WO2016135263A1

WO2016135263A1 - No donors for the treatment of stress-induced pulmonary haemorrhage in animals

Info

Publication number: WO2016135263A1
Application number: PCT/EP2016/054021
Authority: WO
Inventors: Armin Scherhag; Hubert STÜCKLER; Pierre Vankan
Original assignee: Armin Scherhag
Priority date: 2015-02-27
Filing date: 2016-02-25
Publication date: 2016-09-01

Abstract

The invention relates to the field of veterinary medicine and particularly to the use of exogenous NO donors for the prevention and treatment of physical stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, CamelicJae or Canidae. More particularly the invention relates to amino-C₂- C₆-alkyl nitrates or amino-C₂-C₄-alkyl nitrates and the pharmaceutically acceptable salt thereof, for the acute or chronic treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae with the objective to accelerate the recovery and the healing process after a lung bleeding event. The invention also relates to the use of said amino-alkyl nitrates and the pharmaceutically acceptable salt thereof as an oral dosage form or in the form of an extended release composition. The most preferred amino-alkyl nitrate in accordance with the invention is 2-aminoethyl nitrate.

Description

NO donors for the treatment of stress-induced pulmonary haemorrhage in animals

The present invention relates to the field of veterinary medicine and particularly to the use of exogenous NO donors for the prevention and treatment of physical stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae, such as e.g. in racehorses, in racing camels or in racing dogs.

Background of the invention

It has been observed that when some non-human mammals undergo maximum physical stress, e.g. during a race, this may lead to a significant increase in the heart rate and the respiratory rate. Thus, e.g. during a horse race it is not uncommon that the heart rate of the horse may increase to approx. 220 / min. and the respiratory rate to about 130 / min. The blood pressure increases accordingly. In this way the body attempts to bring a maximum amount of fresh oxygen in the form of oxygenated haemoglobin through the lungs into the rest of the body and in particular to the running muscles of the horse. This ensures an appropriate oxygen delivery which enables an excellence in running performance. After extreme physical performance during a horse race nose bleeding is observed in approximately 5 to 20% of the animals. This phenomenon has been known since ancient times and affected horses are called "bleeders". Bronchoscopy studies have shown that bleedings in the lungs occurs in 50 to 70% of racehorses, and especially in Arabs. Such bleedings can be detected by analysing bronchial washing fluid. Thus it is clear that the nose bleeding observed in " bleeders" is not a harmless side effect under maximal performance activity but represents a recurrent pulmonary bleeding, which may have an impact on the short term as well as long term performance of the affected horses. Stress-induced pulmonary haemorrhage in animals at high energy is also known as stress-induced

pulmonary hypertension haemorrhage and/or associated dyspnoea. This associated dyspnoea results in a decreased performance of the animal and may require an increased recovery period after the race (e.g. due to the lung bleeding event).

As has been noted above, the physical peak power during a race leads to a massive increase in the pressure in the pulmonary arteries to values between 80- 100 mm Hg (the normal value in horses being 20-30 mm Hg).The tripling of the pulmonary arterial pressure together with the massive increase in heart rate allows the body of the horse to pump a high amount of blood through the right side of the heart to the lungs, where it is supplied with oxygen. The oxygenated blood is then pumped via the left side of the heart to the body in order to provide it to the running muscles. The oxygen required by the lungs is pumped under race conditions by an over a five-fold increase in respiratory rate in the lungs. The rapid exchange of inhalation and exhalation leads to an on average negative (sub-) pressure in the pulmonary capillaries and alveoli, which is so great that it causes a rupture of small pulmonary capillaries and a burst of small alveoli. This leads to a penetration of blood and plasma into the bronchi resulting in a decreased performance until the animal has recovered from the exercise induced lung bleeding event.

