TECHNICAL FIELD

This invention relates to a combination for simultaneous, separate or sequential therapy comprising a therapeutically effective amount of (a) one or more bile acids and/or bile salts, (b) a compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety and (c) a phosphodiesterase inhibitor. This invention also relates to uses and methods of using this combination in preventing or treating a microbial infection or a chronic inflammatory condition.

BACKGROUND

The integrity of immune system to combat microbial infections in the first instance (otherwise known as innate immunity) depends on a number of factors. Firstly, an appropriate response of white cells (neutrophils and polymorphs) is required. In particular, the production of toxic granulations within the white cells, enabling the cells to destroy the offending invaders, is of significant importance. Digestion of microbes through phagocytosis, mediated by macrophages, is also an important mechanism for fighting infections.

The production of toxic granules within the white cells in turn is dependent on functioning mitochondrial activity to provide the energy source. Functioning mitochondria are also of importance to enable optimum lysosome activity, this organelle being responsible for fusing with phagosomes after the phagosome has engulfed a microbe and resulting in the digestion of the microbe. This is discussed in Baixauli et al. (2015) Cell Metab., 22, pp.485- 98.

Firstly, there is evidence that the plasma component of whole blood also has anti-microbial properties. This inhibiting activity in part may be due to surface tension of plasma. Bile acids and bile salts, which are natural surfactants, contribute to lowering of plasma surface tension and inhibit bacterial and virus replication, as discussed in Yadav et al. (2020) J. Med. Eng. Tech., 44, pp.227-236 and Kumar et al. (2021) Future Virol., 15, pp 779-782. Whilst bile acids are made by the liver naturally, it has been reported that bile acid levels can deteriorate in subjects suffering from an infection or a chronic inflammatory disease, and it is thought that this is due to the condition itself affecting the diet of the subjects concerned.

Secondly, nicotinamide adenine dinucleotide (NAD, NAD ⁺ or NADH) is a fundamental housekeeping molecule that catalyses electron transfer within mitochondria. Silent Information Regulator (SIRT) activity is also dependent on NAD activity. It has been hypothesised that patients with a deficiency in NAD are more likely to experience worse symptoms from an infection compared to patients with healthy levels of NAD. Miller et al. (2020) Medical Hypotheses, 144, 110044 and Radenkovic et al. (2020), Pharmaceuticals, 13, 247 discuss such a hypothesis in relation to COVID-19 infections.

Thirdly, it has been reported that endothelial cells which form the inner layer of blood vessels have an important role to play in relation to fighting an infection, and dysfunction of these cells can participate in the pathogenesis of an infection, such as a COVID-19 infection, through altering the integrity of the vessel barrier, promoting a pro-coagulative state, inducing endothelial inflammation, and even mediating leukocyte infiltration, as discussed in Jin et al. (2020) Signal Transd. Tar. Therapy, 5, 293. In this regard, it has been shown that phosphodiesterase inhibitors are effective at maintaining endothelial function and could be beneficial in preventing and treating infections such as a COVID-19 infection, as discussed in Giorgi et al. (2020) Int. J. Mol. Sci., 21, 5338. Inhibitors of phosphodiesterase 5 has been shown to be particularly effective in maintaining normal endothelial function, particularly through activating endothelial nitric oxide synthase (as well as inducible nitric oxide synthase present in dendritic cells, T cells and neutrophils), leading to an increase in nitric oxide and a favourable immune response, as discussed in Kniotek & Boguska (2017) J Immunol. Res., Article ID: 4541958.

Whilst the lack of toxic granule production, the increase of surface tension within blood plasma and the dysfunction of the endothelial cells have all been reported separately to contribute to a poor physiological response to a microbial infection, no reports have investigated the benefit of addressing all of these issues simultaneously.

There is therefore a need for effective therapies that are able to prevent or treat microbial infections and chronic inflammation.

SUMMARY OF THE INVENTION

The inventor has surprisingly found that the combination of (a) one or more bile acids and/or bile salts, (b) a compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety and (c) a phosphodiesterase inhibitor works in synergy to treat microbial infections where one of these components in isolation, or even any two of these components, are unable to lead to the same result.

According to a first aspect, the present invention provides a combination for simultaneous, separate or sequential therapy comprising a therapeutically effective amount of (a) one or more bile acids, bile salts or derivatives thereof; (b) a compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety, wherein the compound has a molecular mass of less than 1000 g-mol’ ¹ ; and (c) a phosphodiesterase inhibitor or pharmaceutically acceptable salt thereof. In a further aspect, the present invention provides a pharmaceutical pack comprising (a) one or more bile acids, bile salts or derivatives thereof, (b) a compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety and (c) a phosphodiesterase inhibitor or pharmaceutically acceptable salt thereof as described herein.

In a further aspect, the present invention provides a pharmaceutical composition comprising (a) one or more bile acids, bile salts or derivatives thereof, (b) a compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety and (c) a phosphodiesterase inhibitor or pharmaceutically acceptable salt thereof as described herein.

The present invention is characterised by the novel combination of the three components (the "triple therapy") that had not previously been combined for any medical indication. This invention shows that the triple therapy leads to a surprising increase in toxic granulation and phagocytosis, and through at least these mechanisms is able to clear microbial infections.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to", and do not exclude other components, integers or steps. Moreover the singular encompasses the plural unless the context otherwise requires: in particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Within the scope of this application it is expressly intended that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, in the claims and/or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and/or features of any embodiment can be combined in any way and/or combination, unless such features are incompatible.

