WO2011028112A1

WO2011028112A1 - Imidazole-4-carboxylic acid for use in treating a disease related to extracellular reactive oxygen species

Info

Publication number: WO2011028112A1
Application number: PCT/NL2010/050551
Authority: WO
Inventors: Arthur Kammeijer; Joannes Dositheus Bos
Original assignee: Valletta Health B.V.
Priority date: 2009-09-02
Filing date: 2010-09-02
Publication date: 2011-03-10
Also published as: TW201124138A

Abstract

The invention relates to the field of medicine and in particular to the use of imidazole-4-carboxylic acid or a pharmaceutically acceptable salt thereof for use in treating a disease related to extracellular reactive oxygen species, such as acne, atopic dermatitis, rosacea, pyoderma gangrenosum, necrotizing fasciitis, Sweet's syndrome, Behçet's disease, septic shock, rheumatoid arthritis, or colitis. The present invention also relates to the field of pharmacy and in particular to the pharmaceutical use of imidazole-4-carboxylic acid.

Description

Title: Imidazole-4-carboxylic acid for use in treating a disease related to extracellular reactive oxygen species.

The invention relates to the field of medicine and in particular to the use of imidazole-4-carboxylic acid for use in treating a disease related to

extracellular reactive oxygen species, such as acne, atopic dermatitis, rosacea, pyoderma gangrenosum, necrotizing fasciitis, Sweet's syndrome, Behcet's disease, septic shock, rheumatoid arthritis, or colitis. The present invention also relates to the field of pharmacy and in particular to pharmaceutical use of imidazole-4-carboxylic acid.

Acne vulgaris is a chronic disease of the pilosebacea gland associated with an increase of fat secretion, ductal hyperkeratosis with pilosebaceous follicle block and secondary inflammation caused by bacterial colonization of Propionibacterium acnes. Acne risk factors are, amongst other, androgens, heredity and menstruation. A correlation exists between acne severity and stress episodes. Acne is the most frequent cutaneous disorder in teenagers, with a prevalence of 79% to 95% of 16 to 18 year olds. It is generally more severe in males than females.

The diagnosis of acne is a clinical diagnosis. It is characterized by open- closed comedones and inflammatory lesions affecting mainly face, back, high part of trunk, arms and shoulders. Clinically it can be classified as: comedonic acne, pustulous acne, cystic acne or acne fulminans.

Use of fatty cosmetics must be avoided and instead, oil free products should be used. No evidence exists that dietetic changes may influence the severity of the process.

Choice of treatment will typically depend on the type and severity of the acne. Benzoyl peroxide at 2.5%, 5 or 10%, has shown beneficial effect on acne with different degree of severity and is at present the treatment of first choice. However, some studies have reported contact sensitization to the active ingredient in up to 76% of subjects using benzoyl peroxide, resulting in red, dry, itchy and/or painful skin. Another disadvantage of benzoyl peroxide is that it is a powerful bleaching agent. Contact with fabrics or hair can cause permanent color dampening almost immediately.

Other treatments of acne include the use of topic retinoids, such as retinoic acid or adapalene, topic antibiotics such as erythromycin or

clindamycin, systemic antibiotics such as doxycycline and erythromycin, oral combined contraceptives, such as ethinylestradiol with changeable doses of progestagens or cyproterone acetate, and oral isotretinoins.

Disadvantages of most retinoids are for example that retinoids can cause skin irritation and increase the risk of sunburn when the skin is exposed to sunlight. The use of antibiotics, topically or systemically, bears the risk of induction of resistance and, in particular when used systemically, antibiotics can for instance cause antibiotic-associated diarrhea (AAD) by irritating the bowel directly, changing the levels of gut flora, or allowing pathogenic bacteria to grow.

Hormone therapy, such as oral combined contraceptives, which is typically only prescribed to female subjects suffering from acne, may for instance increase the risk on developing breast or cervical cancer. Other documented side-effects of hormone therapy include: nausea, vomiting, abdominal cramps, bloating, severe headaches, spotting, weight gain, mood swings, anxiety, depression, tenderness of the breasts.

Sometimes isotretinoin is used for treating acne. However, it is a teratogen and highly likely to cause birth defects if taken during pregnancy. Furthermore isotretinoin has a broad spectrum of mild to severe side effects ranging from dryness of skin, lips and mucous membranes to all kinds of organ inflammation, such as optic neuritis, inflammatory bowel disease, pancreatitis, and hepatitis. It is even believed that severe depression can occur as a result of isotretinoin treatment, although the underlying disease, e.g. acne, may contribute to the depression as well. Considering the side effects, isotretinoins are only given as a last resort. One object of the present invention is to provide a treatment for acne, atopic dermatitis, rosacea, pyoderma gangrenosum, necrotizing fasciitis, Sweet's syndrome, Behcet's disease, septic shock, rheumatoid arthritis, and/or colitis. Another object of the present invention is to provide a treatment for a disease that is accompanied by the presence of a superoxide anion radical.

In one aspect, the present invention provides a treatment for acne, atopic dermatitis, rosacea, pyoderma gangrenosum, necrotizing fasciitis, Sweet's syndrome, Behcet's disease, septic shock, rheumatoid arthritis, and/or colitis.

