WO2010035047A1

WO2010035047A1 - Binary compositions comprising an no-donor and an a-smase inhibitor for the treatment of respiratory diseases

Info

Publication number: WO2010035047A1
Application number: PCT/GB2009/051262
Authority: WO
Inventors: Keith Mccormack
Original assignee: PharmaPatents Global Ltd
Current assignee: PharmaPatents Global Ltd
Priority date: 2008-09-25
Filing date: 2009-09-25
Publication date: 2010-04-01
Anticipated expiration: 2011-03-25
Also published as: GB0817585D0; GB2463883A

Abstract

The present invention relates to a pharmaceutical composition comprising an A-SMase inhibitor and/or a NO-donor. Preferred A-SMase inhibitors are tricyclic antidepressants, most preferably amitriptyline. A preferred NO-donor is arginine. Preferably the pharmaceutical composition comprises an A-SMase inhibitor and a NO-donor, which preferably act synergistically to inhibit A-SMase. The pharmaceutical composition is suitable for treating or preventing respiratory disorders such as chronic cough, cystic fibrosis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and emphysema. Preferably the pharmaceutical composition is administered by inhalation. The present invention also relates to methods of treating or preventing respiratory disorders, methods of inhibiting A-SMase, and assays for investigating the effect of test compounds upon A-SMase activity in vitro.

Description

BINARY COMPOSITIONS COMPRISING AN NO-DONOR AND AN A-SMASE INHIBITOR FOR THE TREATMENT OF RESPIRATORY DISEASES

Field of the invention

The present invention relates to a pharmaceutical composition comprising an A-SMase inhibitor and/or a NO-donor. Preferred A-SMase inhibitors are tricyclic antidepressants, most preferably amitriptyline. A preferred NO-donor is arginine. Preferably the pharmaceutical composition comprises an A-SMase inhibitor and a NO-donor, which preferably act synergistically to inhibit A-SMase. The pharmaceutical composition is suitable for treating or preventing respiratory disorders such as chronic cough, cystic fibrosis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and emphysema. Preferably the pharmaceutical composition is administered by inhalation. The present invention also relates to methods of treating or preventing respiratory disorders, methods of inhibiting A-SMase, and assays for investigating the effect of test compounds upon A-SMase activity in vitro.

Background of the invention

A wide and varied range of agents are in clinical use for the treatment of respiratory disorders. In the search for further therapies, classes of drugs first developed for other purposes have come under increasing scrutiny.

Amongst these, tricyclic antidepressants such as maprotiline have surprisingly recently found application in the treatment of asthma (WO 96/37198), whilst other studies have suggested that oral doses of lOmg amitriptyline may be used to treat chronic cough (Bastian et al., Otolaryngology - Head and Neck Surgery, 2006, vol. 135, pp. 17-21). The biological effects of tricyclic antidepressants are numerous; they are believed to act for instance as norepinephrine and serotonin re-uptake inhibitors, as modulators of muscarinic and histamine Hl receptors, and as inhibitors of acid sphingomyelinase (A-SMase).

Acid sphingomyelinase is a water-soluble, lysosomal glycoprotein that catalyses the degradation of membrane-bound sphingomyelin into phosphorylcholine and ceramide. A deficiency of A-SMase results in Niemann-Pick disease. Mammalian A-SMases exist in two forms, lysosomal A-SMase (L-ASMase) and secretory A-SMase (S-ASMase). L- and S-ASMase are very similar proteins, and cells transfected with an A-SMase cDNA overexpress both L-ASMase and S-ASMase, indicating that S-ASMase does not arise by alternative processing of the A-SMase gene. In addition, antibodies made against L-ASMase recognize S-ASMase, and the molecular weights of the enzymes on Western blot are similar. However, S-ASMase requires exogenously added zinc for activation, whereas L-ASMase isolated from cell or tissue homogenates does not. The lack of stimulation of L-ASMase by any cations and its lack of inhibition by ethylenediaminetetraacetic acid (EDTA) has led to the labelling of L-ASMase as a "cation- independent" enzyme (see, for example, J. Biol. Chem., 1998, vol. 273(29), pp. 18250- 18259).

Stimuli, that include cytokines, activate A-SMase and induce a translocation of this enzyme onto the extracellular leaflet of the cell membrane. A-SMase has been shown to be activated in response to various stress stimuli that include ligation of death receptors (tumour necrosis factor (TNF)-alpha, CD95, and TNF-related apoptosis-inducing ligand (TRAIL), radiation (UV-C and ionizing radiation), chemo therapeutic agents (cisplatin, doxorubicin, paclitaxel, and histone deacetylase inhibitors), and viral, bacterial, and parasitic pathogens and cytokines.

Studies have described rapid activation of A-SMase, most likely via a post-translational modification (Newrzella and Stoffel, J. Biol. Chem., 1996, vol. 271(50), pp. 32089-32095; Ferlinz et al, Eur. J. Biochem., 1997, vol. 243(1-2), pp. 511-517). However, transcriptional change is also a regulator of A-SMase activity. For example, differentiation of monocytic cells to macrophages requires up-regulation of the enzyme at a transcriptional level, and transcription factors SPl and AP-2 mediate the A-SMase response during differentiation (Langmann et al, J. Lipid Res., 1999, vol. 40(5), pp. 870-880). Additional studies demonstrate that AP-2 also up-regulates A-SMase in leukaemia cells treated with retinoic acid (Murate et al, J. Biol. Chem., 2002, vol. 277(12), pp. 9936-43).

It has also been demonstrated that phorbol esters such as phorbol myristate acetate (PMA), induce activation of A-SMase through protein kinase C (PKC) delta-dependent phosphorylation at serine 508. This phosphorylation is essential for activation of the enzyme, and appears to be necessary for the PMA-induced translocation of A-SMase from the lysosome to the plasma membrane. Ultraviolet radiation has also been demonstrated to phosphorylate A-SMase at serine 508 (Zeidan and Hannun, J. Biol. Chem., 2007, vol. 282(15), pp. 11549-61).

In addition, cysteine 629 situated toward the C terminus of A-SMase has been proposed as an important residue for regulation of the enzyme such that modification (mutation or deletion) of this residue results in a 5-fold activation of the enzyme (Qiu et al., J. Biol. Chem., 2003, vol. 278(35), pp. 32744-52).

Whilst the precise modus operandi of tricyclic antidepressants in the treatment of respiratory disorders is not well understood, a recent study has demonstrated that, in relation to acute neonatal inflammatory lung injury at least, it is the role of tricyclic antidepressants as A-SMase inhibitors, and hence as inhibitors of ceramide generation, that is crucial (von Bismarck et al, American Journal of Respiratory and Critical Care Medicine, 2008, vol. 177, pp. 1233-1241).

It is thought that the inhibition of A-SMase by tricyclic antidepressants occurs in two phases, the first involving intercalation of the tricyclic antidepressant within the lipid leaflet of the cell membrane resulting in separation of A-SMase from its substrate, sphingomyelin, and the second involving proteolytic degradation of the displaced A-SMase (J. Immunol., 2004, vol. 173, pp. 4452-4453; Biochim. Biophys. Acta, 1995, vol. 1266(1), pp. 1-8; J. Med. Chem., 2008, vol. 51(2), pp. 219-37).

An entirely different drug class of interest is represented by nitric oxide (NO) and donors of NO such as glyceryl trinitrate (nitro-glycerine). NO is an important cellular signalling molecule, having a vital role in many biological processes. NO is generated from L-arginine by the enzymatic action of nitric oxide synthase (NOS). There are two endothelial forms of NOS: constitutive NOS (cNOS) and inducible NOS (iNOS). There is also a neural NOS (nNOS), which acts as a transmitter in the brain and peripheral nervous system. - A -

NO is generated by a variety of stimuli in many physiological and pathological conditions. In many of these situations, the inducible isoform of nitric oxide synthase (iNOS), expressed after induction by cytokines or endotoxins, converts L-arginine into NO and L- citrulline.

NO-donors have been employed for well over 100 years to treat conditions such as angina and heart failure. They are effective due to the vasodilatory effect of NO. More recently, this effect has been employed to treat pulmonary vasoconstriction and asthma using NO or NO-donors such as S-nitroso-N-acetylpenicillamine by inhalation (WO 92/10228).

In spite of the above, however, there is a continuing need for new and improved agents and formulations.

In a separate advancement, recent studies on cell apoptosis have demonstrated that nitric oxide is also able to inhibit A-SMase. This action by NO was found to be mediated through stimulation of guanylate cyclase, to be cyclic-GMP (c-GMP) -dependent and to require activation of protein kinase G (PKG) (Barsacchi et al, Cell Death and Differentiation, 2002, vol. 9, pp. 1248-1255).

Additional studies have shown that basal sphingomyelin hydrolysis was not modified when NO-donors, inhibitors or stimulators of guanylate cyclase, and an inhibitor of PKG were administered without an inducing agent (Falcone et al., ]. Immunol., 2004, vol. 173(7), pp. 4452-63; Barsacchi et al., E. CeU Death Differ., 2002, vol. 9(11), pp. 1248-55). These studies indicate that NO inhibits activated but not basal A-SMase and A-SMase activities via a mechanism involving activation of guanylate cyclase, formation of cGMP and activation of PKG.

It has been surprisingly appreciated by the present inventors that conventional A-SMase inhibitors and nitric oxide or donors thereof are able to act synergistically to inhibit A-SMase and to treat respiratory disorders such as chronic cough, cystic fibrosis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis or emphysema. Without wishing to be bound by a particular theory, it is thought that inhibiting upstream processes of A-SMase activation during induction using nitric oxide, or a nitric oxide donor, non-linearly augments the downstream effects of an inhibitor such as a tricyclic antidepressant, which takes effect at the substrate level.

Summary of the invention

A first aspect of the present invention relates to a pharmaceutical composition comprising an A-SMase inhibitor and a NO-donor, and optionally comprising one or more pharmaceutically acceptable excipients. Preferably the A-SMase inhibitor and the NO- donor act synergistically to inhibit A-SMase and/or to treat respiratory diseases.

As used herein, the term "A-SMase" refers to acid sphingomyelinase. A-SMase can exist as lysosomal A-SMase (L-ASMase) or secretory A-SMase (S-ASMase). The term "A-SMase" can refer to L-ASMase or S-ASMase or both. An "A-SMase inhibitor" refers to any compound or substance that is not a NO-donor and that inhibits A-SMase. Preferably the term "A-SMase inhibitor" refers to any compound or substance that is capable of inhibiting A-SMase by disrupting its attachment to a lysosomal membrane, more preferably by disrupting its attachment to an intralysosomal membrane.

As used herein, a "NO-donor" refers to a nitric oxide donor. As used herein, a "nitric oxide donor" refers to any compound or substance that has the capacity to augment or increase the bioavailability of nitric oxide in vivo or in vitro. The term "NO-donor group" is similarly defined as a group comprising a nitric oxide donor.

In one embodiment of the first aspect of the present invention, the A-SMase inhibitor is selected from alimemazine, ami trip tyline, trans-l,4-bis(2-chlorobenzaminomethyl)- cyclohexane (AY9944), chlorpromazine, clomipramine, cocaine, cyamemazine, desipramine, dibucaine, imipramine, mianserin, perhexiline, prochlorperazine, propericiazine, quinacrine, tamoxifen, thioproperazine, thioridazine, trifluoperazine, trihexyphenidyl, trimipramine, N-(6-aminohexyl)-l-naphthalenesulfonamide (W- 5), N-(6- aminohexyl)-l-[5'-chloro-naphthalene-l'-sulfonamide] (W-7), amlodipine, astemizole, benztropine, bepridil, camylofin, clomiphene, cloperastine, cyclobenzaprine, cyproheptadine, doxepin, drofenine, pimethixene, promazine, protriptyline, chlorprothixene, fendiline, fluoxetine, maprotiline, norfluoxetine, nortriptyline, paroxetine, promethazine, sertraline, suloctidil, terfenadine, triflupromazine, 8-bromoguanosine-3',5'- cyclic monophosphate, tricyclo-decan-Q-yl-xanthate, monensin, or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

As used herein, "prodrug" refers to any derivative of a drug that is metabolized or otherwise converted into an active form upon introduction into the body of an animal. Prodrugs are well known to those skilled in the art of pharmaceutical chemistry, and provide benefits such as increased absorption and half-life. Prodrugs of this invention may be formed when, for example, hydroxy groups are esterified or alkylated, or when carboxyl groups are esterified. Those skilled in the art of drug delivery will readily appreciate that the pharmacokinetic properties of the compounds of the invention may be controlled by an appropriate choice of moieties to produce prodrug derivatives.

The compounds of the present invention can be used both, in their free acid or base form and their salt form. For the purposes of this invention, a "salt" of a compound of the present invention may be an acid addition salt, or a salt formed between an acidic functionality of a compound of the present invention (e.g. a carboxylic acid group) and a suitable cation.

Acid addition salts are preferably pharmaceutically acceptable, non-toxic addition salts with suitable acids, including but not limited to inorganic acids such as hydrohalogenic acids (for example, hydrofluoric, hydrochloric, hydrobromic or hydroiodic acid) or other inorganic acids (for example, nitric, perchloric, sulphuric or phosphoric acid); or organic acids such as organic carboxylic acids (for example, propionic, butyric, glycolic, lactic, mandelic, citric, acetic, benzoic, salicylic, succinic, malic or hydroxysuccinic, tartaric, fumaric, maleic, hydroxymaleic, mucic or galactaric, gluconic, pantothenic or pamoic acid), organic sulphonic acids (for example, methanesulphonic, trifluoromethanesulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, benzenesulphonic, toluene-p-sulphonic, naphthalene-2-sulphonic or camphorsulphonic acid) or amino acids (for example, ornithinic, glutamic or aspartic acid). The acid addition salt may be a mono- or di-acid addition salt. A preferred salt is a hydrohalogenic, sulphuric, phosphoric or organic acid addition salt. A more preferred salt is a hydrochloric acid addition salt.

Suitable cations for forming a salt with an acidic functionality include, but are not limited to lithium, sodium, potassium, magnesium, calcium and ammonium. The salt may be a mono-, di- or tri-salt. Preferably the salt is a mono- or di-lithium, sodium, potassium, magnesium, calcium or ammonium salt. More preferably the salt is a mono- or di-sodium salt.

In a second embodiment of the first aspect of the present invention, the A-SMase inhibitor comprises a nitrogen atom. Preferably the A-SMase inhibitor has a pK_a of at least 5, such as at least 6, at least 8 or at least 8.45. Preferably the A-SMase inhibitor has a pK_a of between 5 and 20, more preferably of between 6 and 15, and most preferably of between 8 and 12.

As used herein, the pK_a of an A-SMase inhibitor refers to the pK_a of the conjugate acid of that inhibitor in dilute aqueous solution at 25°C.

Preferably in the second embodiment of the first aspect of the present invention, the A-SMase inhibitor has a log P of at least 0, such as at least 1, at least 2 or at least 3.61. Preferably the A-SMase inhibitor has a log P of between 0.5 and 30, more preferably of between 1 and 15, and most preferably of between 2 and 8.

As used herein, the term "log P" in relation to a compound refers to the logarithm of the ratio of the compound's concentration in the octanol phase to its concentration in the aqueous phase of a two-phase octanol/water system at 25°C, i.e.

log P = log ([compound]_octanol/[compound]_water)

Preferably in the second embodiment of the first aspect of the present invention, the A-SMase inhibitor has a steric factor k ≤ 20, such as ≤ 15 or ≤ 10. More preferably the A-SMase inhibitor has a steric factor k ≤ 6 such as ≤ 5. Most preferably the A-SMase inhibitor has a steric factor k ≤ 4 or ≤ 3. The steric factor "k" in relation to a nitrogen-containing compound is calculated in accordance with the method outlined in Kornhuber et al., J. Med. Chem., 2008, vol. 51, pp. 219-237, as follows: (1) the most basic nitrogen atom in the compound is identified; (2) the largest substituent at the most basic nitrogen atom, as assessed by heavy atom count, is disregarded; (3) k is the sum of the heavy atom counts of the remaining substituents at the most basic nitrogen atom. As used herein, the term "heavy atom count" in relation to a substituent refers to the number of non-hydrogen atoms in that substituent, e.g. 1 for a methyl group, 3 for a -COMe group etc. Where the most basic nitrogen atom is part of a single ring and does not carry a double bond, k is assessed by hypothetically cleaving the nitrogen atom from the chemical structure to leave two fragments; k is the heavy atom count of the smaller of the two fragments (as assessed by heavy atom count); e.g. 1 for 1- methyl-pyrrolidine, 4 for 1-hexyl-pyrrolidine etc. Where the most basic nitrogen atom is simultaneously part of two or three rings, or is part of a single ring and carries a double bond, k is assigned a value of 6.

In a third embodiment of the first aspect of the present invention, the A-SMase inhibitor is selected from alimemazine, ami trip tyline, trans-l,4-bis(2-chlorobenzaminomethyl)- cyclohexane (AY9944), chlorpromazine, clomipramine, cocaine, cyamemazine, desipramine, dibucaine, imipramine, mianserin, perhexiline, prochlorperazine, propericiazine, quinacrine, tamoxifen, thioproperazine, thioridazine, trifluoperazine, trihexyphenidyl, trimipramine, N-(6-aminohexyl)-l-naphthalenesulfonamide (W- 5), N-(6- aminohexyl)-l-[5'-chloro-naphthalene-l'-sulfonamide] (W-7), amlodipine, astemizole, benztropine, bepridil, camylofin, clomiphene, cloperastine, cyclobenzaprine, cyproheptadine, doxepin, drofenine, pimethixene, promazine, protriptyline, chlorprothixene, fendiline, fluoxetine, maprotiline, norfluoxetine, nortriptyline, paroxetine, promethazine, sertraline, suloctidil, terfenadine, triflupromazine, or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof. Preferably the A-SMase inhibitor is selected from alimemazine, amitriptyline, chlorpromazine, clomipramine, desipramine, dibucaine, imipramine, prochlorperazine, tamoxifen, thioridazine, trifluoperazine, trihexyphenidyl, trimipramine, amlodipine, benztropine, bepridil, clomiphene, cyclobenzaprine, cyproheptadine, doxepin, promazine, protriptyline, chlorprothixene, fluoxetine, maprotiline, nortriptyline, paroxetine, promethazine, sertraline, or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof. In a fourth embodiment of the first aspect of the present invention, the A-SMase inhibitor is a cationic amphophilic drug.

In a fifth embodiment of the first aspect of the present invention, the A-SMase inhibitor is an antidepressant, such as alimemazine, amitriptyline, chlorpromazine, clomipramine, cocaine, cyamemazine, desipramine, imipramine, mianserin, prochlorperazine, propericiazine, thioproperazine, thioridazine, trifluoperazine, trihexyphenidyl, trimipramine, cyclobenzaprine, cyproheptadine, doxepin, pimethixene, promazine, protriptyline, chlorprothixene, fluoxetine, maprotiline, norfluoxetine, nortriptyline, paroxetine, promethazine, sertraline, triflupromazine, or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

Preferably in the fifth embodiment of the first aspect of the present invention, the antidepressant is a selective serotonin reuptake inhibitor (SSRI), a serotonin- norepinephrine reuptake inhibitor (SNRI), a noradrenergic and specific serotonergic antidepressant (NASSA), a norepinephrine (noradrenaline) reuptake inhibitor (NRI), a norepinephrine-dopamine reuptake inhibitor, a tricyclic antidepressant (TCAD) or a monoamine oxidase inhibitor (MAOI). Most preferably the antidepressant is a tricyclic antidepressant (TCAD).

