and congenital diaphragmatic hernia.

Inhaled NO in gaseous form has been shown to be useful in the treatment of pulmonary hypertension, in mammals, when delivered to the lung ' via an endotracheal route during mechanical ventilation (Nelin et al., Pediatric Research 1994, 35:20-24; Etches et al., Pediatric Research 1994, 35:15-19). NO/nucleophile adducts or- "NONOates", soluble nitric oxide donors, have also been shown to be useful in the treatment of pulmonary hypertension, again in mammals, when administered as an aerosol or direct intratracheal injection (Brilli et al., J Appl Physiol, 1997 Dec; 83(6):1968-75; Brilli et al., Crit Care Med, 1998 Aug; 26(8):1390-6; Jacobs et al., AM J Respir Crit Care Med, 1998 Nov; 158 (5 Pt 1 ): 1536-42; Jacobs et al., Nitro Oxide, 2000 Aug: 4(4): 412-22). NO has been used previously to inhibit growth factors, in animals, when delivered by the inhaled route as a gas, but not as a NO donor (Hart CM., Chest

_. 1999:115:1407; Yanagisawa M., Circulation 1994:89;1320; Bousette N,

D'Orleans-Juste P, Shennib H, Giaid A., Harrison's Principals of Internal Medicine, www.harrisonsonline.com).

The exact mechanism by which inhaled NO dilates the pulmonary vascular bed is unknown. It is presumed that NO is distributed to distal ventilated alveolar segments, where is passes readily due to its great lipophilicity, through the epithelium into the interstitial space. From there, NO passes through the vascular adventitia and reaches the cytosol of the arteriolar vascular smooth muscle, where it interacts with iron in the heme centre of guanylyl cyclase. NO binding induces a conformational change in the enzyme which permits the catalysis of GTP to cGMP, with a subsequent alteration in intracellular calcium ion concentration and vascular smooth muscle relaxation.

NO that alternately passes through the vascular smooth muscle and endothelium and into the vascular lumen, is believed to be inactivated by its interaction with haemoglobin. As such, NO is believed to be a selective pulmonary vasodilator, since it becomes ineffective before reaching the systemic circulation. Whilst some recent evidence suggests that NO may circulate as a relatively stable adduct, in the form of a nitrosylated haemoglobin species, which could in theory ^• cause systemic vasodilation, in practice, it is not apparent that inhaled NO has any direct effect on systemic vascular resistance.

US Patent 5958427 to Salzman, et al describes a class of nitric oxide donor compounds and several pharmaceutical compositions incorporating these compounds. Further described is their use in the treatment of pulmonary hypertension and impotence.

None of the available literature provides information relating to use of NO donor compounds in the treatment of the inhibition of growth factors in the pulmonary vasculature, the prevention and treatment of remodelling of the pulmonary vasculature and the right heart, and to the improvement of effort tolerance in patients suffering from smoking-induced lung injury and other lung disease and heart disease processes resulting in pulmonary hypertension in humans.

NONOates or diazeniumdiolates are chemical compounds that carry the [N(O)NO]^" functional group. They are generally synthesized by exposing a nucleόphile, which is usually an amine, to several atmospheres of nitric oxide (NO), under anaerobic conditions. When NONOates dissolve in physiological solutions, buffers or cell culture rnedium, they continuously release 2_. moles of NO per mole of parent compound decomposed. The half-lives of NO generation of NONOate vary from 1 minute to 1 day under normal physiological conditions. NONOates may be superior to gaseous NO in the clinical therapeutics with respect to: (1 ) Due to long half-lives in liberating of NO, NONOates- enable intermittent therapy or even once daily therapy. Rebound pulmonary hypertension, which is always a concern with discontinuation of gaseous NO therapy, is less likely to happen. (2) NONOates are stable in solid for and highly water-soluble, the complex delivery and monitoring systems used in gaseous NO delivery are not necessary.

The preceding discussion of the background art is intended to facilitate an understanding of the present invention only. It should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to was part of the common general knowledge in Australia as at the priority date of the application.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Disclosure of the Invention

The term "mucosally impermeant" refers to the compounds reduced ability, and possibly inability, to cross a mucous membrane. The term "NONOate"„ refers to a compound that includes an N₂O₂ ^" moiety (a NONOate moiety) and which is capable of releasing nitric oxide under physiological conditions.

