WO2014096958A1 - Inosine monophosphate and salts thereof for use in the treatment of complement-related disorders - Google Patents

Abstract

The present invention relates to the identification of novel compounds that counteract the formation of the Membrane Attack Complex (MAC) which is the terminal process during complement activation of the immune system, and that is involved in the breakdown of nerves during neurodegenerative diseases. MAC is also deposited after nerve disorders such as traumatic brain injuries and the identified compounds according to the invention are also suitable for the treatment of such disorders. The invention further relates to inosine monophosphate (IMP) and functional equivalent s thereof for use in the treatment, prevention, alleviation or inhibition of disorders mediated by undesired effects of the complement system, such as acute and chronic nerve injuries, preferably to promote axonal regeneration after such injuries have occurred.

Description

INOSINE MONOPHOSPHATE AND SALTS THEREOF FOR USE IN THE TREATMENT OF COMPLEMENT-RELATED DISORDERS

FIELD OF THE INVENTION

The present invention relates to the field of medicine. In particular, the invention relates to pharmaceutical compounds and compositions for use in the treatment of disorders that involve complement activation, more specifically to disorders in which the Membrane Attack Complex (MAC) is deposited. More in particular, the invention relates to inosine monophosphate (IMP) and salts thereof for use as a medicament, preferably in disorders mediated by an undesired activity of the complement system.

BACKGROUND OF THE INVENTION

Complement plays a key role in the immune system. Activation of

complement generally occurs via distinct pathways: the so-called 'classical pathway', 'the lectin pathway' and the 'alternative pathway' . The complement system includes a system of about 30 proteins and is constantly active within the body. Several proteins are involved in the regulation of complement activation through inhibition and activation processes. Both classical and alternative pathways are identical in the formation of the so-called Membrane Attack Complex (MAC) which is a set of proteins that assemble and adhere to cell surfaces and subsequently create lytic holes in the cell membrane often resulting in the death of the cell. MAC is formed by the sequential assembly of complement proteins C6, C7, C8 and (C9)_n along with C5b. CD59 is a protein that - within the tight control of complement activation in vivo - prevents the assembly of MAC.

Complement activation, including MAC formation, has been identified to play a very important role in determining the severity of a number of disorders, especially in hemolytic uremic syndrome, complement mediated kidney disease, ischemia reperfusion disorders, transplant rejection, meningitis, neurodegenerative diseases such as Alzheimer's disease (AD), age-related macular degeneration and Parkinson's disease (PD). It was also found that after a nerve injury, for instance in traumatic brain trauma or in the disorder referred to as Wallerian Degeneration (WD), MAC formation plays a role in the breakdown of the injured nerves. One of the inventors of the present invention has previously shown that in the process during which a nerve attempts to re-grow along the path of the formerly present axon, such re-growth is inhibited by MAC formation (WO 2008/044298). This MAC formation could in turn be inhibited by inhibitors known to interfere with the activity/expression of proteins involved in MAC formation. Examples are oligonucleotides that inhibit the expression of C6 (WO 2010/005310), C8-alpha (WO 2011/105900, also referred to as C8a or C8a), C8-beta (WO 2011/105902, also referred to as C8b or C8P) and C9 (WO 2011/105901). As a consequence such inhibition results in the promotion of axonal regeneration which is clearly beneficial, for example in the case of (traumatic) nerve injuries and WD.

Although many natural and synthetic inhibitors have been identified that may prevent or diminish complement activation and MAC formation, as outlined above, many of these promising compounds are in early phases of development and at present it is uncertain whether such compounds will be (commercially) viable in the long term. Hence, there is an ongoing need for pharmaceutical compositions comprising compounds that can be used in the prophylaxis and/or treatment of disorders mediated by an undesired activity of the complement system, which includes MAC deposition.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the computational design of membrane attack complex (MAC) inhibitors. A) X-ray crystallographic complex of C8a (right) and C8y (left top) in PDB 2rd7, and predicted coordinates of CD59 (left low) that binds to TMHD2 region on C8a; B) Close-up view of CD59- C8a interactions. Interaction hot-spots are shown in stick model and labeled; C) a representative sample of ligand that binds to CD59 binding region on C8a. Figure 2 shows the selection of IMP as an inhibitor of complement mediated lysis. Compound RGS2022 (identified as inosine monophosphate: IMP) inhibits complement mediated lysis of red blood cells after activation of the classical pathway in vitro. Erythrocyte lysis was measured in a spectrum photometer at 405 nM. Other molecules (depicted as RGS2005, RGS2054, RGS2049 and RGS2048) that were previously selected in the same in silico screen as IMP appeared not to inhibit complement activation.

Figure 3 displays: A) a schematic representation of complement activation through the MBL pathway, in a mannan activated complement ELISA (MAC formation is measured using an antibody that detects the neo-epitope C5b-9 of MAC); B) the effect of 25 mM IMP (RGS2022) on the deposition of C4 with 10% human serum in the complement ELISA; C) the effect of 25 mM IMP (RGS2022) on the deposition of MAC with 10% human serum in the complement ELISA; and D) MAC formation in a graph showing that IMP (RGS2022) inhibits MAC formation in a dose-dependent manner.

Figure 4 is a diagram showing that intraperitoneal (i.p.) injection of IMP (RGS2022) prevents complement activation in serum. IMP was injected i.p. into mice and serum was collected after 30 min and used in a hemolytic assay to determine the ability to lyse rabbit erythrocytes. Erythrocyte lysis was measured in a spectrum photometer at 405 nM.

Figure 5 is a diagram that shows that IMP (RGS2022) reduces neurological damage in vivo when administered (i.p.) before- or 30 min after the trauma (i.p. administration), as measured in a neurological severity score (NSS). The NSS is a 0 to 10-point system, wherein 0 means healthy and 10 means total failure in all tasks.

Figure 6 displays histological data showing that IMP administration (i.p. injection) prevents MAC formation and macrophage infiltration after traumatic brain injury.

Figure 7 shows the comparison of IMP (RGS2022) activity versus adenosine monophosphate (AMP; RGS2065) activity in the mouse TBI model. IMP and AMP were both administered in a dose of 1.5 g/kg body weight directly prior to the injury. The NSS (A) and the percentage of weight loss (B) was measured.

Figure 8 is a graph comparing the recovery of sensory function after sciatic nerve crush injury in subjects administered IMP and subjects administered a control (PBS). Figures 9a and 9b are graphs showing the serum levels and biodistribution, respectively, of H IMP (diammonium salt) after i.p. injection.

