Formulation and method of manufacture

The present invention relates to a formulation. The formulation may be particularly suitable for treatment of various disorders. The invention also relates to a method of producing the formulation, and to methods of treatment using said formulation.

WO 2006/021814 describes a serum composition comprising corticotropin releasing factor (CRF) and a method of manufacture for that composition. In brief, an ungulate (such as a goat) is subjected to immunogenic treatment via injection with HIV in some form (such as a viral lysate).

WO 2006/021814 also describes the use of the serum composition comprising CRF for treating a number of disorders, in particular multiple sclerosis and inflammatory disorders such as rheumatoid arthritis; optic neuritis; motor neuron disease; autoimmune diseases; axonal or nerve damage; and cancers. Particular cancers of interest include myelomas, melanomas and lymphomas. Other disorders include cardiovascular diseases; and neural disorders, both demyelinating and non- demyelinating. Examples of particular disorders which may be treated with CRF include cerebrovascular ischaemic disease; Alzheimer's disease; Huntingdon's chorea; mixed connective tissue diseases; scleroderma; anaphylaxis; septic shock; carditis and endocarditis; wound healing; contact dermatitis; occupational lung diseases; glomerulonephritis; transplant rejection; temporal arteritis; vasculitic diseases; hepatitis; and burns. Particular non-demyelinating disorders which may be treated include multiple system atrophy; epilepsy; muscular dystrophy; schizophrenia; bipolar disorder; depression; channelopathies; myasthenia gravis; pain due to malignant neoplasia; chronic fatigue syndrome; fibromyositis; irritable bowel syndrome; work related upper limb disorder; cluster headache; migraine; and chronic daily headache. Particular demyelinating disorders which may be treated include infections of the nervous system; nerve entrapment and focal injury; traumatic spinal cord injury; brachial plexopathy (idiopathic and traumatic, brachial neuritis, Parsonage Turner syndrome, neuralgic amyotrophy); radiculopathy; channelopathies; and tic douloureux. Particular autoimmune disorders which may be treated include lupus; psoriasis; eczema; thyroiditis; and polymyositis. Particular peripheral neuropathy of axonal and demyelinating type which may be treated include hereditary motor and sensor neuropathy of all types; Charcot-Marie-Tooth disease (CMT) types CMT1A, CMT1 B, CMT2, CMT3 (Dejerine Sottas disease), CMT4 (Types A, B, C and D), X-linked Charcot-Marie-Tooth disease (CMTX); Hereditary Neuropathy with liability to pressure palsies (HNPP), also called Tomaculous neuropathy; Hereditary Motor and Sensory Neuropathy with Deafness - Lorn (HMSNL); Proximal Hereditary Motor and Sensory Neuropathy/Neuronopathy (HMSNP); Hereditary Neuralgic Amyotrophy; Hereditary Sensory and Autonomic Neuropathies (HSAN1 , HSAN2, HSAN3 (also called Riley-Day syndrome or familial dysautonomia), HSAN4, HSAN5); Familial Amyloid polyneuropathies (Type I, Type II, Type III, Type IV); Metachromatic Leukodystrophy; Krabbe's Disease; Fabry's Disease; Adrenoleukodystrophy; Refsum's disease (HMSN IV); Tangier Disease; Friedreich's ataxia; Spinal cerebellar ataxia (SCA) all types - SCA1 , SCA2, SCA3, SCA4, SCA5, SCA6, SCA7, SCA8, SCA10, SCA1 1 , SCA12, SCA13, SCA14, SCA16; Spinocerebellar Ataxia; Cockayne's syndrome and Giant axonal neuropathy. CRF is also identified as being useful in the treatment of chronic inflammatory demyelinating polyneuropathy (CIDP) and Guillain-Barre syndrome. CRF is also described as having anti-angiogenic properties. It is therefore understood that use of CRF, also known as corticotropin releasing hormone (CRH) also known as corticoliberin, is advantageous in treating the above- mentioned disorders in a patient.

The present inventors are however investigating alternatives to using HIV as the immunogen.

The present invention accordingly provides a method of manufacturing a composition comprising CRH (and optionally further comprising alpha-2 macroglobulin), the method comprising:

(i) providing serum from an ungulate; and

(ii) aliquoting said serum into a vial, wherein said serum is in a form for administration to a patient and comprises CRH and optionally alpha-2 macroglobulin; wherein the ungulate has been inoculated with:

(a) a virus other than HIV;

(b) a virus-free cell lysate which is immunogenic for said ungulate; or

(c) at least one virus-like particle (VLP).

The virus may be attenuated or non-attenuated. The virus may be present in intact host cells, or cell-free extracts (e.g. from in vitro synthesis), viral fragments, a viral lysate (e.g. from a lysate of an infected cell), or a mixture thereof. The virus may be from any suitable virus, such as Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviradae, Flaviviridae Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papoviridae, Paramyxoviridae, Parvoviridae (e.g. adeno- associated virus), Picornaviridae, Poxviridae, Reoviridae, Retroviridae (but not HIV), Rhabdoviridae, and Togaviridae. The cell lysate may be from a cell line, which may be a human cell line such as Hut78 or H9. A lysate can be created from said cell line through conventional means, such as treatment with detergent. The lysate can then be precipitated and injected into an animal as an immunogen. Pre-treatment of the lysate (e.g. with an adjuvant) prior to administration to the ungulate is also envisaged. The cell lysate may be from any species that will give rise to an immunogenic response in the ungulate. In one embodiment, the cell lysate is from a non-ungulate cell.

