WO2010142997A1

WO2010142997A1 - Use of a serum composition to reduce the levels of tnf and/or vegf in mammals

Info

Publication number: WO2010142997A1
Application number: PCT/GB2010/050977
Authority: WO
Inventors: Bryan Youl
Original assignee: Aimsco Limited
Priority date: 2009-06-11
Filing date: 2010-06-10
Publication date: 2010-12-16
Also published as: GB0910032D0

Abstract

The invention relates to methods for reducing the plasma levels of TNF, VEGF, anti-inflammatory cytokines and chemokines. The invention also relates to methods and compositions for use in the treatment of a disorder characterised by abnormally high TNF and/or VEGF levels.

Description

Use of a serum composition to reduce the levels of TNF and/or VEGF in mammals

FIELD OF THE INVENTION

The present invention relates to methods and compositions for use in the treatment of a disorder characterised by abnormally high TNF and/or VEGF levels.

BACKGROUND OF THE INVENTION

PCT publications WO03/004049, WO03/064472, WO 2005/056053 and WO2005/097183 describe therapeutic agents and treatments that are based on a serum composition obtained following challenge with an immunogen with many surprising beneficial effects. The respective content of each of these texts is incorporated in full by specific reference. In particular, the reader is referred to them for an understanding of how the therapeutic agent can be prepared, and for the indications which can be treated.

Characterisation of the above-described serum composition has revealed a high titre of anti-HLA class 2 antibodies, which are able to inhibit a variety of mixed lymphocyte reactions (Cadogan and Dalgleish, unpublished observations). Thus, it was postulated that the serum would be useful in the treatment of multiple sclerosis and similar conditions. The treatment of multiple sclerosis and other similar conditions is described in the above publications. The composition is also believed to cause a reduction in viral load in HIV patients and an increase in CD4+ cells.

In addition, PCT publication WO 2006/021814 describes that the serum composition may also comprise proopiomelanocortin (POMC) peptides and corticotrophin releasing factor binding protein (CRF-BP).

The present application describes the applicant's surprising findings that the serum composition can reduce the levels of both TNF and/or VEGF in mammals.

The TNF family (tumour necrosis factor family) is a group of cytokines, of which the most well-known family member is TNF alpha. The main role of these cytokines is to regulate immune cells, although TNF can also induce apoptosis and inflammation and inhibit tumorigenesis and viral replication. In particular, local increases in TNF, for example within a joint capsule, will cause heat, swelling, pain and redness, all of which are classic signs of inflammation. Greater increases of TNF in the blood can cause shock.

Accordingly, it has been demonstrated that dysregulation of TNF causes a number of human disorders, including cancer. In particular, a number of disorders are characterised by an increase in the levels of TNF. Specific examples include Alzheimer's disease, Crohn's disease, Amyotrophic Lateral Sclerosis, myasthenia gravis, systemic lupus erythematosus, chronic inflammatory demyelinating polyradiculoneuropathy, Guillain-Barre syndrome, multiple sclerosis, cystic fibrosis, ankylosing spondylitis, adhesive arachnoiditis, acute and chronic hepatitis (either infective - including hepatitis due to the Hepatitis B and C viruses or non-infective - including alcoholic hepatitis), Graft-versus-host disease and other disorders with an inflammatory component.

VEGF (Vascular endothelial growth factor) is a member of the platelet-derived growth factor family. VEGF is a signalling protein that has an important role in both vasculogenesis and angiogenesis. Again, an increase in the levels of VEGF has been associated with a number of disorders, including cancer, in particular breast cancer, inflammatory arthritis (such as rheumatoid arthritis, psoriatic arthritis, arthritis associated with Inflammatory Bowel Disease and ankylosing spondylitis), adhesive arachnoiditis, acute and chronic hepatitis (either infective - including hepatitis due to the Hepatitis B and C viruses or non-infective - including alcoholic hepatitis), amyotrophic lateral sclerosis, myasthenia gravis, systemic lupus erythematosus, chronic inflammatory demyelinating polyradiculoneuropathy, Guillain-Barre syndrome, multiple sclerosis, cystic fibrosis, diabetic retinopathy, age-related wet macular degeneration, retinal vein occlusion, rubeosis iridis, the early formation of atherosclerotic plaques in diabetic and non-diabetic patients with or without tissue ischaemia, patellar tendonopathy and other disorders characterised by neovascularisation.