In the past affected animals have usually been treated as summarized below: a) Nasal strip (called "flair): This adhesive tape will be attached to the nostrils with the idea to prevent collapse of the nostrils by large negative pressure of the rapid inhalation. This is a simple method which has shown some success in some animals presumably because it leads to a decrease of the aspiratory negative pressure in the small airways.

b) Lasix™ (furosemide): This so-called loop diuretic has been widely applied to increase fluid excretion (urine) and to lower the pulmonary arterial pressure. Lasix has been used quite often in countries where it is not on the list of prohibited drugs. Whether Lasix has a beneficial effect is still controversial because no beneficial effect has clearly been demonstrated in appropriate studies up to now. c) Cortisone: steroids are on the doping list in almost all countries and cannot be used in race horses. Cortisones are believed to act via capillary sealing effect. d) Citrus bioflavonoids, hesperidin and vitamin C: have been proposed to be beneficial due to a capillary stabilizing effect, which has however not been scientifically proven.

e) Aspirin, aminocaproic acid (as fibrin degradation inhibitor), vitamin K: No effect detected.

0 NO-donors: so far no positive effects have been demonstrated (study design, however flawed (dose, duration of treatment, etc ).

g) Sildenafil: not allowed, possibly because of observed side effects.

Since nitrates can expand the coronary arteries (coronary) also it has also been tried to treat the affected animals with exogenous NO (nitric oxide)-donators. Nitric oxide is a naturally and continuously formed vessel-expanding substance produced by the vascular membrane lining (endothelium) with a half-life of a few seconds. In chronic vascular disease or under maximum stress which leads to a relative deficit of oxygen, the body is not in a position to produce enough NO in the affected vascular territories. The resulting local lack of NO leads to a consequent vasoconstriction. In equine animals, no positive effects of oral and / or chronic administration have been demonstrated in the treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea with nitrates until today. In one study the effects of nitroglycerin (but not 2-aminoethyl nitrates or the tosylate salts thereof - which are much more effective than nitroglycerine), has been examined over four minutes as single intravenous infusions with escalating doses to examine the acute haemodynamic effects in thoroughbred horses at rest

(Manohar ML, " Effects of glyceryl trinitrate (nitroglycerin) on pulmonary vascular pressure in standing thoroughbred horses" , Equine Vet. J. 1995 Jul; 27(4): 275- 80). Only the highest dose (2100 g/min) showed some acute, haemodynamic efficacy on pulmonary pressure. However, no exercise testing with respect to physical performance and the efficacy of nitroglycerin (which is known to rapidly cause tolerance) on the prevention and treatment of lung bleeding were

conducted. Nitrates are well established since a long time in the treatment of coronary heart disease and acute myocardial infarction in humans. Such nitrates are however difficult to dose and may provoke tolerance after a certain time of use. So far only one nitrate is known which does not provoke tolerance in humans, viz. AEN (2-aminoethyl nitrate). The corresponding drug product, Nilatil™, has been marketed by Pharmacia AB, but was later again withdrawn from the market.

Whereas AEN snows an excellent pharmacological profile, in particular no development of nitrate tolerance, the short half-live of approx. 2 hours requires frequent dosing and causes high peak-to-trough ratios, which is not desirable since it compromises appropriate patient compliance in humans.

In summary, it can be said that there has been no convincing therapeutic approach for the prevention and treatment of stress-induced pulmonary

haemorrhage and associated dyspnoea in animals of the family Equinea,

Camelidae or Canidae to date. There is therefore a need for new treatment options.