Virus-like particles (VLP) are lipid enveloped particles which contain viral proteins. Certain viral proteins have an inherent ability to self-assemble, and in this process bud out from cellular membranes as independent membrane-enveloped particles. VLPs are simple to purify and can, for example, be used to present viral antigens. The VLP may be from any suitable virus, such as Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviradae, Flaviviridae Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papoviridae, Paramyxoviridae, Parvoviridae (e.g. adeno-associated virus), Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, and Togaviridae. Specific viruses include HIV, particularly HIV-IIIB, Hepatitis C virus or Hepatitis B virus (HBV). The VLP may be from HIV, particularly HIV 1MB. Alternatively, in another embodiment, the VLP are not from HIV or specifically not from H IV- 1MB.

The method may be carried out such that the serum remains unfrozen throughout the method up to and including the aliquoting step. The method may also be carried out such that molecules having a size of greater than 0.2 microns (or greater than 200 kDa) are retained in the composition.

Thus, the manufacture method of the present invention may proactively retain molecules having a size greater than 0.2 microns (or greater than 200 kDa). In one embodiment, the method does not include a 0.2 micron pore size filtration step. For example, the method does not include any filtration step employing a filter pore size of 0.2 microns or less that removes molecules having a size greater than 0.2 microns (or greater than 200 kDa). Thus, the method proactively retains large molecules (e.g. alpha-2 macroglobulin, and preferably stabilized 'CRH - alpha-2 macroglobulin' complex though the latter may re-associate post-filtration).

In a variation of the above method, a nanofiltration step may be employed. Said nanofiltration may remove molecules having a size greater than 35 nanometres and may be carried out using a 35 nanometre filter, which may be a 35 nanometre hollow fibre filter. Other size filters may be employed. Thus the invention encompasses a method where molecules having a size greater than 30 nanometres, greater than 25 nanometres, or greater than 20 nanometres are removed. The CRH may be present in the composition provided by the above method at a concentration of more than 60 pg/ml. Thus, the method of the invention may provide CRH in an amount that is preferred for complexing with alpha-2 macroglobulin.

The alpha-2 macroglobulin may be present in the composition provided by the above method at a concentration of more than 150,000 picograms per ml for example more than 160,000 picograms per ml, or more than 170,000 picograms per ml, or more than 180,000 picograms per ml, or more than 190,000 picograms per mi, or more than 200,000 picograms per ml, or more than 210,000 picograms per ml, or more than 220,000 picograms per ml, or more than 230,000 picograms per ml, or more than 250,000 picograms per ml. Thus, alpha-2 macroglobulin may be present in a suitable amount for complexing with CRH. In one embodiment, alpha-2 macroglobulin is present in the composition of the invention at a concentration of at least 1 μΜ (micro molar), at least 1 .5 μΜ, at least 2 μΜ, typically in the range 2-5 μΜ.

The CRH and alpha-2 macroglobulin may be present in a stabilised complex. In more detail, the composition provided by the above method may comprise a stabilised complex of CRH and alpha-2 macroglobulin. The complex may have an approximate molecular weight of 800 kDa.

The alpha-2 macroglobulin may be present in the composition provided by the above method at a concentration of more than 150,000 picograms per millilitre, and the CRH may be present at a concentration of more than 80 pg/ml. The alpha-2 macroglobulin may be present in the composition of the invention at a concentration of more than 150,000 pg/ml (or more than 1 micro molar), and the CRH may be present at a concentration of more than 100 pg/ml (such as more than 120 pg/ml). CRH binding protein (CRH-BP) may also be present in the composition provided by the above method. The CRH-BP may be present at a concentration of more than 60 pg/ml, for example more than 70 pg/ml, or more than 70 pg/ml, or more than 80 pg/ml, or more than 90 pg/ml, or more than 100 pg/ml, or more than 120 pg/ml, or more than 150 pg/ml. Lower concentrations of CRH-BP may be employed such as more than 40 or more than 50 pg/ml. A typical upper range for CRH-BP would be in the region of 300 pg/ml.