In a working example, the invention shows that imidazole-4-carboxylic acid, but not cis-urocanic acid or ethyl-imidazole-4-acetic acid scavenges superoxide anion radicals produced by a cell.

Without being bound to theory, it is thought that imidazole- 4- carboxylic acid, through removal of extracellular reactive oxygen species produced by a cell, for instance neutrophils or keratinocytes, has a beneficial effect on a disease that is accompanied by the presence of an extracellular reactive oxygen species. Examples of such a disease are for instance acne, atopic dermatitis, rosacea, pyoderma gangrenosum, necrotizing fasciitis, Sweet's syndrome, Behcet's disease, septic shock, rheumatoid arthritis, and colitis.

In a first embodiment, the present invention therefore provides a method of treating an individual suffering from acne, atopic dermatitis, rosacea, pyoderma gangrenosum, necrotizing fasciitis, Sweet's syndrome, Behcet's disease, septic shock, rheumatoid arthritis or colitis, comprising providing said individual with imidazole-4-carboxylic acid and/or a

pharmaceutically acceptable salt thereof. In another working example, the present invention shows that in a clinical pilot study, a majority of subjects suffering from acne reported and/or showed a beneficial effect of topically applied imidazole-4-carboxylic acid. No serious or severe adverse effects were reported in this study or in the foregoing safety study, wherein the safety and pharmacokinetic parameters of topical treatment with imidazole-4-carboxylic acid were assessed.

It has been postulated that granulocytes may play a role in inflamed acne lesions¹. Recently, involvement of reactive oxygen species, and in particular superoxide anions, produced and secreted by keratinocytes has been reported in the pathogenesis of acne².

In a more preferred embodiment, the invention provides a method of treating an individual suffering from acne, comprising providing said individual with imidazole-4-carboxylic acid and/or a pharmaceutically acceptable salt thereof.

Atopic dermatitis is an inflammatory, chronically relapsing, noncontagious and pruritic (itching) skin disorder. Atopic dermatitis has a strong genetic component. Currently there is no cure for atopic eczema and treatment mainly involves avoiding triggers, such as allergens, and the use of topical hydrating and/or anti-inflammatory ointments. It has been reported that reactive oxygen species play a role in the etiology of atopic dermatitis. For example, monocytes from patients with atopic dermatitis are primed to generate ROS in response to zymosan, a Toll-like receptor 2 (TLR2) ligand⁶. This indicates that in individuals suffering from atopic dermatitis, TLR2 ligands such as for instance bacterial lipoproteins induce the production of ROS. Further provided is therefore a method of treating an individual suffering from atopic dermatitis, comprising providing said individual with imidazole-4-carboxylic acid and/or a pharmaceutically acceptable salt thereof. Behcet's disease is a form of vasculitis that can lead to ulceration and other lesions. Significant neutrophil infiltration is a hallmark of early lesions in Behcet's disease. It is unknown whether neutrophil hyper-activity, i.e.

excessive production of extracellular reactive oxygen species, is genetically determined or due to persistent activation by external priming agents.

However, it is postulated that neutrophil activity in Behcet's disease is under genetic control³.

Sweet's syndrome or acute febrile neutrophilic dermatosis is a condition characterized by the sudden onset of fever, leukocytosis, and tender, erythematous, well-demarcated papules and plaques which show dense neutrophilic infiltrates on histological examination. Current first line treatment of Sweet's syndrome is immune suppression with corticosteroids. Patients who have a potential systemic infection or in whom corticosteroids are contraindicated generally use oral potassium iodide or colchicine.

Other alternatives to corticosteroid treatment include dapsone, doxycycline, clofazimine, and cyclosporine. All of these drugs influence migration and other functions of neutrophils. Imidazole-4-carboxylic acid is also very useful as a first line treatment of Sweet's syndrome, as imidazole-4-carboxylic acid is able to remove an extracellular reactive oxygen species, preferably a superoxide anion radical. Further, imidazole-4-carboxylic acid does not interfere significantly with intracellular reactive oxygen species that are useful for combating an infection. Reactive oxygen species (ROS) have also been reported to play a role in the pathophysiology of the skin disease rosacea, a chronic, genetically- determined and UV-triggered disease⁴. Pyoderma gangrenosum is a disease that causes tissue to become necrotic, causing deep ulcers that usually occur on the legs. When they occur, they can lead to chronic wounds. Though the etiology is not well understood, the disease is thought to be due to immune system dysfunction, and

particularly improper functioning of neutrophils, (i.e., defects in chemotaxis or hyperactivity). Anti-inflammatory treatment, for instance with dapsone, which inhibits neutrophil migration and production of reactive oxygen species, has been shown useful in treatment of pyoderma gangrenosum.

Necrotizing fasciitis (NF), commonly known as flesh-eating disease or flesh-eating bacteria, is a rare infection of the deeper layers of skin and subcutaneous tissues, easily spreading across the fascial plane within the subcutaneous tissue. The term flesh-eating bacteria is in fact not correct, the bacteria do not actually "eat" flesh, but cause destruction of skin and muscle by releasing toxins (virulence factors). Some of these toxins are so called super antigens and capable of activating T-cells non- specifically, which causes the overproduction of cytokines. These cytokines subsequently attract and activate other immune cells, amongst which, granulocytes. Imidazole- 4- carboxylic acid is especially useful in the treatment of necrotizing fasciitis, as it is able to prevent tissue damage by removing an extracellular reactive oxygen species, preferably a superoxide anion radical.