Summary of invention

The present invention relates to the use of an amino-C₂-C₆-alkyl nitrate or a pharmaceutically acceptable salt thereof in the prevention or treatment of stress- induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae and particularly in race horses. It has now been found that amino-C₂-C₆-alkyl nitrate compounds, such as e.g. 2- aminoethyl nitrate or the tosylate salt thereof (CLC1011) can surprisingly reduce the pulmonary arterial and venous blood pressure in these animals and thus can be used for the prevention and avoidance of stress-induced pulmonary

haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae as it may occur after peak-exercise such as in a race. The treatment of the animals with amino-Ga-C₆-alkyl nitrate compounds, such as e.g. 2- aminoethyl nitrate or the tosylate salt thereof (CLC 1011) leads to a decrease of the blood pressure in the pulmonary circulation "before" the heart and thus relieve the heart and the lungs. This again leads to a reduction in pulmonary arterial and capillary pressure in the pulmonary vessels. By this nitrate-induced pulmonary blood pressure lowering the stress-induced pulmonary bleeding can be

significantly reduced because the pressure gradient between the alveoli (air sacs) and the pulmonary capillaries decreases and it will not come to an inspiratory rupture of these delicate vessels. In this way lung bleeding can be minimized which again leads to an accelerated healing process and a corresponding reduced recovery period after the physical stress caused under race conditions. It may also be that a lung bleeding may be avoided in the animals by the preventive treatment with the amino-C₂-C₆-alkyl nitrates.

As shown in the examples below particular amino-C₂-C₆-alkyl nitrates in free base form or in form of a pharmaceutically acceptable salt thereof can be used for the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae, such as e.g. racehorses, race camels or race dogs. These amino-C₂-C₆-alkyl nitrates therefore may accelerate the recovery and healing process in animals experiencing exercise induced stress symptoms after a race. Suitable amino-C₂-C₆-alkyl nitrates in free base form or in form of a

pharmaceutically acceptable salt thereof have been described in detail in

PCT/WO 2014029841 A (CARDIOLYNX AG) 27.02.2014. In accordance with the present invention these amino-C₂-C₆-a1kyl nitrates may be used for acute treatment or chronic treatment of animals of the family Equinea, Camelidae or Canidae, such as e.g. racehorses, race camels or race dogs.

The amino-C₂-C₆-alkyl nitrates are preferably prepared and used in the form of a pharmaceutical composition suitable for oral administration to animals, either in form of an immediate release formulation or in form of an extended (or prolonged) release formulation. Such pharmaceutical compositions may be in liquid or in solid form as outlined further below.

Brief description of drawings Fig. 1 shows the release profile (ng/ml) after administration of a 10 mg dose of CLC-1011 to a dog in form of an extended release formulation in accordance with the present invention over a period of 24 hours.

Fig. 2 shows the release profile (ng/ml) after administration of a 60 mg dose of CLC-1011 to two different dogs in form of an extended release formulation in accordance with the present invention over a period of 8 hours.

Description of the embodiments

The present invention provides amino-C₂-C₆-alkyl nitrates (the therapeutically active ingredient) or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Came!idae or Canidae. C₂-C₆-a!kyl is an alkyl group of consisting of 2 to 6 carbon atoms, in particular ethyl, propyl, butyi, pentyl and hexyl. The alkyl group may be linear, as in n-propyl, n-butyl, n-pentyl and n-hexyl, or branched, as for example in iso-propyl, iso-butyl, sec-butyl, tert-butyl, iso-pentyl, 1-or 2-methylbutyl, 1-ethylpropyl, 1,2- dimethylpropyl, tert-pentyl, and corresponding branched hexyl groups, e.g. iso- hexyl and 1, 2, and 3-methyl-pentyl.

A C₂-C₆-alkyl nitrate is sometimes also called C₂-C₆-alkanol nitrate, indicating more clearly that a nitrate is an ester of nitric acid with the corresponding alkanol, or also a nitrooxyalkane. In the context of the present invention it is understood that in an alkyl nitrate the nitrate function is covalently bonded to the alkyl residue by an oxygen atom.