In accordance with the present invention there is provided the use of nitric oxide donor compounds in the preparation of a medicament for the inhibition of growth factors in the pulmonary vasculature of a mammal,.

In accordance with the present invention there is further provided the use of nitric oxide donor compounds in the preparation of a medicament for the prevention and/or treatment of remodelling of the pulmonary vasculature and/or right heart of a mammal.

The remodelling treated in accordance with the present invention may be the result of pulmonary hypertension, acute hypoxaemia, and/or chronic hypoxaemia such as is present in smoking-induced lung injury and other lung disease and heart disease processes resulting in pulmonary hypertension in humans.

In accordance with the present invention there is still further provided the use of nitric oxide donor compounds in the preparation of a medicament for improving effort tolerance in humans suffering smoking induced lung injury and other lung disease and heart disease processes resulting in pulmonary hypertension.

The nitric oxide donor compounds utilised in the present invention are preferably selected from the mucosally impermeant NO donor compounds including tertiary and quaternary amino aliphatic NO donor compounds, and polyalkyleneamine NONOates.

In accordance with a further aspect of the present invention there is provided a method for the treatment of one or more of the conditions referred to above, the method comprising the administration of a pharmacologically effective amount of a nitric oxide donor compound to a patient in need thereof by way of a nebuliser or metered dose inhaler. Preferably, administration of the compound is intermittent and is directed to the tracheobronchial tree.

The nebuliser is preferably in the form of a jet nebuliser or ultrasonic nebuliser. The metered dose inhaler is preferably a _. pressurised canister containing a solution or suspension of the compound which is released in precisely metered doses.

Brief Description of the Drawings

The present invention will now be described, by way of example only, with reference to a number of embodiments thereof and the accompanying drawings, in which:

Figure 1 is a graphical representation of the pulmonary vascular resistance index (PVRI) change for control (n=4), DETA/NO treated (n=3), and DPTA/NO treated (n=4) groups after aerosol administration in Example 1. The significant difference of hemodynamic parameters in DETA/NO treated (0) and DPTA/NO treated (▼) groups from the values in controls (•) at the same time points are indicated by * (p<0.05) and #

(p<0.05) signs, respectively. Data are shown as mean ±SD;

Figure 2 is a graphical representation of the pulmonary arterial pressure (PAP) change for control (n=4), DETA/NO treated (n=3), and DPTA NO treated (n=4) groups after aerosol administration;

Figure 3 is a graphical representation of the systemic vascular resistance index (SVRI) change for control (n=4), DETA/NO treated (n=3), and DPTA/NO treated (n=4) groups after aerosol administration;

Figure 4 is a graphical representation of the systemic blood pressure (SBP) change for control (n=4), DETA/NO treated (n=3), and DPTA/NO treated (n=4) groups after aerosol administration. Figure 5 is a graphical representation of the total serum nitrate levels in a DETA/NO treated group in accordance with Example 2;

• Figure 6 is a graphical representation of the body weight gained/growth rate in Example 2;

Figure 7 is a graphical representation of the LWDR of control and treated groups in Example 2; and

Figure 8 is a graphical representation of the PMN ratio (%) comparison of those two groups for lung parenchyma in Example 2.

Best Mode(s) for Carrying Out the Invention

The present invention is directed to the use of a particular class of compounds in the treatment of a specific range of ailments or conditions, as well as to specific methods of treatment of these ailments or conditions.

The class of compounds include tertiary and quaternary amino aliphatic nitric oxide donor compounds as described in detail in US Patent 5958427, the content of which is incorporated^' herein by reference. Also included are the polyalkyleneamine NONOates. Preferably, the compounds utilised in the present invention are selected from the mucosally impermeant NO donor compounds.

