WO2021023977A1 - Formulations comprising corticotropin releasing hormone (crh) and alpha-2 macroglobulin - Google Patents

Abstract

The present invention is directed to a method for manufacturing a modified formulation comprising corticotropin releasing hormone (CRH) and alpha-2 macroglobulin, the method comprising: a. providing a nano-filtered serum obtained by a method comprising: i. subjecting hyperimmune serum from an ungulate to a microfiltration step that removes molecules having a size greater than 0.2 microns, thereby providing a micro-filtered serum; and ii. subjecting said micro-filtered serum to a nanofiltration step that removes molecules having a size greater than 35 nanometres, thereby providing the nano-filtered serum; and b. adding alpha-2 macroglobulin to the nano-filtered serum, thereby providing the modified formulation comprising CRH and alpha-2 macroglobulin. The invention is also directed to said formulations as well as therapeutic uses thereof.

Description

FORMULATIONS COMPRISING CORTICOTROPIN RELEASING HORMONE (CRH) AND ALPHA-2

MACROGLOBULIN

The present invention relates to formulations comprising corticotropin releasing hormone (CRH), methods of manufacturing, and uses thereof.

WO 2006/021814 describes a serum composition comprising corticotropin releasing factor (CRF). WO 2006/021814 also describes the use of 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 ischemic 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, CMT1B, 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, SCA11, 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 (Cl DP) and Guillain-Barre syndrome. CRF is also described as having anti-angiogenic properties, caused by the molecules thrombospondin-1 (TSP-1) and platelet factor-4 (PF-4).

It is therefore understood that use of CRH is advantageous in treating the above-mentioned disorders in a subject.

WO 2014/001749 A1 (incorporated herein by reference) describes a method for manufacturing a hyperimmune serum (a.k.a. Purified Hyperimmune Caprine Serum (PHICS)) comprising 140+ proteins and peptides. The hyperimmune serum is a mixture principally comprising caprine immunoglobulins but also various cytokines, melanocortins and small molecular weight peptides. The proteins at the time believed to be of principal interest for their biological activity within the hyperimmune serum of WO 2014/001749 A1 include POMC (proopiomelanocortin), IL-10 (Interleukin-10) and CRH (Corticotrophin Releasing Hormone, also referred to as CRF or corticoliberin). The cytokines, arginine vasopressin and the hypothalamo-pituitary-adrenal axis (HPA)-associated proteins, POMC precursor and its regulator CRH are believed to be the key active ingredients of the hyperimmune serum. The cytokines include, but are not limited to, IL-4, IL-1beta, TGF beta, IL-6 and IL-10. The formulation has proven both safe and effective in the treatment of a number of complex debilitating inflammatory and neurodegenerative conditions. An overview of a manufacturing process according to WO 2014/001749 A1 is provided at Figure 1 herein. Important features in the manufacture of the hyperimmune serum of WO 2014/001749 A1 are a 0.2 micron microfiltration step and a 35 nm nanofiltration step for viral clearance. These steps are required by regulatory authorities for rendering a serum-based formulation suitable for administration to a human subject. However, the nanofiltration step risks altering the concentrations of active components of the hyperimmune serum, for example owing to non specific adherence of any active components to the filtration media and/or removal of high- weight proteins/protein complexes.

Moreover, one drawback associated with CRH formulations is that, following administration to a subject, the CRH protein has a limited effective biological half-life - by way of example, the CRH protein has a very low effective plasma half-life (approximately 4 minutes), and thus a low bioavailability/ efficacy, relying on a pulsatile stimulus.

Attempts to achieve long-term persistent levels of CRH in vivo have been reported, e.g. by Walker JJ et al (Walker et al (2012) “The origin of glucocorticoid hormone oscillations; PLoS Biol 10(6):e1001341). Even with constant infusion of CRH over a six-hour period, however, elevated levels of CRH can be maintained in vivo for no longer than an hour before falling back toward base-line levels; this effect appears to persist regardless of the dosage used. Moreover, constant infusion of CRH is undesirable and an impractical therapeutic method outside of a hospital or laboratory setting.

There is therefore a need for a CRH formulation having a longer effective biological half-life and thus a longer effective therapeutic window of efficacy.

Another drawback associated with CRH formulations is that said formulations have limited stability, and thus sub-optimal efficacy. There is therefore a need for a CRH formulation having improved stability and thus a longer effective therapeutic window of efficacy.

The present invention overcomes one or more of the above-mentioned problems.

The present inventor has surprisingly developed a manufacturing method that includes a 35 nm nanofiltration step for viral clearance (and thus complies with regulatory requirements/renders the formulation suitable for human administration) but that provides a formulation exhibiting improved activity/efficacy and/or immunomodulatory potency when compared to prior art formulations. Therapeutic uses of such formulations are also provided for prevention or treatment of one or more diseases (including, but not limited to, said hereinbefore listed disorders), including Alzheimer's disease, vasculitis, systemic sclerosis, inflammatory bowel disease, various forms of arthritis, diabetes mellitus, multiple sclerosis and obesity.

Thus, in one aspect the invention provides a method for manufacturing a modified formulation comprising corticotropin releasing hormone (CRH) and alpha-2 macroglobulin, the method comprising: a. providing a nano-filtered serum obtained by a method comprising: i. subjecting hyperimmune serum from an ungulate to a microfiltration step that removes molecules having a size greater than 0.2 microns, thereby providing a micro-filtered serum; and ii. subjecting said micro-filtered serum to a nanofiltration step that removes molecules having a size greater than 35 nanometres, thereby providing the nano- filtered serum; and b. adding alpha-2 macroglobulin to the nano-filtered serum, thereby providing the modified formulation comprising CRH and alpha-2 macroglobulin.

In one embodiment a method comprises: a. providing a nano-filtered serum obtained by a method comprising: i. providing hyperimmune serum from an ungulate that has been immunised; ii. subjecting said hyperimmune serum to a microfiltration step that removes molecules having a size greater than 0.2 microns, thereby providing a micro-filtered serum; and iii. subjecting said micro-filtered serum to a nanofiltration step that removes molecules having a size greater than 35 nanometres, thereby providing a nano- filtered serum; and b. adding alpha-2 macroglobulin to the nano-filtered serum, thereby providing the modified formulation comprising CRH and alpha-2 macroglobulin.

In one embodiment a method of the invention comprises: a. subjecting hyperimmune serum from an ungulate to a microfiltration step that removes molecules having a size greater than 0.2 microns, thereby providing a micro-filtered serum; b. subjecting said micro-filtered serum to a nanofiltration step that removes molecules having a size greater than 35 nanometres, thereby providing the nano-filtered serum; and c. adding alpha-2 macroglobulin to the nano-filtered serum, thereby providing the modified formulation comprising CRH and alpha-2 macroglobulin.

The “hyperimmune serum from an ungulate” is serum from an ungulate that has been immunised.

Thus, in one embodiment a method of the invention comprises: a. providing hyperimmune serum from an ungulate that has been immunised; b. subjecting said hyperimmune serum to a microfiltration step that removes molecules having a size greater than 0.2 microns, thereby providing a micro-filtered serum; and c. subjecting said micro-filtered serum to a nanofiltration step that removes molecules having a size greater than 35 nanometres, thereby providing a nano-filtered serum; and d. adding alpha-2 macroglobulin to the nano-filtered serum, thereby providing the modified formulation comprising CRH and alpha-2 macroglobulin.

In one aspect the present invention provides a modified formulation obtainable by a method of the present invention.

