Benzimidazole compound with anthelmintic activity and PDE-5-inhibitor for treating chronic inflammatory skin disorder

Abstract

The present invention relates to the use of benzimidazole compounds with anthelmintic activity and Phosphodiesterase 5 inhibitors in the treatment of Chronic Inflammatory Skin Disorders in a vertebrate subject.

Description

[0001] TREATMENTS

[0002] Provided herein are methods and compositions for treating Chronic inflammatory skin diseases (ISDs). In particular, compositions are provided comprising a benzimidazole compound with anthelmintic activity and / or a Phosphodiesterase 5 Inhibitor for use in such methods.

[0003] BACKGROUND

[0004] Chronic ISD, including psoriasis, eczema, and atopic dermatitis (AD), encompass a range of conditions characterised by chronic or recurrent inflammation, immune dysregulation, and varying degrees of skin barrier dysfunction. These disorders cause significant physical symptoms, including pruritus, pain, and discomfort, as well as psychological distress, leading to a substantial impact on the quality of life. Despite differences in clinical presentation and underlying mechanisms, these conditions share several overlapping features, including immune-mediated inflammation, compromised skin barrier function, and increased susceptibility to environmental triggers and secondary infections.

[0005] By way of example: Psoriasis is associated with the formation of we 11 -demarcated, thick, scaly plaques driven by hyperproliferation of keratinocytes due to Thl / Thl7-mediated immune responses. Eczema is characterised by erythematous, oozing lesions in acute stages, which can progress to thickened, lichenified skin, reflecting a shift from Th2 -dominated inflammation to Thl / Th22 pathways. Atopic Dermatitis (AD) involves severe Th2-skewed immune responses, significant barrier dysfunction, and intense pruritus, which increase the risk of secondary bacterial and viral infections.

[0006] These conditions impose a considerable burden on healthcare systems worldwide due to their chronicity, frequent relapses, and the challenges associated with effective long-term management.

[0007] Current therapies for ISD include topical agents, systemic immunosuppressants such as selective Janus Kinase Inhibitors, and biologies. While these options provide symptomatic relief, significant limitations have been observed, leaving many patients inadequately treated or facing adverse effects.

[0008] State of the art topical treatments include corticosteroids, calcineurin inhibitors and emollients and are widely used for milder cases. These treatments offer temporary relief but do not address the underlying immune dysregulation. Long-term use, particularly of corticosteroids, is associated with side effects such as skin atrophy and reduced efficacy over time.

[0009] Other known systemic immunosuppressants include agents such as methotrexate, cyclosporin, and azathioprine and are employed in moderate to severe cases. However, these drugs are non-specificin their actions, broadly suppressing the immune system, which increases the risk of infections, organ toxicity, and other systemic side effects.

[0010] Biologies targeting specific cytokines, such as IL- 17 (psoriasis) or IL-4 / IL-13 (AD), have improved outcomes for some patients. However, these treatments are costly, require regular administration, and may not be effective for patients with complex or overlapping inflammatory profiles. Furthermore, they do not address the skin barrier dysfunction that is particularly critical in eczema and AD.

[0011] There are also a number of general limitations which are associated with known therapies. Many existing therapies focus solely on suppressing inflammation or modulating immune responses, neglecting the repair of skin barrier function. Furthermore, a substantial proportion of patients experience incomplete responses, adverse effects, or relapses, indicating a need for more comprehensive and durable therapeutic options.

[0012] It will therefore be appreciated that there is an unmet clinical need for innovative treatments that address both immune dysregulation and skin barrier dysfunction across ISD and it is an object of the present invention to address this need.

[0013] The drug treatments described herein represent novel treatments for Chronic ISD and a significant advancement in the treatment thereof. By simultaneously modulating multiple pathological pathways and improving skin barrier function, the treatment is positioned to deliver superior and long-lasting therapeutic outcomes compared to existing treatments.

[0014] SUMMARY OF THE INVENTION

[0015] The inventors have established that benzimidazole compounds with anthelmintic activity may be combined with a Phosphodiesterase 5 (PDE5) inhibitor to effectively treat, reverse, arrest or slow down Chronic ISD.

[0016] Furthermore, the inventors have demonstrated that combinations of a benzimidazole compound and a PDE5 inhibitor produce a synergistic anti-inflammatory effect. This is illustrated (see Example 3) in an IL-17-stimulated normal human epidermal keratinocyte (NHEK) model representative of psoriatic inflammation. In this model, treatment with a benzimidazole compound and a PDE5 inhibitor resulted in suppression of IL-17- and TNFa-responsive inflammatory signaling pathways, including NF-KB and AP-1. The combinations further exhibited transcriptomic synergy, with a ABliss synergy score greater than 0.3, indicating that the suppressive effect of the combination exceeded that expectedfrom the individual components. These findings confirm the suitability of the compositions for treating ISDs driven by IL- 17 or related cytokines.

[0017] According to a first aspect of the invention there is provided a composition comprising a benzimidazole compound with anthelmintic activity and a PDE5 inhibitor for use as a medicament to treat Chronic ISD in a vertebrate subject.

[0018] According to a second aspect of the invention there is provided a composition comprising a benzimidazole compound with anthelmintic activity and a further composition comprising a PDE5 inhibitor for use in combination as medicaments to treat a Chronic ISD in a vertebrate subject.

[0019] According to a third aspect of the invention there is provided a method of treating Chronic ISD in a vertebrate subject in need of such treatment comprising administering to the subject a therapeutically effective amount of a benzimidazole compound with anthelmintic activity and a therapeutically effective amount of a PDE5 inhibitor.

[0020] In preferred embodiments of the invention the benzimidazole compound and PDE5 inhibitor are formulated in separate compositions with vehicles suitable for pharmaceutical, veterinary or nutraceutical use.

[0021] PDE5 inhibitors have been shown to improve the morphology of ageing skin (see US2013 / 0030174). However, ause ofPDE5 inhibitors for reversing, arresting or slowing down Chronic ISD was not contemplated and most notably, novel combinations of benzimidazole compounds with anthelmintic activity and PDE5 inhibitors have not been described.

[0022] The inventors are the first to show synergy between these classes of compounds as treatments for Chronic ISD. The inventors previously showed that anthelmintic benzimidazoles and PDE5 inhibitors could be used in the prevention of age-related pathologies (WO2023 / 242599) whereas the present invention concerns their utility in treating inflammatory skin conditions such as eczema, psoriasis, atopic dermatitis, vitiligo, rosacea and lupus associated skin disorders which are not restricted to old age.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed compositions and methods, there are shown in the drawings exemplary embodiments of the compositions and methods;however, the compositions and methods are not limited to the specific embodiments disclosed. In the drawings:

[0025] FIG. 1 represents photographs of a middle-aged canine subject before, during and after being treated according to the invention as discussed in Example 1.

[0026] FIG.2, is a graph illustrating the extent to which specified genes are downregulated in NHEK’s that have been treated with FA and TAD as discussed in Example 2.

[0027] FIG. 3, represents bar charts showing: (A) signed fold change expression of key TNFa-responsive genes in NHEKs treated with IL- 17 in the presence of the indicated compounds; and (B) signed fold-change expression profiles for canonical inflammatory responsive genes in NHEKs treated with IL- 17 in the presence of the indicated compounds as discussed in Example 3.

[0028] DETAILED DESCRIPTION

[0029] The disclosed compositions and methods may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed compositions and methods are not limited to the specific compositions and methods described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed compositions and methods.

[0030] Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. When a range of values is expressed, another embodiment includes from the one particular value and / or to the other particular value. Further reference to values stated in ranges, include each and every value within that range. All ranges are inclusive and combinable.

[0031] It is to be appreciated that certain features of the disclosed compositions and methods which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed compositions and methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination.

[0032] The following abbreviations are used herein: Chronic Inflammatory Skin Disease (ISD) ; Active Pharmaceutical Ingredient (API); Atopic Dermatitis (AD); phosphodiesterase type 5 (PDE5); Fenbendazole (FA); Albendazole (AL); Tadalafil (TAD); Type 2 Diabetes (T2D); glucose transporter 4 (GLUT4); hexokinase II (HK11); adenosine monophosphate kinase (AMPK); mammalian target of rapamycin (mTOR); glyceraldehyde phosphate dehydrogenase (GAPDH); German Shepherd Dog(GSD); Interleukin 17 (IL- 17); Nuclear Factor kappa-light-chain-enhancer od activated B cells (NF-KB); Activator protein 1 (AP-1); Tumour Necrosis Factor alpha (TNFa); normalised enrichment scores (NES); and normal human epidermal keratinocyte (NHEK) cells.