BRIEF DESCRIPTION OF THE FIGURES

One or more embodiments of the invention will be described, by way of example only, with reference to the accompanying figures, in which:

Figure 1 presents images of Escherichia co I i -infected whole blood cell samples before or sixty minutes after treatment with the combination of nicotinamide adenine dinucleotide at 100 pmol/L, ursodeoxycholic acid at 100 pmol/L and tadalafil at 100 pg/L. Before treatment no toxic granulation or phagocytosis is seen, whilst sixty minutes after treatment these activities are visible. Figure 2 presents images of infected whole blood cell samples before or sixty minutes after treatment with the combination of nicotinamide adenine dinucleotide at 100 pmol/L, ursodeoxycholic acid at 100 pmol/L and tadalafil at 100 pg/L. Before treatment no toxic granulation or phagocytosis is seen, whilst sixty minutes after treatment these activities are visible. In the "toxic granulation after treatment" column, the image show toxic granulation after both Staphylococcus aureas and E coli. In the "phagocytosis after treatment" column, the top and bottom images shows phagocytosis after infection with S aureas and the middle image shows phagocytosis after infection with E. coli.

Figure 3 presents images of infected whole blood cell samples with both E. coli and S. aureas. A shows a stained neutrophil before the samples were infected. B shows a large increase in bacteria after two hours at 37°C and after a further twenty four hours at ambient room temperature without any treatment. C shows evidence of toxic granulation two hours after treatment with the combination of nicotinamide adenine dinucleotide at 100 pmol/L, ursodeoxycholic acid at 100 pmol/L and tadalafil at 100 pg/L at 37°C. D shows evidence of phagocytosis after treatment with this combination at 37°C. E shows almost complete clearance of the bacteria when treated with this combination and then incubated for two hours at 37°C and after a further twenty-four hours at ambient room temperature.

Figure 4 presents images of whole blood cell samples infected with either E. coli or S. aureas. A shows a stained neutrophil before the samples were infected. B shows a large increase in toxic granulation after two hours at 37°C in uninfected blood after the application of the combination of nicotinamide adenine dinucleotide at 100 pmol/L, ursodeoxycholic acid at 100 pmol/L and tadalafil at 100 pg/L. C shows evidence of phagocytosis two hours after treatment of this combination at 37°C in E. coli -infected whole blood. D shows evidence of phagocytosis two hours after treatment of this combination at 37°C in S. aureus- infected whole blood. E shows evidence of phagocytosis with neutrophil collaboration two hours after treatment of this combination at 37°C in E. coli -infected whole blood. F shows evidence of phagocytosis with neutrophil collaboration two hours after treatment of this combination at 37°C in S. aureus- infected whole blood. It is noted that phagocytosis takes place against one bacterial species when in a group.

DETAILED DESCRIPTION

The present invention relates to how, surprisingly, the combination of following three components leads to synergistic antimicrobial and anti-inflammatory effects: a. one or more bile acids, bile salts or derivatives thereof; b. a compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety, wherein the compound has a molecular mass of less than 1000 g-mol’ ¹ ; and c. a phosphodiesterase inhibitor or pharmaceutically acceptable salt thereof.

This is referred to herein as the "triple therapy".

A combination for simultaneous, separate or sequential therapy

The term "combination" is used herein to cover scenarios where all or some of the components of the triple therapy are within the same solution, mixture or formulation, such as a pharmaceutical composition, but also scenarios where all or some of the components of the triple therapy are in separate solutions, mixtures or formulations, such as in a pharmaceutical pack. It is envisaged that two of the components of the triple therapy may be within the same solution, mixture or formulation with the third component in a separate solution, mixture or formulation. Preferably all components of the triple therapy are within the same solution, mixture or formulation, such as a pharmaceutical composition.

It is understood that the synergistic effects of the triple therapy would not only occur when all components of the triple therapy are administered simultaneously (as would be the case if all of the components of the triple therapy are within the same solution, mixture or formulation, for example) but also when the components are administered separately or sequentially. Preferably, when the components are administered separately or sequentially, the second and third components of the triple therapy are administered before the first component has been cleared from the body, and so preferably all three components are administered within a twenty-four-hour period, more preferably a twelve hour period, more preferably a six hour period, more preferably a four hour period. Preferably, all three components are administered simultaneously or immediately sequentially, i.e. with little or no break between administration of each component.

One or more bile acids, bile salts or derivatives thereof

Bile acids are known in the art as steroid acids found predominantly in the bile produced by the liver that aid the digestion of lipids in the small intestine. But bile acids and their salts have far-reaching effects beyond the small intestines and, as discussed above, are known to lower the surface tension of blood plasma through the basic surfactant properties of the molecules, which in turn improves the plasma's ability to fight infections and reduce inflammation. Based on this, it is considered that bile acids and bile salt derivatives that are able to reduce this blood plasma surface tension (in addition to unmodified bile acids and bile salts) would be effective also and fall within the scope of the claimed invention. Blood plasma surface tension is routinely measured using, for example, a tensiometer, and so it would be straightforward for the skilled person to determine which derivatives led to a decrease in the surface tension of the blood plasma and so fell within the scope of the claims and which did not. Such studies can be carried out in vitro where the bile acid or bile salt derivative is directly applied to blood samples taken from a subject, or in vivo where the bile acid or bile salt derivative is administered to a subject and then blood samples are taken and tested from that subject.

Bile acids synthesised in the liver are known as primary bile acids. In humans, the main primary bile acids are cholic acid and chenodeoxycholic acid, although children with infections have also been reported to produce o-muricholic acid and p-muricholic acid also, suggesting that these muricholic acids are of antimicrobial importance. In other mammals, such as mice, ursodeoxycholic acid is synthesised, and this bile acid has been shown extensively to be effective in humans also. Prior to secretion of these bile acids, liver cells conjugate them with either a taurine group, , or a glycine group, Taurine salts of any of any bile acids, preferably any of cholic acid, chenodeoxycholic acid, o-muricholic acid, p-muricholic acid, co-muricholic acid and ursodeoxycholic acid, and glycine salts of any bile acids, preferably any of cholic acid, chenodeoxycholic acid, o-muricholic acid, p-muricholic acid, co-muricholic acid and ursodeoxycholic acid fall within the scope of the invention.

Once secreted into the lumen of the intestine, the bile salts are partially dehydroxylated by gut bacteria to give secondary bile acids. Such secondary bile acids in humans include deoxycholic acid, iso-deoxycholic acid, lithocholic acid, murideoxycholic acid, iso-lithocholic acid and ursocholic acid. Additional secondary bile acids in animals other than human, such as mice, include co-muricholic acid, hyocholic acid, hyodeoxycholic acid and murideoxycholic acid. These bile acids are recycled through enterohepatic circulation.