As used herein, the term "tricyclic antidepressant" refers to any antidepressant comprising at least three fused rings, such as amitriptyline or clomipramine. Thus, the term also includes antidepressants comprising more than three rings, such as tetracyclic antidepressants including mianserin, prochlorperazine and maprotiline. Optionally, the term "tricyclic antidepressant" may refer to any antidepressant containing three fused rings and no other cyclic groups.

Preferably the tricyclic antidepressant is selected from alimemazine, amitriptyline, chlorpromazine, clomipramine, cyamemazine, desipramine, imipramine, mianserin, prochlorperazine, propericiazine, thioproperazine, thioridazine, trifluoperazine, trimipramine, cyclobenzaprine, cyproheptadine, doxepin, pimethixene, promazine, protriptyline, chlorprothixene, maprotiline, nortriptyline, paroxetine, promethazine, tfiflupromazine, or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof. Most preferably the tricyclic antidepressant is amitriptyline or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

In a sixth embodiment of the first aspect of the present invention, the A-SMase inhibitor is selected from a compound of formula (I) or QI):

or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof, wherein at each occurrence:

X is independently selected from N

_

-C(R^π)₂-N- __NR ^{i i}-CR¹¹- -CR^{1 L}NR^π- -N=C- -C=N- -Q-CR¹¹-

> l h —

-C=CR¹¹- or R C C

— X'— > i i is independentiy selected from C— -NR^U-C- — C— NR¹¹-

— o — c— — c — o— — s — c— — c — s- — - cC —- (C"r(τR)i¹h¹ — ih _^_

)^2 - or -C(R J₂

-Y- is independently selected from a chemical bond, -O-, -S-, -NR¹²-, -C(R¹²)₂-, -C(R¹²)₂-C(R¹²)₂-, -R¹²C=CR¹²-, -NR¹²-C(R¹²)₂-, -C(R¹²)₂-NR¹²-, -N=CR¹²-, -R¹²C=N-, -O-C(R¹²)₂-, -C(R¹²)₂-O-, -S-C(R¹²)₂- ^or -C(R¹²)₂-S-; n is independently 1, 2, 3, 4, 5 or 6; m is independently 0, 1, 2, 3, 4 or 5;

R¹, R², R³, R⁴, R⁵, R⁶, R⁷_ R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independentiy selected from hydrogen, -F, -Cl, -Br, -I, -CF₃, -CCl₃, -CBr₃, -CI₃, -OH, -SH, -NH₂, -CN, -NO₂, -COOH, -R¹³-O-R¹⁴, -R¹³-S-R¹⁴, -R¹³-SO-R¹⁴, -R¹³-SO₂-R¹⁴, -R¹³-SO₂-OR¹⁴, -R¹³-O-SO₂-R¹⁴,

-R¹³-SO₂-N(R¹⁴)₂, -R¹³-NR¹⁴-SO₂-R¹⁴, -R¹³-O-SO₂-OR¹⁴, -R¹³-O-SO₂-N(R¹⁴)₂, -R¹³-NR¹⁴-SO₂-OR¹⁴, -R¹³-NR¹⁴-SO₂-N(R¹⁴)₂, -R¹³-N(R¹⁴)₂, -R¹³-N(R¹⁴)₃ ⁺, -R¹³-P(R¹⁴)₂, -R¹³-Si(R¹⁴)₃, -R¹³-CO-R¹⁴, -R¹³-CO-OR¹⁴, -R¹³-O-CO-R¹⁴, -R¹³-CO-N(R¹⁴)₂, -R¹³-NR¹⁴-CO-R¹⁴, -R¹³-O-CO-OR¹⁴, -R¹³-O-CO-N(R¹⁴)₂, -R¹³-NR¹⁴-CO-OR¹⁴, -R¹³-NR¹⁴-CO-N(R¹⁴)₂, -R¹³-CS-R¹⁴, -R¹³-CS-OR¹⁴, -R¹³-O-CS-R¹⁴, -R¹³-CS-N(R¹⁴)₂, -R¹³-NR¹⁴-CS-R¹⁴, -R¹³O-CS-OR¹⁴, -R¹³-O-CS-N(R¹⁴)₂, -R¹³-NR¹⁴-CS-OR¹⁴,

-R¹³-NR¹⁴-CS-N(R¹⁴)₂ or -R¹⁴;

-R¹³- is independently a chemical bond, or an optionally substituted C₁-C₁₀ alkylene, C₂-C₁₀ alkenylene or C₂-C₁₀ alkynylene group;

-R¹⁴ is independently hydrogen or a C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₂- C₁₀ aryl, C₃-C₁₀ arylalkyl, C₄-C₁₀ arylalkenyl, C₄-C₁₀ arylalkynyl, C₃-C₁₀ alkylaryl, C₄-C₁₀ alkenylaryl or C₄-C₁₀ alkynylaryl group, each of which may optionally be substituted; and wherein any two or more R¹, R², R³, R⁴, R⁵, R⁶, R⁷ R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ or R¹⁴ may, together with the atom or atoms to which they are attached, form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more heteroatoms N, O or S in its carbon skeleton.

For the purposes of the present invention, an "alkyl" group is defined as a monovalent saturated hydrocarbon, which may be straight-chained or branched, or be or include cyclic groups. An alkyl group may optionally include one or more heteroatoms N, O or S in its carbon skeleton. Examples of alkyl groups are methyl, ethyl, »-propyl, 2-propyl, »-butyl, i- butyl, /-butyl and »-pentyl groups. Preferably an alkyl group is straight-chained or branched and does not include any heteroatoms in its carbon skeleton. Preferably an alkyl group is a C₁-C₁₂ alkyl group, which is defined as an alkyl group containing from 1 to 12 carbon atoms. More preferably an alkyl group is a C₁-C₆ alkyl group, which is defined as an alkyl group containing from 1 to 6 carbon atoms. An "alkylene" group is similarly defined as a divalent alkyl group.

An "alkenyl" group is defined as a monovalent hydrocarbon, which comprises at least one carbon-carbon double bond, which may be straight-chained or branched, or be or include cyclic groups. An alkenyl group may optionally include one or more heteroatoms N, O or S in its carbon skeleton. Examples of alkenyl groups are vinyl, allyl, but-1-enyl and but-2-enyl groups. Preferably an alkenyl group is straight-chained or branched and does not include any heteroatoms in its carbon skeleton. Preferably an alkenyl group is a C₂-C₁₂ alkenyl group, which is defined as an alkenyl group containing from 2 to 12 carbon atoms. More preferably an alkenyl group is a C₂-C₆ alkenyl group, which is defined as an alkenyl group containing from 2 to 6 carbon atoms. An "alkenylene" group is similarly defined as a divalent alkenyl group.

An "alkynyl" group is defined as a monovalent hydrocarbon, which comprises at least one carbon-carbon triple bond, which may be straight-chained or branched, or be or include cyclic groups. An alkynyl group may optionally include one or more heteroatoms N, O or S in its carbon skeleton. Examples of alkynyl groups are ethynyl, propargyl, but-1-ynyl and but-2-ynyl groups. Preferably an alkynyl group is straight-chained or branched and does not include any heteroatoms in its carbon skeleton. Preferably an alkynyl group is a C₂-C₁₂ alkynyl group, which is defined as an alkynyl group containing from 2 to 12 carbon atoms. More preferably an alkynyl group is a C₂-C₆ alkynyl group, which is defined as an alkynyl group containing from 2 to 6 carbon atoms. An "alkynylene" group is similarly defined as a divalent alkynyl group.

A cyclic "hydrocarbyl" group includes cyclic alkyl, alkenyl, alkynyl and aryl groups.

An "aryl" group is defined as a monovalent aromatic hydrocarbon. An aryl group may optionally include one or more heteroatoms N, O or S in its carbon skeleton. Examples of aryl groups are phenyl, naphthyl, anthracenyl and phenanthrenyl groups. Preferably an aryl group does not include any heteroatoms in its carbon skeleton. Preferably an aryl group is a C₄-C₁₄ aryl group, which is defined as an aryl group containing from 4 to 14 carbon atoms. More preferably an aryl group is a C₆-C₁₀ aryl group, which is defined as an aryl group containing from 6 to 10 carbon atoms. An "arylene" group is similarly defined as a divalent aryl group.

For the purposes of the present invention, where a combination of groups is referred to as one moiety, for example, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl, the last mentioned group contains the atom by which the moiety is attached to the rest of the molecule. A typical example of an arylalkyl group is benzyl. For the purposes of this invention, an optionally substituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylene, alkenylene, alkynylene, arylene or hydrocarbyl group may be substituted with one or more of -F, -Cl, -Br, -I, -CF₃, -CCl₃, -CBr₃, -CI₃, -OH, -SH, -NH₂, -CN, -NO₂, -COOH, -R^α-O-R^β, -R^α-S-R^β, -R^α-SO-R^β, -R^α-SO₂-R^β, -R^α-SO₂-OR^β, -RO-SO₂-R^β, -R^α-SO₂-N(R^β)₂, -R^α-NR^β-SO₂-R^β, -RO-SO₂-OR^β, -RO-SO₂-N(R^β)₂, -R^α-NR^β-SO₂-OR^β, -R^α-NR^β-SO₂-N(R^β)₂, -R^α-N(R^β)₂, -R^α-N(R^β)₃ ⁺, -R^α-P(R^β)₂, -R^α-Si(R^β)₃, -R^α-CO-R^β, -R^α-CO-OR^β, -RO-CO-R^β, -R^α-CO-N(R^β)₂, -R^α-NR^β-CO-R^β, -RO-CO-OR^β, -RO-CO-N(R^β)₂, -R^α-NR^β-CO-OR^β, -R^α-NR^β-CO-N(R^β)₂, -R^α-CS-R^β, -R^α-CS-OR^β, -RO-CS-R^β, -R^α-CS-N(R^β)₂, -R^α-NR^β-CS-R^β, -RO-CS-OR^β, -RO-CS-N(R^β)₂, -R^α-NR^β-CS-OR^β, -R^α-NR^β-CS-N(R^β)₂, -R^β, a bridging substituent such as -O-, -S-, -NR^β- or -R"-, or a π-bonded substituent such as =O, =S or =NR^β. In this context, -R^α- is independently a chemical bond, a C₁-C₁₀ alkylene, C₂-C₁₀ alkenylene or C₂-C₁₀ alkynylene group. -R^β is independently hydrogen, unsubstituted C₁-C₆ alkyl or unsubstituted C₆-C₁₀ aryl. Optional substituent(s) are not taken into account when calculating the total number of carbon atoms in the parent group substituted with the optional substituent(s). Preferably an optionally substituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylene, alkenylene, alkynylene, arylene or hydrocarbyl group is not substituted with a bridging substituent. Preferably an optionally substituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylene, alkenylene, alkynylene, arylene or hydrocarbyl group is not substituted with a π-bonded substituent. Preferably a substituted group comprises 1, 2 or 3 substituents, more preferably 1 or 2 substituents, and even more preferably 1 substituent.

In one embodiment of any aspect of the present invention, any optional substituent contains 1 to 12 atoms. Preferably any optional substituent contains 1 to 6 atoms. More preferably any optional substituent contains 1 to 4 atoms.

Any optional substituent may be protected. Suitable protecting groups for protecting optional substituents are known in the art, for example from "Protective Groups in Organic Synthesis" by T.W. Greene and P.G.M. Wuts (Wiley-Inters cience, 4th edition, 2006). Preferably in the sixth embodiment of the first aspect of the present invention,

(a) — X— is independently selected from -N- -CR¹¹- -N-C(R¹^₂- or

(b) -^χ'^~ is ^~c~ .

Preferably, -Y- is independently selected from -S-, -C(R¹²)₂-, -C(R¹²)₂-C(R¹²)₂-, -R¹²C=CR¹²-, -O-C(R¹²)₂- or -C(R¹²)₂-O-. Preferably n is 2 or 3 and/or m is 1 or 2.

Optionally in the sixth embodiment of the first aspect of the present invention, one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is selected from hydrogen, -F, -Cl, -Br, -I, -CF₃, -CCl₃, -CBr₃, -CI₃, -OH, -SH, -NH₂, -CN, -NO₂, -COOH, -R¹³-O-R¹⁴, -R¹³-S-R¹⁴, -R¹³-SO-R¹⁴, -R¹³-SO₂-R¹⁴, -R¹³-SO₂-OR¹⁴, -R¹³-O-SO₂-R¹⁴, -R¹³-SO₂-N(R¹⁴)₂, -R¹³-NR¹⁴-SO₂-R¹⁴, -R¹³-O-SO₂-OR¹⁴, -R¹³-O-SO₂-N(R¹⁴)₂, -R¹³-NR¹⁴-SO₂-OR¹⁴, -R¹³-NR¹⁴-SO₂-N(R¹⁴)₂, -R¹³-N(R¹⁴)₂, -R¹³-N(R¹⁴)₃ ⁺, -R¹³-P(R¹⁴)₂, -R¹³-Si(R¹⁴)₃, -R¹³-CO-R¹⁴, -R¹³-CO-OR¹⁴, -R¹³-O-CO-R¹⁴, -R¹³-CO-N(R¹⁴)₂, -R¹³-NR¹⁴-CO-R¹⁴, -R¹³-O-CO-OR¹⁴, -R¹³-O-CO-N(R¹⁴)₂, -R¹³-NR¹⁴-CO-OR¹⁴, -R¹³-NR¹⁴-CO-N(R¹⁴)₂, -R¹³-CS-R¹⁴, -R¹³-CS-OR¹⁴, -R¹³-O-CS-R¹⁴, -R¹³-CS-N(R¹⁴)₂, -R¹³-NR¹⁴-CS-R¹⁴, -R¹³O-CS-OR¹⁴, -R¹³-O-CS-N(R¹⁴)₂, -R¹³-NR¹⁴-CS-OR¹⁴, -R¹³-NR¹⁴-CS-N(R¹⁴)₂ or -R¹⁴, and aU other R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are hydrogen. In one alternative, R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are all hydrogen. In another alternative, one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is not hydrogen.

Preferably in the sixth embodiment of the first aspect of the present invention, at each occurrence R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently selected from hydrogen, -F, -Cl, -Br, -I, -CF₃, -CCl₃, -CBr₃, -CI₃, -CN, -R¹³-O-R¹⁴, -R¹³-S-R¹⁴, -R¹³-SO-R¹⁴, -R¹³-SO₂-R¹⁴,

-R¹³-SO₂-OR¹⁴, -R¹³-O-SO₂-R¹⁴, -R¹³-SO₂-N(R¹⁴)₂, -R¹³-NR¹⁴-SO₂-R¹⁴, -R¹³-O-SO₂-OR¹⁴,

-R¹³-O-SO₂-N(R¹⁴)₂, -R¹³-NR¹⁴-SO₂-OR¹⁴, -R¹³-NR¹⁴-SO₂-N(R¹⁴)₂, -R¹³-N(R¹⁴)₂, or -R¹⁴.

Preferably at each occurrence R⁹ is independently selected from hydrogen or a C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₂-C₁₀ aryl, C₃-C₁₀ arylalkyl, C₄-C₁₀ arylalkenyl, C₄-C₁₀ arylalkynyl, C₃-C₁₀ alkylaryl, C₄-C₁₀ alkenylaryl or C₄-C₁₀ alkynylaryl group, each of which may optionally be substituted. Most preferably at each occurrence R is independently selected from hydrogen or methyl.

Preferably at each occurrence R¹⁰ is independently selected from hydrogen or a C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₂-C₁₀ aryl, C₃-C₁₀ arylalkyl, C₄-C₁₀ arylalkenyl, C₄-C₁₀ arylalkynyl, C₃-C₁₀ alkylaryl, C₄-C₁₀ alkenylaryl or C₄-C₁₀ alkynylaryl group, each of which may optionally be substituted. Most preferably at each occurrence R¹⁰ is independently selected from hydrogen or methyl.

Optionally in the sixth embodiment of the first aspect of the present invention, at each occurrence R¹¹ is hydrogen, and/or at each occurrence R¹² is hydrogen, and/or at each occurrence -R¹³- is a chemical bond. Preferably at each occurrence -R¹⁴ is independently hydrogen or a C₁-C₄ alkyl, C₂-C₄ alkenyl or C₂-C₄ alkynyl group, each of which may optionally be substituted.

Optionally in the sixth embodiment of the first aspect of the present invention, one R⁹ and one R¹⁰, together with the atoms to which they are attached, form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more additional heteroatoms N, O or S in its carbon skeleton.

Optionally in the sixth embodiment of the first aspect of the present invention, the two R¹⁰, together with the nitrogen atom to which they are attached, form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more additional heteroatoms N, O or S in its carbon skeleton.

Optionally in the sixth embodiment of the first aspect of the present invention, one R¹¹ and one R¹², together with the atoms to which they are attached, form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more additional heteroatoms N, O or S in its carbon skeleton.

Optionally in the sixth embodiment of the first aspect of the present invention, one R¹⁰ and one R¹¹, together with the atoms to which they are attached, form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more additional heteroatoms N, O or S in its carbon skeleton.

Preferably in the sixth embodiment of the first aspect of the present invention, an optionally substituted cyclic hydrocarbyl group is substituted with one or more of -F, -Cl,

-Br, -I, -CF₃, -CCl₃, -CBr₃, -CI₃, -CN, -NO₂, -O-R¹⁵, -S-R¹⁵, -N-(R¹⁵)₂ or -R¹⁵, or one or more π-bonded substituents such as =O, =S or =NR¹ , wherein -R¹ is independently hydrogen or an unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₂-C₁₀ aryl, C₃-C₁₀ arylalkyl, C₄-C₁₀ arylalkenyl, C₄-C₁₀ arylalkynyl, C₃-C₁₀ alkylaryl, C₄-C₁₀ alkenylaryl or C₄-C₁₀ alkynylaryl group. Most preferably an optionally substituted cyclic hydrocarbyl group is substituted with -OH or a methyl group.

In a seventh embodiment of the first aspect of the present invention, the NO-donor is nitric oxide, a NO-synthase substrate, a NO-synthase agonist, or a NO-releaser.

A "NO-synthase substrate" refers to a compound or substance from which a nitric oxide synthase is capable of generating nitric oxide. Preferably it refers to a compound or substance from which endothelial nitric oxide synthase is capable of generating nitric oxide.

A "NO-synthase agonist" refers to a compound or substance which is capable of increasing the generation of nitric oxide by a nitric oxide synthase but that is not in itself a NO-synthase substrate. Such compounds include the essential co-factor tetrahydrobiopterin. Preferably the nitric oxide synthase is endothelial nitric oxide synthase.

A "NO-releaser" refers to a compound or substance that is capable of releasing nitric oxide in vivo or in vitro. The term "NO-releasing group" is similarly defined as a group comprising a moiety that is capable of releasing nitric oxide in vivo or in vitro.