A “modified formulation” is one to which alpha-2 macroglobulin has been added, thereby distinguishing said formulation from a nano-filtered serum to which alpha-2 macroglobulin has not been added. Thus, a “nano-filtered serum” may be referred to as an “unmodified formulation”. The term “formulation” is used herein to refer to the “modified formulation” of the invention unless context indicates otherwise.

The CRH and alpha-2 macroglobulin of the invention are preferably present in a stabilised complex, e.g. wherein the latter is believed to protect CRH in vivo.

An ungulate described herein may be one or more selected from the following families: Bovidae, Equidae, Tapiridae, Rhinocerotidae, Camelidae, Tayassuidae, Suidae, Hippopotamidae, Tragulidae, Antilocapridae, Giraffidae, Cervidae, and Moschidae. Suitably, the ungulate may be one or more selected from a goat, a horse, a zebra, a donkey, cattle, bison, a pig, a moose, an elk, a deer, a llama, a camel, an alpaca, an antelope or a gazelle. In one embodiment an ungulate described herein is not an ovine. Preferably an ungulate described herein is a goat.

By virtue of the microfiltration step and nanofiltration steps, the formulations of the present invention are suitable for administration to a human subject.

A formulation (e.g. modified formulation) of the present invention preferably comprises a hyperimmune serum (preferably obtainable from an ungulate) that has been subjected to microfiltration and nanofiltration, and to which alpha-2 macroglobulin has been added. In other words, the formulation of the invention may be derived from a hyperimmune serum. In one embodiment, a formulation comprises hyperimmune serum components (e.g. CRH) and recombinant components, such as recombinant alpha-2 macroglobulin.

The formulations of the invention are suitably liquid formulations (e.g. when at room temperature).

In one aspect the invention provides a formulation for administration to a human subject, the formulation comprising:

80-120 mg/ml corticotropin releasing hormone (CRH); and alpha-2 macroglobulin at a concentration of greater than 0.2 mM, preferably at least 0.3 mM or 0.4 mM.

In one embodiment a formulation comprises:

80-120 mg/ml corticotropin releasing hormone (CRH); and alpha-2 macroglobulin at a concentration of at least 0.5 mM.

In a related aspect, the invention provides a formulation for administration to a human subject, the formulation comprising corticotropin releasing hormone (CRH) and alpha-2 macroglobulin, wherein the alpha-2 macroglobulin is present at a concentration of greater than 5 mM.

A formulation of the invention may comprise alpha-2 macroglobulin at a concentration of at least 0.3 mM, 0.4 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM or 5 mM. Preferably a formulation according to the invention comprises alpha-2 macroglobulin at a concentration of greater than 5 mM.

Similarly, a method of the invention may comprise adding alpha-2 macroglobulin to a final concentration of at least 0.3 mM, 0.4 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM or 5 mM. Preferably a method of the invention comprises adding alpha-2 macroglobulin to a final concentration of greater than 5 mM.

In one embodiment alpha-2 macroglobulin may be present in a formulation at a concentration of 0.3-500 mM, 0.4-500 mM, 0.5-500 mM, 1-250 mM, 2.5-200 mM, 5-150 mM, preferably 6-100 mM, e.g. 7.5-50 mM. More preferably, alpha-2 macroglobulin may be present at a concentration of 10-30 mM, such as about 20 mM. Similarly, in one embodiment a method of the invention comprises adding alpha-2 macroglobulin to a final concentration of 0.3-500 mM, 0.4-500 mM, 0.5-500 mM, 1-250 mM, 2.5-200 mM, 5-150 mM, preferably 6-100 mM, e.g. 7.5-50 mM. More preferably, a method of the invention comprises adding alpha-2 macroglobulin to a final concentration of 10-30 mM, such as about 20 mM.

In one aspect, the invention provides a formulation (e.g. obtainable by the method of any one of the preceding claims) for administration to a human subject, the formulation comprising corticotropin releasing hormone (CRH) and alpha-2 macroglobulin, wherein a concentration ratio of alpha-2 macroglobulin to CRH is greater than 1,088,333:1. The concentration ratio may refer to a mg/ml alpha-2 macroglobulin to mg/ml CRH ratio.

In one embodiment a concentration ratio of alpha-2 macroglobulin to CRH is at least 1,250,000:1, 1,500,000:1, 2,000,000:1, 2,500,000:1, 3,000,000:1, 3,500,000:1, 4,000,000:1, 4,500,000:1, 5,000,000:1, 5,500,000:1, 6,000,000:1, 6,500,000:1, 7,000,000:1, 7,500,000:1, 8,000,000:1, 8,500,000:1, 9,000,000:1, 9,500,000:1. 10,000,000, 15,000,000:1, 20,000,000:1, 25,000,000:1, 50,000,000:1 or 100,000,000:1.

In one embodiment a concentration ratio of alpha-2 macroglobulin to CRH is at least 2,725,000:1, 5,441,667:1, 10,883,334:1, 16,325,001:1, 21,766,668:1 or 27,208,335:1. Preferably a formulation according to the invention comprises a concentration ratio of alpha-2 macroglobulin to CRH that is greater than 27,208,335:1.

Similarly, a method of the invention may comprise adding alpha-2 macroglobulin to a final concentration ratio of alpha-2 macroglobulin to CRH of at least 2,725,000:1, 5,441,667:1, 10,883,334:1, 16,325,001:1, 21,766,668:1 or 27,208,335:1. Preferably a method of the invention may comprise adding alpha-2 macroglobulin to a final concentration ratio of alpha-2 macroglobulin to CRH of greater than 27,208,335:1.

In one embodiment a concentration ratio of alpha-2 macroglobulin to CRH is 2,725,000:1 to 2,720,833,500:1, 5,441,667:1 to 1,360,416,700, 13,604,167:1 to 1,088,333,330:1,

27,208,355:1 to 816,250,050, preferably 32,650,002:1 to 544,166,700:1, e.g. 40,812,502:1 to 272,083,350:1. More preferably, alpha-2 macroglobulin may be present at a concentration of 54,416,670:1 to 181,250,010:1, such as about 108,833,333:1 or 120,833,333:1. Similarly, a method of the invention may comprise adding alpha-2 macroglobulin to a final concentration ratio of alpha-2 macroglobulin to CRH of 2,725,000:1 to 2,720,833,500:1, 5,441,667:1 to 1,360,416,700, 13,604,167:1 to 1,088,333,330:1, 27,208,355:1 to

816,250,050, preferably 32,650,002:1 to 544,166,700:1, e.g. 40,812,502:1 to 272,083,350:1. Preferably a method of the invention may comprise adding alpha-2 macroglobulin to a final concentration ratio of alpha-2 macroglobulin to CRH of 54,416,670:1 to 181,250,010:1, such as about 108,833,333:1 or 120,833,333:1.

In one embodiment alpha-2 macroglobulin is present in a formulation of the invention at a concentration of greater than 131 mg/ml (micrograms per ml), for example at least 140 mg/ml, 150 mg/ml, 200 mg/ml, 300 mg/ml, 400 mg/ml, 500 mg/ml, 600 mg/ml, 700 mg/ml, 800 mg/ml, 900 mg/ml, 1000 mg/ml, 1100 mg/ml, 1200 mg/ml, 1300 mg/ml, 1400 mg/ml, 1500 mg/ml, 2000 mg/ml, 2500 mg/ml, 3000 mg/ml, 3500 mg/ml, 4000 mg/ml, 4500 mg/ml, 5000 mg/ml, 6000 mg/ml, 7000 mg/ml, 8000 mg/ml, 9000 mg/ml, 10,000 mg/ml, 11,000 mg/ml, 12,000 mg/ml, 13,000 mg/ml or 14000 mg/ml.