The manufacture method of the invention may include an optional step of treating the serum prior to aliquoting. By way of example, a suitable precipitation step may be introduced, such as (conventional) ammonium sulphate precipitation. This may be followed by resuspension of the precipitate in an aqueous solution (e.g. PBS buffer), and dialysis - by way of example, a 10 kDa membrane cut-off diafiltration step may be employed (thereby retaining all molecules having a molecular weight greater than 10 kDa). Alternate means known to a person skilled in the art (e.g. separation columns, etc.) may equally be employed. The manufacture method of the invention may include an optional microfiltration step (to remove large macromolecules and any other undesirable large structures) whilst retaining molecules having a size (e.g. average diameter) greater than 0.2 microns (or greater than 200 kDa), which pass through the filter as filtrate. A microfiltration step may be included in combination with or separate from any 'treating of serum' step (described above). If employed, a microfiltration step is performed before the 'aliquoting' step (and after any 'treating of serum' step, if present). In one embodiment, molecules having a size greater than 0.3 microns (or greater than 300kDa) are retained (e.g. by employing a 0.3 micron pore size microfilter). An alternative microfiltration step may be employed to retain molecules having a size greater than 0.4 microns (or greater than 400 kDa), or to retain molecules having a size greater than 0.5 microns (or greater than 500 kDa), or to retain molecules having a size greater than 0.6 microns (or greater than 600 kDa), or to retain molecules having a size greater than 0.7 microns (or greater than 700 kDa), or to retain molecules having a size greater than 0.75 microns (or greater than 750 kDa), or to retain molecules having a size greater than 0.8 microns (or greater than 800 kDa), or to retain molecules having a size greater than 0.85 microns (or greater than 850 kDa), or to retain molecules having a size greater than 0.9 microns (or greater than 900 kDa). Without wishing to be bound by any theory, by employing a high microfilter (such as a large pore size filter as hereinbefore described, for example greater than 0.2 microns, such as in the range of 0.3 to 0.9 microns), a stabilised complex (i.e. 'CRH - alpha-2 macroglobulin' complex) in an optimal form and/ or stabilised complex having improved efficacy may be provided in the formulation of the present invention. At the same time, any undesirable macromolecules having a size greater than the defined pore size are captured by the microfilter. In this regard, microfiltration to retain molecules in the filtrate having a molecular weight of more than 800 kDa or more than 850 kDa (e.g. employing a filter pore size of at least Ο.δμιη filter, or at least 0.85μιη) is desirable. The manufacture method of the invention includes an aliquoting step in which the serum is aliquoted into vials, optionally with protein concentration adjustment, to provide a single dose amount. At this stage it may be frozen (e.g. at minus 22 degrees C) prior to use. In this regard, prior to use, the aliquoted serum is thawed, followed by prompt administration (e.g. within 6 hours, preferably within 4 hours, preferably within 1 hour, preferably within 5 minutes, preferably within 1 minute) to a patient.

In one embodiment, the CRH formulation of the present invention is prepared by a method that comprises: providing isolated blood from an ungulate (e.g. a goat), wherein said ungulate has been treated with an uninfected cell lysate and/or VLP, and obtaining serum from the blood (e.g. by centrifugation); treating the serum to separate the CRH and other active components of interest; diafiltration (e.g. using a l OkDa cut off membrane) of the separated serum comprising CRH and other active components of interest, thereby retaining molecules having a molecular weight of at least 10 kDa; wherein all of the above steps are performed under cooled conditions (e.g. less than 22 degrees C, such as less than 10 degrees C, or less than 5 degrees C); whilst ensuring that the serum remains unfrozen following treatment to separate the CRH and other active components. A nanofiltration step may also be employed. Said nanofiltration may remove molecules having a size greater than 35 nanometres and may be carried out using a 35 nanometre filter, which may be a 35 nanometre hollow fibre filter. Other size filters may be employed. Thus the invention encompasses a method where molecules having a size greater than 30 nanometres, greater than 25 nanometres, or greater than 20 nanometres are removed.

Thus, the basic methodology as described in WO 2006/021814, which is hereby incorporated in its entirety by reference thereto, may be employed, though without the immunisation with an immunodeficiency virus and instead using a different virus, or a virus-free cell lysate and/or VLP. The method as described in WO 2006/021814 may also include one or more additional steps selected from (i) specific retention of molecules having a size greater than 0.2 microns (greater than 200 kDa); (ii) avoidance of multiple freeze-thaw steps and/ or minimising ambient temperature exposure; (iii) a mixing/ agitation step (to enhance CRH: alpha-2 macroglobulin complex formation); and/or (iv) a nanofiltration step to remove molecules having a size greater than 35 nanometres, greater than 30 nanometres, greater than 25 nanometres, or greater than 20 nanometres. A cell lysate is the solution produced when cells are destroyed by disrupting their cell membranes, often with detergent or other agents that break down the cell wall; (sometimes by freeze-thawing (liquid nitrogen, 37°C, 1 minute vortex), in a process known as cytolysis. This releases the contents within the cell. After a crude lysate has been generated, the first step in processing the lysate is often ultracentrifugation, which separates the solution into distinct bands containing organelles, membrane fragments, various proteins and nucleic acids.

For the purposes of vaccinating the ungulate, the lysate is preferably mixed with an adjuvant immediately before injecting it into the ungulate.

The lysate can be from any cell, that is preferably a non-ungulate cell or from a different species than the ungulate, in order to present the ungulate with foreign antigens that provoke an immune response. The cell may be from a human cell line. Two possible human cell lines are Hut78 (ATCC Accession Number TIB-161 ) and its derivative H9 (ATCC Accession Number HTB-176). Both cell lines are publically available.