[0033] As used herein, the singular forms “a,” “an,” and “the” include the plural.

[0034] When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Furthermore, the term “about” when used in reference to numerical ranges, cut-offs, or specific values is used to indicate that the recited values may vary by up to as much as 10% from the listed value. As many of the numerical values used herein are experimentally determined, it should be understood by those skilled in the art that such determinations can, and often times will, vary among different experiments. The values used herein should not be considered unduly limiting by virtue of this inherent variation. Thus, the term “about” is used to encompass variations of ± 10% or less, variations of ± 5% or less, variations of ± 1% or less, variations of ± 0.5% or less, or variations of ± 0.1% or less from the specified value.

[0035] As used herein, by the term “Chronic ISD” we mean disorders which include psoriasis, eczema, and atopic dermatitis (AD), that are characterised by chronic or recurrent inflammation, immune dysregulation, and varying degrees of skin barrier dysfunction. These disorders are associated with pruritus, pain, and discomfort, as well as psychological distress, which can lead to a substantial impact on the quality of life.

[0036] As used herein, by the terms “treatment” or “treating”, when compared to untreated controls, we mean, a therapeutically effective amount of an API (or APIs) resolves, reverses, arrests, or slows down the development of a Chronic ISD. “Treating” and like terms may also refer to reducing the severity and / or frequency of symptoms; eliminating symptoms and / or the underlying cause of said symptoms; reducing the frequency or likelihood of symptoms and / or their underlying cause; delaying, preventing and / or slowing the development of a Chronic ISD.

[0037] As used herein, the phrase “therapeutically effective dose” refers to an amount of a composition comprising a composition comprising a benzimidazole compound with anthelmintic activity (and optionally a PDE 5 inhibitor) as described herein, effective to achieve a particular biological or therapeutic result such as, but not limited to, biological or therapeutic results disclosed, described, or exemplified herein. The therapeutically effective dose may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to cause a desired response in a subject. Such results include, but are not limited to, reversing, arresting or slowing Chronic ISD as determined by any means suitable in the art.As used herein, “vertebrate subject” includes any animal of veterinary interest (e.g. sheep, cattle, horses, fish, dogs, cats, rabbits and most reptiles) and also humans. Preferably the subject is a mammal. In one embodiment of the invention the subject may be a domestic animal (e.g. a dog or cat) or animal used in leisure or sporting pursuits (e.g. a horse). In another embodiment of the invention the subject may be a farm animal. In preferred embodiments of the invention the subject is a dog, cat or human being.

[0038] As used herein “a benzimidazole compound with anthelmintic activity” means a compound containing the structure of formula I (see below) that has activity as an antiparasitic drug for expelling or killing parasitic worms (helminths) and other internal parasites from a subject. Preferred benzimidazole compounds that may be used according to the invention are those known for use in the treatment of animals and humans who are infected by helminths.

[0039] As used herein, reference to the API of the invention means a benzimidazole compound with anthelmintic activity and / or a PDE5 inhibitor depending on the context.

[0040] A “pharmaceutically acceptable vehicle” may be any physiological vehicle known to those of ordinary skill in the art useful in formulating pharmaceutical compositions.

[0041] The inventors recognized that benzimidazole compounds with anthelmintic activity and / or PDE5 inhibitors had previously been shown to downregulate specific age-associated inflammatory factors and to extend lifespan in rodents consistent with their use for the treatment of age-related pathologies. However inflammatory pathologies of the skin are complex multi-system disorders which may or may not be related to biological age and it was not obvious that these APIs would be useful as described herein. For example, AD, psoriasis and vitiligo very often occur in childhood and persist into adulthood. Conditions such as rosacea and lupus are not age-related where the latter is an autoimmune condition which predisposes to skin rashes and lesions. In light of the previously described anti-ageing activity of the APIs, the inventors hypothesised these compounds may also have activity against ISDs which are not age-related such as AD, psoriasis, vitiligo, rosacea and lupus. In order to test this hypothesis, a German Shepherd dog who developed AD at 6 years old, was successfully treated when aged 8 years with the FA and TAD combination as shown in Example 1.

[0042] Further support for the invention is provided by a series of experimental examples. These include: RNA-Seq transcriptomic characterisation of benzimidazole and PDE5 inhibitor effects in unstimulated human keratinocytes (Example 2); and an evaluation of the combination in an IL-17-activated keratinocyte model (Example 3). Collectively, the results show that the compositions of theinvention act at multiple points in the inflammatory cascade and are capable of reversing cytokine-driven transcriptional programmes characteristic of ISDs.

[0043] APIs used according to the invention

[0044] Benzimidazole compounds with anthelmintic activity used according to the invention comprise a benzimidazole structure of formula I with substitutions at positions 2, 5 and / or 6 of the structure of formula I.

[0045]

[0046] Benzimidazoles compounds used according to the invention are preferably selected from the group comprising: FA, Mebendazole, Flubendazole, Parbendazole, Oxfendazole, Oxibendazole, Albendazole, Ricobendazole, Albendazole sulfoxide, Thiabendazole, Thiophanate, Febantel, Netobimin, and Triclabendazole.

[0047] It is more preferred that the benzimidazole used according to the invention is selected from the group comprising Triclabendazole, Mebendazole, Albendazole and FA and functional analogues thereof. These inhibitors have the structures set out in formula II - V.

[0048] Triclabendazole:

[0049]

[0050] Albendazole:

[0051]

[0052] It is most preferred that the benzimidazole compound used according to the invention is Fenbendazole (Formula V) or a structural equivalent thereof. The chemical name of FA is methyl 5-(phenylthio)-2 -benzimidazole carbamate (CAS Registry Number is 43210-67-9).

[0053] In one embodiment the benzimidazole compound used according to the invention is Albendazole (AL) (Formula III) or a structural equivalent thereof. The chemical name of AL is methyl [6-(propylsulfanyl)-lH-benzimidazol-2-yl] carbamate (CAS Registry Number is 54965-21-8).

[0054] A structural equivalent of FA or AL may have a drugbank similarity threshold (Tanimoto Score) of 0.6; more preferably of 0.7 and most preferably of 0.8.

[0055] In some embodiments compositions according to the invention may comprise a benzimidazole compound and a PDE5 inhibitor. In other preferred embodiments a first composition comprising a benzimidazole compound may be co-administered with a further composition comprising a PDE5 inhibitor.

[0056] PDE5 inhibitors are vasodilators which work by preventing the degradative action of cGMP-specific PDE5 on cyclic GMP in smooth muscle cells lining the blood vessels supplying various tissues. Their medical uses include the treatment of erectile dysfunction, pulmonary hypertension and benign prostatic hyperplasia.PDE5 Inhibitors that may be used according to the invention are preferably selected from the group comprising vardenafd, sildenafil, avanafil, Tadalafil (TAD) and functional equivalents thereof. These inhibitors have the structures set out in formula VI - IX.

[0057] Sildenafil:

[0058]

[0059] It is most preferred that the PDE5 inhibitor used according to the invention is TAD (Formula IX) or a structural equivalent thereof. The chemical name of TAD is pyrazino[l',2': l,6]pyrido[3,4-b]indole- 1,4-dione, 6-(l,3- benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methyl-, (6R,12aR). (CAS Registry Number is 171596-25-5).A structural equivalent of TAD may have a drugbank similarity threshold (Tanimoto Score) of 0.6; more preferrable of 0.7 and most preferably of 0.8.

[0060] Preferred APIs for use according to the invention

[0061] The inventors have found that any benzimidazole compound with anthelmintic activity with a benzimidazole structure of formula I may be combined with a PDE5 inhibitor to have beneficial effects in the treatment of chronic ISD. Preferably the benzimidazole used according to the invention is selected from the group comprising Triclabendazole, Mebendazole, AL and FA. More preferably the benzimidazole compound is FA or AL and most preferably the benzimidazole compound is FA.

