The present invention relates to the treatment of acute inflammatory disorders characterised by the overproduction of pro-inflammatory cytokines, such as cerebral malaria, sepsis, systemic inflammatory response syndrome (SIRS), ischemic stroke and multiple sclerosis. More particularly, this invention relates to the use of vascular endothelial growth factor (VEGF) receptor agonists, alone and in combination with agents that induce the synthesis of Nrf2, in methods for the treatment of acute inflammatory disorders.

Background of the invention

An acute inflammatory response, for example following tissue damage caused by physical injury, is initiated by the activation of tissue macrophages which starts the inflammation process with the sequential production of the cytokines interferon gamma (IFN-γ) and tumour necrosis factor alpha (TNF-a) and the enzyme indoleamine 2,3- dioxygenase (IDO), followed by permeabilization and controlled damage (apoptosis) of the endothelial cells. This mechanism is tuned to generate a prompt amplified response and recruit additional cells at the site of damage.

Whilst pro-inflammatory cytokines, such as IFN- _Y and TNF-a, have an important role to play during acute inflammation, they are implicated in the pathology of a range of acute inflammatory disorders during which overproduction or dis-regulation of these proteins can lead to tissue and organ damage. The pathology of P. falciparum malaria is the result of a combination of factors that involve the systemic activation of the inflammatory response and hypoxia from blood vessel obstruction leading to endothelial damage that bears a number of analogies with sepsis. The consequent disruption of the blood-brain barrier leads to cerebral oedema, coma and death. In susceptible na ^' ive individuals the sequestration of infected erythrocytes to the cerebral microvasculature and the concomitant activation of the inflammatory processes are responsible for the development of a severe, often fatal, condition known as cerebral malaria (CM). In CM patients the cerebral capillaries are damaged, lined with apoptotic cells and filled with parasitized erythrocytes, while the surrounding brain tissue shows severe signs of focal hypoxia, monocyte infiltration and glial proliferation. The consequent disruption of the blood-brain barrier leads to cerebral oedema, coma and death.

The rodent parasite Plasmodium berghei ANKA strain (PbA) induces in the brain of susceptible mice, pathological changes that are very similar to human CM. The utilization of this experimental cerebral malaria (ECM) model has provided a better understanding of the malaria pathology in the brain and supported the notion that the severity of the condition is linked to a dis-regulation of the inflammation process. Compelling evidence implicates the cytokines IFN- _Y and TNF-a in driving the inflammatory response leading to ECM. IFN- _Y is required for up-regulating the expression of endothelial adhesion molecules, which bind to infected erythrocytes in the brain vessels, and for inducing the synthesis of macrophage- derived TNF-a that in turn enhances the inflammatory response. Mice in which either the genes coding for IFN- _Y and TNF-a or their receptors are disrupted fail to develop ECM. While activation of the inflammatory response is clearly necessary for developing ECM, several lines of evidence suggest that this alone may not be sufficient to fully explain experimental and human brain pathology. Mice in which the genes coding for the endothelial adhesion molecules ICAM-1 , VCAM-1 , and P-selectin have been disrupted do not develop ECM. Normally, these genes are highly induced during ECM and have been implicated in enhancing the binding of leukocytes, platelets and parasitized red blood cells to endothelial cells. In particular the disruption of ICAM-1 and VCAM-1 would prevent the binding of platelets to endothelium, a process that has been shown to induce TGF-beta1 mediated apoptosis of these cells. Furthermore, the observation that high levels of TNF-a and IFN-Y are detected in non-lethal cases of P. vivax infection casts doubts on the inflammation only hypothesis of cerebral malaria. Accordingly, and even more convincingly, attempts to treat CM with anti-inflammatory agents such as anti-TNF monoclonal antibodies and dexamethasone, rather than ameliorating, exacerbated the course of malaria.

Currently more than 20% of children with cerebral malaria are at risk of either dying or acquiring severe learning deficits in spite of being administered effective anti-parasitic treatment thus highlighting the need to develop an effective support therapy.

The pathology of cerebral malaria is known to share a number of similarities with sepsis. This is a clinical condition that is triggered by infection and is associated with high levels of mortality despite appropriate antibiotic therapy. It has been demonstrated that sepsis results from an immune response, for example to bacterial antigens, which leads to inflammation in tissues which may be remote from the site of infection. The antigens stimulate high levels of release of pro-inflammatory cytokines which can lead to multiple organ failure and death. It is known that TNF-a is a key mediator in this inflammatory response, however therapeutic approaches targeting this cytokine have so far proved ineffective or provide only a small survival benefit (Van Amersfoort, E.S., Berkel, T.J.C., Kuiper J. Clin Microbiol Rev., 2003, 16(3), 379-414; Reinhart, K., Karzai, W Crit Care Med., 2001 , 29 (7 Suppl.), S121 -5). Inflammatory disorders resulting over-production of pro- inflammatory mediators may also be prompted by non-infectious 'insults', such as ischemia, trauma, burns, pancreatitis, etc. Such disorders may be clinically defined as systemic inflammatory response syndrome (SIRS).

Initiation of an inflammatory response also plays an important role in the

pathogenesis of ischemic stroke and other types of brain injury. Ischemic damage leads to a complex inflammatory response involving the production of pro-inflammatory cytokines, such as TNF-a, IL-1 β and IL-6, by a variety of activated cell types, and an acute and prolonged inflammatory response in the brain. It is hypothesised that TNF-a plays a key role in the acute propagation of inflammatory processes and cell death in stroke patients. Current approved therapies for stroke which involve pharmacologically induced

thrombolysis are limited in efficacy and there is a need to develop new treatment strategies.

Multiple sclerosis (MS) is a chronic neuroinflammatory disease which is

characterised by acute inflammatory episodes during recurrent relapses and / or progression, which may be linked to brain or spinal cord inflammation. Pro-inflammatory cytokines, such as Interleukin 17 (IL-17), are known to be involved in the initiation of autoimmune tissue inflammation, and have an established role in the pathogenesis of this disorder. The role of TNF-a in demyelinating diseases such as experimental allergic encephalomyelitis (EAE) is well established and increasing evidence exists regarding a role for TNF-a in the pathogenesis of MS. Clinical trials of statins in multiple sclerosis have been carried out, alone and in combination with interferon-β, with conflicting results (Ciurleo, R., Bramanti, P., Marino, S., Pharmacological Research, 87, 2014, 133-143). Despite clear evidence of the role of pro-inflammatory cytokines in the pathogenesis of a range of disorders, current pharmacological strategies using anti-inflammatory agents have typically been shown to have limited efficacy. There is therefore the need to develop new therapeutic approaches for the treatment of acute inflammatory disorders, such as CM, sepsis, systemic inflammatory response syndrome (SIRS), ischemic stroke and multiple sclerosis, which are characterised by the overproduction of pro-inflammatory cytokines.

Summary of the invention

It has been surprisingly found that VEGF-receptor agonists provide therapeutic benefits for the treatment of acute inflammatory disorders, and that the combination of VEGF-receptor agonists and agents that induce the synthesis of Nrf2 provides synergistic therapeutic benefits for the treatment of acute inflammatory disorders. It has been observed that the use of VEGF-receptor agonists, alone or in combination with agents that induce the synthesis of Nrf2, prevents or reduces the dis-regulated activation of an inflammatory response and reduces endothelial damage. Accordingly, in a first aspect of the invention there is provided a VEGF receptor agonist for use in a method for the treatment of an acute inflammatory disorder associated with the overproduction of pro-inflammatory cytokines. Preferably, the VEGF receptor agonist is administered simultaneously, separately or sequentially with an agent that induces the synthesis of Nrf2. In a second aspect of the invention there is provided a pharmaceutical combination comprising a VEGF receptor agonist and an agent that induces the synthesis of Nrf2 for simultaneous, separate or sequential administration.

In a third aspect of the invention there is provided a combination of a VEGF receptor agonist and an agent that induces the synthesis of Nrf2 for use in a method for the treatment of an acute inflammatory disorder associated with the overproduction of proinflammatory cytokines, wherein the VEGF receptor agonist and the agent that induces the synthesis of Nrf2 are for simultaneous, separate or sequential administration. In a fourth aspect of the invention there is provided a method of treatment of an acute inflammatory disorder associated with the overproduction of pro-inflammatory cytokines in a patient, comprising the administration of a therapeutically effective amount of a VEGF-R receptor agonist to the patient. Preferably, the method of treatment additionally comprises the administration of a therapeutically effective amount of an agent that induces the synthesis of Nrf2.

In a fifth aspect of the invention there is provided a method of treatment of an acute inflammatory disorder associated with the overproduction of pro-inflammatory cytokines in a patient, comprising administration of a therapeutically effective amount of a VEGF receptor agonist to the patient, and furthermore comprising administration of a therapeutically effective amount of an agent that induces the synthesis of Nrf2.