SUMMARY OF THE INVENTION

The present invention relates to inosine monophosphate (IMP), or a functional equivalent thereof, for use as a medicament. In one embodiment, said functional equivalent is a salt of IMP (e.g., disodium inosinate). The invention also relates to IMP, or a functional equivalent thereof, for use in the treatment, prevention and/or inhibition of a disorder mediated by an undesired activity of the complement system, preferably wherein said undesired activity of the complement system comprises complement activation, including formation of a membrane attack complex (MAC). In another embodiment the invention relates to the use of inosine monophosphate, or a functional equivalent thereof, in the preparation of a medicament for the treatment, prevention and/or inhibition of a disorder mediated by an undesired activity of the complement system. In one embodiment, said disorder is an acute or chronic injury of the nervous system. The IMP may be administered prior to or after the occurrence of the acute injury. In one embodiment, IMP is administered by intraperitoneal injection. In another embodiment, IMP is administered within 24, 12, 6, 3, 2, 1, or less hours, preferably within 5, 10, 20, 30 or 40 minutes before or after the trauma. In a particular embodiment, a single dose of IMP is administered within 30 minutes before or 30 minutes after the trauma with no subsequent administration of IMP. Preferred disorders that can now be treated with IMP according to the present invention include, e.g., multiple sclerosis, traumatic brain injuries, chronic demyelinating neuropathies, ischemia-reperfusion injuries, atherosclerosis, coronary heart diseases and/or osteoarthritis. According to the present invention, IMP inhibit the formation of MAC and through this prevents the unwanted effects of such MAC formation and activation, thereby promoting axonal regeneration, IMP is preferably used to treat or prevent a disorder that is a condition requiring axonal regeneration.

The invention also relates to pharmaceutical compositions for the treatment of a disorder mediated by an undesired activity of the complement system, wherein said composition comprises IMP, or a functional equivalent thereof, and a

pharmaceutically acceptable diluent, carrier or adjuvant. In one embodiment, the composition comprises IMP in dosage range of about 0.25 - 8.0 gram/kilogram (g/kg) of the host body weight, for example, about 0.50 - 7.75g/kg, 0.75 - 7.50g/kg, 1.0 - 7.25g/kg, 1.25 - 7.0g/kg, 1.50 - 6.75g/kg, 1.75 - 6.5g/kg, 2.0 - 6.25g/kg, 2.25 - 6.0g/kg, 2.50 - 5.75g/kg, 2.75 - 5.50g/kg, 3.0 - 5.25g/kg, 3.25 - 5.0g/kg, 3.5 - 4.75g/kg, 3.75 - 4.5g/kg, or about 4.0 - 4.25g/kg. In another embodiment said pharmaceutical composition further comprises a compound selected from the group consisting of: a complement regulator, a complement receptor such as soluble CRl or Crry-Ig, an antibody or antibody-fragment directed against a complement component, cobra venom factor, a poly-anionic inhibitor of complement, K-76COOH, rosmaric acid, nafamastat mesilate, Cls-INH-248, compstatin, PMX53, PMX205, a CI inhibitor, and derivatives thereof, or comprises an inhibitor such as an anti- sense oligonucleotide, aptamer, miRNA, ribozyme, or siRNA that blocks expression of one or more of C3 convertase, C5, C6, C7, C8a, C8b and C9.

The invention also relates to methods of treating, preventing or inhibiting a disorder mediated by an undesired activity of the complement system (e.g., complement activation, including MAC assembly) in a subject suffering from said disorder, or wherein said subject is at risk of developing said disorder, comprising the steps of administering a pharmaceutical composition of the invention to said subject; and optionally monitoring the progress of the disorder in said subject. The subject is preferably human, such as a human patient suffering from, or that is at risk of suffering from, said disorder.

The present invention furthermore relates to a use of IMP, or a functional equivalent thereof, in the treatment of a disorder mediated by an undesired activity of the complement system (e.g., complement activation, including MAC assembly), said disorder preferably requiring axonal regeneration. In another embodiment, the invention relates to methods of reducing, preventing or inhibiting the production and/or deposition of MAC on a cell membrane or tissue in vitro or in vivo, the method comprising the step of contacting said cell or tissue with inosine monophosphate such that the formation of MAC is reduced or inhibited. DETAILED DESCRIPTION

Despite the fact there is a myriad of causes resulting in injury and disease of the nervous system there is a common feature that is found in all types of neuronal damage and neuronal disease: Activation of the complement system. Such activation can always be detected in damaged neuronal tissue. Complement regulators provide "eat me" and "don't eat me" signals on damaged axons for invading macrophages. Genome- wide association studies have shown that the complement system is strongly associated with neurological disease in the central nervous system (Harold et al. 2009). It is known that complement is synthesized within the nervous system.

Previously it has been shown that especially mRNA and protein levels in the classical complement pathway (Clq and C4) and downstream components of this pathway (C3 and MAC) are elevated after injury of the nervous system. In addition, it was determined that complement activation precedes neuro-degeneration. The extent of activation of the complement pathway in neuronal damage models in rodents has been studied using whole genome transcriptional profiling. A longitudinal, unbiased transcriptome analysis identified a significant, contemporaneous up-regulation of the alternative and classical complement pathways. These patterns of gene expression are consistent with a regulated activation of the complement pathway in response to injury. These results have shown that activation of the complement system hampers recovery after neuronal injury (Ramaglia et al. 2008). Using C6 deficient animals it was established earlier that the terminal part of the complement system, the formation of the Membrane Attack Complex appeared to be essential in this process (Ramaglia et al. 2007).

The Membrane Attack Complex (MAC) is a complex of C5b, C6, C7, C8

(C8a and C8b) and C9 proteins. C8a is the most important component of MAC as it plays an essential role in the stepwise formation of the complex, whereas C9 is highly important for MAC activity. The most prominent inhibitor of MAC formation, CD59, being a natural regulator of MAC formation, binds to both C8a and C9 simultaneously during the process of the formation of MAC. Two experimental structures of C8a are reported in the protein databank (PDB codes: 2qqh and 2rd7). None have been reported for C9. The hypothesis for the study performed by the inventors of the present invention was that natural or synthetic ligands mimicking CD59 binding to C8a could be putative MAC inhibitors. Such ligands may then be useful in the treatment of any disorder related to complement activation in which MAC formation attributes significantly to the disease. Such ligands should counteract the formation of MAC and may therefore be useful in the reduction or prevention and/or treatment of disorders that occur through complement activation and specifically through the activity of formed MAC. As outlined in the non-limiting examples below, the inventors of the present invention, during the performance of their screen, identified one particular compound known in the art, but that never had been associated with the regulation of

complement activation, MAC formation or deposition. This compound, initially referred to as RGS2022, was identified as inosine monophosphate.

Inosine monophosphate (IMP) is a known natural compound with the following chemical formula:

IMP has been produced by different methods and can be purified from natural sources. In the food industry, IMP and its salts such as disodium inosinate, are commonly used as flavour enhancers, and are often produced with the aid of genetically modified organisms, such as yeasts. Functional equivalent forms of ΓΜΡ include, e.g., disodium IMP (E631), dipotassium IMP (E632) and dicalcium IMP (E633) which are used in soups, sauces, and seasonings for the intensification and balance of meat taste.

As known by the person skilled in the art, ΓΜΡ inhibits the synthesis of 5- phosphorybosilamine from 5-phosphoribosyl-l -pyrophosphate (PRPP), disabling glutamine-5-phosphoribosyl-l-pyrophosphate-amidotransferase that catalyzes the reaction. When levels of IMP are high, glutamine-5-phosphoribosyl-l-pyrophosphate- amidotransferase is inhibited and, as a consequence, IMP levels decrease. Also, as a result adenylate and guanylate are not produced, which means that RNA synthesis cannot be completed because of the lack of these two important RNA nucleotides. IMP dehydrogenase (Inosine-5'-monophosphate dehydrogenase) is an enzyme that converts inosine monophosphate to xanthosine monophosphate. IMP dehydrogenase is associated with cell proliferation and is a possible target for cancer chemotherapy. IMP dehydrogenase is inhibited by Mycophenolic acid (Cellcept) and Ribavirin.