[0062] The inventors do not wish to be bound by any hypothesis, but they have come to realise that the surprising efficacy of FA can be explained by observations they have made reviewing art in the field. They have noted that Fusco et al. 2020 (DOI: 10.1042 / CS20191215) reports that topical application of the antihelminitic benzimidazole compound albendazole, has some effect in a murine model of psoriasis (albeit Fusco and colleagues did not contemplate combining albendazole with a PDE5 Inhibitor). However, they came to the view that FA represents a better alternative to albendazole for orally administered treatment of widespread chronic ISD. The inventors believe this is due to the superior safety profile of FA and also its ability to directly inhibit aerobic glycolysis, (Nguyen et al.

[0063] 2024 DOI: 10.21873 / anticanres.17197). The inventorshave noted that orally administered albendazole, has been shown to cause hepatotoxicity and myelosuppression (Chai et al. 2021 DOI: 10.3347 / kjp.2021.59.3.189) which makes it unsuitable for long-term systemic use whereas FA is well known for its low toxicity and excellent long-term safety profile in numerous animal species (Nguyen et al. 2024 DOI: 10.21873 / anticanres.17197). Furthermore, the inventors noted that FA also targets hexokinase 2 in immune cells, reducing their glycolytic activity and potentially lowering the inflammatory response, whereas albendazole lacks this ability (Dogra etal. 2018 DOI: 10.1038 / s41598-018-30158-6;. Thus, since FA is safer and more effective than albendazole, it represents the most preferred benzimidazole for systemic treatment (suitably by oral administration) of chronic ISD. In preferred embodiments of the invention, the FA is formulated for oral consumption as a powder (for mixing with food or making into a drink), granules, aqueous suspensions, pastes, tablets or capsules.

[0064] Although Fenbendazole represents a most preferred benzimidazole compound, the inventors have further shown that the beneficial effects of the invention are not restricted to a single member of the class of Benzimidazole compounds defined by Formula I. In particular, Example 3 illustrates that both Fenbendazole and, irrespective of the points made above, also Albendazole produced cooperative and synergistic suppression of IL-17-driven inflammatory gene expression when combined with a PDE5 inhibitor. This demonstrates that multiple benzimidazole compounds are suitable for use in theinvention (albeit routes of administration and dosing need to be considered in view of toxicity profiles), enabling broad applicability across the antihelminth benzimidazole chemotype. Accordingly, Albendazole will have a role in the treatment of chronic ISD when combined with a PDE5 inhibitor and when formulations are made that take into account potential toxicity. In this respect, Albendazole is useful because the inventors have found that the combination of albendazole and a PDE5 inhibitor is synergistic and this unexpectedly allows for the concentration of Albendazole to be reduced and thereby reduce its potential toxicity to acceptable levels.

[0065] The most preferred PDE5 inhibitor used according to the invention is TAD (Formula IX) or a structural equivalent thereof. Accordingly, a most preferred composition according to the first aspect of the invention may comprise FA and TAD. In a preferred embodiment of the second aspect of the invention a composition may comprise FA and may be co-administered with a further composition comprising TAD. Each of these compositions is most preferably formulated for oral consumption as a powder (for mixing with food or making into a drink), granules, aqueous suspensions, pastes, tablets or capsules.

[0066] Compositions according to first or second aspects of the invention may be used in combination with other anti-inflammatory drugs such as steroids or non-steroidal anti-inflammatory agents (e.g. Ibuprofen, Meloxicam) or alternatively, the selective Janus kinase inhibitor Apoquel, for short-term relief of pruritic symptoms.

[0067] Pharmaceutical and Nutraceutical formulations

[0068] The compositions used according to invention may comprise the API or APIs without any additional components (e.g. a powder of the API which is used by diluting in a liquid or used to fill a capsule). However, in preferred embodiments, the APIs are formulated with other agents, as discussed below, to improve their commercial properties (e.g. to improve delivery, shelf-life, taste and the like).

[0069] Compositions for Oral Administration

[0070] The compositions of the invention may be formulated as a pharmaceutical or nutraceutical composition for oral administration. As such, they can be formulated as gels, solutions, suspensions, syrups, tablets, capsules, lozenges and snack bars or beverages by way of example. Such formulations can be prepared in accordance with methods well known to the art. For example, the API or APIs may be formulated in a syrup or other solution for administration orally, for example as a health drink. One or more excipients selected from sugars, vitamins, flavouring agents, colouring agents, preservatives and thickeners may be included in such syrups or solutions. Tonicity adjusting agents such as sodium chloride, or sugars, can be added to provide a solution of a particular osmotic strength. One or more pH-adjusting agents, such as buffering agents can also be used to adjust the pH to a particular value, and preferablymaintain it at that value. Examples of buffering agents include sodium citrate / citric acid buffers and phosphate buffers.

[0071] In preferred embodiments the API or APIs are formulated as a tablet for oral consumption. For tablet formation, the API or APIs may be typically mixed with a diluent such as a sugar, e.g. sucrose and lactose, and sugar alcohols such as xylitol, sorbitol and mannitol; or modified cellulose or cellulose derivative such as powdered cellulose or microcrystalline cellulose or carboxymethyl cellulose. The tablets will also typically contain one or more excipients selected from granulating agents, binders, lubricants and disintegrating agents. Examples of disintegrants include starch and starch derivatives, and other swellable polymers, for example crosslinked polymeric disintegrants such as cross-linked carboxymethylcellulose, crosslinked polyvinylpyrrolidone and starch glycolates. Examples of lubricants include stearates such as magnesium stearate and stearic acid. Examples of binders and granulating agents include polyvinylpyrrolidone. Where the diluent is not naturally very sweet, a sweetener can be added, for example ammonium glycyrrhizinate or an artificial sweetener such as aspartame, or sodium saccharinate.

[0072] The API or APIs can also be formulated as powders, granules, gels or semisolids for incorporation into capsules. When used in the form of powders, the API can be formulated together with any one or more of the excipients defined above in relation to tablets, or can be presented in an undiluted form. For presentation in the form of a gel or semisolid, the API or APIs can be dissolved or suspended in a viscous liquid or semisolid vehicle such as a polyethylene glycol, or a liquid carrier such as a glycol, e.g. propylene glycol, or glycerol or a vegetable or fish oil, for example an oil selected from olive oil, sunflower oil, safflower oil, evening primrose oil, soya oil, cod liver oil, herring oil, etc. These can then be filled into capsules of either the hard gelatine or soft gelatine type or made from hard or soft gelatine equivalents, soft gelatine or gelatine-equivalent capsules being preferred for viscous liquid or semisolid fillings. In one preferred embodiment, a composition according to the invention is provided in powder form optionally together with a preferred solid (e.g. powdered) excipient for incorporation into capsules, for example a hard gelatine capsule.

[0073] Preferred Formulations for Oral Administration

[0074] FA is used in this section to exemplify specific doses and regimens. However, it will be appreciated that a skilled person may substitute other benzimidazole compounds according to the invention into the formulations discussed below.

[0075] In one embodiment of the invention a composition comprises between 1 - 880 mg of FA made up in a tablet or powder formulated with anhydrous colloidal silica, com starch, sodium carboxymethyl starch (type A), hydroxyethylcellulose, lactose monohydrate and Magnesium Stearate.In another, embodiment of the invention a most preferred formulation for human use is a capsule essentially containing pure FA. Such capsules may comprise: 1 to 1500 mg FA; 1 to 1,000 mg FA; 1 to 880 mg FA; preferably 5 to 500 mg FA; and more preferably 10 to 300 mg FA. In a preferred embodiment the capsule comprises between about 10 to 100 mgs FA (e.g. about 40mg).

[0076] In a further embodiment of the invention, FA may be taken as solid granules which may be mixed with food and then consumed. By way of example, granulated formulations are marketed as Panacur® for use as an anthelmintic for treating animals. Panacur® Granules containing 22.2% (w / w) FA in addition to the excipients lactose monohydrate, Povidone 2500 and maize starch. These granules are used as a dewormer for cats or dogs (by mixing Panacur® granules with their food) and may be repurposed and / or adapted for use according to the present invention.

[0077] In a further embodiment of the invention FA may be formulated as a suspension (to be drunk or mixed with food). FA is also available as Panacur® Equine Guard aqueous 10% w / v oral suspension combined with the excipients Sodium Methyl Parahydroxybenzoate, Sodium Propyl Parahydroxybenzoate, Benzyl alcohol, Silica colloidal anhydrous, Carmellose sodium, Povidone K25, Sodium Citrate Dihydrate, Citric Acid Monohydrate and Water Purified. It is used for treating both small animals and horses and may be repurposed and / or adapted for use according to the present invention.