In addition to natural bile acids and bile salts described above, and derivatives thereof, the term bile acids, bile salts and derivatives thereof as used herein is also intended to encompass synthetic compounds, such as semi-synthetic bile acids. Examples are described in Lazarevic et al. (2019) Pharmacol. Res., 146, 104333. These include 7o-hydroxy-5p- cholan-24-sulfate, 6o-ethyl-3a,7o-dihydroxy-24-nor-5p-cholan-23-ol, 3p-azido-6o-ethyl-7o- hydroxy-5p-cholan-24-oic acid, 3o-amino-5p-cholane-24-ol, 3o,7p-dihydroxy-25,26-bis- homo-5p- cholan-26-oic acid, 6p-ethyl-3o,7p-dihydroxy-5p-cholan-24-ol and 6o-ethyl- 3o,7o-dihydroxy-5p-cholan-24-yl-24-sodium sulfate. This article teaches that sulfate salts, as well as taurine and glycine salts, can be effective also.

In one embodiment of the claimed invention, the one or more bile acids, bile salts or derivatives thereof is of general Formula (I): optionally wherein one, two, three or four hydroxyl, ethyl, amine or azide groups, preferably hydroxyl groups, are present on rings A, B and/or C at one, two, three or four of positions 3, 6, 7 and/or 12, preferably positions 3 and 7, and wherein the radical R ¹ is selected from hydrogen, a methanoic acid group, a hydroxyl group, bile salts). In a preferred embodiment, no more than one non-hydrogen group is present at any one position. In a preferred embodiment, R ¹ is a methanoic acid group and an a hydroxyl group is present at position 3. In a further preferred embodiment, R ¹ is a methanoic acid group and a hydroxyl group is present at positions 3 and 7.

It is noted that positions 3, 6, 7 and 12 are chiral centres once one non-hydrogen group (i.e. an hydroxyl, ethyl, amine or azide group) is present. Where a non-hydrogen group is present, that non-hydrogen group may be in either the R or S configuration, as it is appreciated that this would have little effect on the surfactant properties of the molecule. In one embodiment, the one or more bile acids, bile salts or derivatives thereof is selected from the group consisting of ursodeoxycholic acid, cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, hyodeoxycholic acid, ursocholic acid, hyocholic acid, isodeoxycholic acid, o-muricholic acid, p-muricholic acid, co-muricholic acid, murideoxycholic acid, 7o-hydroxy-5p-cholan-24-sulfate, 6o-ethyl-3o,7a-dihydroxy-24-nor-5p-cholan-23-ol, 3p-azido-6a-ethyl-7a-hydroxy-5p-cholan-24-oic acid, 3o-amino-5p-cholane-24-ol, 3o,7g- dihydroxy-25,26-bis-homo-50- cholan-26-oic acid, 6p-ethyl-3o,7p-dihydroxy-5p-cholan-24- ol and 6a-ethyl-3o,7a-dihydroxy-50-cholan-24-yl-24-sodium sulfate, taurine salts thereof, glycine salts thereof and sulfate salts thereof, preferably ursodeoxycholic acid. All of these molecules fall within the structural definition of Formula (I) above, indicating that there is a high level of structural similarity amongst bile acids in general.

It is envisaged that the use of more than one bile acid, bile salt or derivatives thereof could be effective in the triple therapy also. This is because in paediatrics with septicaemia, production of o-muricholic acid and p-muricholic acid in addition to cholic acid and chenodeoxycholic acid is seen, and as children are known to cope particularly well with septicaemia it is thought that this mixture of numerous bile acids could be beneficial in fighting infections. In one embodiment, the triple therapy comprises two or more bile acids, bile salts or derivatives thereof, preferably two bile acids, bile salts or derivatives thereof. A preferable combination of bile acids is ursodeoxycholic acid with a muricholic acid (o- muricholic acid, p-muricholic acid or co-muricholic acid).

A compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety

One of the components of the triple therapy is a compound comprising a nicotinamide and an adenosine moiety, wherein the compound has a molecular mass of less than 1000 g-mol’ ¹ . In a preferred embodiment, the compound comprises a nicotinamide moiety or a nicotinic acid moiety. Preferably the compound is nicotinamide adenine dinucleotide (NAD, also known as NAD ⁺ and NADH) or a precursor thereof.

As discussed above, NAD is a fundamental mitochondrial housekeeping molecule. NAD consists of two nucleotides joined through their phosphate groups. One nucleotide comprises an adenine nucleobase and the other nucleotide comprises a nicotinamide nucleobase.

It is known in the art that physiologically NAD can be increased not only through administration of NAD itself, but also by NAD precursors that form the structural components of NAD, such as nicotinamide and adenosine-containing compounds. Nicotinic acid (also known as niacin or vitamin B3) is known to be an important nutrient that is used physiologically to form NAD. For this reason, this component of the triple therapy covers not only NAD itself, but compounds that act as NAD precursors. In one embodiment, the compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety is NAD or a precursor thereof, preferably NAD. In a further embodiment, the compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety is selected from the listed consisting of nicotinamide adenine dinucleotide, nicotinamide, nicotinic acid, nicotinamide mononucleotide, nicotinamide riboside and flavin adenine binucleotide, preferably nicotinamide adenine dinucleotide.

The compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety has a molecular mass of less than 1000 g-mol' ¹ . In a preferred embodiment, the compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety has a molecular mass of less than 900 g-mol' ¹ . In a further preferred embodiment, the compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety has a molecular mass of less than 800 g-mol' ¹ . In a further preferred embodiment, the compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety has a molecular mass of less than 700 g-mol' ¹ . salt thereof

As discussed above, it is known that phosphodiesterase inhibitors can play an important role in maintaining endothelial function. In this regard, it is predicted that inhibition of any of the phosphodiesterase families (of which there are eleven, numbered numerically 1 to 11), despite the significant variation in substrate specificity, can be beneficial in maintaining endothelial function, and so all phosphodiesterase inhibitors are covered within the scope of the present invention.