Similarly, a method of the invention may comprise adding alpha-2 macroglobulin to a (final) concentration of greater than 131 mg/ml (micrograms per ml), for example at least 150 mg/ml, 200 mg/ml, 300 mg/ml, 400 mg/ml, 500 mg/ml, 600 mg/ml, 700 mg/ml, 800 mg/ml, 900 mg/ml, 1000 mg/ml, 1100 mg/ml, 1200 mg/ml, 1300 mg/ml, 1400 mg/ml, 1500 mg/ml, 2000 mg/ml, 2500 mg/ml, 3000 mg/ml, 3500 mg/ml, 4000 mg/ml, 4500 mg/ml, 5000 mg/ml, 6000 mg/ml, 7000 mg/ml, 8000 mg/ml, 9000 mg/ml, 10,000 mg/ml, 11,000 mg/ml, 12,000 mg/ml, 13,000 mg/ml or 14000 mg/ml.

In one embodiment alpha-2 macroglobulin may be present in a formulation at a concentration of 325-362,500 mg/ml, 653-181,250 mg/ml, 1633-145,000 mg/ml, 3,265-108,750 mg/ml, preferably 3,918-72,500 mg/ml, e.g. 4,850-36,250 mg/ml. More preferably, alpha-2 macroglobulin may be present at a concentration of 6,530-21,750 mg/ml, such as about 13060 mg/ml or about 14500 mg/ml.

A nano-filtered serum for use in the invention typically already comprises a background level of (ungulate) alpha-2 macroglobulin. Said concentration is typically between 100,000 mg/ml and 150,000 mg/ml, e.g. around 120,000 mg/ml. In one embodiment the concentrations referred to herein are the concentrations of added alpha-2 macroglobulin. In other embodiments the concentrations refer to the total amount of alpha-2 macroglobulin (including that comprised in the nano-filtered serum). In one embodiment, the formulation comprises: a. a nano-filtered serum comprising corticotropin releasing hormone (CRH) obtained by a method comprising: i. subjecting hyperimmune serum from an ungulate to a microfiltration step that removes molecules having a size greater than 0.2 microns, thereby providing a micro-filtered serum; and ii. subjecting said micro-filtered serum to a nanofiltration step that removes molecules having a size greater than 35 nanometres, thereby providing the nano- filtered serum; and b. alpha-2 macroglobulin, wherein the alpha-2 macroglobulin is present at a concentration of greater than 0.2 mM; wherein the CRH is present at a concentration of 80-120 mg/ml.

In one embodiment, the formulation comprises: a. a nano-filtered serum comprising corticotropin releasing hormone (CRH) obtained by a method comprising: i. subjecting hyperimmune serum from an ungulate to a microfiltration step that removes molecules having a size greater than 0.2 microns, thereby providing a micro-filtered serum; and ii. subjecting said micro-filtered serum to a nanofiltration step that removes molecules having a size greater than 35 nanometres, thereby providing the nano- filtered serum; and b. alpha-2 macroglobulin, wherein a concentration ratio of alpha-2 macroglobulin to CRH is greater than 1,088,333:1.

In one embodiment, the formulation comprises: a. a nano-filtered serum comprising corticotropin releasing hormone (CRH) obtained by a method comprising: i. subjecting hyperimmune serum from an ungulate to a microfiltration step that removes molecules having a size greater than 0.2 microns, thereby providing a micro-filtered serum; and ii. subjecting said micro-filtered serum to a nanofiltration step that removes molecules having a size greater than 35 nanometres, thereby providing the nano- filtered serum; and b. alpha-2 macroglobulin, wherein the alpha-2 macroglobulin is present at a concentration of greater than 5 mM.

CRH may be present in a formulation of the invention at a concentration of between 80 picograms per millilitre (mg/ml) and 120 mg/ml, preferably between 90 and 110 mg/ml. In one embodiment CRH may be present in a nano-filtered serum for use in the invention at a concentration of between 80 picograms per millilitre (mg/ml) and 120 mg/ml, preferably between 90 and 110 mg/ml.

The formulation of the invention preferably further comprises CRH binding protein (CRH-BP). The CRH-BP may be present at a concentration of between 30 and 60 picograms per millilitre (mg/ml), preferably between 40 and 50 mg/ml. In one embodiment CRH may be present in a nano-filtered serum for use in the invention at a concentration of between 30 and 60 picograms per millilitre (mg/ml), preferably between 40 and 50 mg/ml.

In one embodiment, a formulation of the invention comprises:

80-120 mg/ml corticotropin releasing hormone (CRH);

30-60 mg/ml CRH binding protein (CRH-BP); and alpha-2 macroglobulin at a concentration of at least 0.5 mM (preferably a concentration of greater than 5 mM, more preferably 6-100 mM.

The nano-filtered serum to which alpha-2 macroglobulin is added may be manufactured as described in WO 2014/001749 A1.

In one embodiment, the stabilised 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 immunised (preferably with an immunodeficiency virus), and obtaining serum from the blood (e.g. by centrifugation); treating the serum to separate the CRH and other active components of interest; diafiltration of the separated serum comprising CRH and other active components of interest, thereby retaining molecules having a molecular weight of at least 10 KDa; filtering to remove molecules having a size greater than 0.2 microns; processing by nanofiltration to remove molecules having a size greater than 35 nanometres; wherein all of the above steps are performed under cooled conditions (e.g. less than 22 degrees C, preferably less than 10 degrees C, preferably less than 5 degrees C); whilst ensuring that the serum remains unfrozen following treatment to separate the CRH and other active components. Alpha-2 macroglobulin may then be added to the serum. The processed serum may be aliquoted into vials, optionally with protein concentration adjustment, to provide a single dose amount. This step can be performed before or after addition of alpha-2 macroglobulin. 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 subject.

In a preferred embodiment a serum for use in the invention may be obtained from an ungulate that has been immunised with an immunodeficiency virus. The use of an immunodeficiency (e.g. HIV-3B) viral lysate as an immunogen is not believed to be essential for the production of active serum; it is believed that a medium which has been used for growth of a viral culture, or which is suitable for such growth, may also produce a suitable response when used as an immunogen. The supernatant of a cell culture growth medium such as PBMC or the cancer immortal cell line as used to grow HIV-3B are given as an example. The HIV or other virus does not need to be present to produce an effective immunogen to create the composition. Other suitable immunogens are recited on pages 12 and 13 of WO 03/064472, which is hereby incorporated in its entirety by reference thereto.

The immunodeficiency virus may be HIV or SIV, which may be HIV 3b; the immunodeficiency virus may be in the form of a lysate or a heat-killed virus.

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

Thus, the basic methodology as described in WO 2006/021814, which is hereby incorporated in its entirety by reference thereto, may be employed to provide a nano-filtered serum for use in the invention, though with the inclusion of a nanofiltration step; and optionally avoidance of multiple freeze-thaw steps and/ or minimising ambient temperature exposure; and/ or a mixing/ agitation step (e.g. to enhance CRH: alpha-2 macroglobulin complex formation).