[0078] In a further embodiment of the invention FA may be formulated as a paste. Panacur® is also available as an 18.75% (w / w) FA paste combined with the excipients Methyl Parahydroxybenzoate, Propyl Parahydroxybenzoate, Propylene Glycol, Apple and Cinnamon Flavour, Carbomer 980, Glycerol (85%), Sorbitol (70%, crystalising), Sodium Hydroxide, Water Purified. It is used for administering orally (by syringe) to rabbits and the like and may be repurposed and / or adapted for use according to the present invention.

[0079] PDE 5 inhibitors may also be formulated as a powder (for mixing with food or making into a drink), granules, aqueous suspensions, pastes, tablets or capsules. In a preferred embodiment PDE 5 inhibitors are formulated as tablets for oral consumption. By way of example, tablets for oral delivery comprising 2.5, 5, 10, or 20 mg of TAD are known to the art for treating erectile dysfunction in humans. An example of such tablets comprises TAD and the following inactive ingredients: croscarmellose sodium, hydroxypropyl cellulose, hypromellose, iron oxide, lactose monohydrate, magnesium stearate, microcrystalline cellulose, sodium lauryl sulfate, talc, titanium dioxide, and triacetin. It will be appreciated that such formulations may be used or adapted for treating animal or human subjects according to the invention and co-administered with a benzimidazole with anthelmintic activity.Any of the abovementioned benzimidazole compound formulations, for example the Panacur® formulations, may be given separately with any of the above PDE 5 inhibitors formulations for use according to the second aspect of the invention.

[0080] Alternatively, the formulations may be adapted by co-formulating a benzimidazole compound (e.g. FA), and a PDE5 inhibitor (e.g. TAD) in the same composition for use according to the first aspect of the invention. This could be at ratios (benzimidazole compound: PDE5 inhibitor) ranging from 1:1 to 1000:1 (w / w). More preferably this will be at ratio of between 20:1 and 100:1 and most preferred between 2:1 and 70:1.

[0081] Dosing and Dose Units for Oral Use

[0082] The amount of a composition used to treat subjects according to the invention will depend upon the species being treated; their size, age and sex; the specific condition being treated or prevented; and the severity of the condition being treated. The required amount can be presented in the form of a unit dosage form containing a defined amount of the API or APIs.

[0083] Benzimidazole compounds with anthelmintic activity may be administered as an oral dose of between 0.1 - 22 mg / Kg / day. It is preferred that a subject receives between 0.2 - lOmg / Kg / day and more preferred subject receives between 0.3 - 5 mg / Kg / day. By way of example a canine or human subject may benefit from receiving between about 0.4 - 2.5 mg / Kg / day. Examples of preferred doses include 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 and l.lmg / Kg / day. In one embodiment about 0.55mg / Kg / day FA may be used to treat canines or humans.

[0084] As single agents, PDE5 inhibitors may be administered as an oral dose of between 10 - 300 pg / Kg / day). It is preferred that a subject receives between 20 - 250 pg / Kg / day and more preferred subject receives between 30 - 200 pg / kg / day. By way of example a human subject may benefit from receiving between about 40 - 150 pg / Kg / day with an equivalent dose used for canines. Examples of preferred doses include 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 and 250 pg / Kg / day. In one embodiment about 125pg / Kg / day TAD may be used to treat canines. In another embodiment humans may receive about 70pg / Kg / day.

[0085] The compositions according to the invention can be presented in the form of unit dosage forms containing a defined amount of the API or APIs. Such unit dosage forms can be selected so as to achieve a desired level of biological activity and / or deliver the daily amounts discussed above. The amount required in a dose unit will depend up the species being treated; their size, age and sex; and the condition being treated.Using FA as an example, a unit dosage form of a benzimidazole compound with anthelmintic activity for oral consumption by humans or canines can contain an amount of up to about 1500 mg (dry weight) of the API, up to about 1000 mg (dry weight), up to about 880 mg (dry weight), up to about 500 mg (dry weight), up to about 250 mg (dry weight) and more typically up to 200 mg, for example between 1 mg and 50 mg (e.g. about 40mg).

[0086] Particular amounts of the benzimidazole compound may be included in a unit dosage form and may be selected from any of the previously defined available liquid suspension, powder or paste formulations.

[0087] Using TAD as an example, a unit dosage form of a PDE5 inhibitor for oral consumption by humans can contain an amount of up to 50 mg (dry weight) of the API, more typically up to 20 mg, and preferably between 1 and 10 mg (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or lOmg). In one embodiment the unit dose may be about 8.75mg. Particular amounts of the PDE5 inhibitor may be included in a unit dosage form may be selected from any of the above formulations

[0088] In one embodiment of the invention, humans may be treated with separate compositions or coformulated compositions providing about 1-50 mg / day dose of FA and about l-20mg / day of TAD; and more preferably the separate compositions or co-formulated compositions provide about 38.5mg / day dose of FA and about 8.75mg / day of TAD.

[0089] Compositions for Topical Administration

[0090] Compositions according to the invention may also be formulated as a medicament or functional cosmetic that is suitable for topical application and may in particular be formulated for administration to the skin.

[0091] Suitable formulations include, but are not limited to, a gel, cream, paste, ointment or lotion. In some aspects, the composition can be formulated as a gel. In some aspects, the composition can be formulated as a cream. In some aspects, the composition can be formulated as a paste. In some aspects, the composition can be formulated as an ointment. In some aspects, the composition can be formulated as a lotion.

[0092] In preferred embodiments, the composition is formulated such that it is suitable for topical delivery of the APIs (e.g. as an ointment, gel, paste lotion or cream) for reversing, arresting or slowing down cellular ageing.

[0093] When used to reverse, arrest or slow down Chronic ISD (e.g. atopic dermatitis), the compositions can be formulated as gels, lotions, paste, creams or ointments that may be applied directly to the skin by techniques known to the art.Preferred compositions for use according to the second aspect of the invention are formulated for topical application to the skin and may comprise FA or AL in a first composition and TAD in a further composition.

[0094] In one embodiment, the pharmaceutically acceptable vehicle can be a liquid and the composition can be a solution. In another embodiment, the vehicle can be a gel and the composition can be a gel for applying to the skin. In a further embodiment, the vehicle can be an emulsion (or other pharmaceutically acceptable base) and the composition can be a cream. In a further embodiment, the vehicle can be smooth and oily and the composition can be an ointment for application to the skin.

[0095] Liquid vehicles may be used in preparing gels, lotions, creams, solutions, suspensions and emulsions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). The vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. The liquid vehicle for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0096] Uses of Compositions according to the invention

[0097] Chronic ISD, including psoriasis, eczema, and AD, have a major influence on human and animal health and new therapeutic regimens using the APIs discussed herein represent an important aspect of the present invention.

[0098] FA has been used as an antihelmintic treatment in animals for approximately 60 years and, after realizing it was useful for treating chronic ISD the inventors first wondered why animal owners had not noticed beneficial effects in their animals. The inventors realised this was due to the fact that antihelminth treatments are recommended for very short periods. For instance, it is recommended that puppies are typically treated for only 3 consecutive days and this is followed by intervals of between 5, 8, 12 and 16 weeks before any further treatments are given. Furthermore, in adult dogs, deworming can occur on a single occasion and intervals are often longer (for instance it is recommended that PANACUR® is given to adult cats or dogs 2 -4 times a year).In contrast to the short term use of FA as an antihelmintic, the inventors have found that it is optimal if the API is administered as a continuous or discontinuous treatment regimen of between 1-7 days / week and given for at least 1 month ranging from periods of between 1-12 months in any 12 month period. More preferably, a regimen for using compounds according to the invention involves treating for 2-7 days per week either continuously or discontinuously for periods of between 1- 6 months.

[0099] In one embodiment, FA can be administered continuously for 1 to 7 days / week for a period of up to 6 months. Depending on symptoms, this can be continued from 1 week to 6 months of continuous treatment or it can be given in episodes of between 1 to 2 weeks followed by a period of 1 to 8 weeks with no treatment and this treatment cycle then repeated as long as is necessary. An example of this type of regimen, as used in dogs, is provided in Example 1 and also as described below.