Phosphodiesterases are enzymes that have been known to be of importance clinically for decades, and the chemical structures necessary to inhibit the enzyme have been extensively researched. As such, off-the-shelf kits for assaying phosphodiesterase activity (and inhibition of said activity) are readily available from companies such as Abeam. As such, it is very straightforward for the skilled person to determine whether a compound inhibits phosphodiesterase and therefore may be a suitable phosphodiesterase inhibitor to be used in this triple therapy.

There are many medicines that have been clinically proven or being developed currently to provide a therapeutic effect through phosphodiesterase inhibition. Thus, in one embodiment, the phosphodiesterase inhibitor of the present invention is selected from the group consisting of medicines for erectile dysfunction, bronchodilators, medicines for analgesia associated with peripheral artery disease, medicines for cardiac failure, medicines for essential thrombocytosis, medicines for intermittent claudication, medicines for heart failure, medicines for analgesia associated with renal colic, medicines for hastening cervical dilatation in labour, medicines for alleviating the symptoms associated with chronic obstructive pulmonary disease, medicines for psoriasis, medicines for psoriatic arthritis, vasodilators, blood-thinning medicines and medicines for treating vasospasm, preferably medicines for erectile dysfunction. In a further embodiment, the phosphodiesterase inhibitor is selected from the group consisting of tadalafil, sildenafil, vardenafil, udenafil, avanafil, mirodenafil, lodenafil, dipyridamole, caffeine, aminophylline, 3-isobutyl-l-methylxanthine, paraxanthine, pentoxifylline, theobromine, theophylline, erythro-9-(2-hydroxy-3- nonyl)adenine, 2-[(3,4-dimethoxyphenyl)methyl]-7-[(lR)-l-hydroxyethyl]-4-ph enylbutyl]- 5-methyl-imidazo[5,l-f][l,2,4]triazin-4(lH)-one, oxindole, 9-(6-Phenyl-2-oxohex-3-yl)-2- (3,4-dimethoxybenzyl)-purin-6-one, inamrinone, milrinone, enoximone, anagrelide, cilostazol, pimobendan, mesembrenone, rolipram, ibudilast, piclamilast, luteolin, drotaverine, reflumilast, apremilast, crisaborole, papaverine and pharmaceutically acceptable salts thereof, preferably tadalafil.

The following medicines have obtained marketing authorization either from the Food and Drug Administration (FDA) or the Medicines and Healthcare products Regulatory Agency (MHRA) for therapeutic use through the inhibition of phosphodiesterase: tadalafil, sildenafil, vardenafil, udenafil, avanafil, mirodenafil, lodenafil, dipyridamole, aminophylline, pentoxifylline, theophylline, inamrinone, milrinone, enoximone, anagrelide, cilostazol, pimobendan, ibudilast, drotaverine, roflumilast, apremilast and papaverine. Thus, in one embodiment the phosphodiesterase inhibitor is selected from the list above and pharmaceutically acceptable salts thereof, preferably tadalafil.

Preferably the phosphodiesterase inhibitor is an inhibitor of phosphodiesterase 5. The following medicines have obtained marketing authorization either from the FDA or the MHRA for therapeutic use through the inhibition of phosphodiesterase 5: tadalafil, sildenafil, vardenafil, udenafil, avanafil, mirodenafil, lodenafil and dipyridamole. Thus, in one embodiment the phosphodiesterase inhibitor is selected from the list above and pharmaceutically acceptable salts thereof, preferably tadalafil.

Therapeutic indications

In one aspect, the present invention provides the triple therapy for use in therapy. Preferably the subject being treated is a human. In one embodiment, the triple therapy is for use in the prevention or treatment of one or more medical conditions selected from the list consisting of a microbial infection, preferably a bacterial or viral infection, and a chronic inflammatory condition.

As discussed above, the triple therapy works through the surprisingly effective combination of components that decrease the surface tension of blood plasma, improve mitochondrial function and maintain endothelial function. All of these components work to optimise the host immune system to fight an infection, the result being either reducing or removing altogether the signs and symptoms associated with an infection (in the case where the triple therapy is being used to treat an infection) or the subject experiencing minimal or no signs or symptoms (in the case of the triple therapy being used prophylactically). This is clearly different to many traditional antibiotic or antiviral therapies that fight infections through direct effects against the invading pathogen. Furthermore, the present examples show that the triple therapy leads to a noticeable increase in toxic granulation (both in infected blood and in non-infected blood) and phagocytosis, and it is readily known that these immune system mechanisms act against not only specific forms of pathogens, but instead against pathogens in general. Based on this, it is clear that the triple therapy would be effective not only against a specific form of pathogen, but against all types of pathogen and fragments thereof. Here, it is noted that activation of neutrophils, an essential part of the innate immune system, is of great importance in reducing the severity of signs and symptoms of patients suffering from respiratory viral infections (Johansson & Kirsebom (2021) Mucosal Immunol., 14, pp 815-27), in particular COVID-19 infections (Hazeldine & Lord (2021) Front. Immunol., 12, 680134), and this triple therapy can assist in such neutrophil activation (as indicated through, for example, an increase in toxic granulation). Also, as this triple therapy works on boosting the innate immune system rather than acting on the microbes directly, it is logical that this therapy would not suffer issues of the microbes evolving resistance against the triple therapy as can be seen with traditional antimicrobials.

The triple therapy may be combined with further antimicrobials, particularly antibiotics and antiviral medicines. As discussed above, the triple therapy does not act on the microbes directly, but instead boosts the immune system, and therefore, without wishing to be bound by theory, it is predicted that the triple therapy could be complementary to traditional antimicrobial treatments and so assist in preventing or treating infections that are hard to treat with antimicrobials alone. Where further antimicrobials are present, these may be administered within the same formulation as one or more of the components of the triple therapy, or alternatively these may be administered separately.