VLP are assembled from viral structural proteins and thus are devoid of any genetic material. They are capable of acting as antigen-generating, immune-cell stimulatory mechanisms. VLPs derived from the Hepatitis B virus and composed of the small HBV derived surface antigen (HBsAg) are known and described in Bayer et al. (1968); Nature 218 (5146): 1057-9 (incorporated herein by reference). VLPs have also been produced from components of a wide variety of virus families including Parvoviridae (e.g. adeno-associated virus), Retroviridae (e.g. HIV), and Flaviviridae (e.g. Hepatitis C virus). VLPs can be produced in a variety of cell culture systems including mammalian cell lines, insect cell lines, yeast, and plant cells. VLPs contain repetitive high density displays of viral surface proteins which present conformational viral epitopes that can elicit strong T cell and B cell immune responses. VLPs are therefore known to be a useful tool for the development of vaccines, as discussed in Akahata et al. (2010); Nature Medicine 16 (3): 334-8 (incorporated herein by reference). The lack of genetic material in VLPs means that they provide a safer alternative to attenuated viruses. VLPs can advantageously be generated in a variety of cell culture types including mammalian and insect cell lines, together with yeast and plant cells. Generation of VLPs is discussed in Santi et al. (2006); Methods 40 (1 ): 66-76; and Landry et al. (2010); In Fouchier, Ron A. M. PLoS ONE 5 (12): e15559 (both of which are incorporated herein by reference). Generation of VLPs from HIV-IIIB is discussed in Rainone et al. (201 1 ) PLoS ONE 6(10): e26979 (incorporated herein by reference).

In this regard, "serum" is defined as that component of the blood from which the blood cells have been removed, e.g. by centrifugation.

Optionally, exposure to ambient temperature (e.g. room temperature, 22 degrees C) is strictly minimised at all stages of the method - by way of example, cold trays (ensuring a maximum temperature of less than 22 degrees C, or less than 10 degrees C, or less than 7 degrees C, or less than 5 degrees C) and other such means are used throughout. Thus the composition may be kept at a constant temperature below 22 degrees C, e.g. at less than 10 degrees C, or less than 7 degrees C, or less than 5 degrees C. It is preferred that the method is carried out as a continuous process, avoiding any freezing steps prior to storage. For example, no freezing step is employed prior to final aliquoting into vials (optionally with adjustment of protein concentration). In particular, it is preferred that no freezing step is carried out once the serum has been treated to separate the CRH and other components of interest.

One or more agitation steps may be carried out during the method; these agitation steps may use cold trays. The manufacture method may comprise a final step (e.g. after any protein concentration adjustment step) of freezing for subsequent storage. In one embodiment, no freezing step is employed prior to protein adjustment, or aliquoting. Without wishing to be bound by any theory, the present inventors believe that the bioactive molecules within the composition comprising CRH are prone to undesirable aggregation upon freezing and thus this step should not be performed more than once prior to use. Thus, multiple freezing-thawing steps should be avoided as they are believed to result in inactivation of key bioactive molecules within the CRH composition and/or lead to removal thereof during the manufacture method.

The ungulate may be a horse, zebra, donkey, camel, cattle, bison, goat, pig, moose, elk, deer, antelope or gazelle. In a most preferred embodiment, the ungulate is a goat.

In a related aspect, the present invention provides a composition comprising CRH (and optionally further comprising alpha-2 macroglobulin) which is obtainable by the above-described method. The CRH and alpha-2 macroglobulin may be present in a stabilised complex. In more detail, the composition may comprise a stabilised complex of CRH and alpha-2 macroglobulin. The complex may have an approximate molecular weight of 800 kDa.

The alpha-2 macroglobulin may be present in the composition of the invention at a concentration of more than 150,000 picograms per ml for example more than 160,000 picograms per ml, or more than 170,000 picograms per ml, or more than 180,000 picograms per ml, or more than 190,000 picograms per mi, or more than 200,000 picograms per ml, or more than 210,000 picograms per ml, or more than 220,000 picograms per ml, or more than 230,000 picograms per ml, or more than 250,000 picograms per ml. Thus, alpha-2 macroglobulin may be present in a suitable amount for complexing with CRH. In one embodiment, alpha-2 macroglobulin is present in the composition of the invention at a concentration of at least 1 μΜ (micro molar), at least 1 .5 μΜ, at least 2 μΜ, typically in the range 2-5 μΜ.

Similarly, the CRH may be present in the composition of the invention at a concentration of more than 60 pg/ml. Thus, the method of the invention may provide CRH in an amount that is preferred for complexing with alpha-2 macroglobulin.

The alpha-2 macroglobulin may be present in the composition of the invention at a concentration of more than 150,000 picograms per millilitre, and the CRH may be present at a concentration of more than 80 pg/ml. The alpha-2 macroglobulin may be present in the composition of the invention at a concentration of more thanl 50,000 pg/ml (or more than 1 micro molar), and the CRH may be present at a concentration of more than 100 pg/ml (such as more than 120 pg/ml). CRH binding protein (CRH-BP) may also be present in the composition. The CRH- BP may be present at a concentration of more than 60 pg/ml, for example more than 70 pg/ml, or more than 70 pg/ml, or more than 80 pg/ml, or more than 90 pg/ml, or more than 100 pg/ml, or more than 120 pg/ml, or more than 150 pg/ml. Lower concentrations of CRH-BP may be employed such as more than 40 or more than 50 pg/ml. A typical upper range for CRH-BP would be in the region of 300 pg/ml.