Preferably the NO-donor is a NO-synthase substrate such as arginine, N-cyclopropyl-N'- hydroxyguanidine, L-canavanine or N^α-benzoyl-L- arginine ethyl ester, or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof. Most preferably the NO-synthase substrate is L- arginine or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof. Alternatively in the seventh embodiment of the first aspect of the present invention, the NO-donor is a NO-releaser. Preferably the NO-releaser is selected from:

(a) Angeli's salt (Na₂N₂O₃), isoniazid, N-hydroxybenzenesulfonamide or P-nitrosophosphate;

(b) an organic nitrate such as glyceryl trinitrate (GTN), [3-(nitrooxymethyl)phenyl]-2- acetyloxybenzoate (NCX-4016), 4-nitrooxybutyl-2-acetyloxybenzoate (NCX-4215), [3-(nitrooxymethyl)phenyl]-2-hydroxybenzoate (B-NOD), isosorbide mononitrate (ISMN), pentaerythrityl tetranitrate (PETN), isosorbide dinitrate (BiDiI), nicorandil, nipradilol, nitro-pravastatin (NCX-6550), O-losartanyl-3-[(nitrooxy)methyl]- benzoate (NO-Losartan A) or methyl 2-[[4-[(nitrooxy)methyl]benzoyl]thio]- benzoate (SE 175);

(c) an organic nitrite such as amyl nitrite or 2-methylbutan-2-yl nitrite;

(d) a NONOate such as diethylenetriamine NONOate (DETA NO), diethylamine NONOate (DEA NO), spermine NONOate (SPER NO), dipropylenetriamine

NONOate (DPTA NO), methylamine hexamethylene methylamine NONOate (MAHMA NO), 3-propylamine-propylamine NONOate (PAPA NO), proline NONOate (PROLI NO), l-[(ethenyloxy)-NNO-azoxy]-pyrrolidine (V-Pyrro NO), O2- (2,4-dinitrophenyl) - 1 - [(4-ethoxycarbonyl)piperazin- 1 -yl] diazen- 1 -ium- 1 ,2-diolate (JS-K), N-(3-aminopropyl-propan-2-ylamino)-N-hydroxynitrous amide (NOC-5),

N-hydroxy-N-(methyl-(3-methylaminopropyl)-amino)-nitrous amide (NOC-7), N- ethyl-2-(l-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC-12), O-β- galactopyrano syl- 1 - (pyrrolidin- 1 -yl) diazen- 1 -ium- 1 ,2-diolate (β -GaI-N ONO ate) or disodium hydroxydiazenesulfonate 1-oxide (Sulfo-NONOate); (e) a S-nitrosothiol such as S-nitroso-N-acetylpenicillamine (SNAP), S-nitroso- glutathione (GSNO), S-nitroso-N-valerylpenicillamine (SNVP), S-nitrosoalbumin, S-nitrosocysteine, N-(N-γ-L-glutamyl-2-amino-2-(4-(4-S-nitrosomercapto-l- methylpiperidine) -acetyl) -glycine) (LA810), nitrosodiclofenac, nitrosocaptopril, N- (β-D-glucopyranosy^-N^acetyl-S-nitroso-D^-penicillaminamide (Glyco-SNAP-1), N-(2-deoxy-α,β-D-glucopyranose-2-)-N²-acetyl-S-nitroso-D,L-penicillaminamide

(Glyco-SNAP-2), 4-phenyl-l,3,2-oxathiazolylium-5-olate, 4-(p- trifluoromethylphenyl)-l,3,2-oxathiazolylium-5-olate, 4-(p-chlorophenyl)-l,3,2- oxathiozolylium-5-olate, 4-(p-methoxyphenyl)-l,3,2-oxathiazolylium-5-olate or N- (l-deoxy-α,β-D-fructopyranose-l-)-N -acetyl-S-nittoso-D^-peniciUaminamide

(Fmctose-SNAP-1); (f) a N-nitrosamine such as streptozocin, N,N'-dimethyl-N,N'-dinitroso-p- phenylenediamine (BNN3) or dephostatin; (g) a C-nitroso compound such as 2-nitro-2-nitrosopropane;

(h) a diazetine dioxide such as 3-bromo-3,4,4-trimethyl-3,4-dihydrodiazete-l,2-dioxide

(DDl) or 3-bromo-4-methyl-3,4-tetramethylene-3,4-dihydrodiazete-l,2-dioxide

(DD2);

(i) a furoxan such as dimethyl 2,6-dimethyl-4-[2'-(3"-methylfuroxan-4"- ylmethoxy)phenyl]-l,4-dihydropyridine-3,5-dicarboxylate, dimethyl 2,6-dimethyl-4-

[2'-(3"-carbamoylfuroxan-4"-ylmethoxy)phenyl]-l,4-dihydropyridine-3,5- dicarboxylate, dimethyl 2,6-dimethyl-4- [2'-(3"-cyanofuroxan-4"-ylmethoxy)phenyI| -

1 ,4-dihydropyridine-3,5-dicarboxylate, 4-methyl-3-phenylsulphonylfuroxan, 4- hydroxymethyl-3-furoxancarboxamide, N-(pyridin-3-yl)-3-phenyl-4- furoxancarboxamide, 4-phenyl-3-(N',N'-dimethyl-2'-aminoethylsulphanyl)furoxan or 4-phenylsulphonyl-3-(N',N'-dimethyl-2'-aminoethylsulphanyl) furoxan; ()) a sydnonimine such as 3-morpholinosydnonimine (SIN-I) or molsidomine; (k) an oxatriazole-5-imine such as 5-amino-3-(3',4'-dichlorophenyl)-l-oxa-2,3,4- triazolium chloride, 5-amino-3-(3'-chloro-2'-methylphenyl)-l-oxa-2,3,4-triazolium chloride or 5-(((cyanomethylamino)carbonyl)amino)-(3'-chloro-2'-methylphenyl)-l- oxa-2,3,4-triazolium chloride; (1) an oxime such as 4-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3- hexenamide (NOR-I), 4-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide

(NOR-2), 4-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), N- [(E,2Z)-4-ethyl-2-hydroxyimino-5-nitrohex-3-enyl] pyridine-3-carboxamide (NOR-

4) or N-[(E,2Z)-4-ethyl-6-methyl-2-hydroxyimino-5-nitrohept-3-enyl]pyridine-3- carboxamide (NOR- 5); or

(m) a metal-NO complex such as sodium nitroprusside (SNP) or a NO zeolite; or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

As used herein, an "organic nitrate" refers to a compound of formula R^a-ONO₂, wherein R^a is an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, optionally with one or more further -ONO₂ groups, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton.

As used herein, an "organic nitrite" refers to a compound of formula R^b-ONO, wherein R^b is an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, optionally with one or more further -ONO groups, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton.

As used herein, a "NONOate" refers to a compound of the formula:

O

R^c— N R^d

W /

N-O wherein R^c is an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton, and wherein R is hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton, or optionally wherein R^c and R^d together with the atoms to which they are attached form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more further heteroatoms N, O or S in its carbon skeleton.

As used herein, a "S-nitrosothiol" refers to a compound of formula R^e-S-NO, wherein R^e is an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, optionally with one or more further -S-NO groups, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton.

As used herein, a "N-nitrosamine" refers to a compound of formula (R^₂N-NO, wherein each R^f is independently hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, optionally with one or more further -NO groups, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton, or wherein the two R groups together with the nitrogen atom to which they are attached form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more heteroatoms N, O or S in its carbon skeleton.

As used herein, a "C-nitroso compound" refers to a compound of formula (R⁸J₃C-NO, wherein each R^g is independently hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, optionally with one or more further -NO groups, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton, and wherein any two R^g groups together with the carbon atom to which they are attached may form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more heteroatoms N, O or S in its carbon skeleton.

As used herein, a "diazetine dioxide" refers to a compound of the formula:

wherein each R is independently hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton, and wherein any two R^h groups together with the carbon atom or atoms to which they are attached may form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more heteroatoms N, O or S in its carbon skeleton.

As used herein, a "furoxan" refers to a compound of the formula:

O

o

^N wherein each R' is independently hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton, or wherein the two R groups together with the carbon atoms to which they are attached form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more heteroatoms N, O or S in its carbon skeleton.

As used herein, a "sydnonimine" refers to a compound of the formula:

and an "oxatriazole-5-imine" refers to a compound of the formula:

wherein each R is independently hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton, and wherein any two R groups together with the atom or atoms to which they are attached may form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more heteroatoms N, O or S in its carbon skeleton.

As used herein, an "oxime" refers to a compound of formula (R )₂C=NOH, wherein each R is independently hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton, or wherein the two R^k groups together with the carbon atom to which they are attached form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more heteroatoms N, O or S in its carbon skeleton. A second aspect of the present invention relates to a compound comprising an A-SMase inhibitor covalently linked to a NO-donor group, or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof. Preferably the A-SMase inhibitor is as specified in any embodiment of the first aspect of the present invention.

In one embodiment of the second aspect of the present invention, the NO-donor group is a NO-releasing group. Preferably the NO-releasing group comprises or is:

(a) a -ONO₂ group;

(b) a -ONO group; (c) a group selected from:

wherein at each occurrence R^c and R are independently selected from hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton;

(d) a -S-NO group;

(e) a -N(R^)NO group, wherein R is independently selected from hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, optionally with one or more further -NO groups, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton;

(f) a

or -NO group, wherein each R^g is independently selected from hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, optionally with one or more further -NO groups, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton, or wherein the two R^g groups together with the carbon atom to which they are attached form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more heteroatoms N, O or S in its carbon skeleton; a group selected from:

wherein each R^h is independently selected from hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton, and wherein any two R groups together with the carbon atom or atoms to which they are attached may form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more heteroatoms N, O or S in its carbon skeleton;

(h) a group selected from:

wherein each R' is independently selected from hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton;

® a group selected from:

wherein at each occurrence R^J is independently selected from hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton, and wherein any two R¹ groups together with the atom or atoms to which they are attached may form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more heteroatoms N, O or S in its carbon skeleton; or ()) a group selected from:

wherein each R is independently selected from hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton.

In a second embodiment of the second aspect of the present invention, the compound further comprises a linker between the A-SMase inhibitor and the NO-donor group. Preferably the linker comprises an alkylene, alkenylene, alkynylene, arylene, arylalkylene, arylalkenylene, arylalkynylene, alkylarylene, alkenylarylene or alkynylarylene group, each of which may optionally be substituted, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton.

In a third embodiment of the second aspect of the present invention, the compound is selected from a compound of formula (III) or (IY):

Q is independently a NO-releasing group;

X is independently selected from N

_

C(Rⁿ)₂— ^ N^T-— — NR 1ⁿ l--rCτR, 11— — r CR^-NR¹¹- -N=C- -C==Nxτ — — Γ OΛ —- /C"TR) I¹l —

I

-CR^1LO- S —- /C"TR) H — — ( C"TR) H^H —-S- -CR¹^C(R¹KO₂ _ -C(R I¹h 0₂ —- rCuRn

C=CR¹¹- or -R¹¹C=C

— X¹— is independently selected from C _^ NR C _^ C NR _^

— o — c— — c — o— — s — c— — c — s- — - rC —- /C"A(R) I¹K¹), —- _or -C(R¹O₂-C- .

-Y- is independently selected from a chemical bond, -O-, -S-, -NR¹²-, -C(R¹²)₂-, -C(R¹²)₂-C(R¹²)₂-, -R¹²C=CR¹²-, -NR¹²-C(R¹²)₂-, -C(R¹²)₂-NR¹²-, -N=CR¹²-, -R¹²C=N-, -O-C(R¹²)₂-, -C(R¹²)₂-O-, -S-C(R¹²)₂- or -C(R¹²)₂-S-; n is independently 1, 2, 3, 4, 5 or 6; m is independently 0, 1, 2, 3, 4 or 5;

R¹, R², R³, R⁴, R⁵, R⁶, R⁷_ R⁸, R⁹, R¹⁰, R" and R¹² are independently selected from hydrogen, -F, -Cl, -Br, -I, -CF₃, -CCl₃, -CBr₃, -CI₃, -OH, -SH, -NH₂, -CN, -NO₂, -COOH, -R¹³-O-R¹⁴, -R¹³-S-R¹⁴, -R¹³-SO-R¹⁴, -R¹³-SO₂-R¹⁴, -R¹³-SO₂-OR¹⁴, -R¹³-O-SO₂-R¹⁴, -R¹³-SO₂-N(R¹⁴)₂, -R¹³-NR¹⁴-SO₂-R¹⁴, -R¹³-O-SO₂-OR¹⁴, -R¹³-O-SO₂-N(R¹⁴)₂, -R¹³-NR¹⁴-SO₂-OR¹⁴, -R¹³-NR¹⁴-SO₂-N(R¹⁴)₂, -R¹³-N(R¹⁴)₂, -R¹³-N(R¹⁴)₃ ⁺, -R¹³-P(R¹⁴)₂, -R¹³-Si(R¹⁴)₃, -R¹³-CO-R¹⁴, -R¹³-CO-OR¹⁴, -R¹³-O-CO-R¹⁴, -R¹³-CO-N(R¹⁴)₂, -R¹³-NR¹⁴-CO-R¹⁴, -R¹³-O-CO-OR¹⁴, -R¹³-O-CO-N(R¹⁴)₂, -R¹³-NR¹⁴-CO-OR¹⁴, -R¹³-NR¹⁴-CO-N(R¹⁴)₂, -R¹³-CS-R¹⁴, -R¹³-CS-OR¹⁴, -R¹³-O-CS-R¹⁴, -R¹³-CS-N(R¹⁴)₂, -R¹³-NR¹⁴-CS-R¹⁴, -R¹³O-CS-OR¹⁴, -R¹³-O-CS-N(R¹⁴)₂, -R¹³-NR¹⁴-CS-OR¹⁴,

-R¹³-NR¹⁴-CS-N(R¹⁴)₂ or -R¹⁴;

-R¹⁴ is independently hydrogen or a C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₂-C₁₀ aryl, C₃-C₁₀ arylalkyl, C₄-C₁₀ arylalkenyl, C₄-C₁₀ arylalkynyl, C₃-C₁₀ alkylaryl, C₄-C₁₀ alkenylaryl or C₄-C₁₀ alkynylaryl group, each of which may optionally be substituted; and wherein any two or more R¹, R², R³, R⁴, R⁵, R⁶, R⁷ R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ or R¹⁴ may, together with the atom or atoms to which they are attached, form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more heteroatoms N, O or S in its carbon skeleton.

Preferred embodiments of X, X', Y, m, n, R¹, R², R³, R⁴, R⁵, R⁶, R⁷ R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ can be found in the sixth embodiment of the first aspect of the present invention.

Preferably in the third embodiment of the second aspect of the present invention, Q is:

O^" f N. R^d

W /

N-O wherein R is independently selected from hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton. Preferably R is hydrogen.

A third aspect of the present invention relates to a compound selected from:

or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

Compounds according to the second or third aspect of the present invention may be prepared using any method known to those skilled in the art, such as those set out in Konter et al., Eur. J. Org. Chem., 2007, pp. 616-624.

For instance, where the A-SMase inhibitor contains or is linked to a nucleophilic group such as a primary or secondary amino group and the NO-releasing group is

O

^"Hi R^d

W /

N-O wherein R^d is as defined above, the compounds may be prepared by exposing the A-SMase inhibitor to nitric oxide. Preferably the nitric oxide is at a pressure of at least 1 bar, at least 2 bar or at least 3 bar. Most preferably the nitric oxide is at a pressure of about 4 bar. In a preferred embodiment, the A-SMase inhibitor is dissolved in a solvent prior to exposure to the nitric oxide gas. Preferably the solvent is a polar protic or a polar aprotic solvent. More preferably the solvent is an alcohol such as an alcohol containing from 1 to 6 carbon atoms. Most preferably the solvent is methanol. An additional base, such as an alkoxide, most preferably a methoxide, may also be added to the solvent.

The above compounds may also be prepared in situ, for example, in an aerosol canister such as one suitable for use in a metered dose inhaler (MDI).

A fourth aspect of the present invention relates to a pharmaceutical composition comprising a compound according to the second or third aspects of the present invention, and optionally comprising one or more pharmaceutically acceptable excipients.

In a preferred embodiment of either of the first or fourth aspects of the present invention, the pharmaceutical composition comprises two or more pharmaceutically acceptable excipients.

In another embodiment of either of the first or fourth aspects of the present invention, the pharmaceutical composition has a pH of 7 or more. Preferably the pharmaceutical composition has a pH of between 7 and 10, more preferably of between 7.5 and 9, most preferably of between 8 and 8.5.

A fifth aspect of the present invention relates to a pharmaceutical composition comprising an A-SMase inhibitor, and optionally comprising one or more pharmaceutically acceptable excipients, wherein the composition has a pH of 7 or more. Preferably the pharmaceutical composition has a pH of between 7 and 10, more preferably of between 7.5 and 9, most preferably of between 8 and 8.5. Preferably the A-SMase inhibitor is as specified in any embodiment of the first aspect of the present invention.

A sixth aspect of the present invention relates to a pharmaceutical composition comprising a NO-donor, and optionally comprising one or more pharmaceutically acceptable excipients, wherein the composition has a pH of 8 or more. Preferably the pharmaceutical composition has a pH of between 8 and 10, more preferably of between 8 and 9, most preferably of between 8 and 8.5. Preferably the NO-donor is as specified in any embodiment of the first aspect of the present invention. In a preferred embodiment of any of the first, fourth, fifth or sixth aspects of the present invention, the composition has a pH of about 8.3.

In one embodiment of any of the first, fourth, fifth or sixth aspects of the present invention, the composition comprises a buffer. Suitable buffers include phosphate buffer systems such as those comprising dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate and/or phosphoric acid, optionally in conjunction with sodium chloride, potassium chloride, calcium chloride, magnesium chloride, citric acid, bovine albumin, potassium hydroxide and/or sodium hydroxide; maleate buffer systems such as those comprising maleic acid and/or maleic anhydride, optionally in conjunction with sodium chloride, tris(hydroxymethyl)aminomethane and/or sodium hydroxide; tetrabutylammonium buffer systems such as those comprising tetrabutylammonium hydroxide, optionally in conjunction with ammonium acetate and/or nitric acid; imidazole buffer systems such as those comprising imidazole in conjunction with hydrochloric acid; barbital buffer systems such as those comprising barbital sodium, optionally in conjunction with sodium acetate, hydrochloric acid, calcium lactate and/or sodium chloride; tris(hydroxymethyl)- aminomethane buffer systems such as those comprising tris(hydroxymethyl)aminomethane in conjunction with hydrochloric acid, sodium chloride, calcium chloride, glycine, sodium edetate, acetic acid and/or bovine albumin; borate buffer systems such as those comprising disodium tetraborate and/or boric acid, optionally in conjunction with sodium chloride, calcium chloride, potassium chloride, sodium hydroxide and/or hydrochloric acid; HEPES buffer systems such as those comprising 2-[4-(2-hydroxyethyl)piperazin-l- yl] ethanesulphonic acid, optionally in conjunction with sodium hydroxide; citrate buffer systems such as those comprising sodium citrate, optionally in conjunction with sodium chloride and/or hydrochloric acid; ammonium buffer systems such as those comprising ammonia, ammonium carbonate and/or ammonium chloride, optionally in conjunction with sodium hydroxide; and diethanolamine buffer systems comprising diethanolamine, optionally in conjunction with magnesium chloride and/or hydrochloric acid. Preferably the buffer is an aqueous buffer.

One preferred buffer for use in the present invention is a tris-glycine buffer system comprising tris(hydroxymethyl)aminomethane, glycine and water. Such a buffer may preferably be prepared by dissolving 6.0 g of tris (hydro xymethyl)aminomethane and 28.8 g of glycine in water and diluting the mixture to 1000 ml with water, diluting a further 10-fold by volume prior to use.