Septic shock is a life-threatening condition resulting from a generalized infection with for instance bacteria or fungi. In septic shock, it is not the infectious agent that makes the condition life-threatening, but the

overshooting immune reaction directed towards it. In septic shock the immune system is deranged, resulting in hyperactivity of many immune cell types, including granulocytes.

Rheumatoid arthritis is a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks synovial joints. Although the cause of rheumatoid arthritis is unknown, autoimmunity plays a pivotal role in both its chronicity and progression. It has been reported that reactive oxygen species contribute to the severity of rheumatoid arthritis.⁸

About 1% of the world's population is afflicted by rheumatoid arthritis and currently no effective cure exists. Further provided is thus a method of treating an individual suffering from rheumatoid arthritis, comprising providing said individual with imidazole-4-carboxylic acid and/or a

pharmaceutically acceptable salt thereof. Colitis refers to an inflammation of the colon and is often used to describe an inflammation of the large intestine (colon, cecum and rectum). It has also been reported that colitis is accompanied by production of ROS, which aggravates the inflammatory reaction observed in colitis. This has for instance been shown in a study, wherein superoxide dismutase ameliorated TNBS- induced colitis by reducing oxidative stress.⁷

Colitis has great impact on an individual's life and currently no effective cure exists. Further provided is thus a method of treating an individual suffering from colitis, comprising providing said individual with imidazole-4- carboxylic acid and/or a pharmaceutically acceptable salt thereof.

As said before, imidazole- 4- carboxylic acid and pharmaceutically acceptable salts thereof are very useful for treating the above mentioned diseases, reported to be accompanied by granulocyte activity. In another embodiment, the invention provides a method of treating a subject suffering from a disease related to the presence of an extracellular reactive oxygen species, preferably a superoxide anion radical. Cellular produced reactive oxygen species are primarily produced by phagocytic cells, such as granulocytes, keratinocytes, monocytes, and macrophages. Almost every cell in the human body is capable of producing superoxide anion radicals. The enzyme superoxide dismutase rapidly catalyzes the removal (dismutation) of superoxide into oxygen and hydrogen peroxide in most cell types. However, some cells, especially those which have a function in primitive defense against microbes, such as granulocytes, keratinocytes, monocytes, and macrophages are capable of storing, for instance in specialized granules, and secreting superoxide anion radicals. Granulocytes,

keratinocytes, monocytes, and macrophages are all considered to have phagocytic activity, and it is believed that their capacity to store and secrete reactive oxygen species is related to their role in immune defense. One function of a phagocyte is to ingest microbes, for instance bacteria, and kill these microbes intracellular by contacting the microbes with, amongst others, reactive oxygen species.

Next to intracellular killing, many phagocytes also secrete reactive oxygen into the extracellular space. When such extracellular reactive oxygen species come into contact with cells of the host, for instance endothelial cells, these cells are damaged, thereby initiating an inflammatory response, which in turn attracts other phagocytes. When uncontrolled, this results in a vicious circle that ultimately results in severe tissue damage. In one embodiment, the invention provides a method for treating a disease that is a result of, or is accompanied by, production, preferably excessive production, of reactive oxygen species with an effective amount of imidazole-4-acetic acid or a pharmaceutically acceptable salt thereof. It is preferred that said disease is accompanied by or a result of tissue damage resulting from superoxide anion production by phagocytic cells. In a preferred embodiment, said disease is colitis. In another preferred embodiment said disease is rheumatoid arthritis. In yet another preferred embodiment said disease is acne, atopic dermatitis, rosacea, pyoderma gangrenosum, necrotizing fasciitis, Sweet's syndrome, or Behcet's disease. In one embodiment said disease is acne, rosacea, pyoderma gangrenosum, necrotizing fasciitis, Sweet's syndrome, or Behcet's disease. In a particularly preferred embodiment, said disease is acne, atopic dermatitis or colitis. In a most preferred embodiment, said disease is acne.

The term "effective amount" refers to a concentration or amount of imidazole-4-carboxylic acid which results in achieving a particular stated purpose. An "effective amount" of imidazole-4-carboxylic acid may be determined empirically. Preferably an effective amount refers to a

concentration or amount of imidazole-4-carboxyilic acid which results in reduction of the concentration of extracellular reactive oxygen species produced by a phagocytic cell, preferably resulting in less inflammation and/or tissue damage. A pharmaceutical composition for topical application, for instance, typically comprises between 0.1% and 20% (w/v) imidazole-4- carboxylic acid, preferably between 1% and 10% (w/v) imidazole-4-carboxylic acid, more preferably about 10%, about 7.5%, about 5%, about 2.5% or about 1% (w/v) imidazole-4-carboxylic acid.

Physiologic plasma concentrations of imidazole-4-carboxylic acid are about 2.5 ng/ml. After systemic administration of imidazole-4-carboxylic acid, e.g. oral or parenteral, a plasma concentration of between 3 ng/ml - 3000 ng/ml is preferably reached. More preferably, said plasma concentration is at between 10 ng/ml - 1000 ng/ml, more preferably between 20 ng/ml and 500 ng/ml, more preferably between 30 ng/ml and 300 ng/ml, even more preferably between 50 ng/ml and 100 ng/ml For oral administration, this typically would result in a pharmaceutical composition for oral use comprising an amount of imidazole-4-carboxylic acid of between 1 and 100 mg, preferably between 5-50 mg, more preferably between 10 and 25 mg.