In amino-C₂-C₆-alkyl nitrate, the amino group and/or the nitrate function may be in a primary, secondary or tertiary position, if possible at all. Preferably, the amino group is not bound to the same carbon atom as the nitrate function. More preferably, both the amino group and the nitrate function are in a primary position, for example as in 2-aminoethyl nitrate, 3-amino-propyl nitrate, 4-aminobutyl nitrate, 5-aminopentyl nitrate, 6-aminohexyl nitrate, 3-amino-2-methylpropyl nitrate, and 3- amino-2,2-dimethylpropyl nitrate. However, other substitution patterns are also considered, for example as in 2-amino-1-methylethyl nitrate, 3-amino-1- methylpropyl nitrate, 2-amino-1 ,1-dimethylethyl nitrate, 2-aminopropyl nitrate, 2- aminobutyl nitrate, and 2-amino-3-methylbutyl nitrate. The said amino-C₂-C₆-alkyl nitrates can be prepared according methods well known in the art. Most of these amino-C₂-C₆-alkyl nitrates are also commercially available in bulk from various suppliers.

Preferred amino-C₂-C₆-alkyl nitrates are amino-C₂-C₄-alkyl nitrates such as 4- aminobutyl nitrate, 3-aminopropyl nitrate, 2-amino-1 -methylethyl nitrate, and 2- aminoethyl nitrate, in particular 4-aminobutyl nitrate and 2-aminoethyl nitrate. Most preferred is 2-aminoethyl nitrate (AEN), also known under the name itramin.

AEN has in the past been marketed by Pharmacia AB under the trade name Nilatil™, but was later again withdrawn from the market. AEN shows an excellent pharmacological profile and in particular does not provoke nitrate tolerance in humans. However, the short half-live of approx. 2 hours requires frequent dosing and causes high peak-to-trough ratios, which is not desirable since it compromises appropriate patient compliance.

Pharmacia has filed for patent protection for certain 2-aminoethylnitrat salts such as e.g. the tosylate salt many decades ago (see SE 168308 C (AB PHARMACIA) 25.08.1959 and US 3065136 B (PHARMACIA AB) 20.11.1962). Pharmaceutically acceptable salts of amino C₂-C₆-alkyl nitrates or amino-C₂-C₄- alkyl nitrates are acid addition salts of pharmaceutically acceptable non-toxic inorganic and organic acids. Preferred pharmaceutically acceptable salts are acetate, benzoate, besylate (benzenesulfonate), bromide, chloride,

camphorsulfonate, chlortheophyllinate, citrate, ethenedisulfonate, fumarate, gluconate, glutamafe, hippurate, 2-hydroxyethanesulfonate, 2- hydroxy-2- phenylacetate, iodide, lactate, laurylsulfate, malate, maleate, mesylate (methane- sulfonate), methylsulfate, napsylate (2-naphthalenesulfonate), nitrate, octadecanoate, oxalate, pamoate, phosphate, polygalacturpnate, succinate, sulfate, tartrate, and tosylate (p-toluenesulfonate).

Most preferred pharmaceutically acceptable salts are sulfate, phosphate, acetate and tosylate, in particular tosylate (e.g. itramin tosilate).

The preferred dosage of the amino-C₂-C₆-alkyl nitrate is between 0.025 to

0.1 mg /kg body weight up to 4 to 5 times daily, with a preferred dosage of 0.05 mg / kg body weight per dose or higher, depending on the species and the respective absorption, bioavailability and efficacy, when dosed as an immediate release formulation. When dosed as an extended release formulation the dosage is between 0.05 and 1.0 mg / kg body weights once or twice a day, again potentially also higher, depending on the species and the respective absorption, bioavailability and efficacy.

Preferred animals of the family Equinea, Camelidae or Canidae in accordance with the present invention are:

- Equinea: Horses in particular race horses, more particularly warm-blooded race horses.

- Camelidae: Camelus bactrianus and Camelus dromedarius.

- Canidae: Racing dogs such as e.g. English Greyhound and Afghan hound.