It has been suggested that these compounds and pharmaceutical compositions containing these compounds are effective delivery vehicles for NO, particularly for site-specific vasodilation, due to their ability to release NO at the epithelial boundary of the mucosa. Charge and polarity prevent the compounds crossing the mucosa whereas the nitric oxide moiety is released over time and diffuses across the epithelium and into the mucosa due to its lipophilic nature. This provides the local vasodilator effect described. In one embodiment of the invention, nitric oxide donor compounds are utilised in the preparation of a medicament for the inhibition of growth factors in the pulmonary vasculature of a mammal.

In a second embodiment of the present invention nitric oxide donor compounds are utilised in the preparation of a medicament for the prevention and/or treatment of remodelling of the pulmonary vasculature and/or right heart of a mammal. The remodelling treated may be the result of pulmonary hypertension, acute hypoxaemia, and/or chronic hypoxaemia such as is present in smoking- induced lung injury and other lung disease and heart disease processes resulting in pulmonary hypertension.

The compounds present, and the drugs that may incorporate them, are designed to release NO within the lung to inhibit the effect of mitogens, such as endothelin - 1 , and similar substances, that affect the layers of the pulmonary vessel below the endothelium. This inhibition is claimed to prevent hypertrophy and narrowing of the pulmonary vessels, particularly those with a diameter of about 100 micron (the precapillary vessels), with subsequent hypertrophy and remodelling of the right ventricle.

In a third embodiment of the present invention nitric oxide donor compounds are utilised in the preparation of a medicament for improving effort tolerance in humans suffering smoking induced lung injury and other lung disease and heart disease processes resulting in pulmonary hypertension.

In a fourth embodiment of the present invention there is provided a method for the treatment of one or more of the conditions referred to above, the method comprising the administration of a pharmacologically effective amount of a nitric oxide donor compound to a patient in need thereof by way of a nebuliser or metered dose inhaler. Administration of the compound is intermittent and is directed to the tracheobronchial tree. The nebuliser is provided in the form of a jet nebuliser or ultrasonic nebuliser. The metered dose inhaler is a pressurised canister containing a solution or suspension of the compound which is released in precisely metered doses. It is envisaged that the drug would be administered to mostly, but not exclusively, ambulant patients over a period of weeks to months. It is further envisaged that patients would need dosing several times a day by either of the devices described above.

The present invention will now be described with reference to two examples. It is to be understood that these examples are exemplary only and the scope of the invention should not be considered as limited thereto.

Example 1

DETA/NO and DPTA/NO are each NONOates. The half-lives of DETA/NO and DPTA/NO for generating NO are approximately 20 hours and 2 hours, respectively. Compared to the extremely short biological half-life of .NO (less

- than 5 seconds), the slow and continuous release . of NO from these two compounds potentially makes them very useful agents for the treatment of pulmonary hypertension and hypoxemia. As noted hereinbefore, the vasodilatory effect of NONOates is mediated by NO released from the parent compounds. In theory, the effect of NONOates will be restricted to the well- ventilated lung units once they are inhaled. This may result in a reduction in the intrapulmonary shunting and improvement of oxygenation. The applicant has therefor hypothesized that inhaled DETA/NO and DPTA/NO may retain the advantages of inhaled NO, as a selective pulmonary vasodilator. Selective pulmonary vasodilatory effect is defined as both a reduction in pulmonary vascular resistance with a lack of effect on systemic circulation, and intrapulmonary shunting. The major differences of the present experimental design with that used in the other similar studies were: (1 ) This study was undertaken in a close-chest condition (the normal physiology of the lung especially the negative pressure between the pleura was retained). (2) This study lasted for 4 hours after inhalation (study duration was usually 1 hour in the open-chest study models). Animals

White landrace cross female piglets (n=8) with body weight ranging from 19 to 25kg were used in this study. They were allocated to control and DETA/NO treated groups: control animals (n=4) received aerosolized normal saline and. treated animals (n=4) received aerosolized DETA/NO.