[0100] When PANACUR® (22.2% FA), is used for deworming animals, it is recommended that a single dose of approximately 1g of FA (4 gm PANACUR®) is administered per 10kg bodyweight for a cat or dog. Further treatment is recommended if natural re-infestation occurs and it is advised that treatments should only occur 2-4 times a year. Thus, in contrast to deworming treatments the inventors have found that more frequent treatment with lower doses will have the beneficial effects described herein for the treatment of ISD. Benzimidazole compounds used according to the invention are preferably used as an oral dose of between 0.1 - 22 mg / Kg / day and most preferably about 0.55mg / Kg / Day.

[0101] TAD is currently not licensed for use in animals. However, during the development of TAD, dogs were treated with doses of 25 mg / kg / day for over a year with minimal side-effects although, at these high doses, it did produce regression of seminiferous tubular epithelium in some dogs. Preferred doses for use according to the invention are much lower and preferably in a dose range of 10 - 300 pg / Kg / day (more preferably about 125 pg / Kg / day). However, the inventors have also noted that, since TAD has a long half-life, it can be administered at a given dose on alternative days to effectively reduce the overall Cmax serum concentration.

[0102] Preferred embodiments

[0103] According to a preferred treatment regimen, the composition or compositions may be used according to the invention in the following way:

[0104] (i) the benzimidazole compound is co-administered every day with a PDE5 inhibitor for a period of between 1 week and 6 months;

[0105] (ii) following period (i) the PDE5 inhibitor may be administered as a single agent for a period of between 2 weeks to 6 months; and(iii) steps (i) and (ii) are repeated until the Chronic Inflammatory Skin Disorder is treated to a subject’s or clinician’s satisfaction.

[0106] It is preferred that the benzimidazole compound is co-administered with the PDE5 inhibitor for between 1 week and 2 months in step (i). It is also preferred that the PDE5 inhibitor is administered for 1 to 2 months in step (ii). In a most preferred embodiment, the benzimidazole compound is coadministered with the PDE5 inhibitor for between 1 week and 2 months in step (i); and the PDE 5 inhibitor is administered for 1 to 2 months in step (ii).

[0107] In one embodiment step (iii) comprises repeated cycles of the benzimidazole compound being co-administered with the PDE5 inhibitor for between 1 week and 2 months and then the PDE 5 inhibitor being administered for 1 to 2 months.

[0108] In another embodiment step (iii) comprises repeated cycles of the benzimidazole compound being co-administered with the PDE5 inhibitor for between 1 to 2 weeks then the PDE 5 inhibitor being administered for between 3 weeks and 2 months.

[0109] According to a most preferred treatment regimen for canine subjects, a dog may be treated with 125pg / kg / day of TAD and 0.55mg / kg / day of FA for a period of 2 months (step (i)). Following this, the dosing may be changed to 125pg / kg of TAD alone for 1 month (step (ii)). Step (iii) may then comprise repeated cycles of 1 week of FA +TAD followed by 3 weeks of TAD alone

[0110] According to a most preferred treatment regimen for human subjects, a person may be treated with 70pg / kg / day of TAD and 0.55mg / kg / day of FA for a period of 2 months (step (i)). Following this, the dosing may be changed to 70pg / kg / day of TAD alone for 1 month (step (ii)). Step (iii) may then comprise repeated cycles of 1 week of FA +TAD followed by 3 weeks of TAD alone

[0111] These regimens may utilise any combination of FA & TAD within the previously defined dose ranges. In a most preferred embodiments about 0.55mg / Kg / day FA and between 70-125pg / Kg / day TAD may be used.

[0112] An exemplar regimen for treating dogs weighing 30-40 kg and above:

[0113] (i) 1 x 5 mg Tadalafil tablet plus 0.1 g of Panacur granules (comprising approximately 20 mg Fenbendazole) per day for 6 weeks.

[0114] (ii) Then 4 weeks 1 x 5 mg Tadalafil / day

[0115] (iii) Then 2 weeks with no treatment.

[0116] (iv) Then 1 x 5 mg Tadalafil plus 0.1 g Panacur (comprising approximately 20 mg Fenbendazole) per day for 1 week then 3 weeks 1 x 5 mg Tadalafil followed by 2 weeks with no treatment.(v) Repeat whole cycle or just (iv) if necessary and considered appropriate after professional assessment.

[0117] The same protocol may be used for smaller dogs (20-3 Og) except 1 x 5mg Tadalafd may be given every alternate day

[0118] For relief of symptoms early in the treatment, other anti-inflammatory agents can also be given (e.g. Apoquel or selected NSAIDs such as meloxicam etc).

[0119] EXAMPLE 1:

[0120] A 40 kg, six year old male German Shepherd Dog (GSD) was diagnosed with AD in April 2022 and was treated with intermittent Apoquel (1 or 2 x 16mg / day) as and when required to control symptoms. Figure 1 A is a photograph of the animal when it first began Apoquel therapy. This was continued for 2 years until April 2024 where his condition had deteriorated such that he was constantly scratching, he had numerous bald patches on his belly and face and he would bite his feet until they bled (see Figure IB).

[0121] In April 2024, he was started on oral 5mg / day (125pg / kg) of TAD plus 22mg / day (0.55mg / kg) of FA (=>100mg Panacur) which was continued for a period of 2 months. Following this, the dosing was changed to 5mg / day TAD (No FA) for 1 month followed by 5mg / day TAD + 22mg / day FA for 1 week then 3 weeks treatment with 5mg / day TAD. This monthly dosing regimen of 1 week of FA +TAD then 3 weeks of TAD as a single agent was subsequently repeated from then on. Figure 1C shows the dog in Oct 2024, 5 months after start of this treatment when there had been a very significant improvement. His scratching had virtually stopped with resolution of bald patches and his skin and coat were visibly much heathier. It is also notable that he appeared to be a much younger animal than when he started the treatment and the owner actually reported he was acting like he did as a puppy and not at all like a 9 year old GSD.EXAMPLE 2

[0122] Having noted the efficacy of the APIs in a canine case study (Example 1), the inventors evaluated whether or not gene markers associated with inflammatory skin conditions were up or down regulated when cultured epidermal cells were treated with APIs according to the invention.

[0123] 2.1 METHODS

[0124] 2.1.1 Materials

[0125] NHEK’s p9 (adult)

[0126] T-75 flasks (2) (Sarstedt Yellow Extra TC coated flasks)

[0127] Promocell Keratinocyte Media 2

[0128] DMSO [stock solution = 2ul in 2ml] [working solution 55ul of stock in 10ml medium]

[0129] FA 50nM [2ul of lOOmM in 2ml medium] [working solution 5ul of stock in 10ml medium] TAD 500nM [2ul of lOOmM in 2ml medium] [working solution = 50ul of stock in 10ml medium] IsoHelix Xtreme RNA isolation kit

[0130] QuickDrop spectrophotometer

[0131] RNA Stabilization Tubes (GENEWIZ)

[0132] 2.1.2 Cells Growth and Treatments

[0133] NHEK’s were cultured according to standard techniques.

[0134] At T = 0, 400,000 NHEK’s were seeded per T-75 flask in 20 mL of Promocell Keratinocyte Media 2 and incubated at 37°C, 5% CO2.

[0135] At T = 48 hrs cells were viewed under a microscope to confirm -70% confluency and the medium was replaced as follows: Control flasks were treated with 20 ml of fresh medium containing 1 lOpl of 0.1% DMSO whereas API treated flasks had 20 ml of fresh medium containing 50nM FA and 500nM TAD dissolved in the same amount of DMSO used in control flasks.

[0136] At T = 96 hrs the medium was changed adding the same amount of DMSO to control flasks whereas API treated flasks had 500nM TAD added as a single agent dissolved in the same amount of DMSO as in control flasks.

[0137] 2.2.3 RNA Harvest

[0138] On the following Monday, RNA was harvested using the IsoHelix Xtreme kit following the manufacturer’s protocol, htps: / / isohelix.com / .

[0139] The concentration and purity of the RNA was established using a QuickDrop spectrophotometer. The RNA was then transferred into 1 RNA Stabilization Tubes (GENEWIZ -htps: / / www.genewiz.com / en-GB / ) for storage and / or prior to any shipment.