Another preferred buffer for use in the present invention is a tris-hydrochloride buffer system. Such a buffer may preferably be prepared by dissolving 9.0 g of tris (hydro xymethyl)aminomethane in 2.9 litres of water, adjusting the pH to 8.3 with IM hydrochloric acid, then adjusting the volume to 3 litres using water.

In one embodiment of any of the first, fourth, fifth or sixth aspects of the present invention, the composition has a chloride ion concentration of between 10 and 1000 mmol/kg. Preferably the composition has a chloride ion concentration of between 50 and 500 mmol/kg, most preferably of between 100 and 200 mmol/kg. In a particularly preferred embodiment, the composition has a chloride ion concentration of about 145 mmol/kg.

The compositions employed in the present invention can be administered by oral, parental (including intravenous, subcutaneous, intramuscular, intradermal, intratracheal, intraperitoneal, intraarticular, intracranial and epidural), transdermal, airway (aerosol), rectal, vaginal or topical (including buccal, mucosal and sublingual) administration. Preferably the compositions employed in the present invention are administered by inhalation.

For oral administration, the compounds and compositions of the invention will generally be provided in the form of tablets, capsules, hard or soft gelatine capsules, caplets, troches or lozenges, as a powder or granules, or as an aqueous solution, suspension or dispersion.

Tablets for oral use may include the active ingredient(s) mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose. Corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatine. The lubricating agent, if present, may be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material, such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

Capsules for oral use include hard gelatine capsules in which the active ingredient(s) are mixed with a solid diluent, and soft gelatine capsules wherein the active ingredient(s) are mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient(s) such carriers as are known in the art to be appropriate.

For parenteral use, the compounds and compositions of the present invention will generally be provided in a sterile aqueous solution or suspension, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride or glucose. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate. The compounds and compositions of the invention may also be presented as liposome formulations.

For topical and transdermal administration, the compounds and compositions of the invention will generally be provided in the form of ointments, cataplasms (poultices), pastes, powders, dressings, creams, plasters or patches.

In one embodiment of any of the first, fourth, fifth or sixth aspects of the present invention, the composition or compound is suitable for inhalation.

The efficiency of an aerosol device, such as an MDI, is a function of the dose deposited at the appropriate site in the respiratory tract. Deposition is affected by several factors, of which one of the most important is the aerodynamic particle size. The distribution of aerodynamic particle sizes of solid particles and/or droplets in an aerosol can be characterized by their mass median aerodynamic diameter (MMAD, the diameter around which the mass aerodynamic diameters are distributed equally) and geometric standard deviation (GSD, the measure of variability of the aerodynamic particle diameters). Aerosol particles of equivalent MMAD and GSD have similar deposition in the respiratory tract irrespective of their composition.

In order for the particles to be carried deep into the lungs, the particles must be very fine, for example having a mass median aerodynamic diameter (MMAD) of less than lOμm.

Particles having aerodynamic diameters greater than about lOμm are likely to impact the walls of the throat and generally do not reach the lung. Particles having aerodynamic diameters in the range of about 5μm to about 3μm will generally be deposited in the respiratory bronchioles, whereas smaller particles having aerodynamic diameters in the range of about 3μm to about 0.05μm are likely to be deposited in the alveoli.

Optionally in one embodiment of any of the first, fourth, fifth or sixth aspects of the present invention, the composition or compound is suitable for use in a dry powder inhaler.

It is favourable for unstable agglomerates of particles to be present in the powder when it is in the inhaler device. For a powder to leave an inhaler device efficiently and reproducibly, the particles of such a powder should be large, preferably ≥ about 40μm. Such a powder may be in the form of individual particles having a size __! about 40μm and/or in the form of agglomerates of finer particles, the agglomerates having a size ≥ about 40μm. The agglomerates formed can have a size of as much as about lOOOμm and, with the addition of a glidant or lubricant, those agglomerates are more likely to be broken down efficiently in the turbulent airstream created on inhalation. Therefore, the formation of unstable or "soft" agglomerates of particles in the powder may be favoured compared with a powder in which there is substantially no agglomeration. Such unstable agglomerates are stable whilst the powder is inside the device, but are then disrupted and broken up when the powder is dispensed. Compositions suitable for use in a dry powder inhaler may further comprise a lubricant or a glidant. Preferably the lubricant or glidant is selected from lauric acid, palmitic acid, stearic acid, erucic acid, behenic acid, or a pharmaceutically acceptable salt or ester thereof, such as sodium stearyl fumarate, sodium stearyl lactylate, sodium lauryl sulphate, magnesium lauryl sulphate or magnesium stearate. Alternatively the lubricant or glidant may be selected from lecithin, leucine, phosphatidylcholines, phosphatidylglycerols, triglycerides, talc, titanium dioxide, aluminium dioxide, silicon dioxide or starch. Preferably the composition comprises 0.05 to 10% by weight of the lubricant or glidant, more preferably 0.1 to 5% by weight of the lubricant or glidant, more preferably 0.2 to 2% by weight, and most preferably about 0.5% by weight of the lubricant or glidant.

Preferably, where the composition is suitable for use in a dry powder inhaler, the composition comprises a carrier material. Preferably the carrier material is selected from lactic acid, polyols such as mannitol, saccharides such as glucose or lactose, or a pharmaceutically acceptable salt thereof such as sodium lactate, or an inorganic salt such as sodium chloride or calcium carbonate. Preferably the carrier material is present in the form of particles with a MMAD of between 1 and 5000μm. More preferably the particles have a MMAD of between 10 and lOOOμm, between 20 and 500μm or between 40 and 200μm. Most preferably the particles have a MMAD of between 60 and lOOμm. Alternatively, the carrier material may be present in the form of particles with a MMAD of between 5 and 30μm, preferably between 10 and 20μm.

In one embodiment where the composition is suitable for use in a dry powder inhaler, the composition comprises up to 99% by weight of the carrier material. Preferably, the composition comprises up to 95%, more preferably up to 90%, 75%, 50%, 30% or 20% by weight of the carrier material.

The lubricant, glidant or carrier material may be in the form of particles which tend to adhere to the surfaces of the active particles, as disclosed in WO 97/03649. Alternatively, the lubricant, glidant or carrier material may be coated on the surface of the active particles by, for example, a co-milling method as disclosed in WO 02/43701. Co-spray drying is another method of producing active particles with lubricant, glidant or carrier material on their surfaces. Other possible methods of manufacturing such "coated" active particles include supercritical fluid processing, spray-freeze drying, various forms of precipitation and crystallisation from bulk solution, and other methods which would be well-known to the person skilled in the art.

Alternatively or in addition, the active particles, optionally coated with a lubricant or glidant as specified above, may adhere to the surfaces of larger carrier particles whilst in the inhaler device, but release and become dispersed upon actuation of the dispensing device and inhalation into the respiratory tract, to give a fine suspension.

In certain embodiments of the present invention, the powder composition is such that a fine particle fraction of at least 35% is generated on actuation of the inhaler device. It is particularly preferred that the fine particle fraction be greater than or equal to 45%, 50% or 60%. Preferably, the fine particle fraction is at least 70%, and most preferably at least 80%.

The emitted dose (ED) is the total mass of the active agent emitted from the device following actuation. It does not include the material left inside or on the surfaces of the device. The ED is measured by collecting the total emitted mass from the device in an apparatus frequently referred to as a dose uniformity sampling apparatus (DUSA), and recovering this by a validated quantitative wet chemical assay.

The fine particle dose (FPD) is the total mass of active agent which is emitted from the device following actuation which is present in an aerodynamic particle size smaller than a defined limit. Where the term "fine particle dose" or "FPD" is used herein, the aerodynamic particle size is smaller than 5μm. The FPD is measured using an impactor or impinger, such as a twin stage impinger (TSI), multi-stage liquid impinger (MSLI), Andersen Cascade Impactor (ACI) or a Next Generation Impactor (NGI). Each impactor or impinger has a pre-determined aerodynamic particle size collection cut point for each stage. The FPD value is obtained by interpretation of the stage-by-stage active agent recovery quantified by a validated quantitative wet chemical assay where either a simple stage cut is used to determine FPD or a more complex mathematical interpolation of the stage-by-stage deposition is used. The fine particle fraction (FPF) is defined as the FPD divided by the ED and expressed as a percentage.

Preferably, the inhaler device used to dispense the powder composition is an active inhaler device, the arrangement being such that a fine particle fraction of at least 35%, preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, and most preferably at least 80% is generated on actuation of the inhaler device. As an active device does not depend on the patient's inhalation for aerosolising the dose, the delivery of the dose is more repeatable than is observed using passive inhaler devices.

As described in WO 01/82906, an additive material may also be provided in a dose which indicates to the patient that the dose has been administered. The additive material, referred to below as indicator material, may be present in the powder as formulated for the dry powder inhaler, or be present in a separate form, such as in a separate location within the inhaler such that the additive becomes entrained in the airflow generated on inhalation simultaneously or sequentially with the powder containing the active material.

In some circumstances, for example, where any carrier particles and/or any fine excipient material present is of a material itself capable of inducing a sensation in the oropharyngeal region, the carrier particles and/or the fine excipient material can constitute the indicator material. For example, the carrier particles and/or any fine particle excipient may comprise mannitol. Alternatively or in addition, where the composition is suitable for use in a dry powder inhaler, the composition may comprise a flavouring agent such as menthol.

In one embodiment where the composition is suitable for use in a dry powder inhaler, the composition comprises at least 50% by weight of the A-SMase inhibitor and the NO- donor. Preferably the composition comprises at least 75%, at least 90%, at least 95% or at least 99% by weight of the A-SMase inhibitor and the NO-donor.

Optionally in another embodiment of any of the first, fourth, fifth or sixth aspects of the present invention, the composition or compound is suitable for use in a metered-dose inhaler (MDI). Preferably such a composition comprises a propellant. The composition suitable for use in a MDI is preferably in the form of a solution or a suspension of the A- SMase inhibitor and/or the NO-donor in the liquefied propellant. Preferably the propellant is a hydrofluorocarbon such as 1,1,1,2-tetrafluoroethane (CF₃CH₂F) (HFA-134a), 1,1,1,2,3,3,3-heptafluoropropane (CF₃CHFCF₃) (HFA-227), 1,1-difluoroethane (CHF₂CH₃) (HFA-152a), trifluoromethane (CHF₃) (HFA-23), difluoromethane (CH₂F₂) (HFA-32), fluoromethane (CH₃F) (HFA-41), 1,1,1,2,2-pentafluoroethane (CF₃CHF₂) (HFA-125), or 1,1,2,2-tetrafluoroethane (CHF₂CHF₂) (HFA-134); a perfluorocarbon such as perfluoroethane (F₃CCF₃), tetrafluoromethane (CF₄) (PFC- 14), or decafluorobutane (CF₃CF₂CF₂CF₃); a hydrochlorofluorocarbon such as monochlorodifluoromethane (ClCHF₂); a chlorofluorocarbon such as Freon 11 (CCl₃F), Freon 12 (CCl₂F₂), or Freon 114 (CF₂ClCF₂Cl); a dialkyl ether such as dimethyl ether; or a low molecular weight hydrocarbon such as »-butane, 2-butane, or propane; or a mixture thereof. Preferably the propellant is a blend of HFA-227 and HFA-134a, most preferably in a volume ratio of about 3:2 HFA-227 to HFA- 134a.

Optionally, where the composition is suitable for use in a metered-dose inhaler, the composition comprises a lubricant, a surfactant, a polar co-solvent and/or a flavouring agent.

Particular lubricants for metered-dose inhalers that may be mentioned include polysorbates, e.g. polysorbate 80, and alkyl aryl polyether alcohols, e.g. tyloxapol. Other lubricating excipients that may be employed include high molecular weight fully halogenated chlorofluorocarbons and esters of medium chain fatty acids. The amount of lubricant in the composition will depend on the other components of the composition, the active ingredient(s), the nature of the valve, etc. Optionally a concentration of 0.01 to 4% w/w and more preferably 0.1 to 2% w/w of lubricant is preferred.

Surfactants are commonly added to aerosol compositions in particular solutions for metered-dose inhalers, for example to lubricate the valve components in the inhaler device and/or improve the physical stability of the aerosol compositions. Suitable surfactants include both non-fluorinated surfactants and fluorinated surfactants known in the art and disclosed, for example, in US 5,849,265 and US 4,352,789. Examples of suitable surfactants which can be used in the compositions include oleic acid; lecithins from synthetic and natural sources; sorbitan trioleate; sorbitan mono-oleate; sorbitan monolaurate; tetrahydrofurfuryl oleate; ethyl oleate; isopropyl myristate; glyceryl trioleate; glyceryl mono- oleate; glyceryl monolaurate; glyceryl monostearate; glyceryl monoricinoleate; cetyl alcohol; stearyl alcohol; and cetyl pyridinium chloride. Preferred surfactants are oleic acid, lecithin, and sorbitan trioleate. Surfactants, if used, are optionally present in amounts not exceeding 5% by weight of the total composition, though higher amounts may be used.

Flavour modifying excipients that may be added to the metered-dose inhaler composition include peppermint oil, menthol, saccharin and saccharin sodium. When the flavour modifying excipient is a solid, preferably it is micronized. The concentration will depend on the individual composition and the flavour modifying excipient. Typically, a concentration of 0.005 to 4% w/w, and more preferably 0.01 to 1% w/w is used.

A polar co-solvent, preferably a polar protic co-solvent such as aliphatic alcohols and polyols, e.g. ethanol, isopropanol and propylene glycol, may be included in the metered- dose inhaler composition. Preferably said polar co-solvent contains 1-6 carbon atoms, more preferably 1-4 carbon atoms, most preferably 1-3 carbon atoms. A preferred co- solvent is ethanol and typically, the aerosol composition may contain 0.01 to 5% w/w of the co-solvent, preferably 0.1 to 5% w/w, and most preferably 0.1 to 1% w/w. For suspension compositions the amount of these polar co-solvents must be controlled so as not to dissolve the active ingredient(s) to any significant extent.

In yet another embodiment of any of the first, fourth, fifth or sixth aspects of the present invention, the composition is suitable for use in a nebulised system. Such systems include conventional ultrasonic nebulised systems and jet nebulised systems, as well as recently introduced handheld devices such as the Respimat (available from Boehringer Ingelheim) or the AERx (available from Aradigm). In such a system, the A-SMase inhibitor and/or the NO-donor could be stabilized in a sterile aqueous solution, for example, with antioxidants such as sodium metabisulfite.

Preferably, in any embodiment where the composition is suitable for inhalation, the A-SMase inhibitor and/or the NO-donor are present in the form of particles with a MMAD of between 0.001 and 500μm, more preferably with a MMAD of between 0.01 and lOOμm. Where the composition is for delivery to the bronchioles, it is preferred that the A-SMase inhibitor and/or the NO-donor are present in the form of particles with a MMAD of between 0.1 and 50μm, more preferably between 0.5 and lOμm, most preferably between 2 and 5μm. Where the composition is for delivery to the throat, it is preferred that the A-SMase inhibitor and/or the NO-donor are present in the form of particles with a MMAD of between 10 and lOOμm, more preferably between 20 and 50μm. Where the composition is for delivery to the alveoli, it is preferred that the A-SMase inhibitor and/or the NO-donor are present in the form of particles with a MMAD of between 0.05 and 3μm.

A seventh aspect of the present invention relates to a pharmaceutical composition according to any of the first, fourth, fifth or sixth aspects of the present invention, or a compound according to either of the second or third aspects of the present invention, for use as a medicament, preferably for treating or preventing a respiratory disorder.

An eighth aspect of the present invention relates to a method of treating or preventing a respiratory disorder, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a pharmaceutical composition according to any of the first, fourth, fifth or sixth aspects of the present invention, or a compound according to either of the second or third aspects of the present invention.

Preferably the respiratory disorder to be treated by the pharmaceutical composition or the compound of the seventh aspect of the present invention or by the method of the eighth aspect of the present invention is:

(a) an acute upper respiratory infection such as acute nasopharyngitis (the common cold), acute sinusitis, acute pharyngitis, acute tonsillitis, acute laryngitis, acute tracheitis, acute obstructive laryngitis, acute epiglottitis, or an acute upper respiratory infection of multiple or unspecified sites;

(b) an acute lower respiratory infection such as influenza, pneumonia, acute bronchitis, acute bronchiolitis, or an unspecified acute lower respiratory infection; (c) a non-infectious disorder of the upper respiratory tract such as vasomotor rhinitis, allergic rhinitis, chronic rhinitis, chronic nasopharyngitis, chronic pharyngitis, chronic sinusitis, nasal polyp, other disorders of the nose or nasal sinuses, chronic diseases of the tonsils or adenoids, peritonsillar abscess, chronic laryngitis, chronic laryngotracheitis, or other disorders of the vocal cords, larynx or upper respiratory tract;

(d) a chronic lower respiratory disease such as catarrhal bronchitis, bronchitis with tracheitis, tracheobronchitis, simple chronic bronchitis, mucopurulent chronic bronchitis, unspecified chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), asthma, status asthmaticus, or bronchiectasis;

(e) respiratory tuberculosis including tuberculosis of the lung, tuberculosis of the intrathoracic lymph nodes, tuberculosis of the larynx, trachea or bronchus, tuberculous pleurisy, and primary respiratory tuberculosis; (f) a lung disease due to external agents such as coal-worker's pneumoconiosis, pneumoconiosis due to asbestos or other mineral fibres, pneumoconiosis due to dust containing silica, pneumoconiosis due to other inorganic dusts, unspecified pneumoconiosis, pneumoconiosis associated with tuberculosis, airway disease due to specific organic dust, hypersensitivity pneumonitis due to organic dust, respiratory conditions due to inhalation of chemicals, gases, fumes or vapours, pneumonitis due to solids or liquids, pneumonitis due to food or vomit, or respiratory conditions due to other external agents;

(g) another respiratory disease principally affecting the interstitium such as adult respiratory distress syndrome (ARDS), pulmonary oedema, pulmonary eosinophilia, or other interstitial pulmonary diseases such as alveolar or parietoalveolar conditions, other interstitial pulmonary diseases with fibrosis including idiopathic pulmonary fibrosis, other specified interstitial pulmonary diseases, or unspecified interstitial pulmonary disease;

(h) a suppurative or necrotic condition of the lower respiratory tract such as abscess of the lung, abscess of the mediastinum, or pyothorax;

(i) another disease of the pleura such as pleural effusion not elsewhere classified, pleural effusion in conditions classified elsewhere, pleural plaque, pneumothorax, or other pleural conditions including chylous effusion, fibrothorax and haemothorax;

()) cystic fibrosis including mucoviscidosis, cystic fibrosis with pulmonary manifestations, cystic fibrosis with intestinal manifestations, cystic fibrosis with other manifestations, and unspecified cystic fibrosis;

(k) cough including chronic cough, psychogenic cough, cough with haemorrhage, and cough caused by poisoning such as with expectorants; (1) symptoms involving the respiratory system such as haemorrhage from respiratory passages, abnormalities of breathing including dyspnoea, stridor, wheezing, periodic breathing, hyperventilation, mouth breathing, hiccough and sneezing, pain in the throat or chest, asphyxia, pleurisy, respiratory arrest, or abnormal sputum; (m) a respiratory disorder specific to the perinatal period such as birth asphyxia, respiratory distress of newborn, congenital pneumonia, neonatal aspiration syndrome, interstitial emphysema and related conditions originating in the perinatal period, pulmonary haemorrhage originating in the perinatal period, chronic respiratory disease originating in the perinatal period, or other respiratory conditions originating in the perinatal period;

(n) a respiratory neoplasm such as a malignant respiratory neoplasm including malignant neoplasms of the lip, tongue, gum, floor of the mouth, palate and other parts of the mouth, malignant neoplasms of the parotid gland, tonsil, oropharynx, nasopharynx, piriform sinus, hypopharynx and other parts of the pharynx, malignant neoplasms of the nasal cavity, middle ear and accessory sinuses, malignant neoplasms of the larynx, trachea, bronchus and lung, and Kaposi's sarcoma of the mouth and other parts of the respiratory system; an in situ respiratory neoplasm including carcinomas in situ of the lip, oral cavity, pharynx, larynx, trachea, bronchus, lung and other parts of the respiratory system; a benign respiratory neoplasm including benign neoplasms of the mouth, pharynx, salivary glands, nasal cavity, middle ear and accessory sinuses, and benign neoplasms of the larynx, trachea, bronchus, lung and other parts of the respiratory system; or a respiratory neoplasm of uncertain or unknown behaviour; or

(o) another disease of the respiratory system such as post-procedural respiratory disorders not elsewhere classified, respiratory failure not elsewhere classified, diseases of the bronchus not elsewhere classified, pulmonary collapse, interstitial emphysema, compensatory emphysema, calcification of the lung, cystic lung disease (acquired), pulmolithiasis, diseases of the mediastinum not elsewhere classified, or disorders of the diaphragm.