In a sixth aspect of the invention there is provided a kit comprising a combination of a VEGF receptor agonist and an agent that induces the synthesis of Nrf2 together with a pharmaceutically acceptable carrier or carriers. In one embodiment, the kit comprises instructions for simultaneous, separate or sequential administration of the combination to a patient for use in the treatment of an acute inflammatory disorder associated with the overproduction of pro-inflammatory cytokines.

In an embodiment of the above aspects the VEGF receptor agonist is VEGF, such as recombinant human VEGF, or a functional variant thereof. In a further embodiment, the VEGF receptor agonist is VEGF165, such as recombinant human VEGF165.

In a further embodiment of the above aspects, the acute inflammatory disorder is sepsis, systemic inflammatory response syndrome (SIRS), cerebral malaria, ischemic stroke or multiple sclerosis.

Brief description of the figures

Figure 1 shows the effect of LPS and VEGF-LPS combination treatment on infected C57BL/6 mice. Figure 2A shows the effect of recombinant VEGF treatment on P. berghei infected Balb/c mice.

Figure 2B shows the effect of recombinant VEGF treatment on P. berghei infected C57BL/6 mice. Figure 3A shows the effect of Axitinib treatment on P. berghei infected C57BL 6 mice.

Figure 3B shows the effect of Axitinib treatment on P. berghei infected Balb/c mice.

Figure 4A shows the effect of statin / VEGF combination treatments on P. berghei infected C57BL/6 mice.

Figure 4B shows the outline of spleens taken from images of spleens dissected from P. berghei infected mice treated with Lovastatin, Simvastatin, LPS+VEGF and Lovastatin- VEGF.

Detailed description of the invention

The present invention relates to vascular endothelial growth factor (VEGF) receptor agonists and to pharmaceutical combinations which comprise a VEGF receptor agonist and an agent that induces the synthesis of the transcription factor nuclear factor erythroid-2 related factor 2 (Nrf2). The term "pharmaceutical combinations" as used herein refers to two or more different pharmacologically active agents, which are intended to produce a specific therapeutic effect in a patient when applied together to the patient, i.e. one or more VEGF receptor agonists and one or more agents that induce the synthesis of Nrf2 in the present invention, and wherein applied together means either simultaneous, separate or sequential administration.

By 'vascular endothelial growth factor receptor (VEGF-R)' it is meant a cellular receptor for VEGF, for example the protein kinase receptors VEGFR-1 , known as Flt-1 , and VEGFR-2, known as KDR or FIK-1 . By 'VEGF-R agonist' it is meant an agent that binds to a VEGF-R and activates or enhances, either partially or fully, the activity of the receptor. Exemplary forms of agonists include, for example proteins, polypeptides, peptides, antibodies or antibody fragments, peptide mimetics and small organic molecules. The VEGF-R agonist of the invention may be a selective agonist of a VEGF receptor, for example the agonist may be a VEGFR-1 selective agonist, i.e. it exclusively or preferentially modulates VEGFR-1 over other VEGF receptors, such as VEGFR-2.

Therefore, in one embodiment of the invention the combinations of the invention comprises a selective agonist of VEGFR-1 and an agent that induces the synthesis of Nrf2. Selective agonists of VEGFR-1 are known, see for example EP151676B1 . Preferred VEGFR-1 selective agonists have binding affinity to VEGFR-1 which is equal to or greater than the binding affinity of native VEGF to VEGFR-1 , and more preferably have less binding affinity to VEGFR-2 than the binding affinity of native VEGF to VEGFR-2.

The VEGF-R agonist of the invention may be VEGF or a functional variant thereof. By 'vascular endothelial growth factor (VEGF)' it is meant a mammalian vascular endothelial growth factor, also termed VEGF-A or vascular permeability factor (VPF). As used herein, the term 'VEGF' includes the various sub-types of VEGF-A that arise by, for example, alternative splicing of the human VEGF gene including VEGF121 , VEGF145, VEGF165, VEGF183, VEGF189 and VEGF206. Preferably, the VEGF is VEGF165.

By "functional variant" of VEGF it is meant a polypeptide having the amino acid sequence of a native VEGF altered in one or more amino acid residues, for example through amino acid substitutions, additions or deletions, but which retains one or more functional attributes of the native ligand, such as the ability to bind to a VEGF-R and activate or enhance, either partially or fully, the activity of the receptor.

VEGF and variants may be prepared using a variety of techniques well known in the art. Amino acid sequence variants of VEGF can be prepared by mutations in the VEGF DNA. Such variants may include, for example deletions from, insertions into and / or substitutions of residues within the native amino acid sequence. The VEGF DNA encoding the variant may then be expressed in a recombinant cell culture.

The VEGF of the current invention may be recombinant human VEGF. The term "recombinant human VEGF" refers to a VEGF of the present invention which is produced by recombinant DNA techniques which are used to transform a host cell to produce the human growth factor. Preferably, the VEGF is recombinant VEGF165. The VEGF receptor agonist may be used in the current invention in combination with an agent that induces the synthesis of Nrf2. Nrf2 (nuclear factor erythroid-2 related factor 2, also known as Nfe2L2) is a cap-and-collar basic leucine transcription factor which plays a key role in cytoprotection against oxidative stress and aptosis. Under basal conditions, Nrf2 levels are retained in the cytoplasm by the cytosolic actin-bound repressor KEAP1 (Kelch-lick ECH associating protein 1 ), which binds to Nrf2 and targets it for degradation. This mechanism helps to maintain steady state levels of Nrf2 and therefore controls Nrf2 mediated transcription. By 'an agent that induces the synthesis of Nrf2' it is meant an isolated pharmacological agent that, after administration, results in an increased level of cytoplasmic Nrf2. Methods of assessing the effect of compounds on the Nrf2 pathway are known to the skilled person, for example as described in EP2680010A1 (Biogen Idee MA Inc.), and include the assessment of the effect of a compound on the level of mRNA corresponding to Nrf2. It will be understood that the agent that induces the synthesis of Nrf2 may, for example, activate existing cellular Nrf2 or may act through an induction of Nrf2 gene expression.

In one embodiment of the invention the agent that induces the synthesis of Nrf2 is an Nrf2 activator. By "Nrf2 activator" it is meant an isolated pharmacological agent that, after administration, results in an activation of existing cellular Nrf2 and therefore an increased level of cellular Nrf2. Such agents may interact, for example, directly with Nrf2, with KEAP1 and / or the Nrf2-KEAP1 complex. Nrf2 activators include bardoxolone methyl (methyl 2-cyano-3,12-dioxo-oleana-1 ,9(1 1 )-dien-28-oic acid, CDDO-Me), ethyl 2-cyano-3, 12-dioxo-oleana-1 ,9(1 1 )-dien-28-oic acid (CDDO-Et), 2-cyano-3,12-dioxo-oleana-1 ,9(1 1 )- dien-28-oic acid (CDDO), 1 [2-Cyano-3,12-dioxooleana-1 ,9(1 1 )-dien-28-oyl]imidazole (CDDO-lm), 2-cyano-N-methyl-3,12-dioxo-oleana-1 ,9(1 1 )-dien-28 amide (CDDO-Methyl amide), 2-cyano-3,12-dioxooleana-1 ,9(1 1 )-dien-28-onitrile (TP-225), [(~)-(4bS,8aR,10aS)- 10a-ethynyl-4b,8,8-trimethyl-3 ,7-dioxo -3 ,4b ,7,8,8a,9,10,10a-octahydrophenanthrene-2,6- dicarbonitrile] (TBE-31 ), gallic acid esters, 3-tert-butyl-4-hydroxyanisole, 2-tert-butyl- hydroxyanisole (BHA), tert-butylquinone (tBQ), tert-butylhydroquinone (tBHQ), 3,5-di-tert- butyl-4-hydroxytoluene (BHT), cinnamic aldehyde, curcumin, cinnamic acid esters, epigallocatechin-3-gallate, carnosol, carnosic acid, phenethyl isothiocyanate, benzyl isothiocyanate, lycopene, 1 ,5-(4-methoxy-phenyl)-1 ,2-dithiole-3-thione (ADT), caffeic acid esters, alkylsulfinylalkyi isothiocyanate, such as 6-methylsulfinylhexyl isothiocyanate, mono- or di-esters of fumaric acid, such as monoalkyl hydrogen fumarates and dialkyl fumarates, for example monomethyl fumarate or dimethyl fumarate (DMF), ethacrynic acid, 4-methyl-5- [2-pyrazinyl]-1 ,2-dithiole-3-thione, sulforaphane, resveratrol, cafestol, kahweol, zerumbone etc.