Without wishing to be bound by any theory, the two principal components of the composition may be present as a stabilised complex. The components may have a ratio of 4: 1 CRH:alpha-2 macroglobulin, 2: 1 CRH:alpha-2 macroglobulin, 1 : 1 CRH:alpha-2 macroglobulin, or combinations thereof. The predominant complexed form of CRH: alpha-2 macroglobulin may be a macromolecular quaternary complex (i.e. 4: 1 ).

The CRH and alpha-2 macroglobulin components may be complexed together via non-covalent bonds such as one or more of hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions. The above-described concentrations typically refer to the concentrations obtained during the manufacture process such as immediately prior to aliquoting and optional freezing for subsequent storage (optionally including any protein concentration adjustment) that yields the ready-to-use formulation (typically having a concentration of 4-5 mg protein per ml).

The CRH may be human or non-human. For example, the CRH may be an ungulate CRH such as horse, camel zebra, donkey, cattle, bison, goat, pig, moose, elk, deer, antelope or gazelle. In one embodiment the CRH is not ovine CRH. In a most preferred embodiment, the CRH is caprine CRH.

Corticotropin releasing hormone (CRH), also known as corticoliberin, is a 41 residue peptide originally isolated from ovine hypothalamus based on its ability to stimulate the hypothalamic-pituitary adrenal axis from cultured anterior pituitary cells. CRH is the principal neuroregulator of the basal and stress-induced secretion of ACTH, β- endorphin, and other pro-opiomelanocortin related peptides from the anterior pituitary gland. In addition to its endocrine function, mediation of CRH specific responses occur via its cognate receptors (they include CRH-R1 , CRH-R2a, CRH- R2p, CRH-R2Y and CRH-binding protein [CRH-BP]). CRH-R1 is a 415 amino acid protein that shows sequence homology across different species (human, mouse and rat). CRH-binding protein represents the smallest receptor at 322 amino acids, and acts as an inhibitor of free CRH. CRH-R1 and CRH-R2 are both ubiquitously expressed on the cell surface of the hypothalamus, cerebellum, cortex, amygdala, subcortex, immune cells, gut and skin. Conversely, CRH-BP is found predominantly in the liver, placenta and brain. Importantly there appears to be no significant overlap in distribution of the said receptors. This likely reflects differing functional roles. An example of this is seen during pregnancy where elevation in peripheral CRH is regulated by an elevation in secreted levels of CRH binding protein. The overall effect of this is to prevent an elevation in peripheral circulating levels of glucocorticoids during pregnancy. A precursor form of CRH has been identified as having 194 amino acids; CRH is a 41 amino acid residue. Alpha-2-macroglobulin, also known as A2M, is a large plasma protein found in blood. It is produced by the liver and is the largest major non-immunoglobulin protein in plasma. A2M is synthesized primarily by the liver and is also produced locally by macrophages, fibroblasts and adrenocortical cells. Alpha-2-macroglobulin acts as an anti-protease and is able to inactivate an enormous variety of proteinases. It also functions as a carrier protein binding to numerous growth factors and cytokines. Examples include transferrin (where A2M regulates the binding of to the surface receptor), binds defensin and myelin basic protein, binds several important cytokines, including basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), nerve growth factor (NGF), interleukin-1 β (IL-Ι β) and interleukin-6 (IL-6), transforming growth factor (TGF-Ι β), and insulin, and modify their biological activity. Human A2M is composed of four identical subunits bound together by disulphide (-S-S-) bonds. The principal mechanism by which A2M inhibits proteases is through steric hindrance. The mechanism involves protease cleavage of the thiol 35 amino acid bait region, a segment of the molecule, which is particularly susceptible to proteolytic cleavage, which initiates conformational change such that A2M collapses about the protease thus resulting in its inhibition. In the resulting A2M-protease complex, the active site of the protease is sterically shielded, thus substantially decreasing access to protein substrates including those that are bound to active A2M. Decreases in A2M have been associated with a variety of diseases, for example a common variant (29.5%) polymorphism of A2M leads to an increase in the risk of Alzheimer's disease.

Stabilisation of CRH by binding to alpha-2 macroglobulin, and the attendant enhanced effective biological half-life may be one effect of compositions of the present invention. Said stability may provide an enhanced (longer) plasma half-life - by way of example, a stabilised complex of CRH and A2M may have a half-life of at least 24 hours. The composition of the invention may be employed in combination with one or more stabilisers, such as fibronectin or albumin. Additionally or alternatively, the composition of the invention may further comprise, or be employed in combination with, a pro-opiomelanocortin (POMC) peptide. The use of POMC peptides in this manner stimulates further in vivo POMC production, and/ or helps to induce a host response before endogenous POMC peptide levels are stimulated. POMC may be present in the composition at a concentration in the range of between 100 picomoles per litre (pmol/l) and 200 pmol/l. In one embodiment, POMC is present at a concentration of at least 140 pmol/l.

Thus, in one embodiment the invention provides a CRH composition having component proportions as defined hereinbefore. For example, the formulation may have between 150,000 and 5000,000 (e.g. at least 250,000) picograms per millilitre (pg/ml) of alpha-2 macroglobulin, 60 pg/ml of CRH, and optionally 140 pmol/l pmol/l of POMC. Without wishing to be bound by any theory, the present inventors believe that alpha- 2 macroglobulin inhibits subtilisin serine endopeptidases (pro-hormone convertases), which may otherwise exert deleterious effects on POMC prior to administration.