Given the importance of the over-expression of these proteins in the pathogenesis of a large number of disorders there is a need to provide alternative methods to reduce their levels and thus, provide alternative treatments for these disorders. SUMMARY OF THE INVENTION

According to a first aspect of the present invention there is provided a method for reducing the plasma levels of TNF in a mammal, the method comprising administering a serum composition obtained from an ungulate following challenge with an immunogen. In another aspect, the invention also provides a method for reducing the plasma levels of VEGF in a mammal, the method comprising administering a serum composition obtained from an ungulate following challenge with an immunogen.

The invention also provides a method for reducing the plasma levels of a proinflammatory cytokine or a chemokine in a mammal, the method comprising administering a serum composition obtained from an ungulate following challenge with an immunogen. In a preferred embodiment the pro-inflammatory cytokine is interleukin- 6 (IL-6) and the chemokine is selected from the group comprising CC chemokines or CXC chemokines. In particular, the chemokine is MCP1-α (monocyte chemoattractant protein 1) (also known as CCL2); RANTES (Regulated on Activation, Normal Expressed and Secreted) (also known as CCL5); or KC (also known as CXCL1).

The invention also provides a method for the treatment of a disorder characterised by an abnormally high level of TNF and/or VEGF, the method comprising administering a serum composition obtained from an ungulate following challenge with an immunogen wherein administration of the serum composition reduces the plasma levels of TNF and/or VEGF.

In a preferred embodiment of the invention, the TNF is TNF alpha. The VEGF may be VEGF-A, VEGF-B, VEGF-C or VEGF-D.

In a preferred embodiment the immunogen may comprise HIV, in intact host cells, cell- free extracts, viral lysate or a mixture thereof. The HIV can be any type, i.e. HIV-1 , HIV- 2 or HIV-3 and any group or sub-type. Preferably, the HIV is HIV-1 SF2 (ARV-2), HIV- 2B or HIV-3B. A cocktail of different HIV viruses may also be used. The cocktail suitably contains 2, 3, 4, 5, 6 or more such viruses. The viruses are preferably in the form of lysates. Examples of preferred lysates include any or all of the following HIV-1 isolates: 91 US056, 92HT593, 92US723, 92US657, 92US660 and 02US714. Preferably the cocktail includes at least 1 ,2, 3, 4, 5 or all 6 of these particular isolates. Alternatively, the immunogen may be a medium that has been used for growth of a viral culture, or which is suitable for growth of a viral culture. For example, the supernate of a cell culture growth medium such as PBMC or the cancer immortal cell line as used to grow HIV-3b. Other suitable immunogens are described in WO 03/004049 and WO03/064472. In addition, the serum composition may be obtained from the serum of an ungulate such as a goat following challenge with human cell line membrane antigens or human white blood cells.

Furthermore, the invention provides a method of treatment of a disorder characterised by an abnormally high level of TNF and/or VEGF, the method comprising administering a serum composition obtained from an ungulate following challenge with an immunogen wherein administration of the serum composition reduces the plasma levels of anti-inflammatory cytokines and chemokines. In a preferred embodiment the pro-inflammatory cytokine is interleukin-6 (IL-6) and the chemokine is selected from the group comprising CC chemokines or CXC chemokines. In particular, the chemokine is MCP1-α (monocyte chemoattractant protein 1) (also known as CCL2); RANTES (Regulated on Activation, Normal Expressed and Secreted) (also known as CCL5); or KC (also known as CXCL1).

The serum composition may also comprise an active component derived from the blood of a suitably challenged ungulate, such as a goat, by a serum extraction technique that is not designed to isolate individual, specific antibodies. In particular, the serum composition may comprise anti-HLA antibodies and/or proopiomelancortin (POMC) peptides and/or corticotrophin releasing factor (CRF).

The ungulate may be a horse, zebra, donkey, bison, cattle, rhinoceros, camel, hippopotamus, giraffe, okapi, moose, deer, tapir, antelope, goat, sheep, pig, llama or gazelle. In one embodiment the ungulate is a goat, horse or sheep. In a more preferred embodiment the ungulate is a goat.

In another aspect of the invention the serum composition may be obtained from the serum of a mammal, wherein the mammal is any species other than the species being treated.