Preferably the respiratory disorder is:

(a) cough including chronic cough, psychogenic cough, cough with haemorrhage, and cough caused by poisoning such as with expectorants; (b) cystic fibrosis including mucoviscidosis, cystic fibrosis with pulmonary manifestations, cystic fibrosis with intestinal manifestations, cystic fibrosis with other manifestations, and unspecified cystic fibrosis;

(c) a chronic obstructive pulmonary disease including chronic asthmatic bronchitis, chronic emphysematous bronchitis, chronic bronchitis with airways obstruction, chronic bronchitis with emphysema, chronic obstructive asthma, chronic obstructive bronchitis, chronic tracheobronchitis, chronic obstructive pulmonary disease with acute lower respiratory infection, chronic obstructive pulmonary disease with acute exacerbation (unspecified), other specified chronic obstructive pulmonary diseases, and chronic obstructive pulmonary disease (unspecified);

(d) an interstitial pulmonary disease with fibrosis including idiopathic pulmonary fibrosis, diffuse pulmonary fibrosis, fibrosing alveolitis (cryptogenic), Hamman- Rich syndrome, chronic pulmonary fibrosis due to inhalation of chemicals, gases, fumes or vapours, and chronic pulmonary fibrosis following radiation; or (e) emphysema including MacLeod's syndrome, panlobular emphysema, centrilobular emphysema, bullous emphysema, vesicular emphysema, emphysematous bleb, compensatory emphysema, emphysema due to inhalation of chemicals, gases, fumes or vapours, interstitial emphysema, interstitial neonatal emphysema, mediastinal emphysema, surgical (subcutaneous) emphysema, traumatic subcutaneous emphysema, emphysema with chronic (obstructive) bronchitis, and emphysematous (obstructive) bronchitis.

More preferably the respiratory disorder is chronic cough, cystic fibrosis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis or emphysema.

A ninth aspect of the present invention relates to an A-SMase inhibitor for treating or preventing a respiratory disorder, wherein the respiratory disorder is:

(a) an interstitial pulmonary disease with fibrosis including idiopathic pulmonary fibrosis, diffuse pulmonary fibrosis, fibrosing alveolitis (cryptogenic), Hamman- Rich syndrome, chronic pulmonary fibrosis due to inhalation of chemicals, gases, fumes or vapours, and chronic pulmonary fibrosis following radiation; or

(b) emphysema including MacLeod's syndrome, panlobular emphysema, centrilobular emphysema, bullous emphysema, vesicular emphysema, emphysematous bleb, compensatory emphysema, emphysema due to inhalation of chemicals, gases, fumes or vapours, interstitial emphysema, interstitial neonatal emphysema, mediastinal emphysema, surgical (subcutaneous) emphysema, traumatic subcutaneous emphysema, emphysema with chronic (obstructive) bronchitis, and emphysematous (obstructive) bronchitis.

(a) A tenth aspect of the present invention relates to a method of treating or preventing a respiratory disorder, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an A-SMase inhibitor, wherein the respiratory disorder is selected from:

(a) an interstitial pulmonary disease with fibrosis including idiopathic pulmonary fibrosis, diffuse pulmonary fibrosis, fibrosing alveolitis (cryptogenic), Hamman- Rich syndrome, chronic pulmonary fibrosis due to inhalation of chemicals, gases, fumes or vapours, and chronic pulmonary fibrosis following radiation; or (b) emphysema including MacLeod's syndrome, panlobular emphysema, centrilobular emphysema, bullous emphysema, vesicular emphysema, emphysematous bleb, compensatory emphysema, emphysema due to inhalation of chemicals, gases, fumes or vapours, interstitial emphysema, interstitial neonatal emphysema, mediastinal emphysema, surgical (subcutaneous) emphysema, traumatic subcutaneous emphysema, emphysema with chronic (obstructive) bronchitis, and emphysematous (obstructive) bronchitis.

Preferably the A-SMase inhibitor of the ninth aspect of the present invention and the method of the tenth aspect of the present invention are suitable for treating or preventing idiopathic pulmonary fibrosis or emphysema. Preferably the A-SMase inhibitor can be administered by inhalation. Preferably the A-SMase inhibitor is as specified in any embodiment of the first aspect of the present invention.

An eleventh aspect of the present invention relates to amitriptyline or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof, for treating or preventing a chronic obstructive pulmonary disease such as chronic asthmatic bronchitis, chronic emphysematous bronchitis, chronic bronchitis with airways obstruction, chronic bronchitis with emphysema, chronic obstructive asthma, chronic obstructive bronchitis, chronic tracheobronchitis, chronic obstructive pulmonary disease with acute lower respiratory infection, chronic obstructive pulmonary disease with acute exacerbation (unspecified), other specified chronic obstructive pulmonary diseases, or chronic obstructive pulmonary disease (unspecified).

A twelfth aspect of the present invention relates to a method of treating or preventing a chronic obstructive pulmonary disease such as chronic asthmatic bronchitis, chronic emphysematous bronchitis, chronic bronchitis with airways obstruction, chronic bronchitis with emphysema, chronic obstructive asthma, chronic obstructive bronchitis, chronic tracheobronchitis, chronic obstructive pulmonary disease with acute lower respiratory infection, chronic obstructive pulmonary disease with acute exacerbation (unspecified), other specified chronic obstructive pulmonary diseases, or chronic obstructive pulmonary disease (unspecified), the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of amitriptyline or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

Preferably the amitriptyline or pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof of the eleventh aspect of the present invention and the amitriptyline or pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof used in the method of the twelfth aspect of the present invention can be administered by inhalation.

A thirteenth aspect of the present invention relates to an A-SMase inhibitor for treating or preventing a respiratory disorder by inhalation, wherein the respiratory disorder is:

(a) cough including chronic cough, psychogenic cough, cough with haemorrhage, and cough caused by poisoning such as with expectorants; or

(b) cystic fibrosis including mucoviscidosis, cystic fibrosis with pulmonary manifestations, cystic fibrosis with intestinal manifestations, cystic fibrosis with other manifestations, and unspecified cystic fibrosis.

A fourteenth aspect of the present invention relates to a method of treating or preventing a respiratory disorder, comprising administering to a subject in need thereof by inhalation a therapeutically or prophylactically effective amount of an A-SMase inhibitor, wherein the respiratory disorder is selected from: (a) cough including chronic cough, psychogenic cough, cough with haemorrhage, and cough caused by poisoning such as with expectorants; or

Preferably the A-SMase inhibitor of the thirteenth aspect of the present invention and the method of the fourteenth aspect of the present invention are suitable for treating or preventing chronic cough or cystic fibrosis. Preferably the A-SMase inhibitor is as specified in any embodiment of the first aspect of the present invention.

A fifteenth aspect of the present invention relates to a NO-donor for treating or preventing cough including chronic cough, psychogenic cough, cough with haemorrhage, and cough caused by poisoning such as with expectorants.

A sixteenth aspect of the present invention relates to a method of treating or preventing cough, including chronic cough, psychogenic cough, cough with haemorrhage, and cough caused by poisoning such as with expectorants, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a NO- donor.

Preferably the NO-donor of the fifteenth aspect of the present invention and the method of the sixteenth aspect of the present invention are for treating or preventing chronic cough.

A seventeenth aspect of the present invention relates to a NO-donor for treating or preventing a respiratory neoplasm.

An eighteenth aspect of the present invention relates to a method of treating or preventing a respiratory neoplasm, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a NO-donor. Preferably the NO-donor of the seventeenth aspect of the present invention and the method of the eighteenth aspect of the present invention are for treating or preventing:

(a) a malignant respiratory neoplasm including malignant neoplasms of the lip, tongue, gum, floor of the mouth, palate and other parts of the mouth, malignant neoplasms of the parotid gland, tonsil, oropharynx, nasopharynx, piriform sinus, hypopharynx and other parts of the pharynx, malignant neoplasms of the nasal cavity, middle ear and accessory sinuses, malignant neoplasms of the larynx, trachea, bronchus and lung, and Kaposi's sarcoma of the mouth and other parts of the respiratory system;

(b) an in situ respiratory neoplasm including carcinomas in situ of the lip, oral cavity, pharynx, larynx, trachea, bronchus, lung and other parts of the respiratory system;

(c) a benign respiratory neoplasm including benign neoplasms of the mouth, pharynx, salivary glands, nasal cavity, middle ear and accessory sinuses, and benign neoplasms of the larynx, trachea, bronchus, lung and other parts of the respiratory system; or (d) a respiratory neoplasm of uncertain or unknown behaviour.

Preferably the NO-donor referred to any of the fifteenth to eighteenth aspects of the present invention can be administered by inhalation. Preferably the NO-donor is as specified in any embodiment of the first aspect of the present invention.

In any of the above aspects of the present invention relating to treating or preventing a respiratory disorder, the subject to be treated is preferably a human. Preferably the human is a non-asthmatic human. Preferably the human is a non-smoking human. As used herein, a "non-smoking human" is a human who has smoked less than ten tobacco products in the last year.

A nineteenth aspect of the present invention relates to an inhaler comprising a pharmaceutical composition according to any of the first, fourth, fifth or sixth aspects of the present invention, or a compound according to either of the second or third aspects of the present invention. Preferably the inhaler is suitable for use in the treatment or prevention of a respiratory disorder. Preferably the respiratory disorder is one of the respiratory disorders mentioned above. In one embodiment of the nineteenth aspect of the present invention, the inhaler is a dry powder inhaler. In a dry powder inhaler, the dose to be administered is stored in the form of a non-pressurized dry powder and, on actuation of the inhaler, the particles of the powder are inhaled by the patient. Dry powder inhalers can be "passive" devices in which the patient's breath is the only source of gas which provides a motive force in the device. Examples of "passive" dry powder inhaler devices include the Rotahaler™ and Diskhaler™ (GlaxoSmithKline) and the Turbohaler™ (Astra-Draco) and Novolizer™ (Viatris GmbH). Alternatively, "active" devices may be used, in which a source of compressed gas or alternative energy source is used. Examples of suitable active devices include Aspirair™ (Vectura Ltd) and the active inhaler device produced by Nektar Therapeutics (as covered by US 6,257,233).

Particularly preferred "active" dry powder inhalers are referred to herein as Aspirair inhalers and are described in more detail in WO 01/00262, WO 02/07805, WO 02/89880 and WO 02/89881, the contents of which are hereby incorporated by reference. It should be appreciated, however, that the compositions of the present invention can be administered with either passive or active inhaler devices.

The dry powder inhaler devices in which the powder compositions of the present invention will commonly be used include "single dose" devices, for example, the Rotahaler™ and the Spinhaler™ in which individual doses of the powder composition are introduced into the device in, for example, single dose capsules or blisters, and also multiple dose devices, for example, the Turbohaler ^M in which, on actuation of the inhaler, one dose of the powder is removed from a reservoir of the powder material contained in the device.

In certain embodiments of the present invention, each dose is stored in a foil "blister" of a blister pack or in a single dose capsule. Accordingly, a twentieth aspect of the present invention relates to a foil blister or a single dose capsule comprising a pharmaceutical composition according to any of the first, fourth, fifth or sixth aspects of the present invention, or a compound according to either of the second or third aspects of the present invention. Preferably the foil blister or single dose capsule is suitable for use in a dry powder inhaler. In accordance with the embodiments of the present invention which utilize foil blisters, exposure of the formulation to air prior to administration is reduced or prevented by storing each dose in a sealed foil blister. In some circumstances, it may be desirable to further protect the formulation by placing a plurality of blisters into a further sealed container, such as a sealed bag made, for example, of a foil such as aluminium foil. Further mechanical protection may also be desirable, to protect the sealed blisters from damage during storage and transportation, etc. The use of the sealed foil blisters (and optional sealed bags and/or other protective packaging) eliminates any need to include anti-oxidants or the like in the formulation.

The blisters which may be used in the present invention consist of a base and a lid. Preferably, the base material is a laminate comprising a polymer layer in contact with the drug, a soft tempered aluminium layer and an external polymer layer. The aluminium provides the moisture and oxygen barrier, whilst the polymer provides a relatively inert layer in contact with the drug. Soft tempered aluminium is ductile so that it can be "cold formed" into a blister shape. It is typically 45-47μm thick. The outer polymer layer provides additional strength to the laminate. The lid material is a laminate comprising a heat seal lacquer, a hard rolled aluminium layer (typically 20-30μm thick) and an external polymer layer. The heat seal lacquer bonds to the polymer layer of the base foil laminate during heat sealing. The aluminium layer is hard rolled to facilitate piercing. Materials for the polymer layer in contact with the drug include polyvinyl chloride (PVC), polypropylene (PP) and polyethylene (PE). The external polymer layer on the base foil is typically oriented poly amide (oPA).

In another embodiment of the nineteenth aspect of the present invention, the inhaler is a metered dose inhaler (MDI). Pressurized metered dose inhalers for use in accordance with the present invention typically have two components: a canister component in which the drug particles are stored under pressure in a suspension or solution form and a receptacle component used to hold and actuate the canister. Typically, a canister will contain multiple doses of the formulation, although it is possible to have single dose canisters as well. The canister component typically includes a valved outlet from which the contents of the canister can be discharged. Aerosol medication is dispensed from the MDI by applying a force on the canister component to push it into the receptacle component thereby opening the valved outlet and causing the medication to be conveyed from the valved outlet through the receptacle component and discharged from an outlet of the receptacle component. Upon discharge from the canister, the medication is "atomised", forming an aerosol.

In certain embodiments, the canister of the MDI may further comprise nitric oxide, such that a compound of the second or third aspect of the present invention is formed by in situ reaction between nitric oxide and an A-SMase inhibitor.

According to a further aspect of the present invention, there is provided an in vitro assay for measuring the effect of one or more compounds, such as an A-SMase inhibitor and/or a NO-donor, upon the activity of A-SMase.

In certain embodiments, the assay is a cell-based assay wherein the cells are human cells, and more preferably human colon adenocarcinoma cells, and most preferably HT29 (human colon adenocarcinoma grade II) cells.

Preferably the A-SMase inhibitor is as specified in any embodiment of the first aspect of the present invention. Most preferably the A-SMase inhibitor is a tricyclic antidepressant, and most preferably the tricyclic antidepressant is amitriptyline.

In preferred embodiments, the A-SMase inhibitor is applied in the assay system at between 0.1 and 100 microM, more preferably between 0.5 and 80 microM, more preferably between 0.8 and 50 microM, more preferably between 0.9 and 45 microM, even more preferably between 1 and 40 microM, and most preferably at about 1, 5, 10, 20, 30 or 40 microM.

Preferably the NO-donor is as specified in any embodiment of the first aspect of the present invention. Preferably the NO-donor is a NO-synthase substrate or a NO-synthase agonist. More preferably the NO-donor is NO-synthase substrate such as arginine or L- arginine. Preferably the NO-donor is not a diethylenetriamine nitric oxide adduct such as DETA NO. Preferably the NO-donor is not a NONOate. Preferably the NO-donor is not a NO- releaser. Preferably the NO-donor is not a synthetic NO-donor.

Most media which are used for cell-based assays contain L-arginine as a nutrient at a concentration of 1140 microM. Since the Km value (numerically, Km equals substrate concentration at 0.5X maximal reaction velocity (Vmax) of the enzyme-catalysed reaction) for iNOS is reported as 1-20 microM, then using such media, the conversion of L-arginine to NO will, practically, be occurring at Vmax. That is, L-arginine is present at a saturating concentration. Such a system lacks sensitivity for assaying the effects of added L-arginine.

Further to this, a phenomenon known as the "arginine paradox" has also been reported, wherein added L-arginine results in increased generation of NO at concentrations of iNOS at which the enzyme should be saturated. Attempts to avoid the arginine paradox by enhancing the activity/induction of iNOS will result in an increase in output of NO. Increased background NO will inhibit activated A-SMase. Moreover, cell viability and function may be compromised at high levels of NO.

The simplest solution to these problems would appear to be the use of an L-arginine depleted/deprived medium, resulting from medium formulation or possibly by arginase treatment. A dose-response curve for the inhibition of A-SMase could then be determined by adding L-arginine to a cell system depleted of L-arginine, in which the activity of iNOS could be induced in order that substrate (L-arginine) concentration was rate-limiting over a sufficient dose-response (inhibition of activated A-SMase) to enable calculation of an IC50 value.

However, arginine starvation can lead to NOS-driven superoxide production, with harmful effects upon cell function and viability. Teleologically, by coupling L-arginine levels to iNOS protein synthesis, a mechanism is provided whereby iNOS is not expressed in L- arginine-depleted cells ensuring superoxide radical production is minimized. Consequently, in many cell lines, L-arginine deprivation results in down-regulation of iNOS protein. Thus, it may be difficult to adequately and reproducibly induce iNOS in L- arginine-depleted cells, resulting in a system with limited or no sensitivity for assaying the effects of exogenously- added L-arginine upon activated A-SMase. Additionally, it could be difficult to control outcomes for the effects of newly-synthesised L-arginine from L-citrulline, and its regulation by L-glutamine in L-arginine-depleted cells capable of this bioconversion.

To address these problems it has now been found that a typical cell-growth medium can be used, which has, for example, a concentration of L-arginine in excess of 1000 microM. This will mean that iNOS with a Km of 1-20 microM will be operating at Vmax. With the concentration of L-arginine at a high and constant level then the kinetics of the reaction (Vmax) will be zero order. That is, the velocity of the reaction will be directly proportional to the concentration of iNOS.

Consequently, given zero order kinetics whereby the concentration of iNOS is rate- limiting, a dose-response curve for the inhibitory effects of L-arginine-derived NO may be successfully derived by the titration of iNOS. That is, iNOS is added to the system in which L-arginine is at a fixed and saturating concentration. Under these conditions, the IC50 of iNOS for the inhibition of A-SMase by iNOS is proportional to the IC50 of L- arginine. That is:

IC50 of iNOS = Constant (Kl) x IC50 of L-arginine = Constant (K2) x IC50 of NO Pquaoon i]

IC50 is the concentration of the agent required to produce a 50% decrease in the rate of the reaction. Thus, from equation 1 above, although the values of the IC50 for iNOS and L-arginine differ numerically (but which algebraically become equal when a proportionality constant is introduced), in both cases the outcome is the same. And in both cases this outcome is achieved by the same concentration of NO.