The agent that induces the synthesis of Nrf2 may be selected from one or more of a lipopolysaccharide (LPS), lovastatin, bardoxolone methyl (methyl 2-cyano-3,12-dioxo- oleana-1 ,9(1 1 )-dien-28-oic acid, CDDO-Me), ethyl 2-cyano-3,12-dioxo-oleana-1 ,9(1 1 )-dien- 28-oic acid (CDDO-Et), 2-cyano-3,12-dioxo-oleana-1 ,9(1 1 )-dien-28-oic acid (CDDO), 1 [2- Cyano-3,12-dioxooleana-1 ,9(1 1 )-dien-28-oyl]imidazole (CDDO-lm), 2-cyano-N-methyl-3,12- dioxo-oleana-1 ,9(1 1 )-dien-28 amide (CDDO-Methyl amide), 2-cyano-3,12-dioxooleana- 1 ,9(1 1 )-dien-28-onitrile (TP-225), [(~)-(4bS,8aR,10aS)-10a-ethynyl-4b,8,8-trimethyl-3 ,7- dioxo -3 ,4b ,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile] (TBE-31 ), gallic acid esters, 3-tert-butyl-4-hydroxyanisole, 2-tert-butyl-hydroxyanisole (BHA), tert- butylquinone (tBQ), tert-butylhydroquinone (tBHQ), 3,5-di-tert-butyl-4-hydroxytoluene (BHT), cinnamic aldehyde, curcumin, cinnamic acid esters, epigallocatechin-3-gallate, carnosol, carnosic acid, phenethyl isothiocyanate, benzyl isothiocyanate, lycopene, 1 ,5-(4- methoxy-phenyl)-1 ,2-dithiole-3-thione (ADT), caffeic acid esters, alkylsulfinylalkyi isothiocyanate, such as 6-methylsulfinylhexyl isothiocyanate, mono- or di-esters of fumaric acid, such as monoalkyl hydrogen fumarates and dialkyl fumarates, for example

monomethyl fumarate or dimethyl fumarate (DMF), ethacrynic acid, 4-methyl-5-[2- pyrazinyl]-1 ,2-dithiole-3-thione, sulforaphane, resveratrol, cafestol, kahweol, and zerumbone.

In one embodiment of the invention, the agent that induces the synthesis of Nrf2 is a statin. Suitable statins may include atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rivastatin, rosuvastatin, velostaiin, and

pharmaceutically acceptable salts, solvates, esters or precursors thereof.

In an embodiment of the invention, the agent that induces the synthesis of Nrf2 is a lipopolysaccharide (LPS). Lipopolysaccharides are the major surface molecule of, and occur exclusively in, the external leaflet of the outer membrane of gram-negative bacteria. Lipopolysaccharides are a group of structurally related complex molecules of approximately 10,000 Daltons in size and consist of three covalently linked regions:

(i) an O-specific polysaccharide chain at the outer region

(ii) an oligosaccharide central region

(iii) a lipid A region - the inner region comprising glucosamine disaccharide units which carry long chain fatty acids.

It will be understood that the LPS used in the combinations of the invention may be synthetic or isolated from bacterial sources. It will also be understood that the agent that induces the synthesis of Nrf2 may be, for example, a synthetic LPS analogue, a truncated variant of an naturally occurring LPS, a chimeric LPS variant etc.

The combinations of the invention may for example comprise a lipopolysaccharide and a VEGF receptor agonist, such as a lipopolysaccharide and a VEGF or a functional variant thereof, for example a lipopolysaccharide and a recombinant human VEGF. In one embodiment of the invention the combination comprises a lipopolysaccharide and recombinant human VEGF-165.

In another embodiment of the invention the combination comprises lovastatin and a VEGF receptor agonist, such as lovastatin and a VEGF or a functional variant thereof, for example lovastatin and a recombinant human VEGF. In one embodiment of the invention the combination comprises lovastatin and recombinant human VEGF-165. In a further embodiment of the invention the combination comprises an Nrf2 activator and a VEGF receptor agonist, such as an Nrf2 activator and a VEGF or a functional variant thereof, for example an Nrf2 activator and a recombinant human VEGF. In one embodiment of the invention the combination comprises an Nrf2 activator and recombinant human VEGF-165. In a further embodiment of the invention the combination comprises an active agent selected from one or more of lipopolysaccharide (LPS), lovastatin, bardoxolone methyl (methyl 2-cyano-3,12-dioxo-oleana-1 ,9(1 1 )-dien-28-oic acid, CDDO-Me), ethyl 2-cyano-3, 12-dioxo-oleana-1 ,9(1 1 )-dien-28-oic acid (CDDO-Et), 2-cyano-3,12-dioxo-oleana-1 ,9(1 1 )- dien-28-oic acid (CDDO), 1 [2-Cyano-3,12-dioxooleana-1 ,9(1 1 )-dien-28-oyl]imidazole (CDDO-lm), 2-cyano-N-methyl-3,12-dioxo-oleana-1 ,9(1 1 )-dien-28 amide (CDDO-Methyl amide), 2-cyano-3,12-dioxooleana-1 ,9(1 1 )-dien-28-onitrile (TP-225), [(~)-(4bS,8aR,10aS)- 10a-ethynyl-4b,8,8-trimethyl-3 ,7-dioxo -3 ,4b ,7,8,8a,9,10,10a-octahydrophenanthrene-2,6- dicarbonitrile] (TBE-31 ), gallic acid esters, 3-tert-butyl-4-hydroxyanisole, 2-tert-butyl- hydroxyanisole (BHA), tert-butylquinone (tBQ), tert-butylhydroquinone (tBHQ), 3,5-di-tert- butyl-4-hydroxytoluene (BHT), cinnamic aldehyde, curcumin, cinnamic acid esters, epigallocatechin-3-gallate, carnosol, carnosic acid, phenethyl isothiocyanate, benzyl isothiocyanate, lycopene, 1 ,5-(4-methoxy-phenyl)-1 ,2-dithiole-3-thione (ADT), caffeic acid esters, alkylsulfinylalkyl isothiocyanate, such as 6-methylsulfinylhexyl isothiocyanate, mono- or di-esters of fumaric acid, such as monoalkyl hydrogen fumarates and dialkyl fumarates, for example monomethyl fumarate or dimethyl fumarate (DMF), ethacrynic acid, 4-methyl-5- [2-pyrazinyl]-1 ,2-dithiole-3-thione, sulforaphane, resveratrol, cafestol, kahweol, a statin, and zerumbone, and a VEGF receptor agonist such as a VEGF or a functional variant thereof, for example a recombinant human VEGF e.g. recombinant human VEGF-165.

It will be understood that the terms "VEGF agonist" and "agent that induces the synthesis of Nrf2" preferably refer to active agents that, if naturally occurring, are purified to isolate the active agents, before being formulated into a pharmaceutical composition, e.g. by conventional means such as chromatography, distillation or crystallisation. The VEGF receptor agonists and the described pharmaceutical combinations of the invention are proposed for use in methods of treatment of acute inflammatory disorders associated with the overproduction of pro-inflammatory cytokines, for example acute inflammatory disorders associated with the overproduction of IFN- _Y and / or TNF-a.

Such disorders include cerebral malaria, sepsis (including severe sepsis and septic shock), ischemic stroke, multiple sclerosis, and systemic inflammatory response syndrome (SIRS), and include acute inflammatory disorders which involve systemic inflammatory responses to infections with bacteria, viruses, fungi, parasites, or their toxins, together with acute inflammatory disorders which involve systemic inflammatory responses of noninfectious origin, for example in response to ischemia, burns, trauma, pancreatitis and stroke (heart and brain). As used herein, the term "multiple sclerosis (MS)" includes one or more of relapsing- remitting MS, primary-progressive MS, secondary-progressive MS, and progressive- relapsing MS. In one embodiment of the invention the acute inflammatory disorder is relapsing-remitting MS. As used herein, "sepsis" refers to a patient's response to a microbial infection, e.g. a widespread inflammatory response caused by an infection, and includes septic shock a condition whereby sepsis has resulted in a significant drop in blood pressure.

The method of treatment of a patient may comprise the administration of a therapeutically effective amount of a VEGF receptor agonist to the patient. The method of treatment of a patient may additionally comprise the administration of a therapeutically effective amount of an agent that induces the synthesis of Nrf2.

"Treating" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, slow down (lessen) or at least partially alleviate an abnormal condition in the subject. Those in need of treatment include those already with the disorder as well as those prone to having the disorder or those in whom the disorder is to be prevented (prophylaxis).

Patients may be selected for treatment using the combination of the invention based on a stratification of patients to identify those patients who are at a high risk of developing an acute inflammatory disorder. For example patients with an infection may be assessed to determine those who are at a high risk of developing an inflammatory disorder, such as, cerebral malaria or sepsis, or those patients with sepsis who are at high risk of developing septic shock. Methods of stratification are known, for example, see Henry K.E., Hager D.N., Pronovost, P.J., Saria, S., Science Translational Medicine, 2015, Vol. 7, Issue 299).

The methods of treatment of current invention may therefore comprise the assessment of a patient to determine the risk of the development of an acute inflammatory disorder and then treatment of those patients considered to be at a high risk of developing the disorder using a method of the invention.