In more detail, a goat may be immunised with HIV-3B viral lysate raised in H9 cells. Approximately 400 cc of blood may then be taken from the goat under sterile technique. The animal may typically be re-bled in 10 to 14 days, once the volume of blood is replenished. A pre-bleeding regime may be useful to stimulate production of the active components of the therapeutic formulation. The blood is then centrifuged to separate the serum, and the serum filtered to remove large clots and particulate matter. The serum is then treated with supersaturated ammonium sulphate (47% solution at 4 degrees C) to precipitate antibodies and other material. The resulting solution is centrifuged in a Beckman J6M/E centrifuge at 3500 rpm for 45 minutes, after which the supernatant fluid is removed. The precipitated immunoglobulin and other solid material are resuspended in PBS buffer (phosphate buffered saline) sufficient to redissolve the precipitate. The solution may then be subjected to diafiltration against a PBS buffer with a molecular weight cut-off of 10,000 Daltons at 4 degrees C. After diafiltration, the product may be filtered through a 0.2 micron filter and then processed by nanofiltration to remove molecules having a size greater than 35 nanometres into a sterile container and optionally adjusted to a protein concentration of ~4 mg/ml. Following addition of alpha-2 macroglobulin, the solution may then be put into vials to give single doses of 1 ml, and stored at minus 22 degrees C prior to use.

In one embodiment a formulation comprises substantially no molecules having a size greater than 0.2 microns, e.g. less than 5%, 1%, 0.1% or 0.01% of total molecules present have a size of greater than 0.2 microns. Preferably, the formulation comprises no molecules having a size greater than 0.2 microns.

In one embodiment a nano-filtered serum comprises substantially no molecules having a size greater than 0.2 microns, e.g. less than 5%, 1%, 0.1% or 0.01% of total molecules present have a size of greater than 0.2 microns. Preferably, the nano-filtered serum comprises no molecules having a size greater than 0.2 microns.

In one embodiment a formulation comprises substantially no molecules having a size greater than 35 nanometres, e.g. less than 5%, 1%, 0.1% or 0.01% of total molecules present have a size of greater than 35 nanometres. Preferably, the formulation comprises no molecules having a size greater than 35 nanometres.

In one embodiment a nano-filtered serum comprises substantially no molecules having a size greater than 35 nanometres, e.g. less than 5%, 1%, 0.1% or 0.01% of total molecules present have a size of greater than 35 nanometres. Preferably, the nano-filtered serum comprises no molecules having a size greater than 35 nanometres.

The embodiments above may be particularly relevant immediately after manufacturing the formulation. Over time, polypeptides may interact to form complexes having a size of greater than 0.2 microns or greater than 35 nanometres. In one embodiment a nano-filtered serum for use in the invention may be manufactured by a method comprising:

(a) providing hyperimmune serum from an ungulate that has been immunised (e.g. with an immunodeficiency virus);

(b) subjecting said hyperimmune serum to a microfiltration step that removes molecules having a size greater than 0.2 microns, thereby providing a micro- filtered serum;

(c) subjecting said micro-filtered serum to a nanofiltration step that removes molecules having a size greater than 35 nanometres, thereby providing a nano- filtered serum;

(d) adding alpha-2 macroglobulin to the nano-filtered serum, thereby providing a modified formulation of the invention; and

(e) aliquoting said formulation into a vial, wherein said formulation is in a form for administration to a subject, comprises the CRH and alpha-2 macroglobulin, and optionally wherein the formulation/serum remains unfrozen throughout steps (b) to (d).

Said microfiltration step may follow an ammonium sulphate precipitation plus (PBS) buffer dialysis step to retain molecules of, for example, at least 10 KDa. Other means of separating the CRH and other components of interest are also contemplated, which are well within the means of the person skilled in the art (separating columns, etc.).

Said nanofiltration step may be carried out using a 35 nanometre filter, which may be a 35 nanometre hollow fibre filter.

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 between the filtration step (e.g. micro- and/or nanofiltration) and final aliquoting into vials (optionally with adjustment of protein concentration). Most especially, 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, e.g. using ammonium sulphate.

One or more agitation steps may be carried out during the method; these agitation steps may use cold trays.

The method may comprise a final step (e.g. after the micro-/ nano- filtration step and any protein concentration adjustment step) of freezing for subsequent storage. In one embodiment, the hyperimmune serum is not frozen prior to micro- and/or nano-filtration.

Without wishing to be bound by any theory, the present inventor believes 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 subsequent filtration.

Alternatively, the formulation of the present invention may be prepared from first principles based on commercially available components (including recombinantly prepared components).

The two principal components of the formulation described herein (when present in the stabilised complex form) 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. In one embodiment, the predominant complexed form of CRH: alpha-2 macroglobulin is 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 concentrations (e.g. in respect of CRH, CRH-BP) etc. referred to herein are typically 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. In one embodiment a nano-filtered serum may be adjusted to a final protein concentration of 4-5 mg/ml (preferably 4.5 mg/ml) prior to the step of adding alpha-2 macroglobulin. In such embodiments, alpha-2 macroglobulin may be added as a powder to ensure that the final concentrations of other components of the formulation are not substantially altered.

The CRH may be human or non-human. For example, the CRH may be an ungulate CRH such as horse, zebra, donkey, cattle, bison, goat, pig, moose, elk, deer, antelope or gazelle CRH. 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, b-endorphin, and other pro opiomelanocortin related peptides from the paraventricular nucleus of 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-R2b, 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 were 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. Several forms of CRH have been identified in nature, they include a high molecular weight form 194 amino acids Mw ~ 30,000, a Mw -18,000, Mw -7,500 and the 41 amino acid residue. All three forms are biologically active and able to stimulate ACTH release.

The nucleotide and amino acid sequences of human CRH are described in GenBank ( see accession numbers BC011031 and AAH 11031.1, respectively, both of which are incorporated herein by reference).

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 b (I L-1 b) and interleukin-6 (IL-6), transforming growth factor (TGF-Ib), and insulin, and modify their biological activity. Human a2M is composed of four identical subunits bound together by -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 may lead to increased risk of Alzheimer's disease.

In one embodiment an alpha-2 macroglobulin is ungulate alpha-2 macroglobulin. Ungulate alpha-2 macroglobulin may be isolated from a hyperimmune serum obtainable from an ungulate.

The term “obtainable” as used herein also encompasses the term “obtained”. In one embodiment the term “obtainable” means obtained.

Thus, in one aspect there is provided a method for obtaining alpha-2 macroglobulin (a2M), the method comprising: a. providing a micro-filtered serum obtained by a method comprising subjecting hyperimmune serum from an ungulate to a microfiltration step that removes molecules having a size greater than 0.2 microns, thereby providing a micro-filtered serum; and b. isolating alpha-2 macroglobulin from the serum.

In one embodiment the method comprises: a. providing a micro-filtered serum obtained by a method comprising: i. providing hyperimmune serum from an ungulate that has been immunised; ii. subjecting said hyperimmune serum to a microfiltration step that removes molecules having a size greater than 0.2 microns, thereby providing a micro-filtered serum; and b. isolating alpha-2 macroglobulin from the serum.

In another aspect there is provided a method for obtaining alpha-2 macroglobulin (a2M), the method comprising: a. providing a nano-filtered serum obtained by a method comprising: i. subjecting hyperimmune serum from an ungulate to a microfiltration step that removes molecules having a size greater than 0.2 microns, thereby providing a micro-filtered serum; and ii. subjecting said micro-filtered serum to a nanofiltration step that removes molecules having a size greater than 35 nanometres, thereby providing the nano- filtered serum; and b. isolating alpha-2 macroglobulin from the serum.