The amino-C₂-C₆-alkyl nitrates are preferably prepared and used in the form of a pharmaceutical composition suitable for administration to animals. Such

pharmaceutical compositions may be in liquid or in solid form. Both the liquid form and the solid form should be stable over a longer period of time, i.e. at least several weeks, preferably 6 to 12 months. The liquid form is preferably an aqueous formulation. Suitable pharmaceutical compositions comprise in addition to the therapeutically effective amount of the pharmaceutically active ingredient suitable excipients such as diluents, stabilizers, wetting agents, emulsifying agents, co-solving agents, buffering agents, surfactants and carrier materials. Suitable pharmaceutically acceptable excipients and appropriate amounts to be used are known to the person skilled in the art (see e.g. Remington s

Pharmaceutical Sciences; Mack Pub. Co., New Jersey, current edition).

Examples for liquid formulations suitable for direct administration to the animal comprise in addition to the amino-C₂-C₆-alkyl nitrate in purified water (preferably isotonic) suitable amounts of:

a) polyethylene glycol (e.g. in the form of macrogol, such as e.g. macrogol 15); b) hydroxystearate (e.g. in a concentration of 0.2 ml/100 kg); and

c) optionally a carrier such as e.g. apple sauce (e.g. 35 ml).

If the pharmaceutical composition is in solid form it is preferably filled in sachets each containing a suitable dose of the amino-C₂-C₆-alkyl nitrate for immediate release. For administration the active ingredient in the sachet is then added directly to the feed concentrate containing e.g. a multivitamin cocktail and optionally an egg shell concentrate (providing calcium carbonate in form for ready uptake). Alternatively the active ingredient in the sachet is combined with a suitable amount of carrier liquid such as e.g. apple sauce or noni juice. Noni juice is derived from the fruit of the Morinda citrifolia tree indigenous to Southeast Asia and Australasia which has beneficial anti-oxidative properties.

The amino-C₂-C₆-alkyl nitrates may also be prepared and used in the form of an extended release formulation as they are also described in detail in PCT/WO 2014029841 A (CARDIOLYNX AG) 27.02.2014. Therefore, in another embodiment the invention provides an amino C₂-C₆-alkyl nitrate or an amino C₂-C₄-alkyl nitrate or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae, wherein the amino-alkyl nitrate is in the form of an extended release composition. Preferably the extended release composition is administered once or twice daily. The term " extended release" as used herein means that the release of the active ingredient does not occur by immediate release, but represents release over a pre-defined, longer time period of time, such as e.g. of up to 24 hours, such as e.g. 6-24 hours, preferably 12-24 hours or also as a chronic treatment. Release characteristics and release time are measured according to standard methods, e.g. those of Ph. Eur., in aqueous solution with a paddle at pH 6.8 and 37X.

The extended-release characteristics for the release of amino-C₂-C₆-alkyl nitrate or the amino-C₂-C₄-alkyl nitrate and pharmaceutically acceptable salts thereof may be varied by modifying the composition of each formulation component, including modifying any of the excipients and coatings or also transdermal patch layers which may be present. In particular the release of the active ingredient may be controlled by changing the composition and/or the amount of the extended-release coating, if such a coating is present. If more than one extended-release

component is present, the coating or matrix former for each of these components may be the same or different. Similarly, when extended-release is governed by an extended-release matrix material, release of the active ingredient may be controlled by the choice and amount of extended-release matrix material utilized. When the extended-release component comprises a modified release matrix material, any suitable extended-release matrix material or suitable combination of extended-release matrix materials may be used. Such materials are known to those skilled in the art. Thus e.g. polymer coated drug-ion exchange resins comprising a very large variety of known drugs, such as e.g. itramin, are described in PCT/WO 2008064163 A (MORTON GROVE PHARMACEUTICALS, INC) 29.02.2008. The term " extended-release matrix material" as used herein includes hydrophilic polymers, hydrophobic polymers and mixtures thereof which are capable of modifying the release of an active ingredient dispersed therein in vitro and in vivo.