Inflammation is a common host response to infections, and chronic inflammation is when the destroyed this response lingers after an infection, leaving the host in a constant state of alert. Whilst inflammatory is often a beneficial immune response, chronic inflammation can have negative effects on the host. Diseases associated with chronic inflammation include arthritis, inflammatory bowel disease, multiple sclerosis, dementia and Alzheimer's disease. There are also conditions that make patients more prone to chronic inflammation, such as obesity and diabetes mellitus. Without wishing to be bound by theory, based on the activities of the three components of the triple therapy as discussed above it is predicted that this therapy could be used to "reset" the immune system and in effect remove the inflammatory triggers, and so either treat or prevent diseases such as arthritis, preferably rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, dementia and Alzheimer's disease, and/or treat or prevent the inflammatory implications associated with obesity and diabetes mellitus, preferably type 2 diabetes mellitus.

The triple therapy may be combined with further medicines used to treat the chronic inflammatory conditions or implications discussed above. As with the antimicrobials discussed above, and wishing to be bound by theory, it is predicted that the triple therapy could be complementary to traditional medicines used to treat the chronic inflammatory conditions or implications and so assist in preventing or treating the conditions or implications that are hard to treat with the medicines currently available. Where further medicines used to treat the chronic inflammatory conditions or implications are present, these may administered within the same formulation as one or more of the components of the triple therapy, or alternatively these may be administered separately.

The triple therapy would be administered at a therapeutically effect amount. This is the amounts of each component which, in combination, are able to treat or prevent the therapeutic indication in a subject, such as a microbial infection or a chronic inflammatory condition. With respect to the one or more bile acids, bile salts or derivatives thereof or the compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety, at least some of these compounds occur naturally in the body, and so a therapeutically effective amount may be the amount necessary to achieve a healthy physiological blood concentration in the subject. However, it may also be beneficial to increase the blood concentration to beyond healthy physiological levels. By contrast, it may also be beneficial to increase the blood concentration not quite to healthy physiological levels. Also, at least some of these compounds are dietary nutrients and so a therapeutically effect amount may be a recommended dietary allowance (RDA), although again it may also be beneficial to increase the dose to beyond the RDA, or to deliver a dose that is not quite as high as the RDA.

Phosphodiesterase inhibitors do not occur naturally. With respect to the phosphodiesterase inhibitors that have been clinically proven or are being developed currently to provide a therapeutic effect through phosphodiesterase inhibition described above, the therapeutically effective amount may be the same as the dose administered for the condition that it was originally developed for. The dosage may be less than the dose administered for the condition that it was originally developed for (for example less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10% of the dose administered for the condition that it was originally developed for), but taken at more regular intervals over a longer period of time compared to what was originally intended, this being particularly the case for medicines with a pro re nata, non-scheduled dosing such as medicines used to treat erectile dysfunction or hastening cervical dilatation in labour.

In a further aspect, the present invention provides one or more bile acids, bile salts or derivatives thereof as described herein for use in therapy by simultaneous, separate or sequential administration with a compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety as described herein and a phosphodiesterase inhibitor or pharmaceutically acceptable salt thereof as described herein.

In a further aspect, the present invention provides a compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety as described herein for use in therapy by simultaneous, separate or sequential administration with one or more bile acids, bile salts or derivatives thereof as described herein and a phosphodiesterase inhibitor or pharmaceutically acceptable salt thereof as described herein.

In a further aspect, the present invention provides a phosphodiesterase inhibitor or pharmaceutically acceptable salt thereof as described herein for use in therapy by simultaneous, separate or sequential administration with one or more bile acids, bile salts or derivatives thereof as described herein and a compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety as described herein.

In a further aspect, the present invention provides a use of a combination, pharmaceutical pack or pharmaceutical composition as described herein in the manufacture of a medicament for the prevention or treatment of one or more medical conditions selected from the list consisting of a microbial infection, preferably a bacterial or viral infection, and a chronic inflammatory condition. In a further aspect, the present invention provides a method of preventing or treating of one or more medical conditions selected from the list consisting of a microbial infection, preferably a bacterial or viral infection, and a chronic inflammatory condition, comprising the step of simultaneous, separate or sequential administration of a therapeutically effective amount of (a) one or more bile acids, bile salts or derivatives thereof as described herein, (b) a compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety as described herein and (c) a phosphodiesterase inhibitor or pharmaceutically acceptable salt thereof as described herein.

Formulation and route of delivery

While it is possible for the components of the triple therapy to be administered as pure compounds, it is preferable to present them as pharmaceutical formulations, i.e. incorporating one or more pharmaceutically acceptable diluents, carriers or excipients.

The components of the triple therapy may be presented, for example, in a form suitable for oral, topical, nasal, transdermal, parenteral, intravenal, intramuscular, subcutaneous or rectal administration, preferably oral administration. Administration by an oral route may be in the form of one or more tablets, capsules, solutions or suspensions. As discussed above, whilst the components may all be within the same formulation for simultaneous administration, the components may also be in separate formulations, and in this case the components may be administered using varying routes of administration. Preferably the components are all within the same formulation suitable for oral administration.

Where the components are in the form of a pharmaceutical pack, preferably the pack comprises the components all in a formulation suitable for oral administration. The pharmaceutical pack can also comprise instructions for administration.

The course of treatment will depend on the indication for which the triple therapy is being given. For the treatment of a microbial infection, administration of all three components may be at least daily (for example one a day, twice a day, three times a day or four times a day, preferably three times a day) for a course of up to two weeks, for example one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, thirteen days or fourteen days. Alternatively, the triple therapy may be administered prophylactically, for example pre-operatively, or for patients with a weakened immune system, or for subjects at risk to becoming infected such as healthcare and service workers during an epidemic or pandemic. Administration for prophylactic treatment may be at least daily over a continuous period where the subject is at risk of an infection. Similarly, for the treatment or prophylaxis of a chronic inflammatory condition as described above, administration may be at least daily over a continuous period where a subject is either suffering from or is at risk of developing the chronic inflammatory condition.