Study procedures

Piglets were anaesthetized by inhalational halothane (0.5 to 1 %) in oxygen- enriched air (FiO₂0.5) and mechanically ventilated (Siemens Servo 900B mechanical ventilator, Siemens Elema, Sweden) via endotracheal tubes throughout the study period. All subjects received a FiO₂ of 0.5 during the study in the Assist Control Ventilation (ACV) mode with a tidal volume of approximately 10ml/kg. Vecuronium bromide was infused intravenously 2 to 6mg/h to facilitate mechanical ventilation. Hydration was maintained infusing isotonic saline solution via the right or left ear vein with an infusion rate of 5ml/kg/hr. A cut- down procedure was performed at the left or right inguinal region to expose the femoral artery and femoral vein. A single lumen catheter was inserted into the femoral artery and connected to the monitor (Siresust 404, Siemens, USA) to continuously monitor the arterial pressure. A 5-lumen 7.5 French gauge balloon- tipped, flow-directed Swan-Ganz catheter (Arrow, Model AH-05050-H, Arrow International, USA) was placed into the femoral vein. The catheter was then advanced into the inferior vena cava and the tip of catheter was eventually placed in the right or left pulmonary artery, verified by the typical pressure-wave forms and pressure changes displayed on the monitor. The proximal end of the 5-lumen catheter measured the pressure at the level of the superior, vena cava (central venous pressure, CVP); while the distal end of the catheter monitored the pressure in the pulmonary arterial pressure (PAP). , Pulmonary capillary wedge pressure (PCWP), which is similar to pulmonary venous pressure, was measured following inflation of the distal balloon of the catheter with not more than 1.5ml of air. PCWP was measured at the end of expiration and immediately before measuring the cardiac output (CO) in each instance. A CO measuring set (Critikit™-SP4500, Becton Dickinson, USA) was connected to the infusion port of the PA. catheter. CO was measured using the standard thermodilution technique by injection of 5ml bolus of cold sterile 5% dextrose water into the right atrium. CO was calculated from the detected change in temperature at the thermistor in the PA by the internal algorithm of the monitor (utilising the Stewart-Hamilton equation). Values of CO was divided by body mass to obtain the cardiac index (Cl), and expressed in a unit of L/min/kg. Blood samples for blood gas analysis were drawn from the femoral arterial line (arterial blood gas sample) and the right atrial port of the PA catheter (mixed venous blood gas sample). These blood samples were analysed by a blood gas analyser (ABL 520, Radiometer, Denmark). The measured and derived variables (i.e. intrapulmonary shunting and A-aDO₂) were obtained from the blood gas measurements. Arterial methaemoglobin levels were concurrently analysed by co-oximetry. Blood samples for analysing serum nitrite/nitrate levels were collected from the arterial line. Once the anaesthetized animals had been stabilized for at least 20min after catheterisation, baseline measurements of hemodynamic parameters and blood samples were taken.

Acute pulmonary hypertension induction protocol

Once all the baseline data had been collected, acute pulmonary hypertension was induced by intravenous infusion of U46619 (Cayman Chemical, Ml, USA) as described in a previous study. Briefly, U46619 was infused continuously via femoral vein at an initial rate of 0.01 μg/kg/min, and increased every 6 min until the target was achieved. The target level of pulmonary hypertension was a pulmonary vascular resistance index (PVRI)≥1.5 times above the baseline value. PVRI is calculated as described by Andrie et al and expressed in a unit of dyne-sec/cm⁵/kg. U46619 was then infused continuously at this constant rate throughout the study.

Study procedures

After target pulmonary hypertension has been achieved and stabilized for at least 20min, the first set of hemodynamic measurements and blood samples were measured or obtained. 10mg DETA/NO ([2,2'-Hydronitrosohydrazino] bisethanamine, Sigma Chemcial, MO, USA) was freshly diluted immediately before inhalation therapy. Normal saline and DETA/NO solution (a final concentration of 12mM) was aerosolised through a Sidestream Jet Nebuliser (Medic-Aid Ltd., UK). The nebuliser was driven by 100% oxygen at a flow rate of 2-3L/min, over 20min into the lungs of piglets by connecting the nebuliser to the inspiratory limb of the breathing circuit. The same hemodynamic measurements were repeated at 5, 10, 15, 20, 25, 30, 45, 60, 75, 90, 105, 120, 180, and 240min after the aerosol therapy commenced. Blood samples were collected at 15, 30, 45, 60, 75, 90, 180, and 240min after the aerosol therapy commenced. At the end of study, the piglets were euthanased with an intravenous injection of overdose pentobarbitone (250mg/kg). Lung and trachea were removed following medial sternotomy.