Certain IMP derivatives (or functional equivalents) have previously been used in medical applications, but importantly, IMP itself has never been applied as a direct medicament or as an active component in a pharmaceutical composition. As far as the inventors of the present invention are aware, IMP has never been disclosed or suggested as being useful for counteracting MAC formation in diseases in which complement activation occurs. It is important to note that IMP is a compound involved in physiological processes that are unrelated to the activation of complement and the formation of MAC therein. Hence, it is therefore highly surprising that IMP is active and can inhibit MAC formation in vitro and in vivo as outlined in the examples below.

The use of IMP as a functional food supplement in mixtures with B group vitamins, phospholipids and gangliosides, sialic acids as well as DHA, and intended to supplement nutrition in the case of patients with neurological or neurodegenerative alterations, has been disclosed in WO 2011/121151. Notably, IMP was not used as a medicament therein but rather as a dietary supplement to act in the cascades that supposedly are involved in plaque formation. There is no suggestion in WO

2011/121151 that IMP is considered an active ingredient that could act as a medicament on its own, or as an inhibitor of complement activation, leave alone the prevention and/or inhibition of MAC formation. Immunopotentiating (adjuvant) effects of 'protected' inosine monophosphate derivatives were disclosed in WO 96/33203. As is well known to the person skilled in the art, adjuvants are not active ingredients that directly act to prevent or counteract a particular disorder. Adjuvants are generally applied to support the activity of the medicament but do not have direct medical effects on their own. Similar to WO 96/33203, WO 03/049670 disclosed pharmaceutical compositions comprising an adjuvant effective amount of a protected IMP derivative: methyl inosine 5 '-monophosphate (MIMP). 'Protected' as used therein refers to the alteration of the compound that makes it resistant to 5'- nucleotidase; a method to render IMP resistant to that enzyme, which method was disclosed in WO 96/33203.

The inventors of the present invention have shown that IMP can act as a compound that interferes with the binding of CD59 to the complement protein C8a, and through this interference can inhibit the formation of MAC which formation is normally regulated by CD59. It was unexpectedly discovered that IMP, a compound that is known to be involved in RNA synthesis as outlined supra, was identified as a complement inhibitor. The complement inhibitory effect of IMP on MAC formation was demonstrated by the inventors in a mouse model for traumatic brain injury (TBI), and nerve crush injury, i.e., disorders that involve complement activation and MAC deposition and activation. It will be appreciated by the person skilled in the art that these models are just two of many models that could have been selected for investigating inhibitory effects on MAC formation, based on the knowledge and skill in the art. Further disorders that are mediated and influenced by undesired effects of complement activation and MAC formation and that can, therefore, also be treated by the administration of inosine monophosphate are outlined in detail below.

The present invention relates to inosine monophosphate (IMP), or a functional equivalent thereof, for use as a medicament. As used herein, the term 'functional equivalent' or 'functionally equivalent' refers to a derivative and/or equivalent of IMP that retains the desired biological activity of IMP of counteracting MAC formation. Preferred functional equivalents of IMP are salts of IMP that, according to the invention, can be employed in therapy and are pharmaceutically acceptable. The term 'pharmaceutically acceptable' refers to functional equivalents that exhibit minimal undesired toxicological effects that are considered acceptable for therapeutic use.

Such boundaries are known to the person skilled in the art. Non-limiting examples of IMP salts can be formed with metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminium, copper, cobalt, nickel, cadmium, sodium, potassium, and the like, or with a cation formed from ammonia, Ν,Ν-dibenzylethylene-diamine, D- glucosamine, tetraethylammonium, or ethylenediamine. Preferred functional equivalent salts of IMP are disodium IMP (E631), dipotassium IMP (E632) and dicalcium IMP (E633). An especially preferred functional equivalent salt of IMP according to the invention is disodium IMP (E631).

The invention also relates to a pharmaceutical composition comprising IMP and a pharmaceutically acceptable diluent, carrier or adjuvant. IMP or a functional equivalent thereof, according to the invention, may be mixed with any material that does not significantly impair the desired action, or with material that supplements the desired action. These could include other drugs, for instance compounds that add to the inhibition of complement activation. Examples of such drugs are disclosed in WO 2007/044928, WO 2010/005310, WO 2011/105900, WO 2011/105901 and WO 2011/105902. For parenteral, subcutaneous, intradermal or topical administration the formulation may include a sterile diluent, buffers, regulators of tonicity and antibacterials. IMP may be prepared with carriers that protect against degradation or immediate elimination from the body, including implants or microparticles with controlled release properties. For intravenous administration the preferred carriers are physiological saline or phosphate buffered saline. Preferably, IMP according to the invention is included in a unit formulation such as in a pharmaceutically acceptable carrier or diluent in an amount effective to deliver to a subject a therapeutically effective amount without causing serious side effects in the treated subject.

Sterile injectable solutions can be prepared by incorporating the active compound (IMP) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, preferably from about 0.1 per cent to about 70 per cent, most preferably from about 1 per cent to about 30 per cent of active ingredient in combination with a pharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response (e.g. , a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

For administration of the present invention, the dosage ranges from about 0.25

- 8.0 gram/kilogram (g/kg) of the host body weight, for example, about 0.50 - 7.75g/kg, 0.75 - 7.50g/kg, 1.0 - 7.25g/kg, 1.25 - 7.0g/kg, 1.50 - 6.75g/kg, 1.75 - 6.5g/kg, 2.0 - 6.25g/kg, 2.25 - 6.0g/kg, 2.50 - 5.75g/kg, 2.75 - 5.50g/kg, 3.0 - 5.25g/kg, 3.25 - 5.0g/kg, 3.5 - 4.75g/kg, 3.75 - 4.5g/kg, or about 4.0 - 4.25g/kg. In another embodiment IMP is administered at about 0.05g/kg/day, 0.04g/kg/day,

0.03g/kg/day, 0.02g/kg/day, O.Olg/kg/day, or less. An exemplary treatment regime entails administration of IMP within 24, 12, 6, 3, 2, 1, or less hours, preferably within 5, 10, 20, 30 or 40 minutes before or after the trauma. In a particular regime, a single dose of IMP is administered within 30 minutes before or 30 minutes after the trauma with no subsequent administration of IMP.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

The pharmaceutical compositions of the present invention may be

administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may for instance be oral, pulmonary, topical, intracranial, or parenteral (including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion administration). In one embodiment the pharmaceutical composition is administered intravenously, intraperitoneally, orally, topically or as a bolus injection or

administered directly into the target organ or tissue. The person skilled in the art of administering pharmaceutical compositions is aware of the formulations,

pharmaceutically acceptable carriers and diluents, substrates, buffers and devices that may be applied in the administration of a compound according to the present invention. In a preferred embodiment, the composition is administered by

intraperitoneal injection (i.p.) or intravenous (i.v.) injection. In another embodiment, the composition is administered within 24, 12, 6, 3, 2, 1, or less hours, preferably within 5, 10, 20, 30 or 40 minutes before or after the trauma, e.g., a single dose of IMP within 30 minutes before or 30 minutes after the trauma with no subsequent administration of IMP. The present invention further relates to a method for treating, preventing or reducing symptoms of a disorder mediated by an undesired activity of the complement system, especially through the deposition of MAC in the final stage of complement activation, the method comprising administering IMP or a

pharmaceutically acceptable salt thereof to a subject suffering from (or at risk of suffering from) said disorder.