By "patient" it is meant a mammal, in particular a human subject. By "therapeutically effective amount" is meant the amount of an active agent, required to treat an acute inflammatory disorder or the symptoms thereof, such as a reduction of an inflammatory response in a patient which is occurring with and / or is resulting from the acute inflammatory disorder. The effective amount of the active agents used to carry out the current invention for therapeutic treatment will vary depending on, for example, the route of administration and the age and size of the patient. The appropriate amount and dosage regime will ultimately be determined by a medical or veterinary professional.

It will be understood by the skilled person the methods of the invention may comprise the administration of a sufficient amount of the active agents of the invention to lead to a reduction in the levels of IFN- _Y and / or TNF-a in a patient with an acute inflammatory disorder.

The active agents of the invention may preferably be administered parenterally, by infusion or injection (intraveneous, intramuscular, subcutaneous, intraperitoneal, intradermal), but other modes of administration such as inhalation, intranasal, buccal and oral may also be applicable. Parenteral administration includes subcutaneous injections, intraveneous, intramuscular, intrasternal injection or infusion techniques. One or more of the active agents of the invention may also be administered as a sustained-release injection, for example using an injectable depot or microsphere technology. The active agents of the invention may be formulated to form pharmaceutical compositions to enable administration to a patient. Such compositions comprise a therapeutically effective amount of one or more of the active agents and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" is meant that the carrier must be compatible with the other ingredients of the formulation and not harmful to the recipient thereof. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the active agents are administered.

The pharmaceutically acceptable carrier, or pharmaceutically acceptable carriers may be a liquid, such as water or oils, with the pharmaceutically composition being, for example, in the form of an injectable liquid. Compositions suitable for administration may also contain one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, and stabiliser. Liquid pharmaceutical compositions may be, for example, solutions or suspensions and can include one or more diluents, such as water, saline solution, for example physiological saline, or aqueous dextrose. There are a number of references that are available to the skilled person which describe pharmaceutically acceptable carriers including the Handbook of Pharmaceutical Excipients (Ed. R.C. Rowe, Pharmaceutical Press) and Remington - The Science and Practice of Pharmacy (Ed. L.V. Allen, Pharmceutical Press).

Active agents used in the methods of the invention may be used in the form of salts, for example as alkali metal or amine salts or as acid addition salts, as prodrugs, solvates, for example as a hydrate, where appropriate to optimise stability and / or physical characteristics, such as solubility.

It will be evident to the skilled person that the optimal dosage of the active ingredients will depend on a number of factors. Relevant factors include the type of patient (e.g. human), the manner of administration, the specific active agents selected and the pharmaceutical composition employed.

The combinations of the invention may be provided as a kit comprising a combination of a VEGF receptor agonist and an agent that induces the synthesis of Nrf2 together with a pharmaceutically acceptable carrier or carriers. When the active agents of the combination are to be administered simultaneously, the kit may contain each active ingredient in a single pharmaceutical composition, or in separate pharmaceutical compositions. When the active agents are not administered simultaneously, the kit will contain the active agents in separate pharmaceutical compositions either in a single package or in separate packages or compartments. The kit may additionally comprise instructions for simultaneous, separate or sequential administration of said combination to a patient for use in the treatment of an acute inflammatory disorder associated with the overproduction of pro-inflammatory cytokines. Other variations and modifications will be apparent to the skilled person. Such variations and modifications may involve equivalent and other features that are already known and which may be used instead of, or in addition to, features described

herein. Features that are described in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, features that are described in the context of a single embodiment may also be provided separately or in any suitable subcombination.

It should be noted that the term "comprising" does not exclude other elements or steps, the term "a" or "an" does not exclude a plurality, a single feature may fulfil the functions of several features recited in the claims and reference signs in the claims shall not be construed as limiting the scope of the claims.

Experimental Results

Lipopolysaccharide (LPS) and VEGF suppress the inflammatory response to P. berghei Anka (PbA) infection and prevent the development of cerebral malaria At low doses, LPS is known to elicit a complex orchestrated counter regulatory response to inflammation known as "tolerance" that involves the up regulation of heme oxygenase-1 (HO-1 ) via the transcription factor Nrf-2 as well as the induction of a number of antioxidant stress response genes including VEGF. When given to P. berghei Anka (PbA) infected C57BL/6 (ECM susceptible) mice, LPS protected 100% of the animals from ECM and prolonged their survival until day +12 post-infection (Figure 1A). No effect was observed on parasite load. Immunohistochemistry analysis of brain sections from these mice showed a marked reduction of the CD3+ cell infiltrate (typical of cerebral malaria pathology) and a similar decrease in the immuno-staining of anti-cleaved caspase 3 antibodies compared to untreated C57BL/6 mice, a marker that reveals the activation of apoptosis (Table 1A). LPS treatment induced from day + 3 post infection to the time of death a significant down regulation to basal levels of both TNF-a and IFN- _Y (p<0.01 ). These expression changes were associated with a significant up-regulation of both HO-1 and Nrf-2 (p<0.01 ). Table 1A shows immunohistochemistry analysis of brain sections after LPS and VEGF-LPS treatment.

The role of obstruction hypoxia and endothelial cell damage associated to brain pathology prompted us to also investigate whether the administration of recombinant VEGF could change the course of PbA infection. We investigated both ECM susceptible C57BL/6 and ECM resistant Balb/c mice treated with recombinant VEGF 200ng/mouse daily starting from day +1 post-infection over a period of 5 days. Under these experimental conditions the treatment prolonged the survival of the C57BL 6 infected mice until day +10 post-infection without affecting the parasite load (Figure 2). None of these animals developed the typical signs of ECM such as: a positive Woolley-White sign and convulsions. However, they showed ruffled fur, pale ears and footpad and red discharge from the nose (which are typical signs of anaemia). Morphological analysis and immunohistochemistry of C57BL/6 brain sections revealed the presence of a moderate infiltrate of CD3 lymphocytes (mainly composed by CD8 rather than CD4 - data not shown) from day +3 to end point. Some apoptotic endothelial cells were also detected, particularly on the day when the animals died. Important differences were detected when analysing the expression of inflammation markers in treated vs non-treated animals. VEGF induced a marked increase of both VEGFR1 and VEGFR2 that was associated with a down regulation of the cytokines IL- 23p19 (p<0.05), TNF-a and IFN-γ (p<0.01 ) (Table 1 B). On the contrary VEGF treatment had little effect on the transcription of inflammation-associated genes in Balb/c mice (Table 1 B).

Table 1 B. Fold change expression of inflammation markers in the brain of VEGF treated mice Expression of inflammation markers in brain tissue of C57BL/6 and Balb/c infected vs. treated mice (VEGF 200ug), +3 days post infection and at the end point (bold). Each value represents the mean of the fold changes obtained from 3 biological replicates. Induction relative to the non-infected control (fold change) was calculated using the comparative Ct method (AACi). Values obtained from non-treated vs treated mice were compared using a one-sample t-test to access statistical significance * (0.01 < p <0.05) and ** ( P < 0.01 ).

Mouse strain

C57BL/6 VEGF VEGFR1 VEGFR2 S1 P IL-6 IL-23p19 TNF-a IFN-y HO-1 Nrf2

Infected 7.43 2.80 4.76 2.30 3.80 20.60 1.30 2.64 1 .10 1 .30

7.50 2.23 7.10 1.60 8.30 21.10 4.30 9.70 0.40 0.88

Infected 7.35 0.50 ^* 1.77 ^* 5.10 1.20 ^* 25.44 ^* 0.89 1 .02 ^** 1 .76 3.76 ^*

+ VEGF 8.11 2.05 9.07** 1.40 0.20** 5.43* 2.10** 2.43** 1.20* 0.90

Mouse strain

Balb/c

Infected 1 .55 0.89 0.85 5.36 0.58 0.27 0.49 0.80 3.48 2.79

0.85 0.61 0.16 3.32 0.72 1.14 0.50 1.13 6.22 6.25

Infected 2.73 ^* 1.32 0.60 3.60 0.09 0.50 0.80 0.40 4.30 2.70

+ VEGF 1.99 1.0** 2.37** 4.60 0.20 0.90 0.30* 0.68 6.70 7.10 The VEGF inhibitor Axitinib exacerbates ECM pathology