Measurement of NO metabolites

The total amount of NO released, which was absorbed into the systemic circulation, from DETA/NO was quantified by its metabolites (i.e. nitrite and nitrate) discovered in the blood. The levels were determined by the modified Griess reaction described by Giovannoni et al. (Adaptation of the nitrate reductase and Griess reaction methods for the measeurement of serum nitrate plus serum nitrate levels. Ann Clin Biochem 1997;34:193-8).

Pulmonary toxicity examinations

The potential pulmonary toxicity of DETA/NO was determined by the lung wet-to- dry ratio (LWDR) and the degree of lung inflammation.

Lung wet-to-dry ratio. The intermediate lobe of the right lung of each subject was excised and weighed immediately. The lobe was dried in an oven at 80^°C for 12h and reweighed. The LWDR was then obtained by dividing the mass of the initial specimen by the mass of the dried specimen. Inflammatory response of lung. Slides of airways and lung parenchyma were taken, as described by van Heerden et al, (Pulmonary toxicity of inhaled aerosolised prostacyclin (IAP) therapy - an observational study. Anaesth Intens

Care 2000;28:161-6) for light microscopic examination. This produced a total of 3 slides of main airway and 10 slides of lung parenchyma from each lung of each subject for light microscopic examination. The degrees of lung inflammatory response after aerosol inhalation were quantified by the ratio of.total number of polymorphonuclear leukocytes (PMN) to total number of non-PMN in each airway and lung parenchymal tissue. PMN ratios were separately measured and calculated for the large (i.e. trachea, right and left main bronchus) and small

^■ airways (defined as all the noncartilaginous airways in the lung parenchyma with nonalveolated walls), and lung parenchyma.

Statistical analysis

Data were analysed using SigmaStat Scientific Software (Version 2.0, Jandel Corp., CA, USA). All data were firstly tested for normality and equivalence of variance by descriptive statistics. ^' Non-normal distributed variables were logarithm transformed. All the data (hemodynamics, blood levels, LWDR, and PMN ratio) were analysed by two-way repeated-measures analysis of variance (ANOVA). Pearson product-moment correlation ^* was used to obtain the correlation coefficient of each pair of continuous variables in the analysis of hemodynamic data. All data are presented at mean ±SEM.

Results

General outcomes

Acute pulmonary hypertension was successfully induced in 4 and 3 piglets in the control and DETA/NO treated groups, respectively. Pulmonary hypertension was not induced in piglet number 8 (in DETA/NO treated group) due to failure of placement of the pulmonary artery catheter. However, all subjects received aerosol therapy, blood analysis, LWDR and lung histologic examinations were therefore performed in all eight subjects. The mean time-periods to achieve target pulmonary hypertension by the infusion of U46619 were 37±10 and 36+16min in the control and DETA/NO treated groups, respectively (=0.6). The mean doses of U46619 infused to achieve target pulmonary hypertension in control and DETA/NO treated groups were 73±8 and 78±15μg, respectively (p=0.65). There was no difference in the cumulative doses of U46619 at 1 , 2, 3, and 4h after the administration of inhaled study drug in both groups.

Hemodynamic effect on the pulmonary circulation

With reference to Figure 1 , the mean baseline PVRI of the control (n=4) and DETA/NO treated animals were 8.6±0.6 and 8.6±0.2dyne'sec/cm⁵/kg, respectively (p=0.93). At the time immediately before aerosol administration (zero time), PVRI were 19±0.6 and 25±2.3dyne se.c/cm⁵/kg in the control and treated groups, respectively (p=0.1). The subsequent PVRI were significantly reduced at 10, 15, 20, 25, 30, 45, and 60min after aerosol inhalation in the DETA/NO treated group. Referring to Figure 2, in considering change in PAP, there were no significant differences between the values of two treated groups at baseline and zero time, but PAP was significantly reduced in DETA/NO treated group compared to control subjects from 10 to 120min after treatment. No significant differences were found in PCWP and Cl at any time point between the two treated groups. The relationships of PVRI to PAP, PCWP and Cl were plotted (figure). PVRI has a significant positive relationship with PAP (r=0.8) and with PCWP (r=0.3), and a significant negative relationship with Cl (r=0.6).