Without being bound to theory, it is thought that imidazole- 4- carboxylic acid and pharmaceutically acceptable salts thereof are especially useful for scavenging an extracellular, but not an intracellular reactive oxygen species. Preferably, imidazole-4-carboxylic acid or a pharmaceutically acceptable salt thereof scavenges an extracellular superoxide anion radical. It is especially useful to only scavenge an extracellular reactive oxygen species in cases where the phagocytic cell producing said species is in fact combating a microbial infection. A non-limiting example of such a situation is for instance septic shock. During septic shock, which is a life-threatening stage of sepsis, also called bacteraemia, the immune system, while fighting the infectious agent, tends to "over react" and severely damages the host's cardiovascular system and organs, mostly resulting in multi-organ failure and possible death. When a patient suffering from, or at risk of suffering from septic shock, is treated with imidazole-4-carboxylic acid, the extracellular produced reactive oxygen species are scavenged, thereby resulting in less inflammation, vascular damage and/or tissue damage, whereas the microbicidal activity of the intracellular produced reactive oxygen species is thereby not affected.

In one embodiment, the invention provides imidazole-4-carboxylic acid or a pharmaceutically acceptable salt thereof for use in the treatment of a disease related to the presence of extracellular reactive oxygen species produced by a cell. In a preferred embodiment, said cell is a phagocyte, preferably a granulocyte or a keratinocyte. Granulocytes are white blood cells characterized by the presence of granules in their cytoplasm. Granulocytes are generally divided into three different types, distinguished by their appearance under Wright's stain:

neutrophil granulocytes, eosinophil granulocytes, and basophile granulocytes. As neutrophil granulocytes are the most abundant granulocyte type in the blood and are responsible for the majority of extracellular produced reactive oxygen species, it is especially useful to inhibit a function of a neutrophil granulocyte. In one embodiment, imidazole- 4- carboxy lie acid for use in the invention is provided, wherein said granulocyte is a neutrophil granulocyte.

In one embodiment, the invention provides an imidazole-4-carboxylic acid or a pharmaceutically acceptable salt thereof for use in the treatment of acne, atopic dermatitis, rosacea, pyoderma gangrenosum, necrotizing fasciitis, Sweet's syndrome, Behcet's disease, septic shock, rheumatoid arthritis, or colitis. In a preferred embodiment, an imidazole- 4- carboxylic is provided for use in the treatment of colitis. In another preferred embodiment, an imidazole- 4-carboxylic is provided for use in the treatment of rheumatoid arthritis. In one embodiment, an imidazole-4-carboxylic is provided for use in the treatment of acne, atopic dermatitis, rosacea, pyoderma gangrenosum, necrotizing fasciitis, or Sweet's syndrome. In a more preferred embodiment, an imidazole- 4-carboxylic is provided for use in the treatment of acne, rosacea, pyoderma gangrenosum, necrotizing fasciitis, or Sweet's syndrome. In an even more preferred embodiment, the imidazole-4-carboxylic acid is used for treatment of acne or colitis or atopic dermatitis. In a most preferred embodiment, the imidazole-4 carboxylic acid is used for the treatment of acne. Imidazole-4- carboxylic acid or a pharmaceutically acceptable salt thereof can be used as such, or firstly be incorporated into a (pharmaceutical) composition, which preferably further comprises a carrier, diluent or excipient. In a further preferred embodiment, the invention thus provides a composition comprising imidazole-4-carboxylic acid, or a pharmaceutically acceptable salt thereof, and a carrier, diluent or excipient for use in the treatment of acne, atopic dermatitis, rosacea, pyoderma gangrenosum, necrotizing fasciitis, Sweet's syndrome, Behcet's disease, septic shock, rheumatoid arthritis, or colitis. Suitable carriers, diluents or excipients are commonly known in the art of pharmaceutical formulation and may be readily found and applied by the skilled artisan, references for instance Remington: The Science and Practice of Pharmacy⁵

A typical carrier for oral formulation of an imidazole-4-carboxylic acid or a pharmaceutically acceptable salt thereof for use in the invention is an aqueous carrier such as for instance water, a buffered aqueous solution comprising but not limited to phosphate buffered saline, or an aqueous alcoholic solution. An auxiliary agent such as a detergent can be added to the aqueous carrier to enhance the solubility of imidazole-4-carboxylic acid for use in the invention.

A typical diluent or excipient for an imidazole-4-carboxylic acid or a pharmaceutically acceptable salt thereof for use in the invention comprises a binder such as starch or a cellulose derivative. Said diluent may also comprise a colored additive or a flavor enhancer.