To better understand the role of VEGF we investigated disease progression, brain pathology and inflammatory gene expression in both C57BL/6 and Balb/c mice treated with Axitinib. This is a potent small-molecule inhibitor of the VEGF receptor tyrosine kinase that induces a significant reduction in the VEGF response to inflammation and hypoxia. Typically, untreated PbA infected C57BL/6 mice showed signs of convulsions, ataxia, coma, and a positive Wooly/White sign and died of ECM within 7 days post infection. All Axitinib treated C57BL/6 mice died one or two days earlier than the untreated-control group, showing severe signs of ECM (Figure 3A). Treatment of Balb/c mice with Axitinib did not induce ECM but these mice also died earlier than untreated control starting from day +8 post infection (survival: day 8: 98%, day 9: 80%, day 10: 50%) (Figure 3B). Brain samples from C57BL/6 and Balb/c mice were collected in a replica experiment and processed for histological examination at day + 3 post infection and at the time of death (end point). Brain sections from C57BL/6 mice stained with H&E showed a considerable monocyte infiltrate and accumulation of malaria pigment that progressively increased from day +3 to the time of death. Immunohistochemistry of brain samples revealed that the monocyte infiltrate was composed of CD3 positive cells, (CD4 ⁺ and CD8 ⁺ mixed subpopulations) with a higher number of CD4, calculated as % of area of the positive signal, (Infected C57BL/6: 4.3%, C57BL/6 treated with Axitinib: 12.4%; Balb/c Infected: 2.3%, Balb/c treated with Axitinib: 4%.) vs. CD8 (Infected C57BL/6: 2.8%, C57BL/6 treated with Axitinib: 2.9%; Balb/c Infected: 0.5%, Balb/c treated with Axitinib: 2.4%.), in both mouse strains. To evaluate the ECs apoptosis we used an antibody directed against the cleaved form of caspase 3. The immunohistochemistry showed an increased staining of brain parenchyma and endothelial cells in Axitinib treated C57BL/6 mice. The morphological examination of brain sections and the results of the immunochemistry analyses are coherent with the worsening of disease progression in Axitinib-treated mice.

Analysis of cytokine and inflammatory markers in Axitinib treated animals

The brains of PbA infected C57BL/6 mice showed consistent high levels of IL-23p19 and a progressive up regulation of TNF-a and IFN- _Y from day +3 to the onset of ECM together with basal levels of both Nrf2 and HO-1 . In these mice Axitinib treatment strongly suppressed the expression of VEGF and both its receptors VEGFR1 and VEGFR2. We also observed that Axitinib significantly down regulated the expression of a number of cytokines including IL-12p40, IL-6 and IL-23p19 (p<0.01 ) while leaving unaffected the induction of TNF-a and IFN-γ. When analysing circulating cytokines we observed that 5 Axitinib treated mice showed significantly higher level of IFN-γ . Balb/c mice responded to PbA infection with a marked up-regulation in the brain of both HO-1 and Nrf2 as shown in previous observations. In these mice Axitinib treatment significantly inhibited this response (p<0.01 ) in agreement with its worsening effect on disease progression (Table 2).

Table 2. Expression of inflammation markers in the brain of Anti-VEGF 0 treated mice

Expression of inflammation markers in brain tissue of C57BL/6 and Balb/c Infected vs. treated mice (Axitinib 25mg/Kg), +3 days post infection and at the end point (bold). Each value represents the mean of the fold changes obtained from 3 biological replicates. Induction relative to the non-infected control (fold change) was calculated using the5 comparative Ct method (AACt).Values obtained from non-treated vs treated mice were compared using a one-sample t-test to access statistical significance ^* (0.01 < p <0.05) and * ^* ( p < 0.01 ).

Mouse strain

C57BL/6 VEGF VEGFR1 VEGFR2 S1 P IL-6 IL-23p19 TNF-a IFN-y HO-1 Nrf2

7.43 2.80 4.76 2.30 3.80 20.60 1.30 2.64 1.10 1 .30

Infected

7.50 2.23 7.10 1.60 8.30 21.10 4.30 9.70 0.40 0.88

0.08 ^* 0.30 ^** 0.30 ^** 1 .10 ^** 1 .90 ^** 0.85 ^** 0.16 ^* 0.74 ^** 0.17 ^* 0.16 ^**

Infected + Axitinib

0.10* 0.10* 0.16** 1.70 0.16** 0.08** 3.87 10.40 0.15** 0.05**

Mouse strain Balb/c

1 .55 0.89 0.85 5.36 0.58 0.27 0.49 0.80 3.48 2.79

Infected

0.85 0.61 0.16 3.32 0.72 1.14 0.50 1.13 6.22 6.25 0.61 ^** 0.38 ^* 0.14 ^* 0.66 ^** 0.06 0.36 1.41 ^** 1.14 0.99 ^* 0.78 ^*

Infected + Axitinib

0.47** 0.34 0.75* 0.41** 0.20* 0.96 1.29 1.47 1.04** 2.27**

LPS and VEGF acts synergistically to completely silence the inflammatory response to PbA infection The exogenous addition of recombinant VEGF to LPS further improved the course of PbA infection in C57BL 6 mice without affecting the parasite load (Figure 4A). Not only did 100 % of the treated mice not develop ECM but also their survival was significantly prolonged. This improvement was mirrored by a complete absence of brain pathology. Very few if any cells were found to react against antibodies directed to CD3 lymphocyte antigen and to cleaved caspase 3 (Table 1 B). VEGF-LPS treatment also induced changes in the inflammation response to parasite infection that were striking when compared to those induced by VEGF or LPS alone. In the brain the genes S1 Pr1 (a molecule involved in endothelial remodelling), Nrf-2 and HO-1 were significantly up regulated by about approximately 1 0, 25 and 9 fold respectively (p<0.01 ), while the expression of TNF-a and IFN-Y was reduced at the level of non-infected control mice (p<0.01 ) (Table 3).

Table 3. Expression of inflammation markers in the brain of LPS-VEGF treated mice.

Expression of inflammation markers in brain tissue of C57BL/6 Infected and treated mice (LPS+VEGF), +3 days post infection and at the end point (bold). Each value is the mean of the fold changes obtained from 3 biological replicates. Induction relative to the non-infected control (fold change) was calculated using the comparative Ct method (AACi) .Values obtained from non-treated vs treated mice were compared using a one- sample t-test to access statistical significance * (0.01 < p <0.05) and ** ( p < 0.01 ).

Mouse IL-

VEGF VEGFR1 VEGFR2 S1 P IL-6 TNF-a IFN HO-1 Nrf2 strain 23p19 C57BL/6

Infected 7.43 2.80 4.76 2.30 3.80 20.60 1.30 2.64 1.10 1 .30

7.50 2.23 7.10 1.60 8.30 21.10 4.30 9.70 0.40 0.88

5.15 ^* 1 .98 ^* 1.80 5.60 23.50 0.98 0.70 ^* 1 .70 ^* 0.60

Infected 7.66

+ LPS

3.16 2.95** 2.20 0.30* 7.90** 1.00** 0.84** 3.42* 3.70*

4.28**

Infected 1.25 ^** 1.20 ^* 1.74 ^** 12.60 ^** 4.00 40.57 0.80 0.95 3.30 ^** 23.01 ^**

+ LPS +

VEGF 5.46* 1.90 7.30 10.30** 0.50** 4.64** 1.40* 0.77** 9.10** 25.21**

Non 0.60 ^** 4.50 ^* 0.90 ^** 0.75 ^* 3.70 6.30 ^* 0.80 ^* 0.50 ^* 2.00 ^** 3.91 ^* infected

+ LPS 0.97** 0.93* 1.33** 2.40 0.40** 10.20 2.92* 1.50* 3.10** 3.81 *

Most surprisingly the spleen of LPS-VEGF treated mice were very similar in term of morphology (size, colour and appearance) to those of non-infected controls. We therefore performed an expression analysis of inflammation markers of the internal organs (liver, spleen and lung) from treated and untreated mice (Table 4). This investigation revealed that LPS-VEGF treatment completely abolished the inflammatory response in all organs analyzed. In the liver, PbA infection induced at day +3 post infection an important activation of the inflammatory response up regulating the expression of both TNF-a and IFN- _Y by approximately 50 and 30 fold respectively. The LPS-VEGF treatment reversed this response to baseline levels while up regulating the Nrf2 gene (p<0.01 ). Notably, by the time the non-treated mice developed ECM, the liver had down regulated the inflammatory response thus suggesting the presence of a potent auto regulatory pathway in this organ. The spleens of untreated C57BL 6 mice showed a dramatic inflammatory response to PbA infection: the development of ECM coincided with a strong up regulation of VEGF, VEGFR1 , VEGFR2, TNF-a and IFN- _Y . Here, LPS-VEGF treatment completely silenced this response to baseline level while significantly up regulating the expression of the Nrf-2 gene close to 40 fold (p<0.01 ). Unlike the brain, we did not observe the induction of HO-1 gene in the liver or in the spleen. The lung was not affected by the inflammatory response elicited by PbA infection.

Table 4. Fold change expression of inflammation markers in internal organs of LPS-VEGF treated mice Fold changes of gene expression in liver, spleen and lung of C57BL/6 Infected vs. treated mice (LPS-VEGF), +3 days post infection and at the end point (bold). Each value represents the mean of the fold changes obtained from 3 biological replicates. Induction relative to the non- infected control (fold change) was calculated using the comparative Ct method (AACt). Values obtained from non- treated vs treated mice were compared using a one-sample t-test to access statistical significance ^* (0.01 < p <0.05) and ^** ( p < 0.01 ).