Consequently, the magnitude of the interaction between amitriptyline and L-arginine upon inhibiting A-SMase activity (in a system in which A-SMase activity is induced) can be demonstrated using iNOS as a surrogate for L-arginine, but only with zero order kinetics where small changes in substrate concentration (consumed by adding iNOS) do not influence the rate of the reaction. Accordingly, in assays according to the invention wherein the NO-donor is arginine, the effect of arginine upon the activity of A-SMase may be established by the use of iNOS as a surrogate for arginine. By this is meant that the effect of arginine upon the activity of A- SMase may be established by the introduction of a known concentration of iNOS to a system wherein the level of arginine within the system is saturating, i.e. wherein the level of arginine within the system is such that the kinetics of the conversion of arginine to NO will be substantially directly proportional to the concentration of iNOS.

In preferred embodiments, the final concentration of iNOS used in the assay is 0.01 to 10 Units (U); in more preferred embodiments, the final concentration of iNOS used in the assay is 0.05 to 5 U; in even more preferred embodiments, the final concentration of iNOS used in the assay is 0.08 to 2 U; and most preferably, the final concentration of iNOS used in the assay is about 0.1, 0.2, 0.3, 0.5, 0.7 or 1 U.

In preferred embodiments, the assay is used to assess the effect of both an A-SMase inhibitor and a NO-donor upon the activity of A-SMase, and is sufficiently sensitive to characterize the magnitude of the interaction between the A-SMase inhibitor and the NO- donor.

As discussed above, NO inhibits activated but not basal A-SMase. Accordingly, the assay involves activating endogenous A-SMase by exposing the cells to an inducing agent such as one or more of a fatty acid, lipopolysaccharide, phorbol myristate acetate or sodium butyrate.

In embodiments wherein the assay comprises the application of exogenous iNOS, assay sensitivity could be compromised by the presence of background iNOS. Accordingly, in preferred embodiments, the assay has little or no induced activity of endogenous iNOS. Accordingly, in preferred embodiments, the inducing agent does not induce the activity of endogenous iNOS, and in even more preferred embodiments, the inducing agent suppresses endogenous iNOS expression. In most preferred embodiments of the invention, the inducing agent is sodium butyrate. The use of sodium butyrate as the inducing agent is particularly advantageous, as sodium butyrate has been reported to suppress iNOS expression and the excretion of NO in HT29 cells at concentrations including 0.9 to 15.4 mM following 24 hour incubation (Li et ah, Ai Zheng, 2004, vol. 23(4), pp. 416-20). Sodium butyrate has also been shown to reduce promoter-dependent iNOS transcriptional activity dose-dependently at concentrations higher than 0.1 mM in colon cancer cells (Sasahara et a/, Cancer Lett., 2002, vol. 177(2), pp. 155-161). Further to this, the use of sodium butyrate reflects the physiological situation, as butyrate has been found in the colon of healthy humans at a concentration of 15-60 mM (Li et a/., Ai Zheng, 2004, vol. 23(4), pp. 416-20).

Accordingly, an assay system which uses HT29 cells and low (i.e. mM) concentrations of sodium butyrate as the inducing agent is particularly advantageous, as it provides greater assay sensitivity and reproducibility by reducing and controlling background levels of iNOS. An assay system in which the inducing agent does not suppress iNOS is far less sensitive since there is no control of endogenous activity of iNOS.

In preferred embodiments, the inducing agent is applied in the assay system at a physiological concentration. Preferably, the inducing agent is applied at between 0.5 and 100 mM, more preferably between 1 and 80 mM, more preferably between 1 and 50 mM, more preferably between 1 and 25 mM, and most preferably between 1 and 12 mM.

Preferably the assay system is substantially free of ethylenediaminetetraacetic acid (EDTA). Preferably the assay system is substantially free of Zn chelating agents. Preferably the assay system is substantially free of divalent cation chelating agents. Preferably the assay system is substantially free of cation chelating agents.

Providing a cell-based assay system for investigating the effect of an A-SMase inhibitor and/or a NO-donor upon the activity of A-SMase and, in particular, for assessing the magnitude of the interaction between an A-SMase inhibitor and a NO-donor upon the activity of A-SMase has several advantages. Most importantly, it reflects a physiological system, and thus is of clinical relevance. This is particularly the case when a human cell line is used; when a physiological NO-donor is used in place of a synthetic NO-donor such as DETA NO; and when A-SMase is activated within the system using a physiological inducing agent such as a fatty acid, at a physiological concentration.

In a further aspect of the present invention, there is provided a method of inhibiting A- SMase, comprising the use of a NO-donor and/or an A-SMase inhibitor. Preferably a NO- donor and an A-SMase inhibitor are used. Preferably the A-SMase inhibitor and/or NO- donor are as specified in any embodiment of the first aspect of the present invention. Most preferably the NO-donor is arginine or L-arginine. Preferably the A-SMase inhibitor is a tricyclic antidepressant, and most preferably is amitriptyline. Alternatively the inhibition may comprise the use of a compound according to the second or third aspect of the present invention. The inhibition may occur in vivo, in vitro or ex vivo.

For the avoidance of doubt, insofar as is practicable any embodiment of a given aspect of the present invention may occur in combination with any other embodiment of the same aspect of the present invention. In addition, insofar as is practicable it is to be understood that any preferred or optional embodiment of any aspect of the present invention should also be considered as a preferred or optional embodiment of any other aspect of the present invention.

The present invention will now be described by way of example with reference to the accompanying drawing in which:

Figure 1 is a graph showing the induction of A-SMase activity by sodium butyrate in HT29 cells.

Example 1: Acid sphingomyelinase activity in butyr ate treated HT29 cells

Objective:

Determination of acid sphingomyelinase activity in human HT29 cells treated with butyrate. Assay Protocol:

2mL of HT29 cells at 0.25 x 10E6 cell/mL were plated into each well of a 12-well plate and incubated for 24 hours in RPMI 5% FBS + 2mM ala-glutamine at 37 ⁰C and 5% CO₂.

Sodium butyrate was added to each well to make a final concentration of 10 mM, 5 mM, 2.5 mM, 1.25 mM, or 0 mM (3 wells /concentration), and the cells were incubated for 24 hours.

After the incubation period, the cells were washed three times with phosphate buffer solution (PBS). The cells were then lysed in 25mM sodium acetate (NaOAc) containing

0.25% triton X 100. The lysate was spun at 20Og and acid sphingomyelinase activity was determined using an ECHELON Acid Sphingomyelinase Assay Kit (K-3200) following the manufacturer's recommendations. 20 microL of cell lysate was tested per well and the activity was interpolated against an acid sphingomyelinase control which was supplied in the kit.

In addition, 5 microL of lysate was used to determine the protein concentration using a Pierce BCA Protein Assay Kit (Cat#23225) in accordance with the manufacturer's instruction.

Results:

A significant change in acid sphingomyelinase activity as a function of butyrate concentration was achieved after a 24 hour incubation. Over 4-fold induction of acid sphingomyelinase activity was seen when HT29 cells were treated with 5 mM butyrate, as can be seen from Figure 1.

The concentration of acid sphingomyelinase did not increase at the highest concentration of butyrate (10 mM). This result is likely to be due to cellular toxicity. Example 2: Inhibition of acid sphingomyelinase activity by amitr iptyline

Assay Protocol:

2mL of HT29 cells at 0.25 x 10E6 cell/mL are plated into each well of a 12-well plate and incubated for 24 hours in RPMI 5% FBS + 2mM ala-glutamine at 37 ⁰C and 5% CO₂.

After 24 hours, the cells are incubated with amitriptyline solubilised in a suitable solvent, such as DMSO at 20 mM and diluted in DMSO to provide 6 concentrations. The amitriptyline titration is further diluted in culture media to a 20X concentration in a daughter plate to be delivered to the cellular plate. Final concentrations of amitriptyline are within the range 0.1 microM-100 microM, with the initial preferred final concentrations being 1, 5, 10, 20, 30 and 40 microM.

After treatment of the cells with amitriptyline for 1 hour, acid sphingomyelinase activity is induced using sodium butyrate at a final concentration of 5 mM, and the cells are incubated for 24 hours.

After the incubation period, the cells are washed three times with PBS, scraped, harvested and lysed by incubation in 25mM sodium acetate (NaOAc) plus 0.25% triton X 100. The lysate is spun at 20Og and acid sphingomyelinase activity is determined using an ECHELON Acid Sphingomyelinase Assay Kit (K-3200) following the manufacturer's recommendations. 20 microL of cell lysate is tested per well and the activity is interpolated against an acid sphingomyelinase control supplied in the kit.

In addition, 5 microL of lysate is used to determine the protein concentration using a Pierce BCA Protein Assay Kit (Cat#23225) in accordance with the manufacturer's instructions.

Interpolated data is normalized to vehicle controls and analysed for an inhibitory concentration 50% (IC50) of amitriptyline using a non-linear regression analysis four point parameter fit. Example 3: Inhibition of acid sphingomyelinase activity by iNOS

Assay Protocol:

After 24 hours, the cells are incubated with iNOS solubilised in a suitable solvent, such as DMSO at 20 mM and diluted in DMSO to provide 6 concentrations. The iNOS titration is further diluted in culture media to a 2OX concentration in a daughter plate to be delivered to the cellular plate. Final concentrations of iNOS are within the range 0.01 Units (U)-IO U, with the initial preferred final concentrations being 0.1, 0.2, 0.3, 0.5, 0.7 and 1 U.

After treatment of the cells with iNOS for 1 hour, acid sphingomyelinase activity is induced using sodium butyrate at a final concentration of 5 mM, and the cells are incubated for 24 hours.

Interpolated data is normalized to vehicle controls and analysed for an inhibitory concentration 50% (IC50) of iNOS using a non-linear regression analysis four point parameter fit. Example 4: Inhibition of acid sphingomyelinase activity by a fixed dose combination of amitriptyline plus iNOS

Assay Protocol: 2mL of HT29 cells at 0.25 x 10E6 cell/mL are plated into each well of a 12-well plate and incubated for 24 hours in RPMI 5% FBS + 2mM ala-glutamine at 37 ⁰C and 5% CO₂.

After 24 hours, the cells are incubated with a fixed dose combination of amitriptyline plus iNOS, solubilised in a suitable solvent, such as DMSO at 20 mM and diluted in DMSO to provide 6 concentrations. The combination titration is further diluted in culture media to a 2OX concentration in a daughter plate to be delivered to the cellular plate. Final concentrations of the combination represent serial dilutions of each drug (amitriptyline and iNOS) at the IC50 that was previously determined in the assay system (see examples 2 and 3). The final concentrations of the combination are derived using doses that represent the fixed dose ratio of the IC50 of each drug, that is serially diluted by xθ.5, xθ.25, xθ.125,

After treatment of the cells with the combination of amitriptyline plus iNOS at each of the fixed dose ratios described above for 1 hour, acid sphingomyelinase activity is induced with sodium butyrate at a final concentration of 5 mM, and the cells are incubated for 24 hours.

In addition, 5 microL of lysate is used to determine the protein concentration using a Pierce BCA Protein Assay Kit (Cat#23225) in accordance with the manufacturer's instructions. Interpolated data is normalized to vehicle controls and analysed for an inhibitory concentration 50% (IC50) using a non-linear regression analysis four point parameter fit. Using the derived value of the IC50 value for both drugs in combination, the magnitude of the interaction between the two drugs is examined using standard isobolographic methods of analysis as described by Tallarida (RJ. Tallarida, J. Pharmacol. Exp. Ther., 2001, vol. 298(3), pp. 865-872). Using this method of analysis the theoretical IC50 value of the combination that would yield an additive interaction is compared at the 5% level of probability with the experimental value. A significant shift to the left upon the constructed isobologram as described by Tallarida will represent a non-linear augmentation in effects, representing a superadditive interaction (synergy).

The assay of the present invention provides a sensitive system for assessing the effects of amitriptyline alone; iNOS alone; and a combination of amitriptyline and L-arginine (as established by the use of exogenous iNOS) upon the activity of A-SMase.

It will be understood that the present invention has been described above by way of example only. The examples are not intended to limit the scope of the present invention. Various modifications and embodiments can be made without departing from the scope and spirit of the invention, which is defined by the following claims only.

Claims

1. A pharmaceutical composition comprising an A-SMase inhibitor and a NO-donor, and optionally comprising one or more pharmaceutically acceptable excipients.

2. A pharmaceutical composition as claimed in claim 1, wherein the A-SMase inhibitor is capable of inhibiting A-SMase by disrupting its attachment to a lysosomal membrane.

3. A pharmaceutical composition as claimed in claim 2, wherein the lysosomal membrane is an intralysosomal membrane.

4. A pharmaceutical composition as claimed in any one of claims 1 to 3, wherein the A-SMase inhibitor is selected from alimemazine, ami trip tyline, trans-l,4-bis(2- chlorobenzaminomethy^cyclohexane (AY9944), chlorpromazine, clomipramine, cocaine, cyamemazine, desipramine, dibucaine, imipramine, mianserin, perhexiline, prochlorperazine, propericiazine, quinacrine, tamoxifen, thioproperazine, thioridazine, trifluoperazine, trihexyphenidyl, trimipramine, N-(6-aminohexyl)-l-naρhthalenesulfonamide (W- 5), N-(6-aminohexyl)-l-[5'-chloro-naρhthalene-l'-sulfonamide] (W-7), amlodipine, astemizole, benztropine, bepridil, camylofin, clomiphene, cloperastine, cyclobenzaprine, cyproheptadine, doxepin, drofenine, pimethixene, promazine, protriptyline, chlorprothixene, fendiline, fluoxetine, maprotiline, norfluoxetine, nortriptyline, paroxetine, promethazine, sertraline, suloctidil, terfenadine, triflupromazine, 8-bromoguanosine-3',5'- cyclic monophosphate, tricyclo-decan-9-yl-xanthate, monensin, or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

5. A pharmaceutical composition as claimed in any one of claims 1 to 4, wherein the A-SMase inhibitor comprises a nitrogen atom.

6. A pharmaceutical composition as claimed in claim 5, wherein the A-SMase inhibitor has:

(a) a ρK_a of at least 5; and/or

(b) a ρK_a of between 6 and 15; and/or (c) a pK_a of between 8 and 12; and/or

(d) a log P of at least 0; and/or

(e) a log P of between 1 and 15; and/or

(f) a log P of between 2 and 8; and/or (g) a steric factor k ≤ 20; and/or

(h) a steric factor k ≤ 10; and/or (i) a steric factor k ≤ 4.

7. A pharmaceutical composition as claimed in any one of claims 1 to 6, wherein the A-SMase inhibitor is selected from alimemazine, amitriptyline, trans-l,4-bis(2- chlorobenzaminomethyl)cyclohexane (AY9944), chlorpromazine, clomipramine, cocaine, cyamemazine, desipramine, dibucaine, imipramine, mianserin, perhexiline, prochlorperazine, propericiazine, quinacrine, tamoxifen, thioproperazine, thioridazine, trifluoperazine, trihexyphenidyl, trimipramine, N-(6-aminohexyl)-l- naphthalenesulfonamide (W- 5), N-(6-aminohexyl)-l-[5'-chloro-naphthalene-l'- sulfonamide] (W- 7), amlodipine, astemizole, benztropine, bepridil, camylofin, clomiphene, cloperastine, cyclobenzaprine, cyproheptadine, doxepin, drofenine, pimethixene, promazine, pro trip tyline, chlorprothixene, fendiline, fluoxetine, maprotiline, norfluoxetine, nortriptyline, paroxetine, promethazine, sertraline, suloctidil, terfenadine, triflupromazine, or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

8. A pharmaceutical composition as claimed in claim 7, wherein the A-SMase inhibitor is selected from alimemazine, amitriptyline, chlorpromazine, clomipramine, desipramine, dibucaine, imipramine, prochlorperazine, tamoxifen, thioridazine, trifluoperazine, trihexyphenidyl, trimipramine, amlodipine, benztropine, bepridil, clomiphene, cyclobenzaprine, cyproheptadine, doxepin, promazine, protriptyline, chlorprothixene, fluoxetine, maprotiline, nortriptyline, paroxetine, promethazine, sertraline, or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

9. A pharmaceutical composition as claimed in any one of claims 1 to 8, wherein the

A-SMase inhibitor is an antidepressant.

10. A pharmaceutical composition as claimed in claim 9, wherein the antidepressant is selected from alimemazine, amitriptyline, chlorpromazine, clomipramine, cocaine, cyamemazine, desipramine, imipramine, mianserin, prochlorperazine, propericiazine, thioproperazine, thioridazine, trifluoperazine, trihexyphenidyl, trimipramine, cyclobenzaprine, cyproheptadine, doxepin, pimethixene, promazine, protriptyline, chlorprothixene, fluoxetine, maprotiline, norfluoxetine, nortriptyline, paroxetine, promethazine, sertraline, triflupromazine, or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

11. A pharmaceutical composition as claimed in claim 9 or 10, wherein the antidepressant is a selective serotonin reuptake inhibitor (SSRI), a serotonin- norepinephrine reuptake inhibitor (SNRI), a noradrenergic and specific serotonergic antidepressant (NASSA), a norepinephrine (noradrenaline) reuptake inhibitor (NRI), a norepinephrine-dopamine reuptake inhibitor, a tricyclic antidepressant (TCAD) or a monoamine oxidase inhibitor (MAOI).

12. A pharmaceutical composition as claimed in claim 11, wherein the antidepressant is a tricyclic antidepressant (TCAD).

13. A pharmaceutical composition as claimed in claim 12, wherein the tricyclic antidepressant is selected from alimemazine, amitriptyline, chlorpromazine, clomipramine, cyamemazine, desipramine, imipramine, mianserin, prochlorperazine, propericiazine, thioproperazine, thioridazine, trifluoperazine, trimipramine, cyclobenzaprine, cyproheptadine, doxepin, pimethixene, promazine, protriptyline, chlorprothixene, maprotiline, nortriptyline, paroxetine, promethazine, triflupromazine, or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

14. A pharmaceutical composition as claimed in claim 13, wherein the tricyclic antidepressant is amitriptyline or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

15. A pharmaceutical composition as claimed in any one of claims 1 to 13, wherein the A-SMase inhibitor is selected from a compound of formula (I) or (II):

X is independently selected from N

_

-CR^{1 L}O- S —- /C"TR) H — — ( C"TR) H^H —-S C(R I¹KO₂ _ - -CR¹^ -C(R I¹h^₂ —- rCuRn

C=CR^{1 1}- _or -R^{1 1}C=C- ;

) 1L

X' is independently selected from C _^ NR C _^ C NR _^

-O— C- -C-O- -S-C C-S- -C-C(R»)₂- -C(R^H)₂-C- .