The invention further provides a pharmaceutical composition

comprising imidazole-4-carboxylic acid, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient for use in the treatment of acne, atopic dermatitis, rosacea, pyoderma gangrenosum, necrotizing fasciitis, Sweet's syndrome, Behcet's disease, septic shock, rheumatoid arthritis, or colitis. In one embodiment said pharmaceutical composition is used for treating acne, atopic dermatitis, rosacea, pyoderma gangrenosum, necrotizing fasciitis, or Sweet's syndrome. More preferably said pharmaceutical composition is used for treating acne, rosacea, pyoderma gangrenosum, necrotizing fasciitis, or Sweet's syndrome. Even more preferred is a pharmaceutical composition of the invention for use in the treatment of acne. In another preferred embodiment, a pharmaceutical composition of the invention is provided for use in the treatment of colitis. In another preferred embodiment, a pharmaceutical composition of the invention is provided for use in the treatment of rheumatoid arthritis.

In yet another preferred embodiment, a pharmaceutical composition of the invention is provided for use in the treatment of atopic dermatitis.

In a preferred embodiment, a pharmaceutical composition comprising imidazole-4-carboxylic acid, or a pharmaceutically acceptable salt thereof, is applied onto the skin. Said pharmaceutical composition can be an ointment, paste, cream, lotion, liquid, aerosol (spray), film or laminate, comprising said imidazole-4-carboxylic acid.

In a further aspect the invention provides an ointment, paste, cream, lotion, liquid, aerosol (spray), film and/or laminate, comprising imidazole-4- carboxylic acid for use in the treatment of acne, atopic dermatitis, rosacea, pyoderma gangrenosum, necrotizing fasciitis, Sweet's syndrome, or Behcet's disease. In one embodiment said ointment, paste, cream, lotion, liquid, aerosol (spray), film and/or laminate is used in the treatment of acne, rosacea, pyoderma gangrenosum, necrotizing fasciitis, Sweet's syndrome, or Behcet's disease. More preferably said ointment, paste, cream, lotion, liquid, aerosol (spray), film and/or laminate is used in the treatment of acne or atopic dermatitis, most preferably acne.

In a preferred embodiment, said pharmaceutical composition is a cream. In an even more preferred embodiment, said cream comprises purified water, sodium hydroxide, sodium chloride, sodium methyl parahydroxybenzoate, sodium propyl parahydroxybenzoate, polysorbate 60, cetyl alcohol, liquid paraffin, and/or white soft paraffin.

In a preferred embodiment, said pharmaceutical composition further comprises between 0.1% and 20% (w/v) imidazole-4-carboxylic acid. In a more preferred embodiment, said cream comprises between 1% and 10% (w/v) imidazole-4-carboxylic acid. In an even more preferred embodiment, said cream comprises about 10%, about 7.5%, about 5%, about 2.5% or about 1% (w/v) imidazole-4-carboxylic acid.

In a preferred embodiment, said pharmaceutical composition further comprises other ingredients, such as beeswax, zinc oxide, allantoin, and/or vitamin A, vitamin D and vitamin E, which may help to protect the skin. In a preferred embodiment, said pharmaceutical composition for topical use further comprises solvents such as alcohol and propylene glycol, which are known to increase the solubility of drugs in the skin layers, and may function as a penetration enhancer for transdermal therapeutic systems. Other penetration enhancers that are known in the art, including but not limited to laurocapram, methol, and vitamin E, can be added to said pharmaceutical composition.

In another preferred embodiment, the compositions comprising imidazole-4-carboxylic acid, or a pharmaceutically acceptable salt thereof, for use in the invention are suitable for oral administration and for instance comprise an enteric coating or a controlled release formulation. Enteric coating and controlled release formulations are well known in the art⁵. Enteric coating compositions in the art may comprise of a solution of a water-soluble enteric coating polymer mixed with the active ingredient(s) such as imidazole-4- carboxylic acid, or a pharmaceutically acceptable salt thereof, and other excipients, which are dispersed in an aqueous solution and which may subsequently be dried and/or pelleted. The enteric coating formed, offers resistance to attack of imidazole-4-carboxylic acid by atmospheric moisture and oxygen during storage and by gastric fluids and low pH after ingestion, while being readily broken down under the alkaline conditions which exist in the lower intestinal tract. A composition comprising imidazole-4-carboxylic acid or a pharmaceutically acceptable salt thereof, suitable for oral

administration is especially useful in treating more generalized (systemic) diseases, such as septic shock, Behcet's disease, rheumatoid arthritis, or colitis. In a situation wherein a patient is hospitalized and critically ill, as for instance a sepsis patient, it may be especially useful to administer a pharmaceutical composition comprising imidazole-4-carboxylic acid, or a pharmaceutically acceptable salt thereof, parenterally. In one embodiment therefore, a composition suitable for parenteral administration is provided for use in the invention.

Now that the invention provides the insight that imidazole- 4- carboxylic acid and pharmaceutically acceptable salts thereof are useful in scavenging an extracellular reactive oxygen species, in particular scavenging of an

extracellular superoxide anion, preferably produced by a phagocyte, in one embodiment the invention provides a method for scavenging extracellular reactive oxygen species, preferably a superoxide anion radical, produced by a cell, preferably a phagocyte, more preferable a neutrophil granulocyte or a keratinocyte, comprising providing the extracellular space of said cell with a sufficient amount of an imidazole-4-carboxylic acid or a pharmaceutically acceptable salt thereof. The invention also provides imidazole-4-carboxylic acid or a pharmaceutically acceptable salt thereof for use in scavenging

extracellular reactive oxygen species produced by a cell. The invention also provides a method of scavenging an extracellular reactive oxygen species, preferably a superoxide anion radical, produced by a cell, preferably a phagocyte, more preferable a neutrophil granulocyte or a keratinocyte, in an individual suffering from a disease related to the presence of such a reactive oxygen species, said method comprising providing said individual with an effective amount of imidazole-4-carboxylic acid, a pharmaceutical acceptable salt thereof or a pharmaceutical composition according to the invention.