The present invention also relates to a use of IMP, or a functional equivalent thereof, in the preparation of a medicament for the treatment, prevention and/or inhibition of a disorder mediated by an undesired activity of the complement system. As used herein, the term "undesired activity of the complement system," refers to complement activity which leads to MAC formation, and includes, e.g., hemolytic uremic syndrome, complement mediated kidney disease, ischemia reperfusion disorders, transplant rejection, meningitis, neurodegenerative diseases such as Alzheimer's disease (AD), age-related macular degeneration, multiple sclerosis (MS), Huntington's disease, and Parkinson's disease (PD), as described further herein.

Other disorders associated with MAC formation include, e.g., traumatic brain trauma and Wallerian Degeneration (WD), also described further herein.

In a preferred embodiment of the present invention, the disorder that is treated with the administration of IMP, or a functional equivalent, is an acute or chronic injury of the nervous system. In a particular embodiment, the disorder is a physical injury of a peripheral or central nerve. In WO 2007/044928 it was disclosed that MAC formation negatively influenced nerve degeneration and that

prevention/inhibition of MAC formation subsequently results in an improved regeneration of nerves along the path of the injured axon. Hence, according to the present invention, IMP or any of its functional equivalents (e.g., salts) can be used in the treatment of any disorder requiring nerve regeneration as disclosed in WO

2007/044928, and references as cited therein. In one preferred embodiment, acute injuries of the nervous system are treated. Acute trauma to peripheral nerves is relatively common, including blunt trauma or from penetrating missiles, such as bullets or other objects. Injuries from stab wounds or foreign bodies (e.g. glass, sheet metal) resulting in clean lacerations of nerves are known as are nerve injuries stemming from bone fractures and fracture-dislocations including ulnar nerve neurapraxia and radial nerve lesions and palsies. In general acute nerve injury often produces a long-lasting neuropathic pain, manifested as allodynia, a decrease in pain threshold and hyperplasia, and an increase in response to noxious stimuli. Further acute nerve injuries that can be treated by IMP administration according to the present invention include traumatic brain injury and acute injuries to the spinal cord and peripheral/sensory nerves, including various sports injuries involving nerve insult.

In embodiments of the present invention in which it is desired to inhibit the formation of MAC and through this, promote axonal regeneration in response to an acute nerve injury, it will be generally preferable to administer at least IMP or any of its functional equivalents (and potentially in addition one or more of the inhibitors of complement activation as widely known in the art, such as for instance disclosed in WO 2007/044928, WO 2010/005310, WO 2011/105900, WO 2011/105901 and WO 2011/105902) as soon as possible after the insult, such as within about 24, 12, 6, 3, 2, 1, or less hours, preferably within 5, 10, 20, 30 or 40 minutes after the acute injury has occurred. Additionally, IMP or any of its functional equivalents, can be administered prophylactically (as a precautionary measure), for instance before a medical intervention (e.g. surgery) associated with a certain risk of nerve damage. In this embodiment, nerve regeneration will be favourably enhanced if needed and recovery times shortened.

The present invention also relates to the use of IMP or any of its functional equivalents in the treatment, prevention or inhibition of symptoms related to activation of the complement cascade such as seen in hemolytic uremic syndrome, complement mediated kidney disease, ischemia reperfusion disorders, transplant rejection, meningitis, acute and chronic injuries to the nervous system. Similar to acute injuries to the nervous system, MAC formation has a negative impact on nerve regeneration in chronic injuries, which negative impact can be diminished by the administration of IMP or any of its functional equivalents. Non-limiting examples of chronic injuries to the nervous system include those listed in WO 2007/044928, and include many chronic demyelinating or axonal neuropathies: HMSN (Charcot Marie Tooth disease types 1, 2, 3, 4 and X-linked forms), HNPP and other pressure palsies, HMSN-Lom, Guillain-Barre syndrome (GBS, also known as acute inflammatory demyelinating polyneuropathy or AIDP), chronic inflammatory demyelinating neuropathies (CIDP), Alzheimer's Disease, Huntington's Disease, multiple sclerosis, leukodystrophy, Parkinson's Disease, motor neuron diseases like amyotrophic lateral sclerosis (ALS), diabetic neuropathies, distal axonopathies such as those resulting from a metabolic or toxic neuronal derangement (e.g. relating to diabetes, renal failure, exposure to a drug or toxin, such as an anti-cancer drug, malnutrition or alcoholism), mononeuropathies, radiculopathies such as of the cranial nerve VII or Facial nerve, Hansen's Disease (leprosy), plexopathies such as brachial neuritis, and focal entrapment neuropathies such as carpal tunnel syndrome. The present invention relates to IMP or a functional equivalent thereof, for use in the treatment, prevention and/or inhibition of a disorder mediated by an undesired activity of the complement system (e.g., complement activation, including MAC formation). An undesired activity of the complement system may come in different aspects, but in a preferred embodiment of the present invention the undesired effect of the complement system is the final stage in complement activation, also referred to as the formation of the membrane attack complex (MAC), accompanied by the proliferation and infiltration of macrophages. This recruitment of macrophages and the formation of MAC (both a result of complement activity and both regarded as undesired effects) cause - for instance - the degeneration of the injured nerve in the case of injuries to the nervous system, and prevent the regeneration of axons.

Interference with IMP counteracts or prevents the formation of MAC and in a preferred embodiment, facilitates the re-growth of nerves (axonal regeneration). In the context of the present invention it should be noted that "facilitating axonal regeneration" should be distinguished from reducing or preventing axonal degeneration. Facilitating (or promotion of) axonal regeneration is herein understood to mean that regeneration of an axon is improved in subjects that are treated with IMP or any of its functional equivalents as compared to non-treated subjects. Improved regeneration of an axon preferably is regeneration that occurs at an earlier point in time (after axonal injury or after start of the treatment) in treated subjects as compared to non-treated subjects. Improved regeneration of an axon may also comprise regeneration that occurs at a higher rate and/or to a larger extent in treated subjects as compared to non-treated subjects. IMP or any of its functional equivalents according to the invention thus preferably produces a gain of sensory or motor function.

Improvement in axonal regeneration is preferably determined by functional tests that are relatively easily conducted in (human) subjects, e.g. recovery of sensory or motor function is preferably determined in a standardized test as is available in the art.

Suitable tests preferably are quantitatively standardized and more preferably have had their psychometric properties evaluated and quantified. Such tests include for instance the Weinstein Enhanced Sensory Test (WEST) or the Semmes-Weinstein

Monofilament Test (SWMT) and the shape-texture identification (STI) test for tactile gnosis. Improved axonal regeneration may also be experimentally determined in test animal by histological examination.