Following administration, in vivo activation of POMC may occur by the direct actions of prohormone convertase 1 (PC1 ), prohormone convertase 2 (PC2), carboxypeptidase E (CPE), peptidyl alpha-amidating monooxygenase (PAM), N- acetyltransferase (N-AT) and procarboxypeptidase (PRCP) acting in a tissue specific manner. All said POMC cleavage sites appear to be acted upon by proteases in the hypothalamus, placenta, epithelium and leucocytes.

Human POMC peptide is described in detail in entry 176830 of OMIM (online Mendelian inheritance in man, accessible through http://www.ncbi. nlm.nih.qovA). The nucleotide and amino acid sequence of human POMC is also known, and has GENBANK accession number BC065832. Human POMC gives rise to a glycosylated protein precursor having a molecular weight of 31 kDa.

By "a POMC peptide" is meant any peptide having a corresponding sequence, structure, or function. It will be apparent to the skilled person that the canonical nucleotide and/or amino acid sequences given for human POMC in the GENBANK entry referenced above may be varied to a certain degree without affecting the structure or function of the peptide. In particular, allelic variants and functional mutants are included within this definition. Mutants may include conservative amino acid substitutions. "A POMC peptide" as used herein refers to any peptide acting as a precursor to at least one form of MSH, ACTH, at least one form of lipotrophin (LPH), β endorphin, met-enkephalin and leu-enkephalin; and preferably all of α, β, and γ MSH; ACTH; β and γ LPH; and β endorphin, met- enkephalin and leu- enkephalin.

The POMC peptide may be human or non-human POMC. In one embodiment the POMC peptide is an ungulate POMC such as horse, camel, zebra, donkey, cattle, bison, goat, pig, moose, elk, deer, antelope or gazelle. In one embodiment the POMC peptide is not a rodent (e.g. mouse or rat) POMC peptide.

Administration of a POMC peptide has a self-sustaining effect, in that administration of an initial amount of POMC peptide leads to endogenous production of POMC in the patient; thus, an initial administration of a low level of POMC has a significant effect on the patient.

The formulation of the invention selectively increases the enzymatic degradation of POMC in vivo. The formulation of the invention increases the release of POMC- derived peptides such as ACTH, alpha-MSH, beta-MSH, CLIP, Lipotrophin-gamma, met-enkephalins and beta-endorphins. Longer-term administration of the formulation typically leads to a sustainable increase in POMC-derived peptides in a patient.

The composition of the invention described above may act to enhance the central CRH-1 regulatory response. This may result in optimisation of key anti-inflammatory cytokines and abrogation of certain pro-inflammatory cytokines related to the Th1 - mediated response. Administration of CRH to a patient stimulates production of endogenous CRH and thus leads to a self-sustaining effect. CRH can therefore be administered at a low concentration to a patient. The composition of the invention may also protect CRH from proteolytic degradation by proteases and accordingly administration of the stabilised complex may provide slow release of CRH in the circulation and a significant increase in CRH levels.

The composition of the invention may lead to persistent, elevated levels of CRH in vivo for at least 12 hours following administration, for at least 24 hours following administration, or for at least 48 hours following administration. Administration of the composition of the invention may lead to an in vivo increase in CRH concentration of between 25% and 50% from patient baseline CRH levels at 24 hours from administration, and an in vivo increase of between 75% and 100% from patient baseline CRH levels at 48 hours from administration.

The composition of the invention may selectively up-regulate the CRH-1 receptor both centrally and peripherally in key target issues and enhance the central CRH-1 regulatory response, while also leading to selective down-regulation of CRH-2 specific receptors and up-regulation of CRH-binding protein in tissues such as the adrenal cortex. Administration of the composition of the invention may thus lead to optimisation of key inflammatory cytokines and down-regulation of certain proinflammatory cytokines related to the Th-1 mediated response, through targeting leucocytes and macrophages.

Thus the present invention may provide a stabilised CRH formulation that works within homeostatic constraints following in vivo administration.