In a preferred embodiment, the extended-release composition of the amino-C^C₆- alkyl nitrate or a pharmaceutically acceptable salt thereof, for use in accordance with the present invention will provide more or less constant plasma levels of amino-C₂-C₆-alkyl nitrate or amino-C₂-C₄-alkyl nitrate and the pharmaceutically acceptable salts thereof over 12 hours, more preferably over 24 hours.

One of the objects of this invention is therefore to provide an extended release oral dosage form of an amino-C₂-C₆-alkyl nitrate or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae. In such an extended release oral dosage form the active ingredient or ingredients may be embedded in a non-ionic polymer matrix, which matrix may optionally be coated as described in detail in PCT/WO 2014029841 A (CARDIOLYNX AG) 27.02.2014.

Another object is to provide a multiparticulate extended release composition of an amino-C₂-C₆-alkyl nitrate or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae. Such multiparticulate extended release composition are described in detail in PCT/WO 2014029841 A (CARDIOLYNX AG) 27.02.2014. Still another object of the present invention is to provide a single-unit extended release tablet-, film coated tablet- or hard capsule formulation of an amino-C₂-C₆- alkyl nitrate or an amino-C₂-C₄-alkyl nitrate and the pharmaceutically acceptable salts thereof, for use in the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae. The term " hard capsules" includes any type of hard capsule made from gelatin or a different material, e.g. hypromellose and gellan gum (Vcaps™) or pullulan and carrageenan (NPcaps™). Such single-unit extended release tablet-, film coated tablet- or hard capsule formulation are described in detail in PCT/WO 2014029841 A (CARDIOLYNX AG) 27.02.2014.

Another object of the present invention is to provide an osmotically controlled oral dosage form of an amino-C₂-C₆-alkyl nitrate or an amino-C₂-C₄-alkyl nitrate and the pharmaceutically acceptable salts thereof, for use in the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae. Such osmotically controlled oral dosage forms are described in detail in PCT/WO 2014029841 A (CARDIOLYNX AG) 27.02,2014.

Mother object of the present invention is to provide an amino C₂-C₆-alkyl nitrate or an amino C₂-C₄-alkyl nitrate or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae, wherein the amino-alkyl nitrate is in the form of a transdermal drug delivery system. Such transdermal drug delivery system allows the release of the active ingredient continuously over a time period of several hours up to several days. Preferably the transdermal drug delivery system releases the active ingredient in accordance with the present invention over 12 to 24 hours. Alternatively, the transdermal drug delivery system releases the active ingredient in accordance with the present invention continuously over a time window of 1 to 7 days. Such transdermal drug delivery systems are described in detail in PCT/WO 2014029841 A (CARDIOLYNX AG) 27.02.2014. Another object of the present invention is to provide transdermal patches wherein the amino-alkyl nitrate in accordance with the present invention is embedded and where upon administering to the skin the therapeutically active ingredient is released over an extended period of time, e.g. within 24 hours and taken up by the body through the skin. Such transdermal patches are described in detail in

PCT/WO 2014029841 A (CARDIOLYNX AG) 27.02.2014.

Another object of the present invention is to provide reservoir patches wherein the amino-alkyl nitrate for use in accordance with the present invention is embedded and where upon administering to the skin the therapeutically active ingredient is released over an extended period of time, e.g. within 24 hours and taken up by the body through the skin. Such reservoir patches and their preparation are described in detail in PCT/WO 2014029841 A (CARDIOLYNX AG) 27.02.2014. Another object of the present invention is to provide pharmaceutical compositions comprising an amino-alkyl nitrate in accordance with the present invention for the treatment of animals are selected from the group of race horses, race camels or racing dogs. Preferably the pharmaceutical compositions comprising an amino- alkyl nitrate in accordance with the present invention are provided for the treatment of race horses, in particular warm-blooded race horses.