In a preferred embodiment, said IMP is used in the form of a disodium salt, a dipotassium salt or a dicalcium salt, which are functional equivalents of IMP itself, and according to another preferred embodiment, IMP or any of its functional equivalents is used to prevent, inhibit or treat a disorder according to the invention, wherein said disorder is an acute or chronic injury of the nervous system. In the event that the disorder is an acute injury of the nervous system, said IMP (salt) is either administered prior to-, or after the occurrence of said acute injury. In the event that the acute injury is unexpected, said IMP (salt) is preferably administered within 24, 12, 6, 3, 2, 1, or less hours, preferably within 5, 10, 20, 30 or 40 minutes after the acute injury has occurred. Such administration may be continued for prolonged periods of time depending on the injury and required prevention of nerve degradation and/or required axonal regeneration. In another preferred embodiment, IMP or any of its functional equivalents, is administered to treat, alleviate, prevent or inhibit the symptoms of a disorder that is selected from the group consisting of: multiple sclerosis, a traumatic brain injury, a chronic demyelinating neuropathy, an ischemia- reperfusion injury, atherosclerosis, coronary heart disease and osteoarthritis. Ischemia-reperfusion injuries are well-known in the art and are generally events in which blood circulation to specific tissue is temporarily stopped and then restarted. Examples are reperfusion in the heart tissue after blockage of the arteries and during and/or after tissue/organs transplantation, such as kidney transplantation in which complement activation may be a serious cause of damage. In a highly preferred embodiment, said disorder is a condition requiring axonal regeneration, as outlined supra.

The present invention also relates to a pharmaceutical composition for the treatment of a disorder mediated by an undesired activity of the complement system, wherein said composition comprises IMP, or a functional equivalent thereof, and a pharmaceutically acceptable diluent, carrier and/or adjuvant. Preferably, said composition further comprises a compound selected from the group consisting of: a complement regulator, a complement receptor such as soluble CR1 or Crry-Ig, an antibody or antibody-fragment directed against a complement component, cobra venom factor, a poly-anionic inhibitor of complement, K-76COOH, rosmaric acid, nafamastat mesilate, Cls-INH-248, compstatin, PMX53, PMX205, a CI inhibitor, and derivatives thereof, and/or said composition further comprises an inhibitor such as an anti- sense oligonucleotide, aptamer, miRNA, ribozyme, or siRNA that blocks expression of one or more of C3 convertase, C5, C6, C7, C8a, C8b and C9.

The present invention also relates to a use of IMP, or a functional equivalent thereof, in the treatment of a disorder mediated by an undesired activity of the complement system (e.g., complement activation, including MAC assembly), said disorder preferably requiring axonal regeneration.

The present invention also relates to the use of inosine monophosphate, or a functional equivalent thereof, in the preparation of a medicament for the treatment, prevention and/or inhibition of a disorder mediated by an undesired activity of the complement system (e.g., complement activation, including MAC assembly).

The present invention also relates to a method of producing a pharmaceutical composition suitable for use in the treatment, prevention and/or inhibition of a disorder mediated by an undesired activity of the complement system (e.g., complement activation, including MAC assembly), said method comprising the step of adding to the comprising as an active ingredient inosine monophosphate, or a functional equivalent thereof, and further introducing a pharmaceutically acceptable diluent, carrier and/or adjuvant. Optionally, said method further comprises the step of adding a compound selected from the group consisting of: a complement regulator, a complement receptor such as soluble CR1 or Crry-Ig, an antibody or antibody- fragment directed against a complement component, cobra venom factor, a poly- anionic inhibitor of complement, K-76COOH, rosmaric acid, nafamastat mesilate, Cls-INH-248, compstatin, PMX53, PMX205, a CI inhibitor, and derivatives thereof, and/or said composition further comprises an inhibitor such as an anti-sense oligonucleotide, aptamer, miRNA, ribozyme, or siRNA that blocks expression of one or more of C3 convertase, C5, C6, C7, C8a, C8b and C9.

The present invention furthermore relates to a method of reducing, preventing or inhibiting the production and/or deposition of a membrane attack complex on a cell membrane or tissue in vitro or in vivo, the method comprising the step of contacting said cell or tissue with inosine monophosphate such that the formation of the membrane attack complex is reduced or inhibited.

In another embodiment, the present invention relates to a method of treating, preventing or inhibiting a disorder mediated by an undesired activity of the complement system (such as comprising the formation of MAC) in a subject suffering from said disorder, or that is at risk of suffering from said disorder, comprising the steps of administering a pharmaceutical composition according the invention to said subject; and monitoring the progress of the disorder in said subject. The subject may be a patient and is preferably human. Disorders that can be treated according to the method of the present invention are those that benefit from the inhibition of MAC formation and are mentioned supra. Monitoring the progress (which includes a decrease of symptoms and preferably a return to a healthy state) can be performed by any of the standardized monitoring methods as mentioned supra.

The disorder that may preferably be treated in a method according to the present invention may be an acute or chronic injury to the nervous system. Non- limiting examples of acute injuries to the nervous system include traumatic brain injury, acute trauma to peripheral nerves such as blunt trauma or those resulting from penetrating missiles, bullets or other objects, stab wounds or insults from foreign bodies (e.g. glass, sheet metal) resulting in clean lacerations, nerve injuries stemming from bone fractures and fracture-dislocations including ulnar nerve neurapraxia and radial nerve lesions and palsies. Non-limiting examples of chronic injuries to the nervous system include those listed in WO 2007/044928, and include many chronic demyelinating or axonal neuropathies: HMSN (Charcot Marie Tooth disease types 1, 2, 3, 4 and X-linked forms), HNPP and other pressure palsies, HMSN-Lom, Guillain- Barre syndrome (GBS, also known as acute inflammatory demyelinating

polyneuropathy or AIDP), chronic inflammatory demyelinating neuropathies (CIDP), Alzheimer's Disease, Huntington's Disease, multiple sclerosis, leukodystrophy, Parkinson's Disease, motor neuron diseases like amyotrophic lateral sclerosis (ALS), diabetic neuropathies, distal axonopathies such as those resulting from a metabolic or toxic neuronal derangement (e.g. relating to diabetes, renal failure, exposure to a drug or toxin, such as an anti-cancer drug, malnutrition or alcoholism), mononeuropathies, radiculopathies such as of the cranial nerve VII or Facial nerve, Hansen's Disease (leprosy), plexopathies such as brachial neuritis, and focal entrapment neuropathies such as carpal tunnel syndrome. Other disorders that may preferably be treated in a method according to the invention include but are not limited to meningitis, atherosclerosis, coronary heart diseases, osteoarthritis, hemolytic uremic syndrome, complement mediated kidney disease, and ischemia-reperfusion injuries such as seen with transplant rejection, myocardial infarction and stroke.

In a preferred embodiment of the present invention IMP is used in the prevention, prophylaxis, treatment or alleviation of symptoms related to traumatic brain injury (TBI), a severe form of brain concussion. TBI is a serious world-wide public health problem. Each year, TBI contributes to a substantial number of deaths and cases of permanent disability. Recent data shows that, on average, approximately 1.7 million people sustain a TBI annually in the US alone (Faul et al. 2010). Of these patients approximately 275,000 are hospitalized and many of them suffer from long term consequences. An estimated number of approximately 52,000 patients die as a result of the injury. Notably, TBI is also one of the most prevalent injuries for military personnel. About 33% of all injuries amongst soldiers consist of TBI. Especially the use of IEDs (improvised explosive devices) in the Iraq and Afghanistan wars has increased the rate of TBI dramatically. Other causes of TBI arise from accidents during sports (especially boxing, rugby, football, etc.) and daily work.