In one embodiment, the one or more bile acids, bile salts or derivatives thereof has daily oral dose of between 300 mg and 6 g, preferably between 600 mg and 2,400 mg, more preferably between 600 mg and 1,200 mg. As discussed above, this dose may be administered once in a day, but preferably this dose is administered in three instalments per day (i.e. of between 100 mg and 2 g, preferably between 200 mg and 800 mg, more preferably between 200 mg and 400 mg). This is considered a high dose, to be administered for acute infection or inflammation for up to a week, i.e. one day, two days, three days, four days, five days, six days or seven days. A lower daily oral maintenance dose of between 100 mg and 2 g, preferably between 200 mg and 800 mg, more preferably between 200 mg and 400 mg could then be administered. This maintenance dose could also be used to treat chronic inflammation or used prophylactically. Where used to treat chronic inflammation administration would typically be for between two weeks and two months, preferably between three weeks and six weeks, more preferably one month.

In one embodiment, the compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety has daily oral dose of between 50 mg and 1 g, preferably between 100 mg and 800 mg, more preferably between 200 mg and 400 mg. As discussed above, this dose may be administered once in a day, but preferably this dose is administered in three instalments per day (i.e. of between 17 mg and 333 mg, preferably between 33 mg and 267 mg, more preferably between 67 mg and 133 mg). This is considered a high dose, to be administered for acute infection or inflammation for up to a week, i.e. one day, two days, three days, four days, five days, six days or seven days. A lower daily oral maintenance dose of between 17 mg and 333 mg, preferably between 33 mg and 267 mg, more preferably between 67 mg and 133 mg could then be administered. This maintenance dose could also be used to treat chronic inflammation or used prophylactically. Where used to treat chronic inflammation administration would typically be for between two week and two months, preferably between three weeks and six weeks, more preferably one month.

In one embodiment, the phosphodiesterase inhibitor or pharmaceutically acceptable salt thereof has daily oral dose of between 25 mg and 500 mg, preferably between 50 mg and 400 mg, more preferably between 100 mg and 200 mg. As discussed above, this dose may be administered once in a day, but preferably this dose is administered in three instalments per day (i.e. of between 8 mg and 167 mg, preferably between 17 mg and 133 mg, more preferably between 33 mg and 67 mg). This is considered a high dose, to be administered for acute infection or inflammation for up to a week, i.e. one day, two days, three days, four days, five days, six days or seven days. A lower daily oral maintenance dose of between 8 mg and 167 mg, preferably between 17 mg and 133 mg, more preferably between 33 mg and 67 mg could then be administered. This maintenance dose could also be used to treat chronic inflammation or used prophylactically. Where used to treat chronic inflammation administration would typically be for between two week and two months, preferably between three weeks and six weeks, more preferably one month.

In a preferred embodiment, the triple therapy comprises 600 mg of the one or more bile acids, bile salts or derivatives thereof, 100 mg of the compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety and 50 mg of the phosphodiesterase inhibitor or pharmaceutically acceptable salt thereof in a single tablet or capsule for oral administration. Preferably this tablet or capsule would be administered three times a day for acute infection for three days, followed by once a day for a further seven days for maintenance. This once-a-day dosing can also be used prophylactically or for chronic inflammation. Where used to treat chronic inflammation administration would typically be for between two weeks and two months, preferably between three and six weeks, more preferably one month.

Preferable combinations of the one or more bile acids, bile salts or derivatives thereof, the a nicotinamide a nicotinic acid or an adenosine and a inhibitor

Optional combinations of the preferable one or more bile acids, bile salts or derivatives thereof with the preferable phosphodiesterase inhibitor are provided in Table 1. All of the preferable phosphodiesterase inhibitors include pharmaceutically acceptable salts thereof. The columns represent the preferable phosphodiesterase inhibitors (P) and the row represent the preferable one or more bile acids, bile salts or derivatives thereof (B). A legend to the lettering used in Table 1 is provided below:

B-l - One or more bile acids, bile salts or derivatives thereof.

B-2 - The one or more bile acids, bile salts or derivatives thereof is of general Formula (I) above, optionally wherein one, two, three or four hydroxyl, ethyl, amine or azide groups, preferably hydroxyl groups, are present on rings A, B and/or C at one, two, three or four of positions 3, 6, 7 and/or 12, preferably positions 3 and 7, and wherein the radical R ¹ is selected from hydrogen, a methanoic acid group, a hydroxyl group, a methanol group, preferably a methanoic acid group. B-3 - The one or more bile acids or derivatives thereof is of general Formula (I) above, optionally wherein one, two, three or four hydroxyl, ethyl, amine or azide groups, preferably hydroxyl groups, are present on rings A, B and/or C at one, two, three or four of positions 3, 6, 7 and/or 12, preferably positions 3 and 7, and wherein the radical R ¹ is a methanoic acid group.

B-4 - The one or more bile acids or derivatives thereof is of general Formula (I) above, wherein a hydroxyl, a ethyl, a amine or a azide group, preferably a hydroxyl group is present at position 3 and optionally wherein one, two or three additional hydroxyl, ethyl, amine or azide groups, preferably hydroxyl groups, are present on rings B and/or C at one, two or three of positions 6, 7 and/or 12, preferably position 7, and wherein the radical R ¹ is a methanoic acid group.

B-5 - The one or more bile acids or derivatives thereof is of general Formula (I) above, wherein a hydroxyl, an ethyl, an amine or an azide group, preferably a hydroxyl group is present at positions 3 and 7 and optionally wherein one or two additional hydroxyl, ethyl, amine or azide groups, preferably hydroxyl groups, are present on rings B and/or C at one or two positions 6 and/or 12, and wherein the radical R ¹ is a methanoic acid group.

B-6 - The one or more bile acids or derivatives thereof is of general Formula (I) above, wherein a hydroxyl group is present at position 3 and optionally wherein one, two or three additional hydroxyl, ethyl, amine or azide groups, preferably hydroxyl groups, are present on rings B and/or C at one, two or three of positions 6, 7 and/or 12, preferably position 7, and wherein the radical R ¹ is a methanoic acid group.