Mouse

strain

LIVER VEGF VEGFR1 VEGFR2 S1 P TNF-a IFN-y HO-1 Nrf2

C57BL/6

132.01 55.40 72.50 12.00 54.29 31.77 0.01 1 .00

Infected

27.11 10.23 17.38 8.99 0.60 0.79 0.30 1.45

Infected 8.55 ^** 3.28 ^** 4.00 ^** 1.24 ^** 3.07 ^** 0.77 ^** 1 .00 ^* 67.50 ^** + LPS +

VEGF 9.81 ** 2.15** 5.21** 1.04** 0.56 0.34* 1.30* 4.11**

Mouse

strain

SPLEEN VEGF VEGFR1 VEGFR2 S1 P TNF-a IFN-y HO-1 Nrf2

C57BL/6

920.1 1 288.74 625.19 2.00 30.93 14.99 0.13 1 .00

Infected

790.88 177.50 548.60 1.00 238.38 223.73 0.79 0.80

Infected 225.20 ^** 98.15 ^** 1 19.12 ^** 6.00 8.45 ^** 3.90 ^* 0.87 ^** 39.07 ^** + LPS +

VEGF 42.15** 0.11** 24.04** 13.00** 1.46

1.50** 0.69** 1.30

Mouse

strain

LUNG VEGF VEGFR1 VEGFR2 S1 P TNF-a IFN-y HO-1 Nrf2

C57BL/6 3.1 1 1 .25 1 .00 1 .00 1 .00 1 .10 0.90 0.80

Infected

0.55 0.22 0.10 0.24 0.10 0.12 0.47 0.20

Infected

0.32 ^** 0.18 ^** 0.10 ^** 1.10 1.20 0.28 1.16 2.10

+ LPS

0.51 0.24 0.20 2.80** 0.30 0.10 6.60**

1.12**

+ VEGF

Discussion

The pathology of cerebral malaria involves the binding of infected erythrocytes to the venular endothelium and the concomitant activation of a strong inflammatory response characterized by lymphocyte infiltration of the brain, up regulation of the cytokines TNF-a and IFN-γ and endothelial cell damage. Efforts to develop supportive therapy to decrease morbidity and mortality of malaria must take in to account such a dual nature of the pathogenesis of cerebral malaria. We show here that the administration of LPS, at doses that normally induce a state of tolerance to further inflammatory stimuli, changed significantly the course of PbA infection in ECM susceptible C57BL/6 mice. The treated animals showed a prolonged survival compared to untreated controls and failed to develop signs of ECM. Morphological analysis as well as immunohistochemistry revealed a marked reduction of the cellular infiltrate in the brains of LPS treated and a substantial decrease of reactivity to CD3 and activated caspase 3 antibodies in agreement with the anticipated effect of LPS at low doses. Gene expression analysis revealed that LPS had a profound inhibitory effect on the induction of inflammatory cytokines TNF-a and IFN-γ while up regulating Nrf-2 and HO-1 to levels comparable to those observed in ECM resistant Balb/c mice.

We hypothesized that VEGF could also exert a protective role against ECM. This originated from the notion that the protective activities on brain vessels of CO and NO, two molecules known to prevent the development of ECM, mirror those mediated by VEGF in response to tissue injury and resolution of inflammation. Indeed the relationship linking nitric oxide, carbon monoxide with VEGF is quite intricate. Different activators of heme oxygenase-1 (HO-1 ) induce VEGF expression. This is mediated by the activity of HO-1 that produces CO starting from free heme. In turn VEGF up-regulates the expression of HO-1 in endothelial cells thereby establishing a positive feedback circuit. Accordingly, endothelial cells from HO-1 knockout mice do not respond optimally to VEGF stimulation. NO and NO donors are also potent inducers of VEGF synthesis and potentiate its effect on endothelial cells. Here we show that treatment of C57BL/6 mice with Axitinib, a potent inhibitor of VEGF receptor 1 , 2, 3, CD1 17 (cKIT) and platelet-derived growth factor receptor (PDGFR) aggravates brain pathology dramatically and decreases the survival of the mice. At day five post-infection treated animals showed an important CD3 lymphocyte infiltrate of the brain and widespread apoptosis of both parenchyma and endothelial cells. In ECM-resistant Balb/c mice, Axitinib significantly reduced the accelerated mortality in 100 % of the treated animals. This was associated with sign of brain pathology such as lymphocyte infiltrate and cell apoptosis that were never observed in PbA infected Balb/c mice. The notion that Axitinib acted on the VEGF receptor pathway is corroborated by the protective effect of VEGF treatment. Compared to untreated control group VEGF treated mice did not develop ECM, showed moderate signs of brain inflammation and damage in terms of lymphocyte infiltrate and cell apoptosis respectively and a decreased up-regulation of both TNF-a and IFN-γ.

The addition of VEGF to LPS treatment had a strong synergistic effect that manifested itself with a complete silencing of the inflammatory response to PbA infection. CD3 lymphocytes were absent in the brain parenchyma of treated C57BL/6 mice in repeated examinations. Similarly, sections stained with antibodies directed to activated caspase 3 did not show any reactivity. Expression analysis revealed that the combination treatment was extremely effective in up regulating the expression of the regulatory genes Nrf-2 and HO-1 to levels much higher than those observed in ECM resistant Balb/c mice or when treating the mice with either LPS or VEGF alone. Accordingly VEGF-LPS treated mice showed a prolonged survival and did not develop ECM. When analysing the internal organs at the time of death we noticed that the spleens of VEGF-LPS treated mice could not be distinguished in term of size, colour and weight from those of non-infected controls. This morphological observation was in agreement with the effect of VEGF-LPS treatment on the expression of inflammation markers. In the spleen of untreated PbA infected animals we observed a progressive very strong up regulation of VEGF, VEGFR1 , VEGFR2, TNF-a and IFN-γ respectively. LPS-VEGF treatment completely silenced TNF-a and IFN- _Y response to baseline level while up regulating the expression of the Nrf-2 gene thus shedding for the first time light on the mechanism leading to splenomegaly, a condition observed in human malaria as well as in several acute and chronic infectious diseases. The synergy achieved by combining LPS and VEGF reflect their respective activities on the two pathogenic mechanisms leading to cerebral malaria: an unregulated activation of the inflammation response and obstruction-hypoxia endothelial cell damage. On one hand LPS induces antioxidant genes thereby protecting endothelial cells from injury while down regulating the inflammatory response on the other hand VEGF induces endothelial cells proliferation and restores blood vessel integrity. These observations all together significantly add to our understanding of the factors regulating the inflammatory response to Plasmodium infection and provide the rationale to develop novel effective supportive therapies to treat cerebral malaria and more in general to protect the endothelium in the presence of life threatening inflammatory processes. Lovastatin potentiates the protective activity of VEGF against ECM

We compared several treatment regimens to investigate whether Lovastatin in combination with VEGF exerted a synergistic activity similar to that observed with LPS in protecting C57BL 6 mice against P. berghei experimental cerebral malaria (ECM). Typically P. berghei ANKA-infected (PbA) C57BL/6 mice start showing signs of convulsion, ataxia, coma at around day 6-7 post infection and within the following 24 hours die (Figure 4A). A treatment with Lovastatin 20mg/kg alone significantly ameliorated the signs of neuro- inflammation and prolonged the lifespan of the mice by two days. The administration of Simvastatin 20mg/kg alone, a statin that like Lovastatin inhibits endogenous cholesterol production by competitive inhibition of HMG-CoA reductase, failed to protect against ECM (Figure 4A). A combination treatment including Lovastatin 20mg/kg and VEGF 200ng/mouse daily starting from the first day post infection completely blocked the development of ECM and prolonged the life span of the mice on average by 7 days without affecting the parasite load (Figure 4A). This time course is identical to that observed in P. berghei infected ECM resistant BALB/c mice. Notably no differences were observed when comparing the course of infection between VEGF-Lovastatin and VEGF-LPS treated animals (Figure 4A). Morphological analysis of internal organs revealed that the Lovastatin- VEGF combination treatment had a significant effect in preventing the development of splenomegaly in infected animals as previously observed when treating infected mice with VEGF-LPS (Figure 4B). VEGF-Lovastatin treatment suppresses the inflammatory response and protects the blood vessels integrity

The internal organs (brain, liver, spleen and lungs) of all experimental groups were collected at day +3 post infection and on the day of death (end point) to carry out histology and immunohistochemistry analysis. The examination of the brains from non-treated infected animals showed a mononuclear cell infiltrate alongside with signs of oedema and blood vessel damage typically observed in ECM susceptible mice. Tissue sections taken from the spleens of these animals also showed oedema and increased cellular content. Similarly a significant cellular infiltrate was also observed in the lungs. Lovastatin and Simvastatin when given alone did not improve the histological features of tissue pathology in all tissues and organs examined. Intriguingly, Simvastatin apparently caused a worsening of the oedema and an increase of the mononuclear cellular infiltrate in the brain, the spleen and the liver, while Lovastatin did not show the expected improvement anticipated on basis of its protective effect on the development of ECM. On the contrary the VEGF -Lovastatin combination treatment completely prevented the formation of the cellular infiltrate in the brain and the development of oedema. Accordingly no signs of inflammation could be detected in other tissues including the spleen. This morphological profile is almost identical to that observed in VEGF-LPS treated infected mice in agreement with the progression of P. berghei infection in these animals. The immunohistochemistry analysis supported the conclusions initially inferred on the basis of the histology. At the time of death infected animals either non-treated or statin-treated shared a similar immunohistochemistry reactivity pattern. The tissues from these animals showed a strong reactivity with an antibody directed against the lymphocyte antigen CD3 thus revealing the T cell nature of the mononuclear cell infiltrate. This analysis also demonstrated that capillaries and small veins were stained with an antibody directed against the activated form of caspase 3, a hallmark of apoptosis thus confirming the involvement of extensive endothelial cell damage. In agreement with the morphological observations, the monoclonal antibodies directed against the lymphocyte CD3 antigen and the activated form of caspase 3 did not show any reactivity in tissue sections from VEGF-Lovastatin treated animals. This reactivity profile is identical to that observed in non-infected controls.