[0140] 2.2.4 RNA-Seq Data Analysis Workflow: Quality Control, Gene Expression, and Pathway Insights Using Galaxy and Enrichr

[0141] The following protocols were followed to analyse RNA sequencing (RNA-seq) data from NHEK’s treated with FA and TAD. The workflow includes data quality checks, alignment, gene quantification, and pathway analysis, providing a full overview of the steps taken to derive biologically meaningful insights.

[0142] 2.2.4.1 IntroductionRNA sequencing (RNA-seq) is a powerful tool for investigating gene activity on a genome-wide scale. By comparing the RNA profiles of samples under different conditions, researchers can identify which genes are upregulated or downregulated in response to treatments. This study utilized RNA-seq to assess the effects of FA and TAD on NHEKs.

[0143] The analysis was conducted using Galaxy, an open-source, web-based platform for data-intensive biological research. Galaxy provides an accessible, user-friendly environment that requires no programming skills, allowing researchers to perform complex bioinformatics analyses using pre-built tools and workflows. For RNA-seq, Galaxy integrates quality control, data cleaning, alignment, and gene quantification into a seamless pipeline. By centralising these steps, Galaxy ensures reproducibility and simplifies the computational challenges of RNA-seq analysis.

[0144] After identifying significantly altered genes, the study utilised Enrichr for pathway analysis.

[0145] Enrichr is an intuitive, web-based resource for gene enrichment analysis. It connects experimental results to biological contexts by identifying overrepresented pathways, processes, and molecular functions in a gene list. For this study, the KEGG 2021 database within Enrichr was used to explore how the experimental treatments influenced key cellular pathways.

[0146] Galaxy htps: / / usegalaxy.org /

[0147] Enrichr htps : / / maayanlab .cloud / Enrichr /

[0148] 2.2.4.2 Data Processing Steps

[0149] Step 1: Uploading and Verifying the Data

[0150] 1. Upload raw data:

[0151] • FASTQ files (R1 and R2) for control and treated samples were uploaded. These files contain raw RNA sequences and quality scores for each base.

[0152] 2. Verify file integrity using MD5 checksum (Galaxy Software Tool):

[0153] • MD5 checksum was performed to ensure no corruption occurred during file transfer.

[0154] Step 2: Quality Control of Raw Data

[0155] 1. Assess data quality using FASTQC (Galaxy Software Tool):

[0156] • FASTQC generated reports to highlight issues such as low-quality bases, adapter contamination, and deviations in GC content.

[0157] 2. Initial findings:

[0158] • Low-quality bases were observed at the sequence ends, and adapter contamination was present. These issues needed to be addressed.

[0159] Step 3: Cleaning the Data (Trimming)

[0160] 1. Trimming with Trimmomatic (Galaxy Software Tool):

[0161] • Adapter sequences and low-quality regions were removed.

[0162] • Sliding window trimming was applied, cuting sequences when the average quality within a 4-base window dropped below 20.

[0163] • Sequences shorter than 36 bases were discarded.

[0164] 2. Re-checking quality:

[0165] • FASTQC was run again on trimmed data to confirm improvements.

[0166] Step 4: Aligning Sequences to the Human Genome

[0167] 1. Align sequences using HISAT2 (Galaxy Software Tool):

[0168] • Cleaned RNA sequences were mapped to the human genome (hg38).

[0169] 2. Handling strand specificity:

[0170] • Based on guidance, R2 was identified as the original RNA strand and specified in HISAT2 setings.

[0171] 3. Output:• The alignment produced BAM files, which were quality-checked using SAMtools Flagstat. High mapping rates (>98%) and proper pairing (>95%) confirmed alignment success.

[0172] Step 5: Quantifying Gene Activity

[0173] 1. Counting gene reads using featureCounts (Galaxy Software Tool):

[0174] • BAM files were processed to generate a table of gene counts.

[0175] • A GTF annotation file was used to map sequences to known genes.

[0176] 2. Output:

[0177] • A table of gene counts, where the number of reads per gene reflected its activity level.

[0178] Step 6: Analysing Gene Counts using Microsoft Excel

[0179] 1. Data preparation :

[0180] • Genes with zero counts were removed.

[0181] • Genes with fewer than 50 counts were initially excluded in order to focus on statistically significant changes .

[0182] 2. Calculating fold changes:

[0183] • Gene activity in treated samples was compared to controls.

[0184] • Fold changes were calculated as:

[0185] „ , , Gene Aniivifev (treated) raid Change ■■■■ - - - - ~

[0186] Gene Activity (control)

[0187] 3. Filtering for significance:

[0188] • Genes with less than a 2-fold increase or 2-fold decrease were excluded, leaving only the most relevant results.

[0189] Step 7: Pathway Analysis Using Enrichr and KEGG 2021

[0190] 1. Preparing input data:

[0191] • Separate lists of upregulated and downregulated genes were uploaded to Enrichr. 2. Pathway enrichment using KEGG 2021:

[0192] • KEG 2021 identified pathways enriched in the gene lists.

[0193] • Pathways were ranked based on their p-values and combined scores.

[0194] 3. Key findings:

[0195] • Upregulated genes were associated with stress response and drug metabolism pathways.

[0196] • Downregulated genes were linked to pathways involved in cell growth and division.

[0197] 2.2.4.3 Key Observations and Outcomes

[0198] • Data quality: High mapping rates and well-paired reads confirmed the reliability of the sequencing and alignment processes.

[0199] • Gene expression changes: Significant alterations in gene activity were observed in response to the treatments.

[0200] • Pathway insights: Pathway analysis provided clues about the mechanisms underlying the experimental drug effects.

[0201] This comprehensive protocol demonstrates the meticulous steps involved in processing RNA-seq data to extract biologically meaningful insights. By integrating quality control, alignment, quantification, and pathway analysis, the study successfully identified key genes and pathways affected by experimental treatments, paving the way for future research.

[0202] 2.2 RESUETS2.2.1 Gene Filtering and Differential Expression Analysis

[0203] A comprehensive RNA sequencing analysis was performed to assess the effects of a novel drug combination of FA and TAD on gene expression NHEK’s. An initial dataset of 64,251 genes was rigorously filtered. Genes with counts below 50 were excluded, resulting in 27,570 remaining genes. Further filtering also removed genes with fold changes below 2 (either upregulated or downregulated), leaving 162 upregulated and 83 downregulated genes. Analysis of the expression of the house -keeping genes P actin, GAPDH and elongation factor la from the same data set, showed no significant difference in gene expression.

[0204] 2.2.2 Prioritisation of Downregulated Genes

[0205] Downregulated genes were prioritised for investigation, as these are more likely to reflect pathways directly inhibited by the treatment. This decision aligns with evidence that downregulation often corresponds to sustained suppression of biological processes rather than transient responses (Xia & Wishart, 2011 DOI: 10.1038 / nprot.2011.319). It was shown that suppressed pathways included those involved in inflammation, cell proliferation, and tissue remodeling.

[0206] 2.2.3 Identification of Disease-Relevant Genes

[0207] A literature review of the 83 downregulated genes surprisingly identified 24 genes that are typically upregulated in ISD, including atopic dermatitis and psoriasis.

[0208] Table 1 (below) identifies the genes that are normally upregulated in inflammatory conditions that were downregulated in cultured normal human epidermal keratinocytes (NHEK’s) treated with Fenbendazole (FA) and Tadalafil (TAD).

[0209] Table 1: Genes Upregulated in Inflammatory Conditions (From a total pool of 83 genes which were significantly Downregulated by FA+TAD)

[0210]

[0211]

[0212]

[0213] Most notably, four of these were key genes which are known to be very specific and causally associated with ISD pathologies:

[0214] • ANO1: Involved in pruritus and inflammatory signalling.

[0215] • TNC: Drives tissue remodelling and chronic inflammation.

[0216] • FOXM1: Promotes keratinocyte hyperactivation and cytokine generation.

[0217] • CDK1: Facilitates excessive keratinocyte proliferation.

[0218] The suppression of these genes indicates that the FA / TAD combination targets molecular pathways central to the causation of ISD and Figure 2 shows the fold-change decrease in gene expression induced by this treatment.

[0219] In view of the overlap between the ageing process and ISD, the inventors were surprised that no significant difference in expression of the key age-related genes mTOR, SIRT1 and GSK3 was found. These only showed marginal FA / TAD induced changes which indicates these compounds may not be acting via these key ageing -related targets. In addition, there was a significant drug -induced upregulation of p21 coupled with downregulation of Ki67 (MK167) which indicates an antiproliferative mode-of-action that is consistent with the observed activity against ISD.