In a further aspect of the invention the serum composition may be obtained from any immunologically naive mammal, i.e. a mammal that has not been challenged by an immunogen. Examples of disorders that may be treated in accordance with the methods of the present invention include chronic inflammatory disorders, pulmonary hypertension, neurodegenerative disorders including Parkinson's disease and Alzheimer's disease, Multiple System Atrophy, cancer - including the formation of metastases in all malignant tumours, as well as specific neoplastic disorders such as (but not limited to) melanoma, angiosarcoma, renal cell cancer, breast cancer and lymphoma (and other haemotolgical malignacies), glomerulopathy and endotoxemia, amyotrophic lateral sclerosis, myasthenia gravis, systemic lupus erythematosus, chronic inflammatory demyelinating polyradiculoneuropathy, inflammatory airway disease (IAD), Guillain- Barre syndrome, multiple sclerosis, cystic fibrosis, Crohn's disease, graft-versus host disease, ulcerative colitis and other forms of Inflammatory Bowel Disease, chronic arthritis disorders (such as psoriatic arthritis and arthritis associated with inflammatory bowel disease, ankylosing spondylitis), adhesive arachnoiditis, acute and chronic hepatitis (either infective - including hepatitis due to the Hepatitis B and C viruses or non-infective - including alcoholic hepatitis), chronic inflammation of the skin and nervous system, diabetic retinopathy, Behςet's disease, kidney disease, age related wet macular degeneration and other disorders characterised by neovascularisation including, but not limited to, retinal vein occlusion, rubeosis iridis, the early formation of Atherosclerotic Plaques in Diabetic and Non-Diabetic Patients with tissue ischaemia, and patellar tendonopathy.

In particular the methods of the present invention are useful in the treatment of disorders induced or caused by LPS (lipopolysaccharide), an endotoxin from gram- negative bacteria. Infection with LPS can cause a change in white blood cell counts, disseminated intravascular coagulation, mitochondrial disease or dysfunction, fever, hypotension, shock and death. In humans, infection with LPS can cause endotoxic shock or endotoxemia.

The compositions of the present invention may be used to treat humans and non- human mammals. Examples of non-human mammals that can be treated with the compositions of the present invention include canines, equines, felines, ovines, porcines and bovines.

The serum composition can be administered by any effective route, preferably by subcutaneous injection, although alternative routes which may be used include intravenous, intramuscular or intralesional injection, per vaginam, per rectum, aerosol, parenteral, or topical.

The serum is preferably administered as a liquid formulation, although other formulations may be used. For example, the serum may be mixed with suitable pharmaceutically acceptable carriers, and may be formulated as solids (tablets, pills, capsules, granules, etc.) in a suitable composition for topical or parenteral administration.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the effect of low-dose endotoxin on plasma TNF-α levels in horses pre- treated with AIMSPRO, saline or flunixin.

Figure 2 shows the effect of low-dose endotoxin on plasma NF-κB levels in horses pre- treated with AIMSPRO, saline or flunixin.

Figure 3 shows the effect of AIMSPRO on mouse survival in a murine model of LPS- induced toxic shock.

Figure 4 shows the effect of AIMSPRO on blood cell numbers in a murine model of LPS-induced toxic shock.

Figure 5 shows the effect of AIMSPRO on cytokines in a murine model of LPS-induced toxic shock.

Figure 6 shows the effect of AIMSPRO on TNF-α levels in a rat model of LPS-induced toxic shock.

Figure 7 shows the the effect of AIMSPRO on VEGF levels in a rat model of LPS- induced toxic shock.

DETAILED DESCRIPTION OF THE FIGURES

Production of Goat Serum A goat was inoculated by intramuscular injection with lysed HIV viral cocktail suspended in a normal commercial supemate, using an intra-muscular injection of HIV- 3b at a concentration of 10⁹ viral particles per ml. The virus was previously heat killed at 6O⁰C for 30 minutes. The HIV lysate was formulated with an adjuvant. The adjuvant was a completely animal-derived component free adjuvant. In this adjuvant, the surfactant component, Ariacel A (Mannide Monoleate) is derived from olive oil rather than tallow.

Blood samples were drawn at one and three weeks after immunisation for initial assessment. The goat is injected every week for four weeks, then at six weeks the animal is bled to obtain the reagent.