In another aspect there is provided a method for obtaining alpha-2 macroglobulin (a2M), the method comprising: a. providing a nano-filtered serum obtained by a method comprising: i. providing hyperimmune serum from an ungulate that has been immunised; ii. subjecting said hyperimmune serum to a microfiltration step that removes molecules having a size greater than 0.2 microns, thereby providing a micro-filtered serum; and iii. subjecting said micro-filtered serum to a nanofiltration step that removes molecules having a size greater than 35 nanometres, thereby providing the nano- filtered serum; and b. isolating alpha-2 macroglobulin from the serum.

Alpha-2 macroglobulin may be isolated from a serum using any technique known in the art, such as chromatography. In one embodiment alpha-2 macroglobulin may be isolated from a serum using a HiTrap chelating column (e.g. as described herein). In some embodiments alpha-2 macroglobulin may comprise one or more tags to facilitate purification thereof, e.g. a His-tag, GST tag, HA tag or FLAG tag.

The isolated alpha-2 macroglobulin may be used in any method described herein.

In one embodiment an alpha-2 macroglobulin is a non-ungulate alpha-2 macroglobulin. In one embodiment a method of the invention comprises adding a non-ungulate alpha-2 macroglobulin to a nano-filtered serum (e.g. wherein the nano-filtered serum comprises an ungulate alpha-2 macroglobulin). In embodiments where the alpha-2 macroglobulin is a non ungulate alpha-2 macroglobulin, said formulation may further comprise an amount of ungulate alpha-2 macroglobulin. Thus, it is preferred that the formulations of the invention comprise an ungulate and a non-ungulate alpha-2 macroglobulin. However, preferably a concentration of non-ungulate alpha-2 macroglobulin is much higher than a concentration of non-ungulate alpha-2 macroglobulin, e.g. at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times higher.

In a preferred embodiment a non-ungulate alpha-2 macroglobulin is human alpha-2 macroglobulin (e.g. human alpha-2 macroglobulin commercially-available from Molecular Innovations, Missouri, USA). An alpha-2 macroglobulin of the invention may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 1. In one embodiment an alpha-2 macroglobulin of the invention may comprise a polypeptide sequence having at least 80% or 90% sequence identity to SEQ ID NO: 1. Preferably an alpha-2 macroglobulin of the invention may comprise (or consist of) a polypeptide sequence shown as SEQ ID NO: 1

In one embodiment an alpha-2 macroglobulin for use in the invention is present as a tetramer. In one embodiment at least 50%, 60%, 70%, 80% or 90% of the alpha-2 macroglobulin present in a formulation of the invention is present as a tetramer. Preferably at least 95%, 98%, 99% or 99.9% (most preferably 100%) of the alpha-2 macroglobulin present in a formulation of the invention is present as a tetramer. Thus, in a preferred embodiment a concentration of alpha-2 macroglobulin referred to herein is a concentration of tetrameric alpha-2 macroglobulin.

Improved stabilisation of the formulation and/or tetrameric form of alpha-2 macroglobulin (e.g. for long-term storage) may be achieved by including one or more excipient(s) that prevent oxidation of the formulation, i.e. antioxidants. Suitable antioxidants are described herein. For example, antioxidants may include sugars (e.g. sucrose), amino acids (e.g. cysteine and/or methionine), peptides (e.g. glutathione), 2-mercaptoethanol (2-ME), dithiothreitol (DTT), carotenes (e.g. beta-carotene), and/or vitamins (e.g. vitamin C and/or E and/or A).

Improved stabilisation of the formulation and/or tetrameric form of alpha-2 macroglobulin (e.g. for long-term storage) may be achieved by including sucrose in a formulation of the invention. In one embodiment sucrose is present in the formulation at a concentration of at least 1 nM, 100 nM, 500 nM or 1000 nM. In one embodiment sucrose is present in the formulation at a concentration of 1-1000nM, such as 10-500 nM. Preferably sucrose is present in the formulation at a concentration of 20-100 nM. Thus, in one embodiment a method of the invention comprises adding sucrose to the formulation.

In one embodiment stabilisation of CRH by binding to alpha-2 macroglobulin, and the attendant enhanced effective biological half-life is one effect of formulations of the present invention. Said stability provides an enhanced (longer) plasma half-life - by way of example, the stabilised complex of the present invention may have a half-life of at least 24 hours.

A formulation of the invention may be employed in combination with one or more other stabilisers, such as fibronectin or albumin. Additionally or alternatively, the formulation of the invention may 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 formulation at a range of between 140 picomoles per litre (pmol/l) and 200 pmol/l.

Without wishing to be bound by any theory, the present inventor believes 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 prolylcarboxypeptidase (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 have GenBank accession numbers BC065832 and AAH65832.1, respectively, which are incorporated herein by reference. 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), b endorphin, met-enkephalin and leu-enkephalin; and preferably all of a, b, and g MSH; ACTH; b and g LPH; and b 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, 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 may have a self-sustaining effect, in that administration of an initial amount of POMC peptide leads to endogenous production of POMC in the subject; thus, an initial administration of a low level of POMC may have a significant effect on the subject.

The formulation of the invention may selectively increase the enzymatic degradation of POMC in vivo. The formulation of the invention may increase 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 subject. The formulation of the invention typically possesses a reduced amount of immunoglobulin component. Thus, the associated method of the present invention may provide a formulation in which the immunoglobulin component has been minimised - for example, in said formulation, the immunoglobulin component has been minimised so that the formulation contains less than 4.5 mg/ml, for example less than 4 mg/ml or less than 3.9 mg/ml. A reduced immunoglobulin component is preferred as this helps to minimise a host immune response against said component (notably against any IgG component). 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.

The formulation of the invention may act to enhance the central CRH-1 regulatory response. This results 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 subject may stimulate production of endogenous CRH and thus lead to a self-sustaining effect. CRH can therefore be administered at a low concentration to a subject.

The formulation of the invention may also protect CRH from proteolytic degradation by proteases and accordingly administration of the formulation of the invention may provide slow release of CRH in the circulation and a significant increase in CRH levels (e.g. due to formation of a stabilised complex between CRH and alpha-2 macroglobulin).

Thus, the formulation of the invention may be associated with 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 formulation of the invention may lead to an in vivo increase in CRH concentration of between 25% and 50% from subject baseline CRH levels at 24 hours from administration, and an in vivo increase of between 75% and 100% from subject baseline CRH levels at 48 hours from administration.

This contrasts with administration of formulations containing CRH in similar concentrations where the CRH is not in a stabilised complex (or in a poorer stabilised complex including, for example CRH aggregates): an in vivo increase in CRH concentration from subject baseline CRH levels may be seen within 24 hours of administration, but the effect does not persist and within 36 hours from administration CRH concentration is falling.

The formulation of the invention is believed to selectively up-regulate the CRH-1 receptor both centrally and peripherally in key target issues and enhances 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. It is believed by the inventor that administration of the formulation will thus lead to optimisation of key inflammatory cytokines and down-regulation of certain pro-inflammatory cytokines related to the Th-1 mediated response, through targeting leucocytes and macrophages.

No secondary glucocorticoid surge from increased levels of ACTH are believed to occur despite the elevated CRH levels, in part due to further processing and cleavage of ACTH and a reduction in “free” CRH levels. Elevated “free” peripheral CRH may also be regulated by a parallel increase in CRH-BP. This again mitigates excessive production of unwanted glucocorticoids. The combination of the peripheral and central dynamic relationship is believed to provide a prolonged activation of the CRH pathway without negative feedback. Central CRH is then believed to be able to further target T-helper immune cells and macrophages that express CRH.