[0220] Furthermore, it is also notable that functional annotation of the downregulated genes listed in Table 1, clearly exemplifies the previously discussed functional overlap between genes related to the ageing process and ISD whereby some are exclusively related to ISD (18 / 24 genes)) whereas others are related to both ISD and the aging process (4 / 24). Surprisingly, increased expression of 3 of these (BUB1B, CCNA2, and F0XM1) reduce effects of the ageing process whereby they act to maintain cell division and genomic integrity (Kim & Kao 2005 DOI 10.18632 / aging.100990; Gygli et al 2016 DOI 10.18632 / aging.100990; Ouchi et al 2022 DOI 10.1038 / s43587-022-00222-y). Thus, it is counterintuitive that FA / TAD suppression of these targets would provide therapeutic benefit for the treatment of ISD.

[0221] 2.2.4 Significance of Down-regulation

[0222] To calculate the probability of FA+TAD targeting upregulated genes associated with Psoriasis and Atopic Dermatitis we use the Hypergeometric Distribution Formula.

[0223] Hypergeometric Distribution Formula:

[0224]

[0225] PROBABILITY OF GETTING 18 specifically upregulated genes associated with Atopic Dermatitis and or Psoriasis. [N= 27570 genes in population, K= estimated 1000 upregulated genes associated with Atopic Dermatitis and Psoriasis, n= 83 downregulated genes by FA+TAD, k = 18 / 83 Atopic Dermatitis and or Psoriasis genes.] P = 7.17 x 10“10, or 0.0000000717%.

[0226] This extremely low probability suggests such an overlap is highly unlikely under random sampling and illustrates, as the inventors suspected from the phenotypical changes they observed in the canine study, that APIs have efficacy for treating the conditions according to the invention because they target and downregulate genes associated with inflammation.

[0227] 2.2.5 Pathway Analysis

[0228] Pathway enrichment analysis using the Enrichr KEGG 2021 database revealed two highly enriched pathways among the 83 downregulated genes:1. Cell Cycle: Drug -induced down-regulated expression of key genes (e.g., CDK1, CCNA2) is consistent with reduced abnormal cellular proliferation which is a key feature of psoriasis. Notably, this is also consistent with the observed FA / TAD induced upregulation p21 and downregulation of Ki67.

[0229] 2. Cellular Senescence: Drug-induced downregulated expression of senescence- associated genes may reduce the chronic inflammation that is a product of senescent or damaged cells.

[0230] Clearly, these findings highlight the dual effects of FA / TAD treatment on inflammation and abnormal cellular growth.

[0231] 2.3 DISCUSSION AND RESULTS

[0232] The results indicate that a novel drug combination, comprising treatment with a mixture of FA and TAD, exerts its effects by suppressing critical pathways which drive inflammation, cellular proliferation, and tissue remodeling in skin. Therefore, the observed drug-induced downregulation of disease-relevant genes provides mechanistic insights into its therapeutic potential.

[0233] Treatments according to the invention demonstrate several advantages over existing therapies:

[0234] 1. Mechanism-focused action: Targets the molecular basis of inflammation and hyperproliferation rather than solely managing symptoms.

[0235] 2. Disease-modifying potential: Reduces the activity of pathways underlying chronic disease progression, offering long-term benefits.

[0236] 3. Broad applicability: Effective against core pathways implicated in multiple inflammatory and proliferative conditions, which is very likely to significantly extend its potential beyond dermatological disorders.

[0237] These findings underscore the market advantage of this combination therapy, addressing unmet needs in the treatment of chronic ISD. Example 3 further evaluates the APIs in a cytokine-activated keratinocyte model representative of inflammatory skin disorders.

[0238] EXAMPLE 3

[0239] This example evaluates the effects of benzimidazole compounds and a phosphodiesterase-5 (PDE5) inhibitor on cytokine -driven inflammatory signalling in normal human epidermal keratinocytes (NHEKs). In contrast to the basal conditions described in Example 2, this model employs interleukin-17 (IL-17) to induce a psoriasis-like inflammatory state, enabling assessment of drug-induced transcriptional reversal and potential synergy under disease-relevant conditions. The results demonstrate that combinations of a benzimidazole compound and Tadalafil produce coordinated suppression of IL-17-responsive pathways and exhibit synergistic activity at both the transcript and pathway levels.

[0240] 3.1 Methods

[0241] IL- 17 is a key pathogenic cytokine in psoriasis, atopic dermatitis and related inflammatory skin disorders. Keratinocytes respond strongly to IL-17 by activating NF-KB, AP-1 and chemokine pathways, making IL- 17 stimulation a clinically relevant in vitro model. The following procedures were used to assess the transcriptomic effects of the APIs under IL-17-stimulated inflammatory conditions.

[0242] 3.1.1 Cell culture and RNA sequencingAdult NHEKs (Promocell, passage 6) were cultured in Keratinocyte Growth Medium 2 (10 mL per T-25 flask; 1.2 x 105cells per flask) and incubated at 37°C, 5 % CO2. Cells were stimulated with interleukin- 17 (IL-17; 50 ng / mL) for 48 h to induce a psoriasis-like inflammatory phenotype, then treated for a further 48 h with vehicle (DMSO), Fenbendazole (F, 50 nM), Albendazole (A, 50 nM), Tadalafil (T, 500 nM), or the combinations F + T and A + T. Two biological replicates were prepared per condition; RNA was pooled before library preparation, and technical triplicates were generated where indicated. Total RNA was extracted using IsoHelix Xtreme RNA Kit, quantified spectrophotometrically, and sequenced (Illumina HiSeq, 150 bp paired-end; > 30 million reads per sample). FASTQ files were MD5-verified and uploaded to the Galaxy platform (ht( s; / / i^ega|t y,org ) via a Python API workflow.

[0243] 3.1.2 Data processing and differential expression.

[0244] FASTQC (vO.11.9) confirmed read quality; Trimmomatic (v0.39) removed adapters (ILLUMINACLIP:TruSeq3-PE.fa:2:30: 10, SLIDINGWINDOW:4:20, MINLEN:50). Reads were aligned to GRCh38 / hg38 using HISAT2 (v2.2.1) with GENCODE v47 annotation. featureCounts (v2.0.3) generated paired-end gene-level counts excluding multi-mapped reads (MAPQ < 10). edgeR (v3.38.4) performed TMM normalisation, dispersion estimation, and exact tests against the IL-17 + DMSO control. Genes with FDR < 0.05 and |log2 fold-change| > 1 were considered significant.

[0245] Synergistic effects were assessed using an internal Ravan ABliss transcriptomic model extending the Bliss-independence principle to RNA-seq data, where positive ABliss values denote synergy.

[0246] 3.1.3 Gene Set Enrichment Analysis (GSEA).

[0247] Rank-based pathway enrichment analysis (KEGG 2021 Human and Hallmark gene sets; 1 000 permutations; gene-set sizes 15-500; weighted statistic; FDR q < 0.25 significant) was applied to ranked log2 fold-change lists. Pathway reversals were interpreted relative to the IL-17-induced signature.

[0248] 3.2 Results

[0249] 3.2.1 IL-17-induced inflammatory transcriptional state

[0250] Stimulation of normal human epidermal keratinocytes (NHEKs) with interleukin- 17 (IL-17) induced a robust inflammatory transcriptional response consistent with a psoriasis-like phenotype. Comparison of IL-17-stimulated cells (IL-17 + DMSO) to unstimulated DMSO-treated cells revealed widespread activation of cytokine -responsive and stress-associated gene networks, with 1,572 genes up-regulated and 1,492 genes down-regulated.

[0251] Drug effects were evaluated under continued IL- 17 stimulation by comparing each drug -treated condition to the IL-17 + DMSO control. All treatment effects described below therefore reflect modulation of an established IL-17-driven inflammatory state, and no drug-only (no IL-17) conditions were included.

[0252] Single-agent treatment with Fenbendazole (F), Albendazole (A), or Tadalafil (T) partially attenuated the IL-17-induced transcriptional profile relative to the IL-17 + DMSO control. In contrast, combination treatments comprising a benzimidazole compound and Tadalafil (F + T or A + T) produced broader suppression of IL-17-responsive transcriptional programmes relative to the IL-17 + DMSO control, indicating enhanced inhibition of cytokine-driven inflammatory signalling under disease-relevant conditions.