The present invention further provides a composition as described above for use in prevention or treatment of one or more diseases selected from systemic sclerosis (SSc), multiple sclerosis; inflammatory disorders such as rheumatoid arthritis, osteoarthritis, inflammatory tendonopathies, inflammatory bowel disease and inflammation of the lung, including emphysema, lung fibrosis, alveolitis and cystic fibrosis; optic neuritis; motor neuron disease; autoimmune diseases; axonal or nerve damage; and cancers (including myelomas, melanomas and lymphomas); cardiovascular diseases; neural disorders, both demyelinating and non- demyelinating; cerebrovascular ischemic disease; Alzheimer's disease; Parkinson's disease; Huntingdon's chorea; mixed connective tissue diseases; scleroderma; anaphylaxis; septic shock; endotoxaemia; carditis and endocarditis; wound healing; contact dermatitis; occupational lung diseases; glomerulonephritis; transplant rejection; temporal arteritis; vasculitic diseases; hepatitis (in particular hepatitis C); burns; multiple system atrophy; epilepsy; muscular dystrophy; schizophrenia; bipolar disorder; depression; channelopathies; myasthenia gravis; pain due to malignant neoplasia; chronic fatigue syndrome; fibromyositis; irritable bowel syndrome; work related upper limb disorder; cluster headache; migraine; chronic daily headache; infections of the nervous system; nerve entrapment and focal injury; traumatic spinal cord injury; brachial plexopathy (idiopathic and traumatic, brachial neuritis, Parsonage Turner syndrome, neuralgic amyotrophy); radiculopathy; channelopathies; tic douloureux; lupus; psoriasis; eczema; thyroiditis; polymysotis; hereditary motor and sensor neuropathy of all types; Charcot-Marie-Tooth disease (CMT) types CMT1A, CMT1 B, CMT2, CMT3 (Dejerine Sottas disease), CMT4 (Types A, B, C and D), X-linked Charcot-Marie-Tooth disease (CMTX); Hereditary Neuropathy with liability to pressure palsies (HNPP), also called Tomaculous neuropathy; Hereditary Motor and Sensory Neuropathy with Deafness - Lorn (HMSNL); Proximal Hereditary Motor and Sensory Neuropathy/Neuronopathy (HMSNP); Hereditary Neuralgic Amyotrophy; Hereditary Sensory and Autonomic Neuropathies (HSAN1 , HSAN2, HSAN3 (also called Riley-Day syndrome or familial dysautonomia), HSAN4, HSAN5); Familial Amyloid polyneuropathies (Type I, Type II, Type III, Type IV); Metachromatic Leukodystrophy; Krabbe's Disease; Fabry's Disease; Adrenoleukodystrophy; Refsum's disease (HMSN IV); Tangier Disease; Friedreich's ataxia; Spinal cerebellar ataxia (SCA) all types - SCA1 , SCA2, SCA3, SCA4, SCA5, SCA6, SCA7, SCA8, SCA10, SCA1 1 , SCA12, SCA13, SCA14, SCA16; Spinocerebellar Ataxia; Cockayne's syndrome; Giant axonal neuropathy; chronic inflammatory demyelinating polyneuropathy (CIDP); and Guillain-Barre syndrome. The composition of the present invention may take the form of a pharmaceutical composition. The invention accordingly provides a pharmaceutical composition comprising the composition of the invention and one or more pharmaceutically acceptable excipients, and the use thereof in preventing or treating of one or more of the above-mentioned diseases.

Administration of the formulation of the invention may be accomplished orally or parenterally. Methods of parenteral delivery include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intra-ventricular, intravenous, intraperitoneal, or intranasal administration.

Suitable pharmaceutically acceptable excipients may facilitate processing of the active compounds into preparations suitable for pharmaceutical administration. Oral formulations may include pharmaceutically acceptable carriers known in the art in dosages suitable for oral administration. Such carriers enable the compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like suitable for ingestion by the subject. Formulation for oral use can be obtained through combination of active compounds with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds if desired to obtain tablets or dragee cores. Suitable excipients include carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methylceilulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen. If desired, disintegrating or solubilising agents may be added, such as cross linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof.

Dragee cores can be provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterise the quantity of active compound. Formulations for oral use include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally stabilisers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilisers.

Formulations for parenteral administration include aqueous solutions of active compounds. For injection, the formulations of the invention may take the form of aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous suspension injections can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension can also contain suitable stabilisers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated may be used in the formulation.

The compositions of the present invention can be manufactured substantially in accordance with standard manufacturing procedures known in the art. The composition may also comprise one or more peptide regulatory or releasing factors, which may induce a cascade of release of further peptides by a variety of cells in the patient. Such additional factors are typically provided from the same animal species as the CRH. Suitable factors include a- HLA, TGF-β, and IL-10, among others.

In one embodiment, the composition may comprise one or more of vasopressin, beta endorphin, and an enkephalin. In certain embodiments, the composition may comprise CRH binding protein, CRH-BP. This binds CRH and acts as a reservoir for subsequent release of CRH to the patient.

The present invention also provides a method of treatment for a disease selected from systemic sclerosis (SSc); multiple sclerosis; rheumatoid arthritis; optic neuritis; Parkinson's disease; motor neuron disease; autoimmune diseases including lupus, psoriasis, eczema, thyroiditis, and polymyositis; axonal or nerve damage; cancers, in particular myelomas, melanomas, and lymphomas; neural disorders, both demyelinating and non-demyelinating; inflammatory conditions; obesity; nerve conduction disorders; and sexual dysfunction, in particular erectile dysfunction; the method comprising administering the composition of the invention to a patient in need thereof.

The optimal dosage will be determined by the clinician. For example, administration may be in a dosage of between 0.01 and 10 mg (total protein) per kg (patient), for example between 0.01 and 5 mg/kg, between 0.025 and 2 mg/kg, or between 0.05 and 1 mg/kg. A product suitable for administration to patients may have a total protein concentration of approximately 4 mg/ml. Improved patient responses have been observed when a staged dosage regimen is employed, for example, based on initial 0.1 ml administrations, followed by 0.5ml administrations, followed by 1 ml administrations.