The combined net effect of all of the above is believed to be an anti-inflammatory, reparative and fundamental immunomodulatory therapeutic that works within homeostatic constraints. The unique targeting and accessibility of the formulation (e.g. stabilised complex) of the invention explains its versatility in a wide range of diseases.

Thus in one embodiment the present invention provides a formulation (e.g. stabilised CRH formulation) that works within homeostatic constraints following in vivo administration.

The present invention provides a formulation of the invention for use in medicine.

In one aspect the invention provides a formulation for use in treating one or more disorder(s) selected from the group consisting of: an inflammatory disorder such as rheumatoid arthritis; optic neuritis; motor neuron disease; autoimmune diseases including lupus, psoriasis, eczema, thyroiditis, and polymyositis; 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; systemic sclerosis (SSc); Parkinson’s disease; Huntingdon’s chorea; mixed connective tissue diseases; scleroderma; anaphylaxis; septic shock; carditis and endocarditis; wound healing; contact dermatitis; occupational lung diseases; obesity; nerve conduction disorders; sexual dysfunction, in particular erectile dysfunction ; 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, CMT1B, 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, SCA11, SCA12, SCA13, SCA14, SCA16; Spinocerebellar Ataxia; Cockayne’s syndrome; Giant axonal neuropathy; chronic inflammatory demyelinating polyneuropathy (CIDP); and Guillain-Barre syndrome.

The present invention accordingly further provides a formulation for use in treating one or more disorders selected from systemic sclerosis (SSc), multiple sclerosis and inflammatory disorders such as rheumatoid arthritis; 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; 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, CMT1B, 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, SCA11, SCA12, SCA13, SCA14, SCA16; Spinocerebellar Ataxia; Cockayne’s syndrome; Giant axonal neuropathy; chronic inflammatory demyelinating polyneuropathy (Cl DP); and Guillain- Barre syndrome.

In one embodiment, a disorder is one or more 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.

In one embodiment a disorder is one or more selected from Alzheimer’s disease; systemic sclerosis (SSc); multiple sclerosis; rheumatoid arthritis; optic neuritis; motor neuron disease; hepatitis, in particular hepatitis C; 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; Parkinson’s disease; inflammatory conditions; obesity; nerve conduction disorders; and sexual dysfunction, in particular erectile dysfunction. Preferably a disorder is one or more selected from Alzheimer's disease, vasculitis, systemic sclerosis, inflammatory bowel disease, arthritis, diabetes mellitus, multiple sclerosis, and obesity.

In a related aspect the invention provides a use of a formulation of the invention in the manufacture of a medicament for treating a disorder herein. There is also provided a method of treating a disorder described herein, the method comprising administering a formulation of the invention to a subject.

A “subject” may be a mammal, such as a human or other animal. Preferably “subject” means a human subject.

The term “disorder” as used herein also encompasses a “disease”. In one embodiment the disorder is a disease.

The term “treat” or “treating” as used herein encompasses prophylactic treatment (e.g. to prevent onset of a disorder) as well as corrective treatment (treatment of a subject already suffering from a disorder). Preferably “treat” or “treating” as used herein means corrective treatment.

The term “treat” or “treating” as used herein refers to the disorder and/or a symptom thereof.

Therefore a composition of the invention may be administered to a subject in a therapeutically effective amount or a prophylactically effective amount.

A “therapeutically effective amount” is any amount of the formulation, which when administered alone or in combination to a subject for treating said disorder (or a symptom thereof) is sufficient to effect such treatment of the disorder, or symptom thereof.

A “prophylactically effective amount” is any amount of the formulation that, when administered alone or in combination to a subject inhibits or delays the onset or reoccurrence of a disorder (or a symptom thereof). In some embodiments, the prophylactically effective amount prevents the onset or reoccurrence of a disorder entirely. “Inhibiting” the onset means either lessening the likelihood of a disorder’s onset (or symptom thereof), or preventing the onset entirely.

The formulation of the present invention may take the form of a pharmaceutical composition. The invention accordingly provides a pharmaceutical composition comprising the formulation (e.g. stabilised complex) of the invention, and the use thereof in preventing or treating one or more of the above-mentioned diseases.

Administration of the formulation of the invention may be accomplished orally or parenterally.

In a particularly preferred embodiment the formulation is administered parenterally. Methods of parenteral delivery include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intra-ventricular, intravenous, intraperitoneal, or intranasal administration. Most preferably, the formulation is administered subcutaneously.

In addition to the active ingredients, the formulation of the invention may comprise suitable pharmaceutically acceptable carriers comprising excipients and other components which 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 formulations of the present invention can be manufactured substantially in accordance with standard manufacturing procedures known in the art. The formulation 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 subject. Such additional factors are typically provided from the same animal species as the CRH. Suitable factors include a- HLA, TGF-b, and IL-10, among others.

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

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 (subject), 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 subjects may have a total protein concentration of approximately 4-5 mg/ml, e.g. 4.5 mg/ml, preferably not including the added alpha-2 macroglobulin concentration (i.e. the alpha-2 macroglobulin may suitably be in addition to said concentration, meaning the concentration is greater than 4-5 mg/ml, preferably greater than 4.5 mg/ml).

The precise dosage to be administered may be varied depending on such factors as the age, sex and weight of the subject, 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 subject, 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. Most preferably, the formulation is administered subcutaneously.

The invention also provides use of the aforementioned formulation in the preparation of a medicament for the treatment of one or more of the diseases recited above. Embodiments related to the various methods of the invention are intended to be applied equally to the formulation, therapeutic uses thereof/methods comprising the use of the same, and vice versa.

SEQUENCE IDENTITY

Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131 ) Science 208-214 (1993); Align- M, see, e.g., Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004).

Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes).

The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, % identity may be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences. Sequence comparisons and the determination of percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which will be familiar to a skilled person.

The percent identity is then calculated as:

Total number of identical matches x 100

[length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino- terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

In addition to the 20 standard amino acids, non-standard amino acids (such as 4- hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and a -methyl serine) may be substituted for amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for polypeptide amino acid residues. The polypeptides of the present invention can also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4- methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo- threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro- glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3- azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al. , J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of polypeptides of the present invention.

Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al. , Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention.

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with a general dictionary of many of the terms used in this disclosure.

This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspects or embodiments of this disclosure.

Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation. The term “protein", as used herein, includes proteins, polypeptides, and peptides. As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “enzyme”. The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The 3- letter code for amino acids as defined in conformity with the lUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be defined only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a formulation" includes a plurality of such candidate agents and reference to "the formulation" includes reference to one or more formulations and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

Figure 1 shows a flowchart summarising the key steps in the manufacture of a nano-filtered serum/bulk product (“Prior Art Formulation” as per WO 2014/001749 A1).

Figure 2 shows an infrared Western blot analysis of a hyperimmune serum after a 35 nm nanofiltration step as well as the 35 nm filter retentate. The band corresponding to alpha-2 macroglobulin is indicated. Samples were taken from the retentate and the filtrate and analysed using the ProteinSimple Simple Western System (Jess) (commercially available from ProteinSimple, USA) in accordance with the manufacturer’s instructions.

Figure 3 shows a process for manufacturing a therapeutic formulation of the invention.