Hemodynamic effect on the systemic circulation

During the effective time-period when pulmonary resistance was reduced by inhalation of DETA/NO, there were no significant differences in the systemic vascular resistance index (SVRI) and systemic blood pressure (SBP) between two treated groups, see Figures 3 and 4 respectively. Effect on intrapulmonary shunting and A-aDO₂

The percentage of intrapulmonary shunting and A-aDO₂ after aerosol inhalation in the DETA/NO group tended towards a reduction (up to 120min) compared to the control animals, but these differences did not reach statistical significance (p=0.07 and 0.78, respectively).

Measurement of NO metabolites

The baseline total serum concentrations of nitrate (including nitrate, which was converted into nitrite by nitrate reductase before reacted with Giress reagents) in the control and DETA/NO treated group were 40±4 and 53+12μmol/L (p=0.14), respectively.. The levels of the metabolites in piglets in the DETA NO treated group were significantly higher than those in the control group from 15min after aerosol administration until 4h (end of study). Within the DETA/NO treated group, total serum nitrite levels after treatment were also significantly elevated compared to the baseline measurement from 15min after aerosol administration until the end of study.

Pulmonary toxicity of DETA/NO

LWDR The mean LWDR of the control and DETA NO treated groups were 4.9±0.2 and 4±0.2 (p=0.2), respectively. Also, there was no significant difference of the LWDR between individual piglets within each group (p=0.09).

Inflammatory response of lung. The mean PMN ratios of large airway in the control and DETA/NO treated groups were 1.6±0.4 and 1.9±2.1 %, respectively (p=0.62). There were no significant differences in PMN ratio between the two treated groups at any level of the large airways (i.e. trachea, right and left main bronchus, p=0.25). In the small airways, the overall means PMN ratio were 0.66+0.05 and 0.62±0.05% in control and DETA/NO treated groups, respectively (p=0.1 ). There were no differences in the small airway PMN ratio at any lung area, except lung area 12 (medial posterior section of left lung), PMN ratio in DETA/NO group was higher than that in control group (0.66±0.14 vs 0.57+0.19%, respectively, p=0.01 ). The overall mean PMN ratios of lung parenchyma in control and DETA/NO treated groups were 4.1 ±0.8 and 3.9±0.6%, respectively (p=0.2). There were no statistically significant differences between these two groups in different lung area.

This study has shown that inhaled DETA/NO aerosol is a selective pulmonary vasodilator, with no detectable systemic hemodynamic effects. The treatment effect at least 60min. There was no evidence of haematological and pulmonary toxicity ascribable to DETA/NO. This study further showed that serum nitrite/nitrate levels are a useful method of estimating systemic absorption of NO released from NONOates.

Many pharmacologic agents, such as calcium channel blockers, infused prostacyclin, are potent pulmonary vasodilators, but their therapeutic use is limited due to causing systemic hypotension and worsening ventilation/perfusion matching during acute lung injury. A selective pulmonary vasodilator is an agent that can selectively dilate the pulmonary vasculature, without causing systemic hypotension and without reflecting deoxygenated blood from the poorly - or unventilated lung units. NO is an ideal candidate for a selective pulmonary vasodilator for its vasodilatory effect is retained in the pulmonary vascular bed of well-ventilated lung regions due to it has an extremely short half-life and is instantly inactivated by haemoglobin once it enters the intravascular spaces. However, gaseous NO has many limitations in the treatment of acute pulmonary hypertension partly due to some of its pharmacological characteristics. It has to be delivered continuously throughout the treatment course and abrupt discontinuation may lead to lethal rebound pulmonary hypertension due to its short half-life. High concentration NO and its oxidants are toxic and pro- inflammatory, therefore NO gas needs complex delivery and environmental monitoring systems during the treatment. DETA/NO belongs to a unique class of "slow-release" NO donor, NONOates. NO continuously released from these compounds mediates the vasoldilatory effect, through causing an increase in the intracellular cGMP concentration. The long half-life in the generation of NO enables DETA/NO to provide intermittent or even once-daily therapy with low risk of causing rebound pulmonary hypertension. DETA/NO needs only a nebulizer for delivery and the intensive monitoring system is not necessary. However, the selective pulmonary vasodilatory effect of DETA/NO in acute pulmonary hypertension model has not been explored. The major aim of this study was therefore to determine the vasodilatory effect of DETA/NO on pulmonary and systemic circulation.