With providing said individual is meant by oral, parenteral and/or topical administration of said effective amount to said individual.

As said before, an effective amount is an amount which may have to be determined empirically, for instance during a clinical trial. For topical administration, pharmaceutical composition, for instance an ointment of cream, is used that typically comprises between 0.1% and 20% (w/v) imidazole- 4-carboxylic acid. Preferably said pharmaceutical composition comprises between 1% and 10% (w/v) imidazole-4-carboxylic acid. In an even more preferred embodiment, said pharmaceutical composition comprises about 10%, about 7.5%, about 5%, about 2.5% or about 1% (w/v) imidazole- 4- carboxylic acid. In another embodiment, a method of treating a human individual suffering from acne, atopic dermatitis, rosacea, pyoderma gangrenosum, necrotizing fasciitis, Sweet's syndrome, Behcet's disease, septic shock, rheumatoid arthritis, or colitis is provided, wherein said method comprises treating said individual with an effective amount of imidazole- 4- carboxylic acid, a pharmaceutical acceptable salt thereof, or a pharmaceutical

composition comprising imidazole-4-carboxylic acid or a pharmaceutically acceptable salt thereof. In a preferred embodiment, said individual suffers from colitis. In another preferred embodiment, said individual suffers from rheumatoid arthritis. In another preferred embodiment, said individual suffers from acne, atopic dermatitis, rosacea, pyoderma gangrenosum, necrotizing fasciitis, or Sweet's syndrome. In one embodiment, said individual suffers from acne, rosacea, pyoderma gangrenosum, necrotizing fasciitis, or Sweet's syndrome. In a more preferred embodiment, said individual suffers from acne. The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.

Brief description of the drawing

Figure 1. Inhibition of granulocyte activity by imidazole derivatives in vitro.

Examples

Example 1

The local irritation, safety, tolerability and pharmacokinetics of multiple topical applications of imidazole-4-carboxylic acid (ImCOOH) cream were investigated in a randomized, double-blind, placebo-controlled, dose escalating trial in healthy subjects comparing the left and right forearm.

Methods

The trial population consisted of 24 Caucasian healthy male subjects divided over 3 panels of 8 subjects each.

Subjects in Panel 1 (Session I), 2 (Session II) and 3 (Session III) were treated with 1%, 5% and 10% ImCOOH cream, respectively, twice daily for 14 days with an additional morning application on Day 15. Dose escalation continued only if the previous dose was safe and tolerable. In each session, 6 subjects received a hydrophilic cream containing ImCOOH and a placebo cream randomized over both forearms and simultaneously applied, and 2 subjects received a placebo cream simultaneously applied on both forearms. Plasma concentrations of ImCOOH were determined on Day 1 up to 12 hours and on Day 15 up to 72 hours post-dose by venous puncture from the fossa cubitis (where no cream had been applied). Local irritation, systemic safety and tolerability were monitored until 14 days after the last application.

A preliminary, blinded, pharmacokinetic analysis was performed per panel on the combined data of subjects receiving ImCOOH cream and/or placebo cream.

Results On Day 1 mean pre- dose endogenous concentrations of ImCOOH were respectively 2.0, 2.4 and 2.1 ng/mL for subjects in Panels 1, 2 and 3, respectively. The highest plasma concentration observed was 11 ng/mL in a subject from Panel 3. After 14 days of twice daily application of ImCOOH cream, mean pre-dose ImCOOH concentrations were slightly increased to 2.5, 2.7 and 3.3 ng/mL for subjects in Panels 1, 2 and 3, respectively. The highest plasma concentration observed was 7.5 ng/mL in a subject from Panel 3. Given the variability of the concentrations between subjects, the concentrations observed are still considered within the normal physiological range. The concentrations are much lower than those observed after 1 month daily application of 1%, 5% or 10% (w/w) in rats (mean Cmax up to 2715 ng/mL) and minipigs (mean Cmax up to 169 ng/mL). At these concentrations no systemic toxicity was observed in rats and minipigs. However, some minor dermal changes were observed after repeated application for all dosing strengths, including the placebo cream, suggesting some dermal effects of the base components of the cream. No severe or serious adverse events (SAEs) were reported in the dose-escalation study in healthy subjects. The preliminary, blinded analysis (no information available about which subject received which treatment; ImCOOH cream and/or placebo cream) indicated that all adverse events (AEs) were mild or moderate in severity. AEs (regardless of the treatment relationship) were reported by 7 (88%) of 8 subjects of Panel 1, 8 (100%) of 8 subjects of Panel 2 and 8 (100%) of 8 subjects of Panel 3. AEs that were possibly or probably related to the trial medication (ImCOOH cream or placebo cream) were reported by 4 (50%) of 8 subjects of Panel 1, 5 (63%) of 8 subjects of Panel 2 and 6 (75%) of 8 subjects of Panel 3. AEs that were possibly or probably related to the trial medication were skin irritation (9 of 24 subjects), papulation (2 of 24 subjects), reaction to (application of) cream (2 of 24 subjects), (general) tiredness (3 of 24 subjects), pain in arms (1 of 24 subjects), general malaise (1 of 24 subjects), and increased appetite (1 of 24 subjects). All these AEs were resolved within the study period. In summary, it was demonstrated that ImCOOH does not accumulate in plasma to relevant concentrations after twice daily topical application of 10% ImCOOH cream for 14 days with an additional morning dose on Day 15. No severe or SAEs have been observed following this treatment regimen.