Figure 4 shows the mean concentration of interleukin-6 (IL-6) in the supernatant following treatment of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) for 24 hours, either in the presence of the “Prior Art Formulation” (225 mg) or a formulation of the invention (225 mg “Prior Art Formulation” plus a2M at a final concentration of 1 mM). Error bars show standard deviation around the mean.

Figure 5 shows the mean concentration of TNFa in the supernatant following treatment of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) for 24 hours, either in the presence of the “Prior Art Formulation” (225 mg) or a formulation of the invention (225 mg “Prior Art Formulation” plus a2M at a final concentration of 1 mM). Error bars show standard deviation around the mean.

Figure 6 shows the mean concentration of IL-6 in the supernatant following treatment of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) for 24 hours, either in the presence of a formulation of the invention (225 mg “Prior Art Formulation” plus a2M at a final concentration of 1 mM) or 225 mg “Prior Art Formulation” plus oxidised a2M at a final concentration of 1 mM. Error bars show standard deviation around the mean.

Figure 7 shows the mean concentration of TNFa in the supernatant following treatment of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) for 24 hours, either in the presence of formulation of the invention (225 mg “Prior Art Formulation” plus a2M at a final concentration of 1 mM) or 225 mg “Prior Art Formulation” plus oxidised a2M at a final concentration of 1 mM. Error bars show standard deviation around the mean.

EXAMPLES

EXAMPLE 1 - Manufacture of a Formulation of the Invention

A summary overview of the manufacturing process for a nano-filtered serum/bulk product (“Prior Art Formulation” as per WO 2014/001749 A1) is provided in Figure 1.

When characterising the formulation produced according to the prior art, it was found that the 35 nm nano-filtration step (necessary for viral clearance and regulatory purposes), depleted a significant fraction of alpha-2 macroglobulin (a2M) from the serum, which was clearly evident in the filter retentate ( see Figure 2). It was decided to add additional a2M to determine if this had any effect on the efficacy of the formulation. Likewise, it was noted that the 35 nm nano-filtration step reduced the concentration of at least CRH binding protein (from 98.84 mg/ml to 41.7 mg/ml).

Subsequent steps to produce a therapeutic formulation of the invention (“Formulation + a2M”) from the nano-filtered serum/bulk product are shown in Figure 3.

Human a2M (commercially-available) or caprine a2M may be used in manufacturing a formulation of the invention. Said caprine a2M can be isolated from caprine hyperimmune serum. In more detail, a HiTrap chelating column (GE Healthcare, Silverwater, Australia) is stripped (stripping buffer; 0.5 mM NaCI, 50 mM EDTA, 20 mM HEPES, pH 7.2), washed using milliQ water, recharged (recharge buffer; 0.1 M ZnS0₄), and then equilibrated in binding buffer (1 M NaCI, 20 mM HEPES, pH 7.2). Nano-filtered serum/bulk product is loaded onto the column and unbound proteins are removed by extensively washing with binding buffer. Loosely bound proteins are eluted from the column by washing with 20 mM imidazole, 0.5 M NaCI, 20 mM HEPES, pH 7.2, and discarded. The remaining bound protein is eluted with 500 mM imidazole, 0.5 M NaCI, 20 mM HEPES, pH 7.2, and dialysed against phosphate buffered saline (PBS; 1.53 M NaCI, 1.45 mM KH₂P0₄, 8.34 mM Na₂HP0₄, pH 7.4). The dialysed protein (2 ml) is fractionated by gel filtration using a HiPrep 26/60 Sephacryl S-300 gel filtration column (GE Healthcare, bed volume 320 mL) and fractions containing purified a2M are pooled and stored at 4°C. To quantify the amount of total protein present in the final solution, a standard bicinchoninic acid (BCA) assay is used. Sucrose may be added to a final concentration of 20nM-100nM to facilitate improved stability of the product once frozen.

Using this method, ~20 mg of purified native a2M is obtained from 100 ml of bulk product. a2M may be added to the nano-filtered serum/bulk product at a desired final concentration, e.g. ImicroM (mM).

EXAMPLE 2 -Efficacy of the Formulation of the Invention Materials & Methods

Protein preparation, oxidation and purification

Human a2M (SEQ ID NO: 1) was supplied lyophilised from Molecular Innovations (Missouri, USA). It was reconstituted in PBS at 10mg/ml. The “Prior Art Formulation”, was supplied at 4.5 mg/ml.

For some experiments (as indicated) a2M was oxidised using 50mM NaCIO for 15 minutes at room temperature.

Both a2M and the “Prior Art Formulation” were de-salted and purified using Pierce Protein Concentrator columns (Thermo Scientific, Illinois, USA). Protein recovery was 85-90% after 45 minutes of centrifugation at 4000g and 4°C.

Reagents

Luminex reagents were purchased from R&D systems (Minneapolis, USA). X-VIV015 medium was from Lonza Biologies Inc. (New Hampshire, USA). Lipopolysaccharide (LPS) was from Sigma Aldrich (Missouri, USA). Lympholyte H® was from Cederlane Labs (North Carolina, USA). Propidium Iodide was from Miltenyi Biotec (Bergisch Gladbach, Germany). Alpha 2 macroglobulin was from Molecular Innovations (Missouri, USA). Sodium hypochlorite was from Merck Millipore (Dramstadt, Germany).

PBMC isolation

Leukocyte cones from 5 healthy donors were sourced from the Manchester Blood Centre. The blood had been screened and was negative for HIV, HCV, HbsAg and Syphilis. PBMCs were immediately purified from cones by Ficoll gradient centrifugation using Lympholyte H®. The PBMC-rich layer was washed twice in serum-free RPMI 1640 and cells were counted.

Culture conditions and supernatant collection

Purified PBMCs were plated, in triplicate, in 96-well tissue culture- treated plates at 37°C and 5% CO2 in X-VIV015 medium at a density of 1x10⁵ cells per well in 50mls. Cells were stimulated with LPS for 24 hours. LPS was used at 1mg/ml and for both the “Prior Art Formulation” and “Formulation + a2M” the amount of nano-filtered serum/bulk product added was 225 mg/ml, with the “Formulation + a2M” further comprising 1 mM a2M.

Study end points

Supernatants were collected after each time point, and frozen immediately at -80°C until analysis. Cells were immediately stained with PI and cell viability was assessed using a MACQuant 10 analyser (Miltenyi Biotec). Results were analysed using MACSQuantify software.

Luminex

Supernatants were thawed on ice and assayed individually as experimental triplicates using the R&D System’s human Luminex kits to measure IL-6 and TNF-a, according to the manufacturer’s instructions. The supernatants were assayed neat and not diluted. Plates were read and analysed using an R&D Magnetic Performance Assay system and the Bio- Rad Bio-Plex® 200 system (Hercules, CA, USA).

Results

Purified PBMCs from 5 healthy donors were cultured with 225mg of “Prior Art Formulation” manufactured according to WO 2014/001749 A1 and the formulation of the invention “Formulation + a2M” (with 1 mM a2M) in the presence of 1 mg/ml LPS. The effects of single treatments on the levels of IL-6 and TNFa in the supernatants after 24 hours of LPS stimulation was measured by Luminex^®.

Results are shown as bar graphs with the mean concentration of protein indicated and error bars showing standard deviation. The results showed that, for each donor, the formulation of the invention “Formulation + a2M” significantly reduced concentrations of IL-6 (Figure 4) and TNF-alpha (Figure 5) in the presence of LPS.

Thus, the addition of 1 mM a2M to the “Prior Art Formulation” renders the final product (“Formulation + a2M”) more efficacious in inhibiting the M1 pro-inflammatory response as evidenced by the above-described results.