A single nebulized dose (60 μmol) of DETA NO effectively reduced pulmonary resistance, from 5 to 60min, after treatment following U-46619 induced pulmonary hypertension. . Compared to controls, there was a reduction in mean PAP, from 10 to 120min after treatment, in the DETA/NO treated group. There are no other significant differences in PCWP or Cl during this period. The corresponding reduction in both PVRI and PAP thus indicated a close and direct relationship between these two pulmonary circulation indices. However, when the changes in PAP after treatment were compared to the baseline measurement within DETA/NO group, inhaled DETA/NO failed to demonstrate a significant reduction in PAP; while it had significantly reduced the PVRI to the baseline value within DETA/NO group (from 5 to 75min after treatment). The relationships between PVRI, PAP, PCWP and Cl were therefore analysed. Results showed that PAP had a strong correlation with PVR (r=0.8,r²=0.64) and Cl had a negative relationship with PVR (r=0.6, 1^=0.36). The inventors thus concluded, that the reduction in PVR was due to both a reduction in PAP and an increase in CO. Pulmonary circulation is characterised by two unique components - low resistance (provided by the pulmonary capillary network) and low pressure (modulated by a high distensibility of pulmonary arteries). These two characteristics enable the pulmonary vasculature to accommodate acute changes of pulmonary blood flow and volume. In other words PAP is not the only determinant in the resistance of the pulmonary circulation. Improvement of CO may be as important as the reduction of PVR in the management of pulmonary hypertension. It reflects an improvement of right heart function and may, in the longer term, reduce the remodelling (hypertrophy) of the right heart. Example 2

The lack of pulmonary and haematological toxicity of a single inhaled dose of DETA/NO has been shown in Example 1. However, the lack of pulmonary toxicity after a longer term, multiple-doses exposure has to be ensured before it is used in clinical trials. The aim of this study was therefore to investigate the pulmonary toxicity of aerosolised DETA/NO after a repeated dose exposure for 7 days. The presence of lung toxicity was examined by the lung wet-to-dry ratio (LWDR), the growth rate of the animal, the degree of inflammation of lung tissues, the ultrastructural change^' of the lung tissues, and the serum surfactant protein (SP) level.

A total of 32 rats with body weight ranging from 200 to 250g were allocated into control (n=16) and DETA/NO (n=16) exposure groups., The rats were placed into a closed, glass container during the study period: Drug or placebo was aerosolized by a small-volume nebulizer and delivered into the container through a side-hole. The control group was exposed to 5ml buffer solution over 20min once daily for 7 consecutive days. The treated group was exposed to aerosolized DETA/NO (60imol) once daily for 7 consecutive days.

Four rats, randomly selected from each group, were weighed before being anaesthetized by inhalation of halothane at 1 , 3, 7 and 14 days after the beginning of exposure. Blood samples were collected by cardiac puncture, which was performed immediately after the rats were deeply anaesthetized. The serum obtained was examined for SP and serum nitrite level. Bilateral lobes of lung were removed and fixed by intratracheal instillation of fixative. The left lung of each subject was examined for LWDR. The rest of the lung tissues were processed for histological examinations.

Total serum nitrite levels

The total serum nitrite levels in the DETA/NO treated group were consistently higher than the levels in controls at 1 , 3 and 7 days after exposure (indicating adequate exposure to the study drug), see Figure 5. Growth rate

The control and treated groups had a similar body-weight-gained curve (figure 2). There was no significant difference in the mean body weights between the two groups at any time point (p=0.71 ) (Indicating growth was not stunted by the treatment), see Figure 6.