Approximately 400 cc of blood is drawn from the goat under sterile technique. The area for needle extraction is shaved and prepared with betadine. An 18-gage needle is used to draw approximately 400 cc of blood from the animal. Of note is that the animal can tolerate approximately 400 cc of blood drawn without the animal suffering any untoward effects. The animal does not have to be sacrificed. The animal can then be re-bled in approximately 10 to 14 days after it replenishes its blood volume.

300ml of serum was then filtered to remove large clots and particulate matter. The liquid serum was thenceforth maintained at a low temperature (typically cooler than 8°C). The serum was then treated with cold supersaturated ammonium sulfate (45% solution) to precipitate antibodies and other material. The resulting solution was centrifuged at 5000 rpm for five minutes, after which the supernatant fluid was removed. The precipitated immunoglobulin was re-suspended in phosphate-buffered saline (¹PBS buffer', see Sambrook et. al. 'Molecular cloning, A Laboratory Manual', 1989) sufficient to re-dissolve the precipitate.

The solution was then dialysed through a membrane with a molecular weight cut off of 10,000 Daltons. Dialysis was carried out in PBS buffer, changed every four hours over a period of 24 hours. Dialysis was carried out at 4°C.

After 24 hours of dialysis the contents of the dialysis bag were emptied into a sterile beaker. The solution was adjusted such that the mass per unit volume = 10 mg per ml. The dilution was carried out using PBS. The resulting solution was then filtered through a 0.2 micron filter into a sterile container. After filtration, the solution was divided into aliquots to give single doses of 1 ml and stored at -22°C prior to use.

The reagent is then ready for use.

Changes may be made in this procedure, such as for example by varying the concentration of the ammonium sulphate or switching to other reagents. Similarly the dialysis cut-off need not be at 10,000 Daltons.ln addition, as an alternative, Freund's adjuvant may be used.

The composition is referred herein as AIMSPRO serum.

Case Study 1 : AIMSPRO reduces TNFα concentrations in an endotoxemia horse model

Method

6 female horses were used in this study, mean age 7 years (range 5 - 12 years). The mean body weight was 402.6 kg (standard deviation 12.5 kg). The animals were purchased from a horse dealer, and were previously in training in the trotting industry. All of the horses were clinically examined on arrival and shown to be healthy and clear of any musculoskeletal injuries or any conditions which would preclude them from the study, such as inflammatory lesions or diseases. Horses were assigned the identification codes SB 1-6.

The horses were wormed on arrival with a broad-spectrum wormer (Equimax; Virbac Animal Health) and underwent a 2 week period of quarantine to ensure that no evidence of infectious disease appeared which may have affected other horses on the property. For the duration of the study, the horses were maintained as a herd of 6 animals in a single paddock, with appropriate shelter. Ad libitum hay and water were made available, and a pelleted concentrate ration (0.75 kg per horse) was fed 3 times per week, which included a vitamin and mineral supplement.

Storage and administration of Aimspro and control diluent

1 ml Vials of Aimspro (Ceremben) and control diluent (phosphate-buffered saline) were collected from the sponsor on 13^th June 2008. The investigators were blinded throughout the study, and the vials were only labelled with one of two batch numbers, either D001 or C001. The vials were transported on dry ice to the investigator's laboratory, and then kept in a -20 ⁰C freezer. The freezer was monitored daily and the temperature recorded. The recorded temperature did not rise above -19 ⁰C at any time.

On each day of the study, 10 vials of each batch were transported to the study facility in an insulated container with dry ice, and were kept frozen until immediately prior to administration. The vials were thawed when required, then the contents were drawn up into two 5 ml syringes, and a total of 10ml of either C001 or D001 was administered to the horses by subcutaneous injection.

Procedures

The study was conducted as a randomised crossover design, using 6 horses. On each day for 5 days, the same two horses were used. One horse received batch C001 , and the other received D001. After the 5 days of treatment, there was a washout period of either 2 or 3 weeks for these particular horses. Then the procedure was repeated in those two horses, with each receiving the alternative batch.

A patch of skin approximately 10 x 10 cm on the side of the neck was clipped 3 days before each horse was used. Aimspro or diluent control was administered by subcutaneous injection (10 ml volume; 21 gauge needle) in the area of clipped skin, after cleaning the skin with surgical spirit. The dose of Aimspro in humans (1 ml) was arbitrarily scaled up to a dose of 10ml for horses; dose-ranging studies have not yet been carried out in the horse.