TBI can cause a wide range of functional short- or long-term changes affecting the person's thinking, sensation, language, and/or emotions. TBI can also cause epilepsy and increases the risk for conditions such as Alzheimer's disease,

Parkinson's disease, and other brain disorders that become more prevalent with age (National Institute of Neurological Disorders and Stroke. "Traumatic brain injury: hope through research." Bethesda (MD): National Institutes of Health; 2002 NIH Publication No. 02-158).

Repeated mild TBI's occurring over an extended period of time (i.e. months to years) and that generally arise during sports such as boxing, rugby and football can result in cumulative neurological and cognitive deficits. Repeated mild TBI's that occur within shorter period of time (i.e. hours, days to weeks) are often catastrophic or even fatal. Repeated mild TBI's are sometimes also referred to as concussions. In the USA there are an estimated 3.5 million sports-related cases of repeated mild TBI per year. These people also face the possibility of long-term neurocognitive problems with increased risk of developing dementia such as Alzheimer's disease or

Amyotrophic lateral sclerosis or Parkinson's disease (Stern et al. 2011).

Currently, there is no effective treatment for TBI except for the prevention of internal brain pressure build up in the most severe and/or acute cases. So any drug that can ameliorate the secondary effects that are generally seen after TBI will be very beneficial. The inventors of the present invention have shown in the non-limiting examples (below) that IMP can lower the effects on the nervous system through inhibition of complement activation (or counteracting such complement activation) when IMP is administered before as well as when it administered after the TBI insult, showing that a novel therapeutic has been found for this type of acute nerve injury.

In embodiments in which the therapeutic goal is to treat a chronic nerve insult, more long term administration protocols will be generally preferred. Thus in one embodiment, at least IMP or any of its functional equivalents will be administered by any acceptable route as mentioned intra, constantly or in bolus administrations for at least 24 hours, preferably for a few days, weeks or months up to a few years as needed to treat or reduce symptoms associated with a disorder mediated by an undesired activity of the complement system as disclosed herein.

The invention is further illustrated by the following examples. These examples are not limiting the invention in any way, but merely serve to clarify the invention.

EXAMPLES

Example 1. Virtual screening and computational design of putative Membrane Attack Complex (MAC) inhibitors The complement protein C8-alpha (C8a) was selected as the model protein to screen for compounds that would possibly inhibit the formation of MAC because two crystal structures have been published on this protein, whereas no structures of C9 are available.

Identification of CD59 binding region on C8a

The known C8a PDB structure 2qqh contains coordinates for only C8a, whereas the structure referred to as 2rd7 contains both C8-alpha and C8-gamma. Therefore 2rd7 was considered to be available in a more biologically relevant form and has therefore been used in this study.

In C8a two transmembrane helical domains (TMHD1 and TMHD2) are inserted into the membrane and form a pore. CD59 binds residues 320 to 415 in C8a (Lockert et al. 1995). This stretch of amino acid residues belongs to TMHD2.

However, sufficient electron density for a 12-residue loop (352-363) in TMHD2 (both in 2rd7 and 2qqh) was not observed in X-ray crystallography studies and is therefore not included in PDB coordinates. Then the following was done to identify the CD59 binding region on C8a: The 12-residue loop (352-363) in TMHD2 of C8a was modeled by the Internal Coordinate Mechanics (ICM) program (ICM Manual, revision 3.0. 2006, MolSoft LLC: La Jolla, CA, USA). The X-ray crystallographic structure of CD59 (PDB code 2ofs) was docked onto the TMHD2 region of C8a by ICM. A careful consideration was made to choose the most likely (near native) conformation of CD59 upon docking. From previous site-directed mutagenesis and structural studies (Huang et al. 2006 and 2007), it was assumed that a loop between β- strands B and C in CD59 interacts with TMHD2 of C8a. Therefore, from the suggested docked conformations of CD59, the lowest energy conformation that showed such interaction with C8a was chosen.

The resulting C8a-CD59 complex (Fig. 1A) was studied by computational alanine scanning to predict the energetically important residue hot spots in the interface of C8a-CD59 complex. Three recognized online servers were used to locate the residues: Roberta (Kortemme et al. 2004), FoldX (Schymkowitz et al. 2005) and KFC (Darnell et al. 2007). Three residues (Y354, K357, and L364) in the TMHD2 were recognized as the hot spots for binding of CD59.

A previous study reported a 6-mer peptide of C9 binding to CD59 (Huang et al. 2006). The structure of this CD59-C9 (6-mer) complex was provided by the author and then a complex of C8a-CD59-C8gamma-C9 could be constructed. This complex was simulated by Molecular Dynamics (MD) simulations in explicit solvent for -15 nanoseconds by AMBER10 (Case et al. 2008). The resulting MD-simulated complex was assumed to have near-native conformation, and the coordinates of MD-simulated C8a was considered for further studies.

Screening of known drugs

Subsequently, the molecules from DrugBank (a database of known drugs; Wishart et al. 2008) were virtually screened onto the CD59 binding region of MD- simulated C8a using Glide 4.5 (Schrodinger, LLC, Portland, OR, USA; Friesner et al. 2004 and 2006; Halgren et al. 2004). Three major categories of known drugs in DrugBank were retrieved via ZINC (Irwin and Shoichet 2005; Irwin 2008) to acquire a virtual screening library. ZINC uses an extensive molecular preparation protocol to accumulate the ligands in proper protonated/deprotonated and tautomeric forms near physiological pH (5.75 - 8.25). The following categories (number of drugs in each category is underlined) of known drugs were screened: a) FDA approved small molecule drugs: 1344 (1613 via ZINC protocol b) Experimental drugs: 3116 (5033 via ZINC protocol)

c) Nutraceuticals: 69 (79 via ZINC protocol)

The number between parentheses shows total number of ligands including

protonated/deprotonated and tautomeres in the mentioned pH range. The ligands screened by using all three scoring functions in Glide: High-Throughput Virtual Screening (HTVS), Standard Precision (SP) and Extra precision (XP). These functions vary in the precision for calculating energies of the docking poses.

Prediction of CD59 mimetics that bind to C8a

On the basis of information obtained from the prediction of CD59 binding region on C8a, two online servers were used to predict the CD59 mimetics that may bind C8a: SuperMimic (Goede et al. 2006) to predict the non-peptide mimetics of CD59 and SUPERFICIAL (Goede et al. 2005) to predict the peptide mimetics of CD59 that may bind of C8a. The predicted non-peptide mimetics (30 ligands) and peptidic mimics (5 ligands) were also docked with Glide. Hit-list from known drugs

The most commonly used quantitative measure of success in virtual screening is 'enrichment', which is defined as the fraction of the actives (hits) found at x% of the database screened (both active and inactive) (Hawkins et al. 2008). Among the dataset that was screened (6725 ligands in DrugBank: FDA approved small molecule drugs, experimental drugs, and nutraceuticals), 6280 ligands were initially found to bind with C8a by Glide docking.