/0 -Y- is independently selected from a chemical bond, -O-, -S-, -NR¹²-, -C(R¹²)₂-,

-C(R¹²)₂-C(R¹²)₂-, -R¹²C=CR¹²-, -NR¹²-C(R¹²)₂-, -C(R¹²)₂-NR¹²-, -N=CR¹²-, -R¹²C=N-, -O-C(R¹²)₂-, -C(R¹²)₂-O-, -S-C(R¹²)₂- or -C(R¹²)₂-S-; n is independently 1, 2, 3, 4, 5 or 6; m is independently 0, 1, 2, 3, 4 or 5;

15 R¹, R², R³, R⁴, R⁵, R⁶, R⁷_ R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independently selected from hydrogen, -F, -Cl, -Br, -I, -CF₃, -CCl₃, -CBr₃, -CI₃, -OH, -SH, -NH₂, -CN, -NO₂, -COOH, -R¹³-O-R¹⁴, -R¹³-S-R¹⁴, -R¹³-SO-R¹⁴, -R¹³-SO₂-R¹⁴, -R¹³-SO₂-OR¹⁴, -R¹³-O-SO₂-R¹⁴, -R¹³-SO₂-N(R¹⁴)₂, -R¹³-NR¹⁴-SO₂-R¹⁴, -R¹³-O-SO₂-OR¹⁴, -R¹³-O-SO₂-N(R¹⁴)₂, -R¹³-NR¹⁴-SO₂-OR¹⁴, -R¹³-NR¹⁴-SO₂-N(R¹⁴)₂, -R¹³-N(R¹⁴)₂, -R¹³-N(R¹⁴)₃ ⁺, -R¹³-P(R¹⁴)₂, 0 -R¹³-Si(R¹⁴)₃, -R¹³-CO-R¹⁴, -R¹³-CO-OR¹⁴, -R¹³-O-CO-R¹⁴, -R¹³-CO-N(R¹⁴)₂, -R¹³-NR¹⁴-CO-R¹⁴, -R¹³-O-CO-OR¹⁴, -R¹³-O-CO-N(R¹⁴)₂, -R¹³-NR¹⁴-CO-OR¹⁴, -R¹³-NR¹⁴-CO-N(R¹⁴)₂, -R¹³-CS-R¹⁴, -R¹³-CS-OR¹⁴, -R¹³-O-CS-R¹⁴, -R¹³-CS-N(R¹⁴)₂, -R¹³-NR¹⁴-CS-R¹⁴, -R¹³O-CS-OR¹⁴, -R¹³-O-CS-N(R¹⁴)₂, -R¹³-NR¹⁴-CS-OR¹⁴,

-R¹³-NR¹⁴-CS-N(R¹⁴)₂ or -R¹⁴;

-R¹³- is independently a chemical bond, or an optionally substituted C₁-C₁₀ alkylene, C₂-C₁₀ alkenylene or C₂-C₁₀ alkynylene group; -R¹⁴ is independently hydrogen or a C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₂-

C₁₀ aryl, C₃-C₁₀ arylalkyl, C₄-C₁₀ arylalkenyl, C₄-C₁₀ arylalkynyl, C₃-C₁₀ alkylaryl, C₄-C₁₀ alkenylaryl or C₄-C₁₀ alkynylaryl group, each of which may optionally be substituted; and wherein any two or more R¹, R², R³, R⁴, R⁵, R⁶, R⁷ R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ or R¹⁴ may, together with the atom or atoms to which they are attached, form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more heteroatoms N, O or S in its carbon skeleton.

16. A pharmaceutical composition as claimed in claim 15, wherein:

(a) — X— is independently selected from -N- -CR¹¹- -N-C(R^π)₂- or

(b) — X'— is -c-

17. A pharmaceutical composition as claimed in claim 15 or 16, wherein -Y- is independently selected from -S-, -C(R¹²)₂-, -C(R¹²)₂-C(R¹²)₂-, -R¹²C=CR¹²-, -O-C(R¹²)₂- or -C(R¹²)₂-O-.

18. A pharmaceutical composition as claimed in any one of claims 15 to 17, wherein n is 2 or 3.

19. A pharmaceutical composition as claimed in any one of claims 15 to 18, wherein m is 1 or 2.

20. A pharmaceutical composition as claimed in any one of claims 15 to 19, wherein one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is selected from hydrogen, -F, -Cl, -Br, -I, -CF₃, -CCl₃, -CBr₃, -CI₃, -OH, -SH, -NH₂, -CN, -NO₂, -COOH, -R¹³-O-R¹⁴, -R¹³-S-R¹⁴, -R¹³-SO-R¹⁴, -R¹³-SO₂-R¹⁴, -R¹³-SO₂-OR¹⁴, -R¹³-O-SO₂-R¹⁴, -R¹³-SO₂-N(R¹⁴)₂, -R¹³-NR¹⁴-SO₂-R¹⁴, -R¹³-O-SO₂-OR¹⁴, -R¹³-O-SO₂-N(R¹⁴)₂, -R¹³-NR¹⁴-SO₂-OR¹⁴, -R¹³-NR¹⁴-SO₂-N(R¹⁴)₂, -R¹³-N(R¹⁴)₂, -R¹³-N(R¹⁴)₃ ⁺, -R¹³-P(R¹⁴)₂, -R¹³-Si(R¹⁴)₃, -R¹³-CO-R¹⁴, -R¹³-CO-OR¹⁴, -R¹³-O-CO-R¹⁴, -R¹³-CO-N(R¹⁴)₂, -R¹³-NR¹⁴-CO-R¹⁴, -R¹³-O-CO-OR¹⁴, -R¹³-O-CO-N(R¹⁴)₂, -R¹³-NR¹⁴-CO-OR¹⁴, -R¹³-NR¹⁴-CO-N(R¹⁴)₂, -R¹³-CS-R¹⁴, -R¹³-CS-OR¹⁴, -R¹³-O-CS-R¹⁴, -R¹³-CS-N(R¹⁴)₂, -R¹³-NR¹⁴-CS-R¹⁴, -R¹³O-CS-OR¹⁴, -R¹³-O-CS-N(R¹⁴)₂, -R¹³-NR¹⁴-CS-OR¹⁴, -R¹³-NR¹⁴-CS-N(R¹⁴)₂ or -R¹⁴, and wherein all other R¹, R², R³, R⁴, R⁵, R , R⁷ and R⁸ are hydrogen.

21. A pharmaceutical composition as claimed in claim 20, wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are all hydrogen.

22. A pharmaceutical composition as claimed in claim 20, wherein one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ is not hydrogen.

23. A pharmaceutical composition as claimed in any one of claims 15 to 22, wherein at each occurrence R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently selected from hydrogen, -F, -Cl, -Br, -I, -CF₃, -CCl₃, -CBr₃, -CI₃, -CN, -R¹³-O-R¹⁴, -R¹³-S-R¹⁴, -R¹³-SO-R¹⁴, -R¹³-SO₂-R¹⁴, -R¹³-SO₂-OR¹⁴, -R¹³-O-SO₂-R¹⁴, -R¹³-SO₂-N(R¹⁴)₂, -R¹³-NR¹⁴-SO₂-R¹⁴, -R¹³-O-SO₂-OR¹⁴, -R¹³-O-SO₂-N(R¹⁴)₂, -R¹³-NR¹⁴-SO₂-OR¹⁴, -R¹³-NR¹⁴-SO₂-N(R¹⁴)₂, -R¹³-N(R¹⁴)₂, or -R¹⁴.

24. A pharmaceutical composition as claimed in any one of claims 15 to 23, wherein at each occurrence R⁹ is independently selected from hydrogen or a C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₂-C₁₀ aryl, C₃-C₁₀ arylalkyl, C₄-C₁₀ arylalkenyl, C₄-C₁₀ arylalkynyl, C₃-C₁₀ alkylaryl, C₄-C₁₀ alkenylaryl or C₄-C₁₀ alkynylaryl group, each of which may optionally be substituted.

25. A pharmaceutical composition as claimed in claim 24, wherein at each occurrence R is independently selected from hydrogen or methyl.

26. A pharmaceutical composition as claimed in any one of claims 15 to 25, wherein at each occurrence R¹⁰ is independently selected from hydrogen or a C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₂-C₁₀ aryl, C₃-C₁₀ arylalkyl, C₄-C₁₀ arylalkenyl, C₄-C₁₀ arylalkynyl, C₃-C₁₀ alkylaryl, C₄-C₁₀ alkenylaryl or C₄-C₁₀ alkynylaryl group, each of which may optionally be substituted.

27. A pharmaceutical composition as claimed in claim 26, wherein at each occurrence R¹⁰ is independently selected from hydrogen or methyl.

28. A pharmaceutical composition as claimed in any one of claims 15 to 27, wherein at each occurrence R¹¹ is hydrogen.

29. A pharmaceutical composition as claimed in any one of claims 15 to 28, wherein at each occurrence R¹² is hydrogen.

30. A pharmaceutical composition as claimed in any one of claims 15 to 29, wherein at each occurrence -R¹³- is a chemical bond.

31. A pharmaceutical composition as claimed in any one of claims 15 to 30, wherein at each occurrence -R¹⁴ is independently hydrogen or a C₁-C₄ alkyl, C₂-C₄ alkenyl or C₂-C₄ alkynyl group, each of which may optionally be substituted.

32. A pharmaceutical composition as claimed in any one of claims 15 to 31, wherein one R⁹ and one R¹⁰, together with the atoms to which they are attached, form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more additional heteroatoms N, O or S in its carbon skeleton.

33. A pharmaceutical composition as claimed in any one of claims 15 to 32, wherein the two R¹⁰, together with the nitrogen atom to which they are attached, form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more additional heteroatoms N, O or S in its carbon skeleton.

34. A pharmaceutical composition as claimed in any one of claims 15 to 33, wherein one R¹¹ and one R¹², together with the atoms to which they are attached, form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more additional heteroatoms N, O or S in its carbon skeleton.

35. A pharmaceutical composition as claimed in any one of claims 15 to 34, wherein one R¹⁰ and one R¹¹, together with the atoms to which they are attached, form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more additional heteroatoms N, O or S in its carbon skeleton.

36. A pharmaceutical composition as claimed in any one of claims 15 to 35, wherein an optionally substituted cyclic hydrocarbyl group is substituted with one or more of -F, -Cl, -Br, -I, -CF₃, -CCl₃, -CBr₃, -CI₃, -CN, -NO₂, -O-R¹⁵, -S-R¹⁵, -N-(R¹⁵)₂ or -R¹⁵, or one or more π-bonded substituents such as =O, =S or =NR¹⁵, wherein -R¹⁵ is independently hydrogen or an unsubstituted C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₂-C₁₀ aryl, C₃-C₁₀ arylalkyl, C₄-C₁₀ arylalkenyl, C₄-C₁₀ arylalkynyl, C₃-C₁₀ alkylaryl, C₄-C₁₀ alkenylaryl or C₄-C₁₀ alkynylaryl group.

37. A pharmaceutical composition as claimed in claim 36, wherein an optionally substituted cyclic hydrocarbyl group is substituted with -OH or a methyl group.

38. A pharmaceutical composition as claimed in any one of claims 1 to 37, wherein the NO-donor is nitric oxide, a NO-synthase substrate, a NO-synthase agonist, or a NO- releaser.

39. A pharmaceutical composition as claimed in claim 38, wherein the NO-donor is a NO-synthase substrate.

40. A pharmaceutical composition as claimed in claim 39, wherein the NO-synthase substrate is arginine, N-cyclopropyl-N'-hydroxyguanidine, L-canavanine or N"-benzoyl-L- arginine ethyl ester, or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

41. A pharmaceutical composition as claimed in claim 40, wherein the NO-synthase substrate is L-arginine or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

42. A pharmaceutical composition as claimed in claim 38, wherein the NO-donor is a NO-releaser.

43. A pharmaceutical composition as claimed in claim 42, wherein the NO-releaser is selected from:

(a) Angeli's salt (Na₂N₂O₃), isoniazid, N-hydroxybenzenesulfonamide or P- nitrosophosphate;

(b) an organic nitrate such as glyceryl trinitrate (GTN), [3-(nitrooxymethyl)phenyl]-2- acetyloxybenzoate (NCX-4016), 4-nitrooxybutyl-2-acetyloxybenzoate (NCX-4215), [3-(nitrooxymethyl)phenyl]-2-hydroxybenzoate (B-NOD), isosorbide mononitrate

(ISMN), pentaerythrityl tetranitrate (PETN), isosorbide dinitrate (BiDiI), nicorandil, nipradilol, nitro-pravastatin (NCX-6550), O-losartanyl-3-[(nitrooxy)methyl]- benzoate (NO-Losartan A) or methyl 2-[[4-[(nitrooxy)methyl]benzoyl]thio]- benzoate (SE 175); (c) an organic nitrite such as amyl nitrite or 2-methylbutan-2-yl nitrite;

(d) a NONOate such as diethylenetriamine NONOate (DETA NO), diethylamine NONOate (DEA NO), spermine NONOate (SPER NO), dipropylenetriamine NONOate (DPTA NO), methylamine hexamethylene methylamine NONOate (MAHMA NO), 3-propylamine-propylamine NONOate (PAPA NO), proline NONOate (PROLI NO), l-[(ethenyloxy)-NNO-azoxy]-pyrrolidine (V-Pyrro NO),

O2- (2,4-dinitrophenyl) - 1 - [(4-ethoxycarbonyl)piperazin- 1 -yl] diazen- 1 -ium- 1 ,2- diolate (JS-K), N-(3-aminopropyl-propan-2-ylamino)-N-hydroxynitrous amide (NOC-5), N-hydroxy-N-(methyl-(3-methylaminopropyl)-amino)-nitrous amide (NOC-7), N-ethyl-2-(l-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC- 12), O-β-galactopyranosyl-l-(pyrrolidin-l-yl)diazen-l-ium-l,2-diolate (β-Gal-

NONOate) or disodium hydroxydiazenesulfonate 1-oxide (Sulfo-NONOate);

(e) a S-nitrosothiol such as S-nitroso-N-acetylpenicillamine (SNAP), S-nitroso- glutathione (GSNO), S-nitroso-N-valerylpenicillamine (SNVP), S-nitrosoalbumin, S-nitrosocysteine, N-(N-γ-L-glutamyl-2-amino-2-(4-(4-S-nitrosomercapto-l- methylpiperidine) -acetyl) -glycine) (LA810), nitrosodiclofenac, nitrosocaptopril, N-

(β-D-glucopyranosyl)-N²-acetyl-S-nitroso-D,L-penicillaminamide (Glyco-SNAP-1), N-(2-deoxy-α,β-D-glucopyranose-2-)-N²-acetyl-S-nitroso-D,L-penicillaminamide (Glyco-SNAP-2), 4-phenyl-l,3,2-oxathiazolylium-5-olate, 4-(p- trifluoromemylphenyl)-l,3,2-oxathiazorylium-5-olate, 4-(p-chlorophenyl)-l,3,2- oxathiozolylium-5-olate, 4-(p-methoxyphenyl)-l,3,2-oxathiazolylium-5-olate or N- (l-deoxy-αβ-D-fructopyranose-l^-N^acetyl-S-nittoso-D^-peniciUaminamide (Fructose-SNAP-1); (f) a N-nitrosamine such as streptozocin, N,N'-dimethyl-N,N'-dinitroso-p- phenylenediamine (BNN3) or dephostatin;

(g) a C-nitroso compound such as 2-nitro-2-nitrosopropane;

(h) a diazetine dioxide such as 3-bromo-3,4,4-trimethyl-3,4-dihydrodiazete-l,2-dioxide (DDl) or 3-bromo-4-methyl-3,4-tetramethylene-3,4-dihydrodiazete-l,2-dioxide (DD2);

(i) a furoxan such as dimethyl 2,6-dimethyl-4-[2'-(3"-methylfuroxan-4"- ylmethoxy)phenyl]-l,4-dihydropyridine-3,5-dicarboxylate, dimethyl 2,6-dimethyl-4- [2'-(3"-carbamoylfuroxan-4"-ylmethoxy)phenyl]-l,4-dihydropyridine-3,5- dicarboxylate, dimethyl 2,6-dimethyl-4- [2'-(3"-cyanofuroxan-4"-ylmethoxy)phenyI| - l,4-dihydropyridine-3,5-dicarboxylate, 4-methyl-3-phenylsulphonylfuroxan, 4- hydroxymethyl-3-furoxancarboxamide, N- (pyridin-3-yl) -3-phenyl-4- furoxancarboxamide, 4-phenyl-3-(N',N'-dimethyl-2'-aminoethylsulphanyl) furoxan or 4-phenylsulphonyl-3-(N',N'-dimethyl-2'-aminoethylsulphanyl)furoxan;

()) a sydnonimine such as 3-morpholinosydnonimine (SIN-I) or molsidomine; (k) an oxatriazole-5-imine such as 5-amino-3-(3',4'-dichlorophenyl)-l-oxa-2,3,4- triazolium chloride, 5-amino-3-(3'-chloro-2'-methylphenyl)-l-oxa-2,3,4-triazolium chloride or 5- (((cyanomethylamino) carbonyl) amino) -3- (3'-chloro-2'-methylphenyl)- l-oxa-2,3,4-triazolium chloride;

(1) an oxime such as 4-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3- hexenamide (NOR-I), 4-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide

(NOR-2), 4-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), N- [(E,2Z)-4-ethyl-2-hydroxyimino-5-nitrohex-3-enyl]pyridine-3-carboxamide (NOR- 4) or N-[(E,2Z)-4-ethyl-6-methyl-2-hydroxyimino-5-nitrohept-3-enyl]pyridine-3- carboxamide (NOR- 5); or (m) a metal-NO complex such as sodium nitroprusside (SNP) or a NO zeolite; or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

44. A compound comprising an A-SMase inhibitor covalently linked to a NO-donor group, or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

45. A compound as claimed in claim 44, wherein the A-SMase inhibitor is as defined in any one of claims 2 to 37.

46. A compound as claimed in claim 44 or 45, wherein the NO-donor group is a NO- releasing group.

47. A compound as claimed in claim 46, wherein the NO-releasing group comprises or is:

(a) a -ONO₂ group;

(b) a -ONO group;

(c) a group selected from:

wherein at each occurrence R^c and R^d are independently selected from hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton;

(d) a -S-NO group;

(e) a -N(R^NO group, wherein R^f is independently selected from hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, optionally with one or more further -NO groups, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton;

(f) a

wherein each R is independently selected from hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton, and wherein any two R groups together with the carbon atom or atoms to which they are attached may form an optionally substituted cyclic hydrocarbyl group that optionally includes one or more heteroatoms N, O or S in its carbon skeleton;

(h) a group selected from:

® a group selected from:

48. A compound as claimed in any one of claims 44 to 47, wherein the compound further comprises a linker between the A-SMase inhibitor and the NO-donor group.

49. A compound as claimed in claim 48, wherein the linker comprises an alkylene, alkenylene, alkynylene, arylene, arylalkylene, arylalkenylene, arylalkynylene, alkylarylene, alkenylarylene or alkynylarylene group, each of which may optionally be substituted, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton.

50. A compound as claimed in any one of claims 44 to 47, wherein the compound is selected from a compound of formula (III) or (IV):

Q is independently a NO-releasing group;

X is independently selected from N

_

I

C=CR¹¹- or -R¹¹C=C

— X¹— is independently selected from C _^ NR C _^ C NR _^

-R¹³-NR¹⁴-CS-N(R¹⁴)₂ or -R¹⁴;

51. A compound as claimed in claim 50, wherein Q is:

wherein R^d is independently selected from hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton.

52. A compound as claimed in claim 51, wherein R^dis hydrogen.

53. A compound selected from:

or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

54. A method of synthesising a compound as claimed in any one of claims 44 to 53, said method comprising exposing an A-SMase inhibitor to nitric oxide, wherein said

A-SMase inhibitor comprises or is linked to a nucleophilic group.