The horses were then monitored closely for the next 6 hours, for any evidence of local skin reactions, systemic reactions, or any signs of allergic or inflammatory response. Horses were allowed free access to hay during the procedure, and access to water for the final 2 hours.

Results

Figure 1 shows the effect of low-dose endotoxin on plasma TNFα concentrations in horses pre-treated (at time -60 min) with either Aimspro, saline or flunixin. Endotoxin

(LPS; 30 ng/kg) was administered by slow infusion from time 0 to 30 min, as indicated (n=6 per group). Each point represents the mean and standard error of the TNF concentration at that time. The ^* denotes significant difference between Aimspro and saline groups at these time points (two-way ANOVA with Bonferroni's post hoc test; P<0.05). Note that the effect of Aimspro on TNF levels was seen at 30 minutes from injection and persisted for the duration of the experiment.

This graph shows that Aimspro reduces the amount of TNFα produced by white blood cells in response to low-dose endotoxin. TNFα is a pro-inflammatory cytokine which causes further cell activation and death as part of the systemic inflammatory response. It is one of the most consistent markers that is increased in endotoxemia.

In contrast to the Aimspro curve, the dashed line shows the TNF concentrations in the horses when they were pre-treated with the anti-inflammatory drug, flunixin. Flunixin did not cause any statistically significant decrease in the peak TNFα concentrations. This is because it inhibits different inflammatory pathways which do not influence the production of this particular inflammatory mediator. In fact, it is becoming increasingly apparent that additional therapies are needed to treat cases of endotoxemia in horses, because drugs like flunixin only block some parts of the inflammatory response and can therefore mask some of the underlying effects of sepsis such as white blood cell activation.

In conclusion, the results of this study demonstrate that Aimspro can reduce the levels of plasma TNFα.

In addition to measuring the effects of AIMSPRO on TNFα concentrations, the effect of AIMSPRO on the endototoxin-induced increase in the inflammatory transcription factor, NFKB was also measured. Figure 2 shows the effect of low-dose endotoxin on plasma TNFα concentrations in horses pre-treated with either Aimspro, saline or flunixin. As can be seen from this figure, there was a consistent reduction in the levels of TNFα over time compared to control levels.

The transcription factor, NFKB is a key downstream mediator of the pro-inflammatory effects of TNFα. Accordingly, we believe that AIMSPRO may act through reduction of NFkB concentration to inhibit inflammation. While this may reduce inflammation via the COX-2 pathways, there is also a reduction in TNF-a concentration. Case Study 2: AIMSPRO reduces levels of pro-inflammatory cytokines and chemokines in a murine model of LPS-induced toxic shock

Method

Six week old male C57BL/6 mice were obtained from Harlan Laboratories (France) and housed in the animal facilities of the Museum National d'Histoire Naturelle. The experimental procedure was performed in accordance with institutional animal care guidelines that comply with European regulations (86/609). Mice were challenged intraperitoneal^ with two LPS injections of 200μg in 0.1 ml saline separated by a 24 h interval (LPS from E. coli 0111 :B4, Sigma). Subcutaneous administration of 25 μg of AIMSPRO per mouse was carried out 12 h before and 12 h after the first LPS challenge. Control mice received saline alone. The condition of the mice was monitored twice daily for 5 days and survival data were recorded.

Serum was obtained at 10 h following the second LPS injection and was stored frozen at -20⁰C. Murine cytokines were measured by specific ELISA (Quantibody Mouse cytokine array 1 , Raybiotech Inc) according to the manufacturer's specifications. Data were analysed using the non-parametric Mann Whitney U test when appropriate. Survival data were presented by the Kaplan Meier method and comparisons were made by the log rank test. Differences were considered significant when the p value was less than 0.05.

Results

Figure 3 shows the effect of AIMSPRO on mouse survival. C57BL/6J mice (6 weeks old) were injected i.p. with a lethal dose of LPS from E. coli 0111 :B4 (Sigma) (10 mg/kg) in a volume of 0.1 ml of sterile saline solution. Subcutaneous administration of 25 μg of AIMSPRO per mouse was then carried out 12 h before and 12 h after the LPS challenge. Control mice received the same volume of sterile saline solution. The percentage of surviving mice was analyzed by using GraphPad Prism 4.0 and the log- rank test P values were calculated. Statistical difference between groups, p < 0.05, n = 10 mice per group.