The top 5% of the bound ligands (by Glide SP- score) was also screened by HTVS and XP mode of Glide, and the final ranking was done according to XP score: the robustness of the energy functions (XP > SP > HTVS). XP scores indicate that predicted ligands might be less active than the known drugs. It will be appreciated by the person skilled in the art that it is not surprising that different scores do not correlate with each other, because they include different energy terms in their scorings.

Example 2. Screening of compounds in an in vitro screening assay for MAC inhibition

It was subsequently determined which of the compounds as found in the in silico screens (example 1) were able to inhibit complement mediated hemolysis of erythrocytes in an in vitro haemolytic assay. This haemolytic assay is a traditional method for measuring the total classical complement activity and is well known by the person skilled in the art. The test is a lysis assay, which uses antibody- sensitized erythrocytes as the activator of the classical complement pathway. The percentage hemolysis is determined by a spectrophotometer. This hemolytic test is a method to determine the amount of MAC being formed, since MAC is directly responsible for the hemolysis of the erythrocytes.

Five compounds were tested: RGS2005, RGS2022, RGS2048, RGS2049 and RGS2054. All compounds, except for RGS2022, were unable to prevent lysis of the erythrocytes in diluted or undiluted form (x-axis), except for RGS2022 that in less diluted form did inhibit the lysis in a dose dependent manner, see Fig. 2. Compound RGS2022 was identified as inosine monophosphate (IMP), also sometimes referred to as inosinic acid. IMP is a nucleoside monophosphate and is important in metabolism. It is the ribonucleotide of hypoxanthine and the first nucleotide formed during the synthesis of purine. IMP is formed by the deamination of adenosine monophosphate, and is hydrolysed from inosine. IMP is an intermediate ribonucleoside

monophosphate in purine metabolism. Important derivatives of IMP include purine nucleotides found in nucleic acids and adenosine triphosphate (ATP), which is used to store chemical energy in muscle and other tissues.

The fact that purine synthesis is a process that is completely unrelated to the formation of MAC in complement activation makes the finding that IMP can inhibit MAC formation even more surprising. Example 3. Inhibition of MAC formation by IMP in vitro

IMP in its salt form disodium IMP (E631) was purchased from Sigma Aldrich, USA (product ID 14625). To further study the inhibitory effect of IMP on MAC formation and activity in addition to the hemolytic assay an ELISA was used. In this ELISA complement in serum is activated by coating the ELISA plates with mannan. Such MBL complement ELISA's (schematically shown in figure 3A) are

commercially available: complement system MBL pathway Wieslab^® (product code COMPL MP320), Eurodiagnostica, Malmo, Sweden. When serum is added to these plates the MBL pathway is activated and terminal complement (MAC formation) can be measured using an antibody that recognizes a neo-epitopes (C5b-9) of MAC. Neo- epitopes are epitopes that become available on the complex after formation. Because C5 is unique to the terminal pathway of complement activation, detection of C5 activation products (C5b-9 or C5a) in a test sample is absolute proof of activation of the terminal pathway of complement in the sample.

IMP was tested in different concentrations in the ELISA. The results depicted in figure 3 show that IMP clearly inhibits MAC formation in a dose dependent manner. C4 levels in the MBL ELISA as detected by a commercially available antibody against C4 are not affected by RGS2022 showing that the initial step of complement activation is not inhibited (figure 3B), whereas the MAC formation is inhibited by IMP (figure 3C). Despite the fact that the concentrations of IMP in this assay are in the mM range, it is anticipated that IMP can also be used in vivo because the toxicity profile of IMP is very moderate. Notably, IMP is a food additive and thus generally considered safe in humans (Lewis et al. 1989. Food Additives Handbook; 1st edition; ISBN 978-0442205089). In rodents the LD₅₀ of IMP are 3.9 g/kg subcutaneously, 4.8 g/kg intraperitoneally (i.p.) and 15.9 g/kg orally. Example 4. Inhibition of MAC formation by IMP in vivo

To determine whether IMP was also capable of preventing the formation of MAC in an in vivo mouse model, IMP was injected i.p. in mice at a dose of 1.5 g/kg body weight. 30 min after injection blood was isolated and the serum was used in the hemolytic system (see example 2) to determine MAC formation and activity. The results depicted in figure 4 show that this single dose of IMP (RGS2022) lowered MAC activity significantly as measured by the hemolytic assay.

Example 5. Inhibition of MAC formation by IMP in vivo in a mouse model for traumatic brain injury (TBI)

Next it was determined whether IMP would be beneficial in inhibiting MAC formation in a disorder in which an increased MAC formation increases the severity of the disorder. As an example, a mouse model of neuronal trauma in the central nervous system was applied. For this, a closed head traumatic brain injury (TBI) mouse model was used as described earlier (Flierl et al. 2009). This model was approved by the ethical committee of the Amsterdam Medical Center, where the experiments were performed. In short, TBI is induced by a weight-drop (333 g) on the closed skull of the mouse from a height of 2 cm. There were 6 mice in each group. Mice were injected with a single IMP injection (i.p. 1.5 g/kg body weight) either immediately prior TBI or 30 min post TBI. The mice were monitored after TBI for 72 hrs and every day the neurological severity score (NSS) was measured as described in literature (Stahel et al. 2000). The NSS score is a 0 to 10 point system: a NSS score of 10 means total failure in all tasks (i.e. paralysis), a NSS score of 0 means healthy. In the IMP-treated animals the NSS score dropped significantly quicker from 10 back to nearly normal values in comparison to animals that were treated with PBS. Animals that were treated before TBI recovered faster than the animals that were treated 30 min post TBI, probably because MAC is being formed in this period directly after the trauma has occurred.

Clearly, a treatment with IMP before TBI occurs is not a situation that would often be feasible in real life, but these data demonstrate that rapid treatment with IMP upon TBI significantly reduces the risk of neuronal damage resulting from the formation of MAC and complement activation that comes in concert with such MAC formation. These results also indicate that prevention of breakdown of neuronal tissue due to the treatment of IMP is beneficial in all settings in which MAC formation plays a role.

The brains of the TBI mice were also examined (figure 6). Brains were fixed in formaline after cardial perfusion with formaline. Paraffin wax sections of 7 μιη thickness were stained using a three-step immunoperoxidase method. All the incubations were performed at room temperature. Following de-paraffination and rehydration, endogenous peroxidase activity was blocked with 1% H₂O₂ in methanol for 20 min. In all cases, antigen retrieval with microwave at 800 W for 3 min followed by 10 min at 440 W in 10 mM Tris/1 mM EDTA (pH 6.5) was used. To block the non-specific binding sites, slides were incubated in 10% normal goat serum (NGS) in Tris buffer for 20 min. Primary antibodies (C9, obtained from the lab of Paul Morgan, Cardiff, United Kingdom or IBA-1, WAKO, Cat. #019-19741) were diluted 1:500 in 1% BSA for 90 min. Sections were incubated for 30 min in biotinylated goat anti- rabbit from DakoCytomation (Glostrup, Denmark) diluted 1:200 in 1% BSA and 30 min in horseradish peroxidase labeled polystreptavidin (1:400 in PBS/BSA;

Sigma- Aldrich). To visualize peroxidase activity, staining was developed by using 3,3- diaminobenzidine tetrahydrochloride (DAB; Vector Laboratories) and

counterstained with hematoxylin. Sections stained with isotype control or primary or secondary conjugate alone were included as negative controls. After dehydration, slides were mounted in Pertex (Histolab). This histological examination revealed a strong reduction in MAC deposition in mice treated with IMP (given as RGS2022; left panels) and a significant reduction of infiltrating macrophages (IMP=RGS2022; right panels). Both the mice treated before TBI and the mice treated 30 min after TBI showed similar reduction in MAC deposition and reduction in macrophage infiltration and activation.