The precise dosage to be administered may be varied depending on such factors as the age, sex and weight of the patient, the method and formulation of administration, as well as the nature and severity of the disorder to be treated. Other factors such as diet, time of administration, condition of the patient, drug combinations, and reaction sensitivity may be taken into account. An effective treatment regimen may be determined by the clinician responsible for the treatment. One or more administrations may be given, and typically the benefits are observed after a series of at least three, five, or more administrations. Repeated administration may be desirable to maintain the beneficial effects of the composition.

The treatment may be administered by any effective route, such as by subcutaneous injection, although alternative routes which may be used include intramuscular or intra-lesional injection, oral, aerosol, parenteral, topical or via a suppository. The treatment may be administered as a liquid formulation, although other formulations may be used. For example, the treatment may be mixed with suitable pharmaceutically acceptable carriers, and may be formulated as solids (tablets, pills, capsules, granules, etc) in a suitable composition for oral, topical or parenteral administration.

The invention also provides use of the aforementioned composition in the preparation of a medicament for the treatment of one or more of the diseases recited above.

These and other aspects of the present invention will now be described by way of example only.

Examples

Example 1: Manufacture of the composition of the invention

Ungulate serum from an ungulate that has been treated with uninfected cell lysate and/or VLP is centrifuged to separate any unwanted components, and the method carried out as a continuous process, avoiding any freezing or thawing step(s) prior to final aliquoting. This avoids any aggregation and loss of the CRH component from the formulation. A cell line (such as Hut78) in solution is treated to disrupt the cell membranes, either with detergent or other agents that break down the cell wall (such as Triton); or by other means such as freeze-thawing (liquid nitrogen, 37°C, 1 minute vortex), in a process known as cytolysis. This releases the contents within the cell.

The lysate is then subjected to ultracentrifugation, which separates the solution into distinct bands containing organelles, membrane fragments, various proteins and nucleic acids. The lysate is subsequently mixed with an adjuvant immediately before injecting it into the goats.

In more detail, a serum composition comprising CRH is stored at 2 to 8 degrees C (and not frozen) and is diluted at a ratio of 1 :2 parts serum:cold PBS, and supersaturated ammonium sulphate is added slowly with constant agitation until a ratio of 47:53 of ammonium sulphate: PBS is reached. This is carried out on a cold tray and the resulting solution was maintained at this temperature for 30 to 60 minutes with constant agitation. The serum solution is then centrifuged in a Beckman J6M/E centrifuge at 3500 rpm for 45 minutes at 4 degrees C. The supernatant is removed and discarded. The precipitated solid material is re-suspended in cold 47% saturated ammonium sulphate:PBS solution and re-centrifuged at 3500 rpm at 4 degrees C for 45 minutes. The supernatant is again discarded and the precipitated solid material re-suspended in ice cold PBS buffer. This solution (the serum component) is then subjected to diafiltration at 4 degrees C against PBS with a molecular weight cut-off of 10,000 Daltons.

The solution is adjusted to a protein concentration of between 4 to 5 milligrams per millilitre with ice-cold PBS. Small batches of the solution (1 .2 millilitres) containing the stabilised composition of the invention are put into vials in an isolator. 1 millilitre single doses are thus obtained and stored at -15 to -25 degrees C prior to use. All steps are carried out as a continuous process in the cold except filling into vials, which is conducted so as to minimise exposure to ambient temperature at all times. Cold trays are used whenever possible. Following treatment of the serum with PBS and supersaturated ammonium sulphate, freezing is avoided until the final filling stage and subsequent storage.

Example 2: Comparison of the composition of the invention with previous CRH formulations

The composition of the invention has been compared with prior art formulations as previously disclosed by applicant.

Applicant has disclosed basic manufacture protocols in WO 2003/004049, WO 2003/064472, WO 2005/056053, WO 2005/097183, WO 2006/021814, WO 2007/077465, and WO 2014/001749. The formulation of these prior art teachings is referred to herein as "Aimspro".

In this Example, a composition of the invention was obtained using the manufacture protocol set out in Example 1 . The uninfected human cell line H9 was lysed using a detergent and was formulated for injection into a goat. An ELISA was carried out on this composition to assess the concentration ranges of various proteins. The results are shown in Table 1 below.

Protein Concentration Units

range

IL-4 > 5 pg/ml

IL-10 > 10 pg/ml

TGF > 500 pg/ml

IFN-γ > 10 pg/ml

CRH > 60 pg/ml

POMC > 140 pmole/L

Table 1 . Concentrations of protein in the composition of the invention.

For comparative purposes, the cytokine content of the composition of the invention (in this case an H9 cell lysate) was assessed using ELISA; this protocol was also carried out on an "Aimspro" composition obtained using the protocols from WO 2014/001749. The results are set out below.

The mean H9 lysate IL-6 concentration was found to be 59.8% ± 18.9 of the Aimspro IL-6 concentration.

The mean H9 lysate IL-6 concentration was found to be 59.8% ± 18.9 of the Aimspro IL-6 concentration. The mean H9 lysate TGF concentration was found to be 124.7% ± 14.4 of the Aimspro TGF concentration.

The mean H9 lysate IL-10 concentration was found to be 105.4% ± 61 .1 of the Aimspro IL-10 concentration.

The mean H9 lysate IFNy concentration was found to be 78.7% ± 20.15 of the Aimspro IFNy concentration.