Figure 4 shows the effect of AIMSPRO on blood cell numbers (Neu = Neutrophils, Lym= Lymphocyte, Eos= Eosinophil, Mono= Monocyte). Blood cell numbers were evaluated 10 hours post LPS injection. Results shown at ± SEM. The statistical difference between groups was p>0.05, n =10 mice.

Figure 5 shows the effect of AISMPRO on cytokine levels in mouse sera. Cytokines were measured in sera from mice at 10 hours post second LPS injection using a Quantibody Mouse cytokine array (Raybiotech Inc.). Results shown are ± SEM analysed using GraphPad Prism 4.0. P* =0.05 and P**= <0.01 (Kruskal Wallis H test, Dunn's multiple comparison test).

As can be seen from the figures, there was a marked effect of AIMSPRO treatment on animal survival (Figure 3) and the cytokine profile was altered radically from that normally seen in animals challenged with LPS. At 120 hours, six of the saline-treated cohort had died but all ten of the AIMSPRO-treated mice survived. Analysis of blood taken 10 hours after the second LPS injection showed a marked reduction in white blood cells and particularly in neutrophils in AIMSPRO-treated animals compared with controls (Figure 4). In AIMSPRO-treated animals there was also a marked reduction in proinflammatory cytokines (TNF-alpha, IL-6) as well as in the chemokines MCP1-a (CCL1), RANTES (CCL5) and KC (Figure 5). Levels of IL-4, a key Th-2 cytokine, were increased. No modification of the pro-inflammatory, Th-1 IFNγ cytokine was shown.

The serum concentrations of IL-10, which is generally regarded as an anti-inflammatory cytokine were reduced in animals treated with LPS and AIMSPRO.

Discussion The response to infection includes the activation of host defence mechanisms that result in the influx of activated neutrophils and monocytes and the release of inflammatory mediators. Local vasodilation and increased endothelial permeability are also induced as well as the activation of coagulation pathways. Sepsis is characterised by a similar response to infection, although at a systemic level.

Septic shock results from excessive stimulation of host immune cells by lipopolysaccharide (LPS) released from gram-negative bacteria. As a result of these interactions, cellular activation occurs with the release of cytokine and non-cytokine mediators. Tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1 beta) and interleukin 6 (IL-6), have all been identified as central mediators in the pathogenesis of septic shock and the resultant mortality (Calandra et al, 1991; Marty et al, 1994; Van Deventer et al, 1990; Esposito & Cuzzocrea, 2009; Herzum & Renz, 2008). Cytokines induce the systemic release of molecules with vasodilatory and endotoxic properties, such as prostaglandins, thromboxane A2 and nitric oxide which in turn results in vasodilation and endothelial damage, leading to hypoperfusion and capillary leakage. Cytokines also activate the coagulation pathway, resulting in the formation of capillary microthrombi and a resulting end-organ ischaemia.

In view of the above scenario, such pro-inflammatory cytokines are considered to be potential targets for novel therapies for septic shock. Because of AIMSPRO's ability to intervene in mechanisms involved in cytokine secretion and its potential antiinflammatory activities, we tested the effect of AIMSPRO on LPS-induced septic shock in mice. The present study demonstrates that AIMSPRO was found to protect mice against high dose LPS-induced lethality. This protection correlates with the medication's ability to reduce LPS-induced TNF-α concentration and IL-6 secretion in serum as well as other pro-inflammatory compounds such as CXCL2 (KC) and RANTES (CCL5), chemokines responsible for neutrophil and eosinophil chemoattraction, respectively.

Since the treatment of peripheral blood monocytes with staphylococcal exotoxin B and toxic shock syndrome toxin-1 has been shown to induce the production of IL-1 beta and TNF-alpha, and since TNF-alpha was reduced by Aimspro administration, the absence of a decrease in IL-1 beta by AIMSPRO treatment was surprising. However, it is known from past studies on the characterisation of Aimspro, that the hypothalamo - pituitary-adrenal axis (HPA axis) is affected by Aimspro administration (Mclntosh et al., unpublished observations) and that the stimulation of the HPA axis, which can occur through immune stimulation, is regulated by cytokines including IL-1 beta (Turnbull &

Rivier 1995). It may be that the concurrent stimulation of the HPA axis of the mice during the induction of toxic shock was the cause of the observation. It has also been reported that corticotropin releasing factor (CRF), also a component of AIMSPRO, exerts an anti-inflammatory effect transiently suppressing the release of TNF-α in LPS- activated macrophages (Tsatsanis et al., 2007).