B-7 - The one or more bile acids or derivatives thereof is of general Formula (I) above, wherein a hydroxyl group is present at positions 3 and 7 and optionally wherein one or two additional hydroxyl, ethyl, amine or azide groups, preferably hydroxyl groups, are present on rings B and/or C at one or two of positions 6 and/or 12 and wherein the radical R ¹ is a methanoic acid group.

B-8 - The one or more bile acids or derivatives thereof is ursodeoxycholic acid.

B-9 - The one or more bile acids or derivatives thereof is ursodeoxycholic acid in combination with a muricholic acid.

P-1 - A phosphodiesterase inhibitor.

P-2 - The phosphodiesterase inhibitor is one clinically proven or being developed currently to provide a therapeutic effect through phosphodiesterase inhibition (tadalafil, sildenafil, vardenafil, udenafil, avanafil, mirodenafil, lodenafil, dipyridamole, caffeine, aminophylline, 3-isobutyl-l-methylxanthine, paraxanthine, pentoxifylline, theobromine, theophylline, erythro-9-(2-hydroxy-3-nonyl)adenine, 2-[(3,4-dimethoxyphenyl)methyl]-7-[(lR)-l- hydroxyethyl]-4-phenylbutyl]-5-methyl-imidazo[5,l-f][l,2,4]t riazin-4(lH)-one, oxindole, 9- (6-Phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one , inamrinone, milrinone, enoximone, anagrelide, cilostazol, pimobendan, mesembrenone, rolipram, ibudilast, piclamilast, luteolin, drotaverine, reflumilast, apremilast, crisaborole, papaverine, preferably tadalafil)

P-3 - The phosphodiesterase inhibitor is an inhibitor of phosphodiesterase 5.

P-4 - The phosphodiesterase inhibitor is a medicine for erectile dysfunction.

P-5 - The phosphodiesterase inhibitor is one a medicine that has obtained marketing authorization either from the FDA or the MHRA for therapeutic use through the inhibition of phosphodiesterase 5 (tadalafil, sildenafil, vardenafil, udenafil, avanafil, mirodenafil, lodenafil and dipyridamole).

P-6 - The phosphodiesterase inhibitor is tadalafil.

Table 1

Each of the numbers in brackets in Table 1 represents a specific combination of one group of one or more bile acids, bile salts or derivatives thereof with one group of phosphodiesterase inhibitor. For example, combination (48) is the combination of B-8 (ursodeoxycholic acid) with P-6 (tadalafil).

In one embodiment, the present invention provides a combination for simultaneous, separate or sequential therapy, a pharmaceutical pack or a pharmaceutical composition comprising a therapeutically effective amount of: a) any one of combinations (1) to (54); and b) a compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety, wherein the compound has a molecular mass of less than 1000 g-mol' ¹ .

In a further embodiment, the present invention provides a combination for simultaneous, separate or sequential therapy, a pharmaceutical pack or a pharmaceutical composition comprising a therapeutically effective amount of: a) any one of combinations (1) to (54); and b) a compound comprising a nicotinamide moiety, a nicotinic acid moiety or an adenosine moiety, wherein the compound has a molecular mass of less than 800 g-mol' ¹ .

In a further embodiment, the present invention provides a combination for simultaneous, separate or sequential therapy, a pharmaceutical pack or a pharmaceutical composition comprising a therapeutically effective amount of: a) any one of combinations (1) to (54); and b) a compound comprising a nicotinamide moiety or a nicotinic acid moiety, wherein the compound has a molecular mass of less than 1000 g-mol' ¹ .

In a further embodiment, the present invention provides a combination for simultaneous, separate or sequential therapy, a pharmaceutical pack or a pharmaceutical composition comprising a therapeutically effective amount of: a) any one of combinations (1) to (54); and b) a compound comprising a nicotinamide moiety, wherein the compound has a molecular mass of less than 1000 g-mol' ¹ .

In a further embodiment, the present invention provides a combination for simultaneous, separate or sequential therapy, a pharmaceutical pack or a pharmaceutical composition comprising a therapeutically effective amount of: a) any one of combinations (1) to (54); and b) a compound comprising a nicotinamide moiety, wherein the compound has a molecular mass of less than 800 g-mol' ¹ . In a further embodiment, the present invention provides a combination for simultaneous, separate or sequential therapy, a pharmaceutical pack or a pharmaceutical composition comprising a therapeutically effective amount of: a) any one of combinations (1) to (54); and b) nicotinamide adenine dinucleotide.

The invention will now be further described in the following Examples:

EXAMPLES

Example 1

Methods

Whole blood from anonymous donors, in combination with ethylenediaminetetraacetic acid in order to prevent clotting, was used for the bacterial inhibition and toxic granulation studies below. Both whole blood with a normal white blood cell count (WBC) (considered to be 5000 to 8000 WBC per pL) and a high WBC (considered to be more than 12000 WBC per pL) were used.

These blood samples were infected either with Gram-positive Escherichia coli ATCC 25922 or Gram-negative Staphylococcus aureas ATCCC 25293.

The samples were then treated with either the combination of nicotinamide adenine dinucleotide (NAD) at 100 pmol/L, ursodeoxycholic acid (UDCA) at 100 pmol/L and tadalafil at 100 pg/L, or as controls one of these components individually or a combination of two of these components. This combination was also tested on whole blood samples that were not infected. The samples were then put on a roller mixer for one hour at 20°C. Blood films were prepared before and after incubations, stained with either Romanowsky or Wright- Giemsa stain and examined for toxic granulation and bacteria. Slides were then looked at by light microscopy at a X 100 magnification. Fifty fields were observed per slide. Toxic granulation enhancement and bactericidal inhibition was observed visually and qualitatively compared against controls.