55. A method as claimed in claim 54, wherein said synthesis occurs in a canister.

56. A pharmaceutical composition comprising a compound as claimed in any one of claims 44 to 53, and optionally comprising one or more pharmaceutically acceptable excipients.

57. A pharmaceutical composition as claimed in any one of claims 1 to 43 or claim 56, wherein the composition comprises two or more pharmaceutically acceptable excipients.

58. A pharmaceutical composition as claimed in any one of claims 1 to 43 or claim 56 or 57, wherein the composition has a pH of 7 or more.

59. A pharmaceutical composition comprising an A-SMase inhibitor, and optionally comprising one or more pharmaceutically acceptable excipients, wherein the composition has a pH of 7 or more.

60. A pharmaceutical composition as claimed in claim 59, wherein the A-SMase inhibitor is as defined in any one of claims 2 to 37.

61. A pharmaceutical composition comprising a NO-donor, and optionally comprising one or more pharmaceutically acceptable excipients, wherein the composition has a pH of 8 or more.

62. A pharmaceutical composition as claimed in claim 61, wherein the NO-donor is as defined in any one of claims 38 to 43.

63. A pharmaceutical composition as claimed in any one of claims 1 to 43 or any one of claims 56 to 62, wherein the composition has a pH of about 8.3.

64. A pharmaceutical composition as claimed in any one of claims 1 to 43 or any one of claims 56 to 63, wherein the composition comprises a buffer.

65. A pharmaceutical composition as claimed in any one of claims 1 to 43 or any one of claims 56 to 64, wherein the composition has a chloride ion concentration of between

10 and 1000 mmol/kg.

66. A pharmaceutical composition as claimed in claim 65, wherein the composition has a chloride ion concentration of about 145 mmol/kg.

67. A pharmaceutical composition as claimed in any one of claims 1 to 43 or any one of claims 56 to 66, wherein the composition is suitable for inhalation.

68. A pharmaceutical composition as claimed in claim 67, wherein the composition is suitable for use in a dry powder inhaler.

69. A pharmaceutical composition as claimed in claim 68, wherein the composition comprises a lubricant or a glidant.

70. A pharmaceutical composition as claimed in claim 69, wherein the lubricant or glidant is selected from lauric acid, palmitic acid, stearic acid, erucic acid, behenic acid, or a pharmaceutically acceptable salt or ester thereof, such as sodium stearyl fumarate, sodium stearyl lactylate, sodium lauryl sulphate, magnesium lauryl sulphate or magnesium stearate.

71. A pharmaceutical composition as claimed in claim 69, wherein the lubricant or glidant is selected from lecithin, leucine, phosphatidylcholines, phosphatidylglycerols, triglycerides, talc, titanium dioxide, aluminium dioxide, silicon dioxide or starch.

72. A pharmaceutical composition as claimed in any one of claims 69 to 71, wherein the composition comprises 0.05 to 10% by weight of the lubricant or glidant.

73. A pharmaceutical composition as claimed in any one of claims 68 to 72, wherein the composition comprises a carrier material.

74. A pharmaceutical composition as claimed in claim 73, wherein the carrier material is selected from lactic acid, polyols such as mannitol, saccharides such as glucose or lactose, or a pharmaceutically acceptable salt thereof such as sodium lactate, or an inorganic salt such as sodium chloride or calcium carbonate.

75. A pharmaceutical composition as claimed in claim 73 or 74, wherein the carrier material is present in the form of particles with a MMAD of between 1 and 5000μm.

76. A pharmaceutical composition as claimed in any one of claims 73 to 75, wherein the composition comprises up to 99% by weight of the carrier material.

77. A pharmaceutical composition as claimed in any one of claims 68 to 76, wherein the composition comprises a flavouring agent such as menthol.

78. A pharmaceutical composition as claimed in any one of claims 68 to 77, wherein the composition comprises at least 50% by weight of the A-SMase inhibitor and the NO- donor.

79. A pharmaceutical composition as claimed in claim 67, wherein the composition is suitable for use in a metered-dose inhaler.

80. A pharmaceutical composition as claimed in claim 79, wherein the composition comprises a propellant.

81. A pharmaceutical composition as claimed in claim 80, wherein the propellant is a hydrofluorocarbon such as 1,1,1,2-tetrafluoroethane (CF₃CH₂F) (HFA-134a), 1,1,1,2,3,3,3- heptafluoropropane (CF₃CHFCF₃) (HFA-227), 1,1-difluoroethane (CHF₂CH₃) (HFA- 152a), trifluoromethane (CHF₃) (HFA-23), difluoromethane (CH₂F₂) (HFA-32), fluoromethane (CH₃F) (HFA-41), 1,1,1,2,2-pentafluoroethane (CF₃CHF₂) (HFA- 125), or 1,1,2,2-tetrafluoroethane (CHF₂CHF₂) (HFA-134); a perfluorocarbon such as perfluoroethane (F₃CCF₃), tetrafluoromethane (CF₄) (PFC-14), or decafluorobutane (CF₃CF₂CF₂CF₃); a hydrochlorofluorocarbon such as monochlorodifluoromethane (ClCHF₂); a chlorofluorocarbon such as Freon 11 (CCl₃F), Freon 12 (CCl₂F₂), or Freon 114 (CF₂ClCF₂Cl); a dialkyl ether such as dimethyl ether; or a low molecular weight hydrocarbon such as »-butane, 2-butane, or propane; or a mixture thereof.

82. A pharmaceutical composition as claimed in any one of claims 79 to 81, wherein the composition comprises a lubricant, a surfactant, a polar co-solvent and/or a flavouring agent.

83. A pharmaceutical composition as claimed in any one of claims 67 to 82, wherein the A-SMase inhibitor and/or the NO-donor are present in the form of particles with a MMAD of between 0.001 and 500μm.

84. A pharmaceutical composition as claimed in any one of claims 1 to 43 or any one of claims 56 to 83, or a compound as claimed in any one of claims 44 to 53, for use as a medicament.

85. A pharmaceutical composition or a compound as claimed in claim 84 for treating or preventing a respiratory disorder.

86. A method of treating or preventing a respiratory disorder, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a pharmaceutical composition as claimed in any one of claims 1 to 43 or any one of claims 56 to 83, or of a compound as claimed in any one of claims 44 to 53.

87. A pharmaceutical composition or a compound as claimed in claim 85, or a method as claimed in claim 86, wherein the respiratory disorder is:

(a) an acute upper respiratory infection such as acute nasopharyngitis (the common cold), acute sinusitis, acute pharyngitis, acute tonsillitis, acute laryngitis, acute tracheitis, acute obstructive laryngitis, acute epiglottitis, or an acute upper respiratory infection of multiple or unspecified sites; (b) an acute lower respiratory infection such as influenza, pneumonia, acute bronchitis, acute bronchiolitis, or an unspecified acute lower respiratory infection;

(c) a non-infectious disorder of the upper respiratory tract such as vasomotor rhinitis, allergic rhinitis, chronic rhinitis, chronic nasopharyngitis, chronic pharyngitis, chronic sinusitis, nasal polyp, other disorders of the nose or nasal sinuses, chronic diseases of the tonsils or adenoids, peritonsillar abscess, chronic laryngitis, chronic laryngotracheitis, or other disorders of the vocal cords, larynx or upper respiratory tract;

(e) respiratory tuberculosis including tuberculosis of the lung, tuberculosis of the intrathoracic lymph nodes, tuberculosis of the larynx, trachea or bronchus, tuberculous pleurisy, and primary respiratory tuberculosis; (f) a lung disease due to external agents such as coal-worker's pneumoconiosis, pneumoconiosis due to asbestos or other mineral fibres, pneumoconiosis due to dust containing silica, pneumoconiosis due to other inorganic dusts, unspecified pneumoconiosis, pneumoconiosis associated with tuberculosis, airway disease due to specific organic dust, hypersensitivity pneumonitis due to organic dust, respiratory conditions due to inhalation of chemicals, gases, fumes or vapours, pneumonitis due to solids or liquids, pneumonitis due to food or vomit, or respiratory conditions due to other external agents; (g) another respiratory disease principally affecting the interstitium such as adult respiratory distress syndrome (ARDS), pulmonary oedema, pulmonary eosinophilia, or other interstitial pulmonary diseases such as alveolar or parietoalveolar conditions, other interstitial pulmonary diseases with fibrosis including idiopathic pulmonary fibrosis, other specified interstitial pulmonary diseases, or unspecified interstitial pulmonary disease; (h) a suppurative or necrotic condition of the lower respiratory tract such as abscess of the lung, abscess of the mediastinum, or pyothorax;

(i) another disease of the pleura such as pleural effusion not elsewhere classified, pleural effusion in conditions classified elsewhere, pleural plaque, pneumothorax, or other pleural conditions including chylous effusion, fibrothorax and haemothorax; ()) cystic fibrosis including mucoviscidosis, cystic fibrosis with pulmonary manifestations, cystic fibrosis with intestinal manifestations, cystic fibrosis with other manifestations, and unspecified cystic fibrosis;

(k) cough including chronic cough, psychogenic cough, cough with haemorrhage, and cough caused by poisoning such as with expectorants; (1) symptoms involving the respiratory system such as haemorrhage from respiratory passages, abnormalities of breathing including dyspnoea, stridor, wheezing, periodic breathing, hyperventilation, mouth breathing, hiccough and sneezing, pain in the throat or chest, asphyxia, pleurisy, respiratory arrest, or abnormal sputum; (m) a respiratory disorder specific to the perinatal period such as birth asphyxia, respiratory distress of newborn, congenital pneumonia, neonatal aspiration syndrome, interstitial emphysema and related conditions originating in the perinatal period, pulmonary haemorrhage originating in the perinatal period, chronic respiratory disease originating in the perinatal period, or other respiratory conditions originating in the perinatal period; (n) a respiratory neoplasm such as a malignant respiratory neoplasm including malignant neoplasms of the lip, tongue, gum, floor of the mouth, palate and other parts of the mouth, malignant neoplasms of the parotid gland, tonsil, oropharynx, nasopharynx, piriform sinus, hypopharynx and other parts of the pharynx, malignant neoplasms of the nasal cavity, middle ear and accessory sinuses, malignant neoplasms of the larynx, trachea, bronchus and lung, and Kaposi's sarcoma of the mouth and other parts of the respiratory system; an in situ respiratory neoplasm including carcinomas in situ of the lip, oral cavity, pharynx, larynx, trachea, bronchus, lung and other parts of the respiratory system; a benign respiratory neoplasm including benign neoplasms of the mouth, pharynx, salivary glands, nasal cavity, middle ear and accessory sinuses, and benign neoplasms of the larynx, trachea, bronchus, lung and other parts of the respiratory system; or a respiratory neoplasm of uncertain or unknown behaviour; or

88. A pharmaceutical composition, compound or method as claimed in claim 87, wherein the respiratory disorder is:

(a) cough including chronic cough, psychogenic cough, cough with haemorrhage, and cough caused by poisoning such as with expectorants;

(b) cystic fibrosis including mucoviscidosis, cystic fibrosis with pulmonary manifestations, cystic fibrosis with intestinal manifestations, cystic fibrosis with other manifestations, and unspecified cystic fibrosis;

(d) an interstitial pulmonary disease with fibrosis including idiopathic pulmonary fibrosis, diffuse pulmonary fibrosis, fibrosing alveolitis (cryptogenic), Hamman-

Rich syndrome, chronic pulmonary fibrosis due to inhalation of chemicals, gases, fumes or vapours, and chronic pulmonary fibrosis following radiation; or (e) emphysema including MacLeod's syndrome, panlobular emphysema, centrilobular emphysema, bullous emphysema, vesicular emphysema, emphysematous bleb, compensatory emphysema, emphysema due to inhalation of chemicals, gases, fumes or vapours, interstitial emphysema, interstitial neonatal emphysema, mediastinal emphysema, surgical (subcutaneous) emphysema, traumatic subcutaneous emphysema, emphysema with chronic (obstructive) bronchitis, and emphysematous (obstructive) bronchitis.

89. A pharmaceutical composition, compound or method as claimed in claim 88, wherein the respiratory disorder is chronic cough, cystic fibrosis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis or emphysema.

90. An A-SMase inhibitor for treating or preventing a respiratory disorder, wherein the respiratory disorder is: (a) an interstitial pulmonary disease with fibrosis including idiopathic pulmonary fibrosis, diffuse pulmonary fibrosis, fibrosing alveolitis (cryptogenic), Hamman-

Rich syndrome, chronic pulmonary fibrosis due to inhalation of chemicals, gases, fumes or vapours, and chronic pulmonary fibrosis following radiation; or

91. A method of treating or preventing a respiratory disorder, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an A- SMase inhibitor, wherein the respiratory disorder is selected from: (a) an interstitial pulmonary disease with fibrosis including idiopathic pulmonary fibrosis, diffuse pulmonary fibrosis, fibrosing alveolitis (cryptogenic), Hamman- Rich syndrome, chronic pulmonary fibrosis due to inhalation of chemicals, gases, fumes or vapours, and chronic pulmonary fibrosis following radiation; or (b) emphysema including MacLeod's syndrome, panlobular emphysema, centrilobular emphysema, bullous emphysema, vesicular emphysema, emphysematous bleb, compensatory emphysema, emphysema due to inhalation of chemicals, gases, fumes or vapours, interstitial emphysema, interstitial neonatal emphysema, mediastinal emphysema, surgical (subcutaneous) emphysema, traumatic subcutaneous emphysema, emphysema with chronic (obstructive) bronchitis, and emphysematous (obstructive) bronchitis.

92. An A-SMase inhibitor as claimed in claim 90 or a method as claimed in claim 91, for treating or preventing idiopathic pulmonary fibrosis or emphysema.

93. An A-SMase inhibitor or a method as claimed in any one of claims 90 to 92, wherein the A-SMase inhibitor is administerable by inhalation.

94. An A-SMase inhibitor or a method as claimed in any one of claims 90 to 93, wherein the A-SMase inhibitor is as defined in any one of claims 2 to 37.

95. Amitriptyline or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof, for treating or preventing a chronic obstructive pulmonary disease such as chronic asthmatic bronchitis, chronic emphysematous bronchitis, chronic bronchitis with airways obstruction, chronic bronchitis with emphysema, chronic obstructive asthma, chronic obstructive bronchitis, chronic tracheobronchitis, chronic obstructive pulmonary disease with acute lower respiratory infection, chronic obstructive pulmonary disease with acute exacerbation (unspecified), other specified chronic obstructive pulmonary diseases, or chronic obstructive pulmonary disease (unspecified).

96. A method of treating or preventing a chronic obstructive pulmonary disease such as chronic asthmatic bronchitis, chronic emphysematous bronchitis, chronic bronchitis with airways obstruction, chronic bronchitis with emphysema, chronic obstructive asthma, chronic obstructive bronchitis, chronic tracheobronchitis, chronic obstructive pulmonary disease with acute lower respiratory infection, chronic obstructive pulmonary disease with acute exacerbation (unspecified), other specified chronic obstructive pulmonary diseases, or chronic obstructive pulmonary disease (unspecified), the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of amitriptyline or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof.

97. Amitriptyline or a pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof as claimed in claim 95, or a method as claimed in claim 96, wherein the amitriptyline or pharmaceutically acceptable tautomer, salt, prodrug or hydrate thereof is administerable by inhalation.

98. An A-SMase inhibitor for treating or preventing a respiratory disorder by inhalation, wherein the respiratory disorder is:

99. A method of treating or preventing a respiratory disorder, comprising administering to a subject in need thereof by inhalation a therapeutically or prophylactically effective amount of an A-SMase inhibitor, wherein the respiratory disorder is selected from: (a) cough including chronic cough, psychogenic cough, cough with haemorrhage, and cough caused by poisoning such as with expectorants; or (b) cystic fibrosis including mucoviscidosis, cystic fibrosis with pulmonary manifestations, cystic fibrosis with intestinal manifestations, cystic fibrosis with other manifestations, and unspecified cystic fibrosis.

100. An A-SMase inhibitor as claimed in claim 98, or a method as claimed in claim 99, for treating or preventing chronic cough or cystic fibrosis.

101. An A-SMase inhibitor or a method as claimed in any one of claims 98 to 100, wherein the A-SMase inhibitor is as defined in any one of claims 2 to 37.

102. A NO-donor for treating or preventing cough including chronic cough, psychogenic cough, cough with haemorrhage, and cough caused by poisoning such as with expectorants.

103. A method of treating or preventing cough, including chronic cough, psychogenic cough, cough with haemorrhage, and cough caused by poisoning such as with expectorants, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a NO-donor.

104. A NO-donor as claimed in claim 102, or a method as claimed in claim 103, for treating or preventing chronic cough.

105. A NO-donor for treating or preventing a respiratory neoplasm.

106. A method of treating or preventing a respiratory neoplasm, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a NO-donor.

107. A NO-donor as claimed in claim 105, or a method as claimed in claim 106, for treating or preventing:

(c) a benign respiratory neoplasm including benign neoplasms of the mouth, pharynx, salivary glands, nasal cavity, middle ear and accessory sinuses, and benign neoplasms of the larynx, trachea, bronchus, lung and other parts of the respiratory system; or

(d) a respiratory neoplasm of uncertain or unknown behaviour.

108. A NO-donor or a method as claimed in any one of claims 102 to 107, wherein the NO-donor is administerable by inhalation.

109. A NO-donor or a method as claimed in any one of claims 102 to 108, wherein the NO-donor is as defined in any one of claims 38 to 43.

110. An inhaler comprising a pharmaceutical composition as claimed in any one of claims 1 to 43 or any one of claims 56 to 83, or comprising a compound as claimed in any one of claims 44 to 53.

111. An inhaler as claimed in claim 110, for use in the treatment or prevention of a respiratory disorder.

112. A foil blister or a single dose capsule comprising a pharmaceutical composition as claimed in any one of claims 1 to 43 or any one of claims 56 to 83, or comprising a compound as claimed in any one of claims 44 to 53.

113. A method of inhibiting A-SMase comprising the use of an A-SMase inhibitor and/or a NO-donor.

114. A method as claimed in claim 113, wherein the A-SMase inhibitor is as defined in any one of claims 2 to 37 and/or wherein the NO-donor is as defined in any one of claims 38 to 43.

115. An in vitro assay for measuring the effect of one or more compounds upon the activity of A-SMase.

116. An in vitro assay as claimed in claim 115, wherein the one or more compounds is an A-SMase inhibitor and/or a NO-donor.

117. An in vitro assay as claimed in claim 116, wherein the A-SMase inhibitor is as defined in any one of claims 2 to 37.

118. An in vitro assay as claimed in claim 116 or 117, wherein the N O -donor is as defined in any one of claims 38 to 43.

119. An in vitro assay as claimed in any one of claims 116 to 118, wherein the A-SMase inhibitor is a tricyclic antidepressant and the NO-donor is arginine.

120. An in vitro assay as claimed in claim 119, wherein the tricyclic antidepressant is amitriptyline.

121. An in vitro assay as claimed in any one of claims 115 to 120, wherein the assay is a cell-based assay and the cells are human cells.

122. An in vitro assay as claimed in claim 121, wherein the cells are human colon adenocarcinoma cells.

123. An in vitro assay as claimed in claim 121 or 122, comprising activating endogenous A-SMase by exposing the cells to an inducing agent.

124. An in vitro assay as claimed in claim 123, wherein the inducing agent does not induce the activity of endogenous iNOS, and more preferably suppresses endogenous iNOS expression.

125. An in vitro assay as claimed in claim 123 or 124, wherein the inducing agent is sodium butyrate.