Although when administered as a sole treatment, both LPS and AIMSPRO have been reported to increase IL-10 production by cells of the monocytes/macrophage lineage, in the present model, the administration of LPS to

AIMSPRO treated mice was found to reduce the production of serum IL-10. This inhibition of IL-10 production may be as a consequence of cellular co-stimulation by various cytokines resulting in as yet undefined inhibitory and stimulatory effects. Although IL-10, an anti-inflammatory cytokine, has an inhibitory effect on the production of cytokines, it is released together with TNF-alpha and IL-6 in patients with septic shock (Friedman et al. 1997). Indeed IL-10 blood levels are directly related to the severity of inflammation and the development of organ failure in septic shock (Friedman 1997). AIMSPRO treatment was also found to decrease the production of the chemokine CCL1 in the present study. It is known that under conditions where intestinal or peritoneal injury and inflammation occurs, there is a strong and specific positive feedback system to induce the CCL1/CCR8 chemokine system for the recruitment of peritoneal macrophages (Hoshino 2008). Measurement of the levels of CCL1 in peritoneal lavage fluid revealed that CCL1 was significantly increased in mice with TNBS-induced colitis (Hoshino 2008).

Our results provide dramatic evidence that AIMSPRO can act as a powerful anti- inflammatory agent in a murine model of LPS-induced endotoxic shock.

Case Study 3: AIMSPRO reduces levels of the pro-inflammatory cytokine TNF-α and VEGF in a rat model of LPS-induced toxic shock

In this study the effect of AISMPRO on TNF-α and VEGF levels in LPS-induced Sprague-Dawley (SD) rats was investigated.

SD rats were challenged intraperitoneally with one LPS injection of 0.2mg/kg at time zero. AIMSPRO was administered by intra-peritoneal injection of 450 micrograms as a volume of 0.1 ml/rat. Control rats received the same volume of phosphate buffered saline. Six animals were in each group (i.e. control and AIMSPRO treated). Blood samples were collected at either 0, 1 hour or 4 hours or 30 minutes, 2 hours and 18 hours. The number of animals in each time-group was 3. The levels of serum TNF- VEGF were then analysed using standard commercial kits such as those manufactured by R&D systems.

As can be seen from figures 6 and 7 there was a marked reduction in both TNF-α and VEGF levels with AIMSPRO treatment.

References Calandra T, Gerain J, Heumann D, Baumgartner JD, Glauser MP: High circulating levels of interleukin-6 in patients with septic shock: evolution during sepsis, prognostic value, and interplay with other cytokines. Am J Med. 91 :23-29, 1991.

van Deventer SJ, Bϋller HR, ten Cate JW, Aarden LA, Hack CE, Sturk A: Experimental endotoxemia in humans: analysis of cytokine release and coagulation, fibrinolytic, and complement pathways. Blood. 76:2520-2526, 1990.

Esposito E, Cuzzocrea S: TNF-alpha as a therapeutic target in inflammatory diseases, ischemia-reperfusion injury and trauma. Curr Med Chem. 16:3152-3167, 2009.

Friedman G, Jankowski S, Marchant A, Goldman M, Kahn RJ, Vincent JL: Blood interleukin 10 levels parallel the severity of septic shock. J Crit Care. 12:183-187, 1997.

Herzum I₁ Renz H: Inflammatory markers in SIRS, sepsis and septic shock. Curr Med Chem. 15:581-587, 2008.

Hoshino A, Kawamura Yl, Yasuhara M, Toyama-Sorimachi N, Yamamoto K, Matsukawa A, Lira SA, Dohi T: Inhibition of CCL1-CCR8 interaction prevents aggregation of macrophages and development of peritoneal adhesions. J Immunol. 178:5296-5304, 2007.

Claims

CLAIMS:

1. A method for reducing the plasma levels of TNF in a mammal, the method comprising administering a serum composition obtained from an ungulate following challenge with an immunogen.