[0253] 3.2.2 Synergistic effects assessed using ABliss transcriptomic analysis

[0254] Synergistic interactions between benzimidazole compounds and Tadalafil were assessed using an internal ABliss transcriptomic model extending the Bliss-independence principle to RNA-sequencing data. Positive ABliss values indicate cooperative effects exceeding those expected under additivity.Both combination treatments demonstrated positive ABliss values across multiple transcriptomic domains. Fenbendazole + Tadalafil yielded a ABliss value of 0.49, while Albendazole + Tadalafd yielded a ABliss value of 0.61. These values are consistent with synergistic behaviour rather than simple additivity, with the Albendazole + Tadalafd combination exhibiting slightly stronger cooperative effects. In each case, the combination treatment outperformed the corresponding singleagent treatments when compared to the IL- 17 + DMSO control.

[0255] 3.2.3 Pathway-level effects summarised by NES matrices

[0256] Pathway-level responses were assessed using Gene Set Enrichment Analysis (GSEA) and are summarised in matrices of Normalised Enrichment Scores (NES) included within this Example. NES values were derived from ranked gene lists comparing drug-treated IL-17-stimulated cells to the IL-17 + DMSO control.

[0257] IL- 17 stimulation produced strong positive enrichment within inflammatory response pathways, including TNFa signalling via NF-KB. Single-agent treatments partially reduced enrichment of these pathways relative to the IL- 17 + DMSO control. In contrast, combination treatments (F + T and A + T) consistently produced larger negative NES values, indicating coordinated suppression of IL- 17-driven inflammatory signalling exceeding that observed with individual compounds.

[0258] Across multiple inflammatory and stress-response pathways, combination treatments produced broader pathway-level suppression relative to the IL- 17 + DMSO control, consistent with transcriptomic normalisation of cytokine-activated signalling networks.

[0259] 3.2.4 Gene-level effects contributing to pathway suppression

[0260] Gene-level expression changes contributing to pathway suppression were examined using representative leading-edge genes identified by GSEA. Leading-edge genes comprise the subset contributing most strongly to pathway enrichment and include canonical mediators of keratinocyte inflammatory activation.

[0261] Combination treatments consistently reduced expression of multiple IL-17-inducible inflammatory transcripts relative to the IL- 17 + DMSO control. These transcripts include, for example, AP-1 complex components (such as JUNB and FOS), angiogenic and leukocyte -trafficking factors (such as VEGFA), inflammasome-associated genes (such as NLRP3), and chemokines and cytokines involved in immune-cell recruitment (including CXCL9 and CSF3).

[0262] Representative examples of gene-level expression changes within these inflammatory pathways are illustrated in the accompanying bar-chart figures (Figures 3A and 3B) and Table 2.

[0263] Table 2: represents normalised enrichment scores (NES) across all Hallmark and KEGG gene sets in IL-17-stimulated NHEKs

[0264]

[0265]

[0266] Legend: ▲ NES s 0.5 (activated); ▼ NES s -0.5; • -0.5 < NES < 0.5 (near-neutral)

[0267] 3.3 Transcript-level summary

[0268] Across NF-KB- and AP-1 -associated gene sets and inflammatory effector genes, combination treatments consistently reduced transcript abundance relative to the IL-17 + DMSO control. These transcript-level effects are concordant with the pathway-level suppression observed in the NES matrices described above.

[0269] Taken together, the transcriptomic and synergy analyses support the conclusion that dual treatment with a benzimidazole compound and Tadalafil yields superior suppression of IL-17-driven inflammatory signalling compared with either agent alone.

[0270] 3.4 Mechanistic interpretation

[0271] Without wishing to be bound by theory, the results of this Example are consistent with cooperative suppression of IL-17-driven NF-KB- and AP-l-centred inflammatory networks in keratinocytes. The observed pathway-level and gene-level effects indicate multi-node attenuation of cytokine -responsive transcriptional circuits rather than modulation of a single upstream effector.

Claims

CLAIMS1. A composition comprising a benzimidazole compound with anthelmintic activity and a Phosphodiesterase 5 inhibitor for use as a medicament to treat a Chronic Inflammatory Skin Disorder in a vertebrate subject.

2. A composition comprising a benzimidazole compound with anthelmintic activity and a further composition comprising a Phosphodiesterase 5 inhibitor for use in combination as medicaments to treat a Chronic Inflammatory Skin Disorder in a vertebrate subject.

3. The composition or compositions for use according to claims 1 or 2 wherein the benzimidazole compound is selected from the group comprising: Fenbendazole, Mebendazole, Flubendazole, Parbendazole, Oxfendazole, Oxibendazole, Albendazole, Ricobendazole, Albendazole sulfoxide, Thiabendazole, Thiophanate, Febantel, Netobimin, Triclabendazole and functional equivalents thereof.

4. The composition or compositions for use according to claim 3 wherein the benzimidazole compound is Fenbendazole.

5. The composition or compositions for use according to any one of claims 1-4 wherein the Phosphodiesterase 5 inhibitor is selected from the group comprising Vardenafd, Sildenafil, Avanafil, Tadalafil and functional equivalents thereof.

6. The composition or compositions for use according to claim 5 wherein the Phosphodiesterase 5 inhibitor is Tadalafil.

7. The composition or compositions for use according to any one of claims 1-6 in an amount effective for downregulating one or more genes listed in Table 1.

8. The composition or compositions for use according to claim 7 wherein the gene is one or more selected from AN01, TNC, F0XM1 and CDK19. The composition or compositions for use according to any preceding claim used in a dosing regimen wherein the benzimidazole compound with anthelmintic activity is administered as an oral dose of between 0.1 - 22 mg / kg / day.

10. The composition or compositions for use according to any preceding claim in a dosing regimen wherein a Phosphodiesterase 5 inhibitor is administered as an oral dose of between 10 - 300 pg / / kg / day.

11. The composition or compositions for use according to claim 9 or 10 wherein the composition or compositions are administered as continuous or discontinuous treatment regimens for 1-7 days / week for intermittent periods of between 1-12 months in any 12 month period.

12. The composition or compositions for use according to any one of claims 2 - 11 wherein:(i) the benzimidazole compound is co-administered every day with a PDE5 inhibitor for a period of between 1 week and 6 months;(ii) following period (i) the PDE5 inhibitor is administered as a single agent for a period of between 2 weeks to 6 months; and(iii) steps (i) and (ii) are repeated until the Chronic Inflammatory Skin Disorder is treated to the subject’s or a clinician’s satisfaction.

13. The composition or compositions for use according to claim 12 wherein the benzimidazole compound is co-administered with the PDE5 inhibitor for between 1 week and 2 months in step (i); and wherein the PDE 5 inhibitor is administered for 1 to 2 months in step (ii).

14. The composition or compositions for use according to claims 12 or 13 wherein step (iii) comprises repeated cycles of: step (i) for 1 to 2 weeks followed by step (ii) for periods of between 3 weeks and 2 months.

15. The composition or compositions for use according to any preceding claim wherein the disorder is one selected from eczema, psoriasis, atopic dermatitis, vitiligo, rosacea and lupus associated skin disorders.

16. The composition or compositions for use according to any preceding claim wherein the subject is an animal of veterinary interest.

17. The composition or compositions for use according to any one of claim 1-16 wherein the subj ect is a canine.

18. The composition or compositions for use according to any one of claim 1-15 wherein the subj ect is a human being.

19. A method of treating Chronic ISD in a vertebrate subject in need of such treatment comprising administering to the subject a therapeutically effective amount of a benzimidazole compound with anthelmintic activity and a therapeutically effective amount of a Phosphodiesterase 5 inhibitor.

20. The composition or method according to any preceding claim, wherein the benzimidazole compound with anthelmintic activity and Phosphodiesterase 5 inhibitor are provided in a dose effective to synergistically suppress inflammatory gene expression as determined by a ABliss synergy score greater than 0.3.

21. The composition or method of any preceding claim, wherein the benzimidazole compound with anthelmintic activity and Phosphodiesterase 5 inhibitor are provided in a dose effective to suppress inflammatory responsive or TNFa-responsive NF-KB / AP-1 signaling in keratinocytes.

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