Another object of the present invention is the use of an amino-alkyl nitrate in accordance with the present invention for the prevention or treatment of stress- induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae. Preferably the amino-alkyl nitrate in accordance with the present invention is used for the treatment of race horses, in particular warm-blooded race horses. Another object of the present invention is the use of an amino-alkyl nitrate in accordance with the present invention for the manufacture of a medicament for the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae, preferably in race horses, in particular warm-blooded race horses.

The invention will be more fully understood by reference to the following

Examples. They should not, however, be construed as limiting the scope of the invention, Examples

Example 1: In this example the effect of a 10 mg dose of 2-aminoethyl nitrate tosylate salt (CLC 1011) in the form of an extended release formulation has been studied in dogs. Figure 1 shows the release profile of CLC-1011 <ng/ml) over a period of 24 hours. The graph shows that a release maximum is reached after about 4 hours rapidly levelling off to a bi-phasic plateau level up to about 24 hours.

Example 2: In this example the effect of a 60 mg dose of 2-aminoethyl nitrate tosylate salt (CLC1011) in the form of an extended release formulation has been studied in two different dogs. Figure 2 shows the release profile of CLC-1011 (ng/mi) over a period of 8 hours. The graph shows that a release maximum is reached in both animals after about 2 hours levelling off within a period of about 4 to 6 hours.

.Example 3: 2-aminoethyl nitrate (AEN) in the form of the tosylate salt {CLC-1011) is administered to two groups of freely fed race horses, which exhibit lung bleeding. The two groups of animals are given once daily by oral administration either vehicle alone (purified water, macrogol 15, hydroxystearate - 0.2 ml/100 kg and approximately 35 mi of apple sauce) or vehicle containing the AEN tosylate salt in increasing doses up to 1 mg/kg for 4 weeks. The treated horses receive a daily dose of AEN tosylate salt at 0.1 mg/kg body weight for the first 7 days, followed by 0.2 mg/kg body weight. From day 20 the dose is increased to 1 mg/kg bodyweight. The dose is then further increased until a safe and effective dose is identified. Lung bleeding is measured by either clinical evidence or bronchoscopy.

Measurement of the performance and lung bleeding at the end of the study reveals a statistical significant tendency for a reduction of lung bleeding and/or increased performance in the horses treated with the 2-aminoethyl nitrate.

These data indicate that treated horses experienced a significant improvement of their tendency for lung bleeding.

Example 4: An initial dose pharmacologic dose finding study was conducted in four horses with the objective to investigate the tolerability, absorption and pharmacokinetic properties of oral administration of increasing doses of 2- aminoethyl nitrate tosylate salt (CLC-1011) in horses.

Since this was, after previous administration of CLC-1011 in raits and dogs within the preclinical pharmacology program designed for the development of CLC-1011 for human use, the doses of CLC-1011 known to result in blood pressure effects in rats and dogs from these studies were chosen as a starting point for this study and were extrapolated to the average weight of a racing horse (700-800 kg). Based on the above cited pharmacologic data from the rat and dog studies, four ascending doses (10 mg, 20 mg, 30 mg and 40 mg) were tested, with the tow doses chosen to carefully investigate initial tolerability and potential side effects of CLC-1011 (e.g. drop of blood pressure and related syncope).

For administration, CLC-1011 was solved in a watery solution and injected with a syringe into the mouth of the horses starting at 8 o' clock in the morning. Four plasma samples were taken at one (09:00), two (10:00), four (12:00) and eight hours (16:00) after oral administration.

The study drug was administered by a veterinary and the horses were carefully observed for any potential side effects. It was found that CLC-1011 was well tolerated at all doses. None of typical side effects of nitrates such as dizziness, nausea or behavioral changes which could point towards as significant lowering of blood pressure were observed.

The date are summarized in Table 1 below. For the 10 mg and 20 mg dose, no significant plasma levels (BDL = below detection limit) could be detected in any of the four samples.

For the 30 mg and the 40 mg doses, significantly elevated plasma levels of CLC- 1011 were found indicating a linear dose effect in two of the four horses.