LWDR

There was no significant difference in LWDR between the two groups at any time point (p=0.67) (Indicating that the treatment does not produce pulmonary oedema), see Figure 7.

Degree of lung tissue inflammation

The overall polymorphonuclear leukocyte (PMN) ratios of lung parenchyma were 1.8±0.2, 2.2±0.5, 2.2±0.3, and 2.4+0.4 in controls and 1.9±0.2, 2.2±0.4, 2±0.3, and 2±0.4 in the DETA/NO treated group at 1 , 3, 7 and 14 days after exposure, respectively, see Figure 8. There were no significant differences in PMN ratio between the two groups at these time periods (p=0.85). The tracheas and large airways of the animals were independently reviewed by two pathologists. No apparent inflammation or tissue destruction was found in the two groups (treatment did not produce lung inflammation).

The increase of total nitrite level in the treated group evidenced the systemic uptake of nitric oxide, released from DETA/NO, at the lung tissues of the rats. The deposition of DETA/NO in the lung tissues after prolonged exposure did not cause growth retardation, lung tissue destruction or inflammation and pulmonary edema in rats. These results indicated that repeated exposure to aerosolized DETA/NO at current dose did not cause any detectable pulmonary toxicity. DETA/NO is thus a safe compound to test clinically in human subjects.

It can be seen from the forgoing description that the NO donor compounds described herein will improve effort tolerance, by improving right ventricular function, and that these drugs will prevent adverse changes (re-modelling) of the right ventricle as a result of prolonged exposure to pulmonary hypertension. Further, it is envisaged that these drugs may improve right ventricular size and shape after prolonged course of therapy. .

Modifications and variations such as would be apparent to the skilled addressee are considered to fall within the scope of the present invention.

Claims

1. The use of nitric oxide donor compounds in the preparation of a medicament for the inhibition of growth factors in the pulmonary vasculature of a mammal.

2. The use of nitric oxide donor compounds in the preparation of a medicament for the prevention and/or treatment of remodelling of the pulmonary vasculature and/or right heart of a mammal.

3. The use of nitric oxide donor compounds according to claim 2, wherein the remodelling . treated is the result of pulmonary hypertension, acute hypoxaemia, and/or chronic hypoxaemia.

4. The use of nitric oxide donor compounds in the preparation of a medicament for improving effort tolerance in humans suffering smoking induced lung injury and other lung disease and heart disease processes resulting in pulmonary hypertension.

5. The use of nitric oxide donor compounds according to any one of the preceding claims, wherein the nitric oxide donor compounds utilised are mucosally impermeant NO donor compounds.

6. The use of nitric oxide donor compounds according claim 5, wherein the NO donor compounds are selected from the group of tertiary and quaternary amino aliphatic NO donor compounds, and polyalkyleneamine

NONOates.

7. A method for the treatment of pulmonary hypertension, acute hypoxaemia, smoking induced lung injury, remodelling of the pulmonary vasculature and/or right heart, and/or chronic hypoxaemia, the method comprising the administration of a pharmacologically effective amount of a nitric oxide donor compound to a patient in need thereof by way of a nebuliser or metered dose inhaler.

8. A method of treatment according to claim 7, wherein administration of the compound is intermittent.

9. A method of treatment according to claim 7 or 8, wherein administration of the compound is directed to the tracheobronchial tree.

10. A method of treatment according to any one of claims 7 to 9, wherein the nebuliser is provided in the form of a jet nebuliser or ultrasonic nebuliser.

11.A method of treatment according to any one of claims 7 to 9, wherein the metered dose inhaler is provided in the form of a pressurised canister containing a solution or suspension of the compound and which is released in precisely metered doses.

12. A method of treatment according to any one of claims 7 to 10, wherein the nitric oxide donor compounds utilised are mucosally impermeant NO donor compounds.

13. A method of treatment according to claim 12, wherein the NO donor compounds are selected from the group of tertiary and quaternary amino aliphatic NO donor compounds, and polyalkyleneamine NONOates.

14. A method of treatment according to any one of claims 7 to 13, wherein the patient is ambulant, and administration takes place over a period of weeks to months.

15. A method of treatment according to claim 14, wherein the administration occurs several times a day.