Analysis of cytokine and inflammatory markers in statin-treated animals To study the activity of statins on P. berghei induced inflammatory response we examined the expression of three groups of genes encoding pro-inflammatory cytokines (TNF-a and IFN- _Y ); inflammation regulatory elements (HO-1 , Nfr-2 and IDO) and VEGF modulated signalling molecules (VEGF-R2 and S1 Pr1 ). In line with previous observations PbA infection induced in the brains of C57BL/6 mice 5 and 10 fold up-regulation of the TNF- a and IFN- _Y genes respectively (Table 5). This was accompanied by a parallel increase of IDO. The treatment with Lovastatin had a contrasting effect on the pro-inflammatory cytokines; it inhibited the induction of the IFN- _Y while up-regulated the transcription of TNF- a. Lovastatin also elicited a significant activation of the anti-inflammatory genes Nrf-2 and HO-1 (Table 5). On the contrary the administration of Simvastatin rather than activating the anti-inflammatory genes Nrf-2 and HO-1 , up-regulated the pro-inflammatory cytokines TNF- α and IFN- _Y as well as IDO to levels higher than those observed in infected non-treated animals. The gene expression profile induced by Simvastatin could explain its lack of protective activity against ECM and the worsening of the pathology in the tissues and organs examined. The VEGF-Lovastatin treatment induced a completely different profile. Both Nrf-2 and HO-1 genes were highly induced already from day +3 post infection mirrored by a parallel increase in transcription levels of S1 Pr1 a gene involved in endothelial remodelling and repair (Table 5). The expression profile was very similar to that previously observed when treating infected mice with VEGF-LPS. As previously demonstrated P. berghei infection unlashed in the spleen a strong activation of pro- inflammatory cytokines reaching 200 and 260 fold induction over non infected controls for TNF-a and IFN- _Y respectively. The genes IDO and VEGF-R2 were also strongly up regulated. The activation of IDO most likely represent a response to the high level of IFN- _Y while the activation of VEGF-R2 could represent a compensatory response to parasite mediated endothelial damage. Unlike what has been observed in the brain, in other organs both Lovastatin and Simvastatin were able to down regulate the induction of TNF-a and IFN- _Y though the activation of VEGF-R2 was only marginally affected. The addition of VEGF to Lovastatin enhanced the inhibitory activity on the inflammatory cytokines genes, reverted the activation of VEGF-R2 to nearly basal levels while up regulating the antiinflammatory gene Nrf-2 (Table 5). The liver of untreated mice showed at day +3 post infection an expression profile that with the exception of VEGF-R2 was similar to that observed in the spleen, however unlike the spleen few days later most pro inflammatory genes were back to baseline levels (Table 5). Here statins treatment activated the antiinflammatory gene Nrf-2 and down regulated the induction of both TNF-a and IFN- _Y already by day +3 post infection however only the addition of VEGF completely reversed the proinflammatory cytokines to baseline levels. In the lung of infected animals we did not observe important changes in the expression levels of the genes analysed. The administration of Lovastatin up regulated the transcription of both Nrf-2 and HO-1 while Simvastatin failed to exert a similar activity (Table 5).

Table 5. Fold change expression of inflammation markers in the brain and internal organs Expression of inflammation markers in brain, liver, spleen and lung tissue of

C57BL/6 Infected vs. treated mice (Lovastatin, Simvastatin, LPS+VEGF and Lovastatin+VEGF), +3 days post infection and at the end point (bold). Each value represents the mean of the fold changes obtained from 3 biological replicates. Induction relative to the non-infected control (fold change) was calculated using the comparative Ct method (AACt).Values obtained from non-treated vs treated mice were compared using a one-sample t-test to access statistical significance ^* (0.01 < p <0.05) and ^** ( p < 0.01 ).

BRAIN

Mouse strain VEGF-R2 S1 PM I DO TNFa IFN-Y HO-1 Nrf2

C57BL6

Infected 5.95 1 .87 3.72 1 .18 4.04 1.51 1.91

10.12 1.01 14.98 5.68 10.13 1.00 0.78

LPS-lnfected- 1.74 ^** 12.60 ^** 1.0 ^* 0.80 0.95 3.30 ^** 23.01 ^**

VEGF 7.30 10.30** 0.90* 1.40* 0.77** 9.10** 25.21** Mouse strain VEGF-R2 S1 P I DO TNFa IFN-Y HO-1 Nrf2 C57BL6

Infected* 5.14 ^** 5.63 ^** 2.97 ^* 7.1 1 ^** 6.48 ^** 3.41 ^** 18.24 ^** Lovastatin 4.64** 3.08** 2.58** 11.28** 2.62** 6.03** 23.78**

Infected- 1 .78 ^** 15.91 ^** 2.22 ^** 1.25 ^* 1.25 ^** 6.1 1 ^** 10.58 ^** Lovastatin-VEGF 5.40** 14.70** 1.50** 3.48** 0.56** 12.57** 13.60**

Infected* 2.10 ^** 0.86 ^** 15.54 ^** 3.85 ^** 14.55 ^** 2.45 ^** 3.25 ^** Simvastatin 5.96** 0.72* 20.11** 6.55* 18.86** 1.15* 2.04**

LIVER

SPLEEN

Mouse strain VEGF-R2 S1 P IDO TNFa IFN-Y HO-1 Nrf2

C57BL6

Infected 597.06 1 .95 18.68 23.31 15.79 0.15 0.70

452.05 0.87 180.01 202.13 262.24 1.58 0.63

LPS-lnfected- 101.27 ^** 7.35 ^** 30.67 ^** 8.41 ^** 3.22 ^** 0.84 61.02 ^**

VEGF 23.68** 11.66** 2.74** 1.13** 0.82** 1.13* 2.78** Mouse strain VEGF-R2 S1 P IDO TNFa IFN-Y HO-1 Nrf2