Results

With respect to whole blood (WB) samples with a normal WBC, Table 1 below shows the level of toxic granulation observed through X100 light microscopy immediately after application ("0 min") of the combination of NAD, UDCA and tadalafil (Triple) and sixty minutes after application ("60 min"). This study was carried out on uninfected samples and samples infected with both E. coli and S. aureas. As controls, uninfected samples and samples infected with both E. coli and S. aureas but not treated with this combination were also analysed.

Toxic granulation within neutrophils was graded as normal (+), moderate (++) and severe (+++)■

Table 2

Table 2 shows normal levels of granulation except when this combination is used, regardless of whether the sample were infected or not. This indicates that this combination is able to prime the immune system for microbial attack, and so this therapy could be used both prophylactically and for treating microbial infections.

With respect to whole blood (WB) samples with a normal WBC, Table 2 below shows the level of bacterial inhibition when samples were infected with either E. coli or S. aureas and then treated either with the combination of NAD, UDCA and tadalafil or single components. Analysis was carried out immediately after application ("0 min") of this combination (Triple) and sixty minutes after application ("60 min").

Through X100 light microscopy, one to two bacteria could be seen per field in the controls. Grading of bacterial inhibition was carried out using the following thresholds per fifty fields: less than ten bacteria = and ten to twenty bacteria = "++".

Table 3

Table 3 shows that none of the single components lead to any form of microbial inhibition, and that only the combination of NAD, UDCA and tadalafil leads to microbial inhibition. Figure 1 shows images of toxic granulation and phagocytosis in the samples infected with E. coli sixty minutes after treatment with the triple therapy. Figure 2 shows images of toxic granulation and phagocytosis in the infected samples sixty minutes after treatment with the combination of NAD, UDCA and tadalafil.

With respect to whole blood (WB) samples with a high WBC, Table 3 below shows the level of bacterial inhibition when samples were infected with either E. coli or S. aureas and then treated either with the combination of NAD, UDCA and tadalafil, single components or combinations of two components. Analysis was carried out immediately after application ("0 min") of this combination (Triple) and sixty minutes after application ("60 min").

Through X100 light microscopy, one to two bacteria could be seen per field in the controls. Grading of bacterial inhibition was carried out using the following thresholds per fifty fields: less than ten bacteria = and ten to twenty bacteria = "++".

Table 4

As with Table 3, Table 4 shows that none of the single components lead to any form of microbial inhibition. Furthermore, any combination of two components does not lead to any form of microbial inhibition. Only the combination of NAD, UDCA and tadalafil leads to microbial inhibition.

A longer-term study was carried out, where the effects of the combination of NAD, UDCA and tadalafil on whole blood samples infected with both E. coli and S. aureas were assessed after two hours at 37 °C and then a further 24 hours at ambient room temperature. Figure 3 shows images of toxic granulation, phagocytosis and almost complete clearance of the bacteria after treatment with this combination. By contrast, in samples that were infected but untreated, minimal toxic granulation or phagocytosis was seen (data not shown).

A longer-term study was carried out, where the effects of the combination of NAD, UDCA and tadalafil on whole blood samples infected with either E. coli or S. aureas were assessed after two hours at 37 °C and then a further 24 hours at ambient room temperature. Figure 4 shows images of toxic granulation, phagocytosis and phagocytosis with neutrophil collaboration after treatment with this combination. Figure 4 also shows an increase in toxic granulation in uninfected blood after application of the triple therapy. By contrast, in samples that were infected but untreated, minimal toxic granulation or phagocytosis was seen (data not shown).

This study shows how the combination of NAD, UDCA and tadalafil decreases the level of neutrophil dysfunction in blood samples infected with the virus SARS-CoV-2. Viral infections can induce the formation of Neutrophil Extracellular Traps (NETs), this process otherwise known as NETosis, as set out in, for example, Gillot et al., Front. Pharmacol., 12, Article 708302. NETs can be beneficial in preventing a pathogen from spreading in an organism releasing antibacterial proteins. However, a failure of the organism to adequately clear the NETs can lead to a cytokine storm and the promotion of a pro-inflammatory and procoagulant state, which in turn can lead to multiorgan failure.

Methods:

Fresh whole blood (taken within four hours) from anonymous donors, in combination with ethylenediaminetetraacetic acid (EDTA) to prevent clotting, was used for demonstration of NETS before and after treatment. Samples were infected with SARS-CoV-2 and had a white blood cell count of greater than 12,000 per pl.

Each sample was divided into four 500 pl aliquots and treated as follows:

1. No treatment (SARS-CoV-2 virus only) (V of Table 5 below)

2. Sample infected with E. coli (ATCC25922) and S. aureas (ATCC25293) in 20 pl amount (SARS-CoV-2 and bacterial infection) (V+B of Table 5).

3. Sample treated with combination of nicotinamide adenine dinucleotide (NAD) at 100 pmol/l, ursodeoxycholic acid (UDCA) at 100 pmol/l and tadalafil at 100 pmol/l in 10 pl amounts (SARS-CoV-2 and triple therapy treatment) (V+T of Table 5). 4. Sample infected with E. coli and S. aureas as above and NAD, UDCA and tadalafil as above (SARS-CoV-2, bacterial infection and triple therapy treatment) (V+B+T of Table 5).

NETosis was visualised before incubation and again after incubation at 37°C for one hour.

Results Grading of bacterial inhibition, phagocytosis and NETs was carried out, the results of which is presented in Table 5 below. With respect to the bacteria, "+" relates to less than ten bacterial cells per field and "++" relates to between ten and fifty bacterial cells per field. With respect to the NETs, "++" relates to between 20% and 50% of the neutrophils showing NETS, "+ ++" relates to between 50% and 70% of the neutrophils showing NETs and "++ ++" relates to more than 70% of the neutrophils showing NETs. With respect to phagocytosis, "+" relates to some phagocytosis whilst "++" relates to substantial and effective phagocytosis taking place.

Table 5

The results clearly show that the triple therapy is effective in reducing excessive NETosis that occurs as a result of a viral infection. The triple therapy is also able to stimulate phagocytosis in blood infected with both viruses and bacteria.