However, the plasma levels observed in this initial tolerability and dosing finding study were below what is known to be pharmacodvnamically efficient plasma concentration in rats and dogs, which is estimated to be at least about 200 pg/ml. In a further tolerability and dose finding study escalating higher doses of CLC- 1011 are investigated in order to achieve the desired pharmacodynamics effect (lowering of pulmonary pressure during exercise) in race horses to prevent exercise-induced lung bleeding.

In summary, this initial tolerability and dose finding study with oral administration of CLC-1011 in four horses showed a very good tolerability with no observable side effects. The data do also demonstrate that a watery solution of CLC-1011 which is orally injected in the mouth is indeed absorbed by horses, with detectable plasma levels starting at doses of 30 mg and 40 mg after 4 to 8 hours suggesting a linear dose effect. The results of this initial study are therefore forming a promising basis to further study CLC-1011 in higher doses in race horses or other animals (e.g. racing camels, racing dogs) suffering from exercise-induced lung bleeding.

Table 1: Concentrations of two doses of aminoethyl nitrate tosytate salt (CLC- 1011) in horse plasma samples

Detection limit (BDL): 80 [pg/mL]; Quantification limit (LLOQ): 200 [pg/mL]; BDL: below the detection limit

Claims

Claim 1. An amino-C₂-C₆-alkyl nitrate or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae.

Claim 2. The amino-C₂-C₆-a!kyl nitrate in accordance with claim 1 , wherein the amino-C₂-C₆-alkyl nitrate is an amino-C₂-C₄-alkyl nitrate selected from the group of 4-aminobutyl nitrate, 3-aminopropyl nitrate, 2-amino-1 -methylethyl nitrate, and 2-aminoethyl nitrate.

Claim 3. The amino-Ca-C₆-alkyl nitrate in accordance with claim 1 , wherein the amino-C₂-C₆-alkyl nitrate is 2-aminoethyl nitrate.

Claim 4. The amino-alkyl nitrate in accordance with any one of claims 1 to 3, wherein the amino-C₂-C₆-alkyl nitrate or the amino-C₂-C₄-alkyl nitrate is in the form of a tosylate salt.

Claim 5. The amino-C₂-CValkyl nitrate or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae in accordance with any one of claims 1 to 4 in the form of an composition suitable for oral administration.

Claim 6. The amino-C₂-C₆-alkyl nitrate or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae in accordance with any one of claims 1 to 5 in the form of an extended release composition.

Claim 7. The amino-C₂-C₆-alkyl nitrate or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae in accordance with claim 6, characterized in that the extended release composition releases the active ingredient over a time period of up to 24 hours, of 6 to 24 hours, or of 12 to 24 hours.

Claim 8. The amino-C₂-C₆-alkyl nitrate or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae in accordance with any one of claims 5 to 7,

characterized in that the composition is an oral dosage form and wherein the active ingredient is embedded in a non-ionic polymer matrix and optionally coated.

Claim 9. The amino-C₂-C₆-alkyl nitrate or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae in accordance with any one of claims 1 to 8, wherein the animals are selected from the group of race horses, race camels or racing dogs.

Claim 10. A pharmaceutical composition comprising an amino-C₂-C₆-alkyl nitrate or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 9 for use in the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae.

Claim 11. The use of an amino-C₂-C₆-alkyl nitrate or a pharmaceutically

acceptable salt thereof as claimed in any one of claims 1 to 9 or a

pharmaceutical composition according to claim 10 in the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae.

Claim 12. The use of an amino-C₂-C₆-alkyl nitrate or a pharmaceutically

acceptable salt thereof as claimed in any one of claims 1 to 9 or a

pharmaceutical composition according to claim 10 for the manufacture of a medicament for the prevention or treatment of stress-induced pulmonary haemorrhage and/or associated dyspnoea in animals of the family Equinea, Camelidae or Canidae.

Claim 13. The invention as herein before described.