C57BL6

Infected* 214.66 ^** 5.87 ^** 27.81 ^** 26.00 ^** 4.12 ^** 1.38 ^* 14.17 ^**

Lovastatin 34.00** 7.45** 16.33** 3.36** 19.33** 3.44** 7.44**

Infected* 82.66 ^** 6.04 ^** 44.00 ^** 9.08 ^** 5.36 ^** 0.76 ^* 16.30 ^**

Lovastatin+VEGF 13.0** 17.33** 4.50** 1.75** 1.18** 1.44 2.42**

Infected* 144.54 ^* 1 .82 1 1.77 ^** 9.69 ^** 9.18 ^** 2.70 ^* 5.05 ^**

Simvastatin 81.54** 1.20* 17.33** 7.54** 31.04** 1.19 2.63**

LUNG

Mouse strain VEGF-R2 S1 P IDO TNFa IFN-Y HO-1 Nrf2

C57BL6

Infected 0.70 0.80 0.75 0.70 0.77 0.75 0.62

0.13 0.16 6.60 0.1 0.14 0.43 0.48

LPS-lnfected- 0.50 0.71 8.33 ^** 0.83 ^* 0.36 ^* 1 .92 ^** 2.87 ^**

VEGF 0.98* 2.93** 0.73** 0.27* 0.23 12.16** 6.47**

Infected* 0.15 ^* 3.57 ^** 14.0 ^** 0.73 0.29 ^** 1.91 ^** 2.10 ^**

Lovastatin 0.31 * 6.82** 2.46** 0.68* 0.15 5.21** 4.39**

Infected- 0.35 ^* 0.34 ^* 7.41 ^** 1.19 ^* 0.51 ^* 2.61 ^** 1 .43 ^**

Lovastatin-VEGF 0.83* 1.21** 1.33** 0.57* 0.12 10.92** 7.20**

Infected* 1.20 ^* 1.25 ^** 1 .89 ^* 1 .36 ^* 0.31 ^** 1 .37 ^** 1.49 ^**

Simvastatin 0.37* 0.77** 3.37** 0.71** 1.74** 0.51 0.35*

Discussion Our findings confirmed the protective activity of Lovastatin against ECM in PbA infected mice. This is in agreement with the ability of statins to reduce inducible nitric oxide (NOS) expression, thereby protecting neuronal cells, while increasing endothelial NOS expression, increasing microcirculatory blood flow via local vasodilatation. However, not all statins share the same protective properties. Simvastatin and Lovastatin are both known to inhibit HMG-CoA reductase but Simvastatin does not exert the same protective activity against ECM. On the contrary Simvastatin treated mice developed ECM and showed a marked worsening of tissue pathology as revealed by morphological and immunohistochemistry examination. Our findings confirmed that Lovastatin had the ability to up regulate the expression of the anti-inflammatory genes Nrf-2 and HO-1 in the brain. However, tissues and organs from treated mice still showed important sign of inflammation pathology including mononuclear cell infiltrate, endothelial damage and oedema. Furthermore Lovastatin treated mice died 7-10 days earlier than ECM resistant Balb/c mice. Similar results were obtained when treating ECM susceptible mice with LPS alone thus suggesting that the induction of Nrf-2 and HO-1 is not sufficient to elicit full protection against cerebral and systemic inflammation.

The addition of VEGF to Lovastatin enhanced its protective and anti-inflammatory activity. The treated animals did not develop cerebral malaria and showed a much prolonged post infection life span (up to seven days). The morphological and immunohistochemistry analysis provided important clues to explain this improvement. Tissue sections did not reveal signs of inflammation pathology in all organs examined including brain, spleen liver and lungs. These tissues also failed to react with antibodies against the CD3 antigen and activated caspase 3 thus confirming the lack of both cellular infiltrate and endothelial damage. The analysis of gene expression showed an enhanced activation of the anti-inflammatory genes Nrf-2 and HO-1 mirrored by a complete silencing of the pro-inflammatory cytokines TNF-a and IFN- _Y . Interestingly unlike statins the VEGF- Lovastatin treatment induced a strong activation of S1 P, a gene encoding a sphingolipid signalling molecule implicated in regulating vascular integrity and endothelial remodelling to injury. This gene was up-regulated in the brain, spleen and lung of mice possibly indicating an improved disease outcome, associated with preserved endothelium integrity, reduced host inflammation and T cell influx into these organs. We have demonstrated that the synergy achieved by combining VEGF and LPS reflect their respective protective activities on the two main pathogenic mechanisms leading to cerebral malaria: an unregulated activation of the inflammation response and endothelial cell damage. Here we show that Lovastatin could replace LPS alongside VEGF, without an obvious loss of activity in blocking the activation of systemic inflammation.

Methods

Animals - C57BL/6 and Balb/c females (age 4-6 weeks) were purchased from Harlan Laboratories, U.K. Ltd. Oxon, England. All animal procedures and care conformed strictly to the United Kingdom Home Office Guidelines under the Animals (Scientific

Procedures) Act 1986 and the protocols were approved by the Home Office of Great Britain (License number: 70/7237).

Parasite and infection procedure - The parasite P. berghei ANKA clone 2.34 was originally obtained from The University of Perugia, Dipartimento di Medicina Sperimentale, Italy. Serial passages of P. berghei were carried out by i.p. Inoculation of na ^' ive C57BL/6 mice with 10 ⁵ parasitized red blood cells to obtain parasitized erythrocytes stocks. In this study C57BL/6 or Balb/c mice were infected by i.p. injection of 10 ⁵ infected erythrocytes at day 0. Parasitemia was first measured 3 days post-infection followed by a daily measurement until the day of death. A drop of blood was collected by venesection of the tail of the mouse and transferred onto the edge of a microscope slide. The blood was drawn evenly across a second slide to make a thin blood film and allowed to dry at room temperature before staining with Giemsa stain. Five fields of approximately 200 cells each were counted and the parasitemia was calculated as the percentage of the total red blood cells containing parasites.

Treatment regimen - Starting from day 1 post infection the mice were treated daily with Axitinib 25mg/Kg (Tocris Bioscience, ft 4350) or Mouse Vascular Endothelial Growth Factor-164 (mVEGF ₁₆₄ ) 200ng/mouse (Cell Signaling, it 521 1 ) over a period of 5 days. Lipopolysaccharide S from Escherichia coli 055:B5 (LPS) 20ug/mouse (Sigma-Aldrich, UK, ft L6529) was injected i.p. route from Escherichia coli 055:B5 5 days, prior infection. Parameters used to assess the development of Cerebral Malaria - The onset of cerebral complications was determined by monitoring a number of clinical signs, such as ruffled fur, hunching, wobbly gait, limb paralysis, convulsion and death. Moreover, a drop in body temperature below 34°C was also used to confirm the onset of cerebral complications. Mice were monitored daily from the day of the infection. At the end of the experiment, mice underwent deep anaesthesia for blood sampling by cardiac puncture. Afterwards, they were euthanized and organs collection (brain, spleen, lung, liver) took place.

Cytokine and Chemokine measurements - Circulating cytokine/chemokine levels were measured using the BD Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 Cytokine kit (R&D System, USA # 560485) on 50 μΙ_ of plasma obtained from blood samples collected from either infected, treated or WT littermates at each of the different time points. The assay was performed in triplicate. For all the soluble markers included in the panels, the detection limit of CBA was 20pg/ml_.

Histopathology and immunohistochemistry - Formalin-fixed samples of brain, liver, spleen collected from infected, treated and WT littermates were embedded in paraffin blocks. Thin tissue sections (5 μΐΎΐ-thick) were obtained and routinely processed for histopathological examination (H&E stain) and for immunohistochemistry using antibodies to detect CD3, (Anti CD3 Antibody, Abeam ab5690/ Antigen Retrival: Citrate buffer 10min- MW, dilution: 1 :100- 4C overnight) and Cleaved Caspase 3 positive cells (Cleaved Caspase 3 Antibody, Cell Signaling 09661 , Antigen Retrival: Citrate buffer 10min- MW, dilution:

1 :100- 4C overnight). All the antibodies were visualized using the Vectastain Elite ABC kit (Vector Laboratories, ftPK-6101 ) and DAB Peroxidase Substrate kit (Vector Laboratories, # SK-4100). Additional serial tissue sections were also processed for indirect

immunoperoxidase staining following the procedures described elsewhere. % of area of the signal (CD3+cells and apoptotic ECs- cleaved caspase 3) has been calculated using Fiji ImageJ. The RGB image was threshold by signal intensity.

Microphotographs of organs were taken using a Canon Power Shot A520 with a close-up lens 250D (5.7 zoom). mRNA transcript quantification - Total RNA was extracted using Trizol (Life Technologies) and reverse transcribed using the Superscript III First Strand Synthesis System kit (Life Technologies). Transcript abundance was measured using an Applied Biosystem Thermocycler and Fast SYBER Green Master Mix (Life Technologies). GAPDH was used as housekeeping reference gene in a qPCR reaction. Induction relative to the non-infected control (fold change) was calculated using the comparative Ct method (AACt).

Mouse primers used in this work to profile the cytokines and other immune-related factors were obtained from commercially available sources. Statistical Analysis - All statistical analyses were performed using GraphPad

Prism 4.0 (GraphPad Software Inc). Analysis of variance (ANOVA) followed bySidak's Multiple comparison test. Values of p<0.05 were considered significant and all data are displayed as mean ± SD of results from single representative experiment performed in duplicate and/or in triplicate. Each experiment was repeated at least twice to confirm reproducibility.

Fold changes obtained from real-time PCR data for each of the genes selected from individuals in different conditions were compared. More specifically, the pairings for comparison included (condition A vs. condition B):

- Not treated (infected) vs. o infected + Axitinib, o infected + VEGF, o non infected + LPS, o infected + LPS, o infected + LPS + VEGF littermates (C57BL/6 or Balb/c) infected + Axitinib vs. infected + LPS + VEGF - infected + VEGF vs. infected + LPS + VEGF

Each condition A vs. condition B pairing was explored with biological triplicates. In order to detect the genes for whom the difference of expression between conditions was significant, the following procedure was adopted, individually onto each selected gene (by means of an Excel spreadsheet): the difference between condition-related expressions was computed as: diff = log2(expression _condA / expression _condB ) so that no difference between expressions (i.e. expression _condA / expression _condB = 1 ) resulted in diff=0; to test for statistical significance of expression difference, a one- sample t-test was constructed, the null hypothesis being: H ₀ : = 0, with a sample formed of the three diff values obtained from the three biological replicates. Rejection of the null hypothesis (p-value <0.05) was used to identify significance genes exhibiting significant differences were labeled with ^* (0.01 =< p < 0.05) and ^** (p < 0.01 )