2. A method for reducing the plasma levels of VEGF in a mammal, the method comprising administering a serum composition obtained from an ungulate following challenge with an immunogen.

3. A method for reducing the plasma levels of a chemokine, the method comprising administering a serum composition obtained from an ungulate following challenge with an immunogen, wherein the chemokine is MCP- 1α, RANTES or KC.

4. The method of claim 1, wherein the TNF is TNFalpha.

5. The method of claim 2, wherein the VEGF is VEGF-A, VEGF-B, VEGF-C or VEGF-D.

6. The method of any preceding claim, wherein the immunogen is HIV.

7. The method of any preceding claim, wherein the ungulate is a goat, horse, sheep, pig, zebra, donkey, bison, cattle, rhinoceros, camel, hippopotamus, giraffe, okapi, moose, deer, tapir, antelope, llama or gazelle.

8. The method of claim 7, wherein the ungulate is a goat.

9. A method of treatment of a disorder characterised by an abnormally high level of TNF and/or VEGF, the method comprising administering a serum composition obtained from an ungulate following challenge with an immunogen wherein administration of the serum composition reduces the plasma levels of TNF and/or VEGF and wherein the disorder is a LPS- induced disorder, ulcerative colitis and other forms of Inflammatory Bowel

Disease, pulmonary hypertension, angiosarcoma, renal cell cancer, breast cancer, glomerulopathy, amyotrophic lateral sclerosis, Crohn's disease, chronic arthritis disorders, adhesive arachnoiditis, diabetic retinopathy, Behcet's disease, kidney disease, age related wet macular degeneration, retinal vein occlusion, rubeosis iridis, the early formation of Atherosclerotic Plaques in Diabetic and Non-Diabetic Patients with tissue ischaemia, and patellar tendonopathy

10. A serum composition obtained from an ungulate following challenge with an immunogen for the treatment of a disorder characterised by an abnormally high level of TNF and/or VEGF, wherein administration of the serum composition reduces the plasma levels of TNF and/or VEGF and wherein the disorder is a LPS-induced disorder, ulcerative colitis and other forms of Inflammatory Bowel Disease, pulmonary hypertension, angiosarcoma, renal cell cancer, breast cancer, glomerulopathy, amyotrophic lateral sclerosis, Crohn's disease, chronic arthritis disorders, adhesive arachnoiditis, diabetic retinopathy, Behget's disease, kidney disease, age related wet macular degeneration, retinal vein occlusion, rubeosis iridis, the early formation of Atherosclerotic Plaques in Diabetic and Non-Diabetic Patients with tissue ischaemia, and patellar tendonopathy

11. The method of claim 9 or the serum composition of claim 10, wherein the disorder is an LPS-induced disorder.

12. The method of claim 9 or the serum composition of claim 10, wherein the TNF is TNFalpha.

13. The method of claim 9 or the serum composition of claim 10, wherein the serum composition further reduces the levels of at least one of the following, IL-6, MCP-1α, RANTES and KC.

14. The method of claim 9 or the serum composition of claim 10, wherein the immunogen is HIV.

15. The method of claim 9, or the serum composition of claim 10, wherein the ungulate is a goat, horse, sheep, pig, zebra, donkey, bison, cattle, rhinoceros, camel, hippopotamus, giraffe, okapi, moose, deer, tapir, antelope, llama or gazelle.

16. The method of claim 15 or the serum composition of claim 15, wherein the ungulate is a goat.

17. The method of claim 9 or the serum composition of 10, wherein the VEGF is VEGF-A, VEGF-B, VEGF-C or VEGF-D.

18. The method or the serum composition of any of claims 9 to 17, wherein the serum composition comprises anti-HLA antibodies.

19. The method or the serum composition of any of claims 9 to 17, wherein the serum composition does not comprise anti-HIV neutralising antibodies.

20. The method or the serum composition of any of claims 9 to 17, wherein the serum composition comprises an active component derived from the blood of a suitably challenged goat by a serum extraction technique that is not designed to isolate individual, specific antibodies.

21. The method or the serum composition of any of claims 9 to 17, wherein the serum composition further comprises corticotrophin releasing factor (CRF) and/or proopiomelanocortin (POMC).

22. The method or the serum composition of any preceding claim, wherein the serum composition is administered by subcutaneous injection.