Therefore, this treatment regimen is regarded to be safe when administered to subjects with acne vulgaris.

Example 2 This clinical pilot study assesses the effect of imidazole-4-carboxylic acid

(ImCOOH) administered as a topical cream twice daily for 10 days to subjects with acne vulgaris.

Methods

Study Population

A total of 10 subjects with acne vulgaris were selected. Only one subject did not complete the 10 day treatment period for reason other than tolerability /safety, and was excluded from the (interim) analysis. Subjects all met the following inclusion criteria:

1. Male or female aged between 18 and 35 years, extremes included.

2. Mild acne vulgaris, confirmed by the (sub) investigator in the face, forehead, neck, breast, upper back, or shoulders.

3. Able to comply with the pilot study protocol requirements.

4. Informed Consent Form (ICF) signed voluntarily before the first pilot study- related activity.

5. General medical condition, in the investigator's opinion, does not interfere with the assessments and the completion of the pilot study.

Subjects were not allowed to meet any of the following exclusion criteria: 1. Female subject of childbearing potential without use of effective birth control methods, or not willing to continue practicing these birth control methods for at least until 30 days after the end of the treatment period.

2. A positive pregnancy test or breastfeeding at screening.

3. Having a target area that is hairy in such extent that in the investigator's opinion it could influence the application.

4. Having a target area that is tattooed.

5. Any current or past condition (including but not limited to allergy, alcohol or drug abuse), use of medication (including but not limited to anti-acne medication or other investigational drug) or disease (including but not limited to uncontrolled diabetes), which in the opinion of the investigator could compromise the patient safety or compliance with the pilot study procedures.

Study medication

ImCOOH was formulated as a cream containing 100 mg/g (10% w/w)

ImCOOH. The cream further contained purified water, sodium hydroxide, sodium chloride, sodium methyl parahydroxybenzoate, sodium propyl parahydroxybenzoate, polysorbate 60, cetyl alcohol, liquid paraffin, and white soft paraffin.

Treatment

All subjects received study medication (cream containingl0% ImCOOH) for application twice daily, preferably once between 7:00 am and 10:00 am and once between 7:00 pm and 10:00 pm.

Digital color pictures

Standardized digital color pictures of the target areas were made at several time points for documentation. All pictures were taken in the macro stand and standardization was done by keeping the angle, the exposure time, and the diaphragm the same for each picture. Adverse events

AEs were checked at every visit and reported from time of first application of the pilot study medication onwards until the last pilot study-related activity and recorded in the source documents.

Results

At day 1, before the first morning medication, the skin area to be treated was photographed. At day 5 and day 11, the subjects were asked to give their subjective opinion on acne severity and on (other) effects of the treatment. Before the morning medication at day 5, a color photograph was taken of the target area. On day 10, the last medication was applied in the evening. On day 11, again, a color photograph was taken of the target area and the subjects were asked for a subjective opinion on the effects of treatment. The color photographs taken at day 1, day 5 an day 11 were clinically inspected and an objective opinion on the clinical picture of acne severity was given. In the table below, the subjective and objective opinions on the acne severity and treatment are given:

Subj 1 Treated target area: right hand side of face (not forehead)

Subjective Clinical picture

Day 5 No difference Treated area less red /

inflamed

Day 11 No difference Worsening; more pustules

Remarks: Subject 1 used very little cream. Subject uses quite some make up.

Subj 2 Treated target area: right hand side of neck

Subjective Clinical picture

Day 5 No difference Treated area more hydrated

Day 11 No difference Treated area more hydrated

Subj 3 Treated target area: right hand sic e of neck / upper back Subjective Clinical picture

Day 5 Warm feeling during a few minutes Treated area less red / after every application inflamed

Day 11 Light itch during the day, feels like Treated area less red / an itch when something is healing inflamed

Subj 4 Treated target area: right hand sic e of chest

Subjective Clinical picture

Day 5 Treated area seems less red / Treated area less red / inflamed inflamed

Day 11 Treated area seems less red / Treated area less red / inflamed inflamed

Subj 5 Treated target area: left hand side of face (not forehead)

Subjective Clinical picture

Day 5 Light itch only after the very first Treated area less inflamed application; treated area seems less

inflamed

Day 11 No difference since Day 5. Normally Treated area more hydrated the acne worsens during the

ovulation, but now it only worsens on

the not treated target area

Subj 6 Treated target area: right hand sic e of face (not forehead)

Subjective Clinical picture

Day 5 Light itch after every application of Treated area less inflamed the cream

Day 11 No improvement, more a little Treated area more hydrated worsening

Subj 7 Treated target area: right hand sic e of face (not forehead)