To check the specificity of the IMP action in the TBI mouse model, a similar experiment was performed using adenosine monophosphate (AMP) that is a closely related compound to IMP. Figure 7 shows the results of these experiments that were performed as outlined above and with identical concentrations of IMP and AMP (1.5 g/kg body weight). As a read-out the NSS and loss of body weight was measured in both sets of mice that clearly showed that the IMP treated mice returned back to near- normal NSS levels (figure 7A) and experienced less body weight loss (figure 7B) whereas the AMP-treated mice performed similar to the control (PBS-treated) mice for both parameters. This shows that the effect of IMP on the MAC formation is a very specific process and cannot be found by using a very similar compound such as AMP. Example 6. Sensory recovery by IMP in vivo in a mouse model for nerve crush of the N. ischiadicus

Recovery of sensory function was assessed during a period of 12 days after the nerve crush with the foot- flick test according to the method of De Koning et al. (1986) J. Neurol. Sci. 74: 237-246. Test mice received IMP (1.6 gram/kg) by intra peritoneal (i.p.) injection immediately before nerve crush and the following two days (once a day). Control treated animals received i.p. injections with PBS. Sensory function following the nerve crush was measured at the indicated time points using the footflick assay as described previously (Ramaglia V et al. 2009, Molecular

Immunology 47: 302-309).

Briefly, a shock source with a variable current of 0.02-0.5 mA was used. The mice were immobilized, and two stimulation electrodes were placed at the same point on the foot sole for every animal and stimulation applied by stepwise increasing the current from 0.02 to 0.5 mA. A response was scored positive when the mouse retracted its paw upon stimulation at a given current. The minimum current (mA) needed to elicit a retraction response was recorded. Values are expressed as percentage of normal function and represent the mean +- SEM. At day 12 (post nerve crush) the animals were sacrificed. Animals were perfused with 4% formalin and sciatic nerves were isolated, fixed o/n and embedded in paraffin.

Nerve crush sensory function recovery was followed in IMP treated and control animals. As shown in Figure 8, a faster recovery was observed in the IMP treated group at day 2. Accelerated recovery continued for the IMP treated group, e.g., at day 7 and 10. In the control group, animals started to recover at day 2, then experienced a decrease in recovery, followed by minimal increase in recovery after day 7 and again after day 10. Two way ANOVA analysis of the data in the two treatment groups showed that there was a statistical significance (P<0.05) between the two treatments (control vs IMP).

Example 7. Serum levels and biodistribution of Inosine 5'-[8-3H] monophosphate diamonium salt Serum levels of H IMP (diamonium salt) after i.p. injection in mice were analyzed. At indicated time points after i.p. injection (4.3.10⁶ DPM per animal) animals were sacrificed and serum samples were counted using liquid scintillation counter. Data is expressed as % of injected dose recovered as shown in Figures 9a. Biodistribution among the tissue samples is shown in Figure 9b.

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Faul M et al. (2010) Traumatic brain injury in the United States: emergency department visits, hospitalizations, and deaths. Atlanta (GA): Centers for Disease Control and Prevention, National Center for Injury Prevention and Control.

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Claims

1. Inosine monophosphate, or a salt thereof, for use in the treatment, prevention and/or inhibition of a disorder mediated by an undesired activity of the complement system.

2. The use according to claim 1, wherein said undesired activity of the complement system comprises formation of the membrane attack complex (MAC).

3. The use according to any one of claim 1 or 2, wherein said inosine monophosphate is formulated in the form of a disodium salt, a dipotassium salt or a dicalcium salt.

4. The use according to any one of claims 1 to 3, wherein said disorder is selected from the group consisting of: an acute injury of the nervous system such a traumatic brain injury, a chronic injury of the nervous system such as a chronic demyelinating neuropathy, multiple sclerosis, Huntington's disease, an ischemia- reperfusion injury, atherosclerosis, coronary heart disease and osteoarthritis.

5. The use according to any one of claims 1 to 4, wherein said disorder is an acute injury of the nervous system, e.g., PNS or CNS.

6. The use according to claim 5, wherein said IMP is administered within 24, 12, 6, 3, 2, 1, or less hours, preferably within 5, 10, 20, 30 or 40 minutes after the acute injury has occurred.

7. The use of claim 6, wherein IMP is administered in a single dose with no subsequent administration of IMP.

8. The use according to any one of claims 1 to 7, wherein said disorder is a condition requiring axonal regeneration.

9. A pharmaceutical composition for the treatment of a disorder mediated by MAC formation, wherein said composition comprises inosine monophosphate, or a salt thereof, and a pharmaceutically acceptable diluent, carrier or adjuvant.

10. The pharmaceutical composition according to claim 9, wherein said composition further comprises a compound selected from the group consisting of: a complement regulator, a complement receptor such as soluble CR1 or Crry-Ig, an antibody or antibody-fragment directed against a complement component, cobra venom factor, a poly-anionic inhibitor of complement, K-76COOH, rosmaric acid, nafamastat mesilate, Cls-INH-248, compstatin, PMX53, PMX205, a CI inhibitor, and derivatives thereof, or comprises an inhibitor such as an anti- sense oligonucleotide, aptamer, miRNA, ribozyme, or siRNA that blocks expression of one or more of C3 convertase, C5, C6, C7, C8a, C8b and C9.

11. Use of inosine monophosphate, or a salt thereof, in the preparation of a medicament for the treatment, prevention and/or inhibition of a disorder mediated by MAC formation.

12. Use of inosine monophosphate, or a salt thereof, to promote axonal regeneration.

13. A method of reducing, preventing or inhibiting the production and/or deposition of a membrane attack complex on a cell membrane or tissue in vitro or in vivo, the method comprising the step of contacting said cell or tissue with inosine monophosphate, or a salt thereof, such that the formation of the membrane attack complex is reduced or inhibited.

14. A method of treating, preventing or inhibiting a disorder mediated by MAC formation in a subject suffering from said disorder, or wherein said subject is at risk of developing said disorder, comprising the steps of administering a pharmaceutical composition according to claim 9 or 10 to said subject; and optionally monitoring the progress of the disorder in said subject.

15. The method according to claim 14, wherein said disorder is an acute or chronic injury of the nervous system.

16. The method according to claim 14 or 15, wherein the pharmaceutical composition is administered by intravenous injection, and preferably at a dose of about 0.25 - 8 g/kg, of the host body weight, optionally, 0.05g/kg/day or less.

17. A method of treating a nerve damaged by a physical injury or trauma in a subject, comprising the steps of administering a pharmaceutical composition according to claim 9 or 10 to said subject; and optionally monitoring the progress of the disorder in said subject.

18. The method of claim 17, wherein the nerve is a peripheral nerve.