Subjective Clinical picture

Day 5 Light itch after every application of Treated area more hydrated the cream; skin of treated area feels

smoother, more hydrated

Day 11 Light itch after every application of Treated area more hydrated the cream; skin of treated area feels

smoother, more hydrated

Subj 8 Treated target area: left hand side of neck /upper back

Subjective Clinical picture

Day 5 Light itch after application of the Treated area less red /

cream during the first few days inflamed

Day 11 Treated area is smoother and is less Treated area less red /

inflamed, has less spots inflamed

Subj 9 Treated target area: right hand sic e of face (no forehead)

Subjective Clinical picture

Day 5 Treated area is less inflamed; skin of Treated area less red /

treated area feels smoother, more inflamed

hydrated

Day 11 The acne has worsened some due to Acne has worsened; more stress because of exam results papules than Day 1

Example 3

This experiment shows the effect of ImCOOH on granulocyte activity in vitro.

Methods

Polymorphonuclear neutrophils (PMNs) suspensions were incubated with imidazole-4-carboxylic acid (ImCOOH), imidazole-4-acetic acid (ImAc), cis- urocanic acid (cis-UCA) and ethyl imidazole-4-acetate (Et-ImAc) at a concentration of 3 mM in triplicate. A few minutes prior to the incubation with test compounds, PMNs were stimulated with phorbol 12-myristate, 13-acetate (PMA), present at 50 μΜ. The absorbance of cytochrome c at 550 nm was monitored at 10-min intervals during 30 minutes. To analyze the effects of the test compounds on the generation of superoxide, the AUC was calculated from the data points and represented as a bar in the graph. Results

The effects of the test compounds are shown in Figure 1. The control was set to 100 % and the suppressive effects of the test compounds on superoxide production, as established as cytochrome c reduction, were presented as parts of 100 %. The largest suppression on superoxide production by PMNs was observed with ImCOOH, which was 54 % of a full response (n = 3). Less suppressive behavior was seen with ImAc and cis-UCA (resp. 75 % and 87 %, n = 3). Et-ImAc did not show a suppressive effect.

In a previous experiment (not shown), ImCOOH did not affect hydrogen peroxide (H2O2) release from activated PMNs. It is therefore concluded that ImCOOH thus does not influence the formation of (intracellular) superoxide but acts only on extracellular available superoxide anion radicals, supposedly by scavenging superoxide anion radicals. This extracellular scavenging ability of ImCOOH for superoxide anion radicals is superior to that of ImAc, cis-UCA and et-ImAc.

References

Akamatsu et al. Increased hydrogen peroxide generation by neutrophils from patients with acne inflammation. Int J Dermatol. 2003

May;42(5):366-9.

Grange et al. Production of superoxide anions by keratinocytes initiates P. acnes-induced inflammation in the skin. PLoS Pathogens 2009, 5(7): epub ahead of print (el000527).

Verity et al. Behcet's disease: from Hippocrates to the third millennium. Br. J. Ophthalmol. 2003; 87:1175-1183.

Jones et al. Reactive oxygen species and rosacea. Cutis 2004 Sep;74(3 Suppl):17-20, 32-4.

Remmington: The Science and Practice of Pharmacy. Lippincott

Williams & Wilkins; Twenty first Edition (May 1, 2005)

Okayama Y. Oxidative stress in allergic and inflammatory skin diseases. Curr Drug Targets Inflamm Allergy. 2005 4(4):517-519.

Segui et al. Superoxide dismutase ameliorates TNBS-induced colitis by reducing oxidative stress, adhesion molecule expression, and leukocyte recruitment into the inflamed intestine. J Leukocyte Biol. 2004;76:537- 544

Ling et al. The rheumatoid arthritis shared epitope increases cellular susceptibility to oxidative stress by antagonizing an adenosine-mediated anti- oxidative pathway. Arthritis Res Ther. 2007;9(1):R5

Claims

1. Imidazole-4-carboxylic acid or a pharmaceutically acceptable salt thereof for use in the treatment of acne, atopic dermatitis, rosacea, pyoderma gangrenosum, necrotizing fasciitis, Sweet's syndrome, Behcet's disease, septic shock, rheumatoid arthritis, or colitis.

2. A method of treating an individual suffering from acne, atopic dermatitis, rosacea, pyoderma gangrenosum, necrotizing fasciitis, Sweet's syndrome, Behcet's disease, septic shock, rheumatoid arthritis, or colitis, said method comprising providing said individual with an imidazole-4-carboxylic acid.

3. Method of scavenging extracellular reactive oxygen species produced by a cell, comprising providing the extracellular space of said cell with a sufficient amount of an imidazole-4-carboxylic acid.

4. Imidazole-4-carboxylic acid or a pharmaceutically acceptable salt thereof for use in scavenging extracellular reactive oxygen species produced by a cell.

5. Imidazole-4-carboxylic acid or a pharmaceutically acceptable salt thereof according to claim 4, for use in the treatment of a disease related to the presence of extracellular reactive oxygen species produced by a cell.

6. A method according to claim 3 or an imidazole-4-carboxylic acid according to claim 4 or 5, wherein said cell is a phagocytic cell.

7. A method according to claim 3 or an imidazole-4-carboxylic acid according to claim 4 or 5, wherein said cell is a neutrophil granulocyte or a keratinocyte.