In vivo studies have shown that circulating alpha-2 macroglobulin is predominantly in a tetrameric form. Experiments were therefore carried out to characterize the nature of the alpha-2 macroglobulin present in a formulation of the invention. Specifically, comparative experiments were carried out using oxidised a2M (which is believed to adopt a dimeric or trimeric form) and showed that oxidising a2M reduces the anti-inflammatory effect of the formulation of the invention ( see Figures 6 and 7). Therefore, it is believed that the a2M in the formulation of the invention is predominantly present as a tetramer and naturally adopts this form when added to a hyperimmune serum. Advantageously, this finding highlights that excipients that minimise/prevent oxidation of a2M (e.g. sucrose) will help stabilise/improve efficacy of the formulations of the invention when stored for a prolonged period of time, where there is a risk of oxidation. However, such excipients are not essential for the improved properties of the formulation of the invention ( see Figures 4 and 5).

The formulations tested had no effect on cell viability (data not shown).

EXAMPLE 3 - Stability of the Formulation

To promote stability of the formulation, and especially the a2M component, sucrose at a concentration of 20-100nM is added. High performance capillary electrophoresis (HPCE) (carried out using standard techniques) is used to confirm the long-term stability of the therapeutic formulation.

Using HPCE on stored material over a period of 12 months indicates that the integrity of the formulation is not subject to deterioration and shows improved stability when compared to a control formulation to which sucrose has not been added. Three independent samples stored for different intervals over a twelve-month interval show no protein degradation, thereby confirming that sucrose is an advantageous excipient for use in the formulation. The results are further confirmed through functional analyses in monocyte derived peripheral blood mononuclear cells that are subject to exposure from lipopolysaccharide (LPS), which shows improved reduction of IL-6 and TNF-alpha.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

Claims

1. A method for manufacturing a modified formulation comprising corticotropin releasing hormone (CRH) and alpha-2 macroglobulin, the method comprising: a. providing a nano-filtered serum obtained by a method comprising: i. subjecting hyperimmune serum from an ungulate to a microfiltration step that removes molecules having a size greater than 0.2 microns, thereby providing a micro-filtered serum; and ii. subjecting said micro-filtered serum to a nanofiltration step that removes molecules having a size greater than 35 nanometres, thereby providing the nano-filtered serum; and b. adding alpha-2 macroglobulin to the nano-filtered serum, thereby providing the modified formulation comprising CRH and alpha-2 macroglobulin.

2. The method according to claim 1, the method comprising: a. subjecting hyperimmune serum from an ungulate to a microfiltration step that removes molecules having a size greater than 0.2 microns, thereby providing a micro-filtered serum; b. subjecting said micro-filtered serum to a nanofiltration step that removes molecules having a size greater than 35 nanometres, thereby providing the nano-filtered serum; and c. adding alpha-2 macroglobulin to the nano-filtered serum, thereby providing the modified formulation comprising CRH and alpha-2 macroglobulin.

3. The method according to claim 1 or 2, further comprising the step of adjusting the final protein concentration of the nano-filtered serum to 4-5 milligrams protein per millilitre.

4. The method according to any one of the preceding claims, wherein alpha-2 macroglobulin is added to the formulation to a final concentration of greater than 0.2 mM, preferably at least 0.5 mM.

5. The method according to any one of the preceding claims, wherein alpha-2 macroglobulin is added to the formulation to a final concentration of at least 1 mM.

6. A formulation (e.g. obtainable by the method of any one of the preceding claims) for administration to a human subject, the formulation comprising:

80-120 mg/ml corticotropin releasing hormone (CRH); and alpha-2 macroglobulin at a concentration of greater than 0.2 mM.

7. A formulation (e.g. obtainable by the method of any one of claims 1-5) for administration to a human subject, the formulation comprising corticotropin releasing hormone (CRH) and alpha-2 macroglobulin, wherein a concentration ratio of alpha-2 macroglobulin to CRH is greater than 1,088,333:1.

8. The method according to any one of claims 1-5 or the formulation according to claim 7, wherein the concentration ratio of alpha-2 macroglobulin to CRH is at least 2,725,000:1.

9. The method according to any one of claims 1-5 or the formulation according to claim 7 or 8, wherein the concentration ratio of alpha-2 macroglobulin to CRH is at least 5,441,667:1.

10. The method or formulation according to any one of the preceding claims, wherein the alpha-2 macroglobulin is present at a concentration of at least 0.5 mM, preferably wherein the alpha-2 macroglobulin is present at a concentration of at least 1 mM, more preferably wherein the alpha-2 macroglobulin is present at a concentration of at least 2.5 mM.

11. The method or formulation according to any one of the preceding claims, wherein the alpha-2 macroglobulin is present at a concentration of greater than 5 mM.

12. A formulation (e.g. obtainable by the method of any one of claims 1-5 or 8-11) for administration to a human subject, the formulation comprising corticotropin releasing hormone (CRH) and alpha-2 macroglobulin, wherein the alpha-2 macroglobulin is present at a concentration of greater than 5 mM.

13. The method or formulation according to any one of the preceding claims, wherein CRH is present in the formulation at a concentration of 80-120 mg/ml.

14. The method or formulation according to any one of the preceding claims, wherein CRH binding protein (CRH-BP) is present in the formulation at a concentration of 30-60 mg/ml.

15. The method or formulation according to any one of the preceding claims, wherein the alpha-2 macroglobulin is present at a concentration of 6-100 mM.

16. The method or formulation according to any one of the preceding claims, wherein the alpha-2 macroglobulin is present at a concentration of 10-30 mM.

17. The formulation according to any one of claims 6-16, comprising: a. a nano-filtered serum comprising corticotropin releasing hormone (CRH) that has been obtained by a method comprising: i. subjecting hyperimmune serum from an ungulate to a microfiltration step that removes molecules having a size greater than 0.2 microns, thereby providing a micro-filtered serum; and ii. subjecting said micro-filtered serum to a nanofiltration step that removes molecules having a size greater than 35 nanometres, thereby providing the nano-filtered serum; and b. alpha-2 macroglobulin.

18. The method or formulation according to any one of the preceding claims, wherein the nanofiltration step has been carried out using a 35 nanometre filter, for example using a 35 nanometre hollow fibre filter.

19. The method or formulation according to any one of the preceding claims, wherein the alpha-2 macroglobulin (e.g. the added alpha-2 macroglobulin) is a non-ungulate alpha-2 macroglobulin.

20. The method or formulation according to any one of the preceding claims, wherein the formulation comprises an ungulate alpha-2 macroglobulin and a non-ungulate (e.g. added) alpha-2 macroglobulin.

21. The method or formulation according to any one of the preceding claims, wherein the alpha-2 macroglobulin (e.g. the added alpha-2 macroglobulin) is a human alpha-2 macroglobulin.

22. The method or formulation according to any one of the preceding claims, wherein the alpha-2 macroglobulin (e.g. the added alpha-2 macroglobulin) is a recombinant alpha-2 macroglobulin.

23. The method or formulation according to any one of the preceding claims, wherein pro-opiomelanocortin (POMC) peptide is present in the formulation at a concentration of between 140 picomoles per litre (pmol/l) and 200 pmol/l.

24. The method or formulation according to any one of the preceding claims, wherein an antioxidant is added to the nano-filtered serum or formulation (preferably to the formulation).

25. The method or formulation according to any one of the preceding claims, wherein the formulation comprises an antioxidant.

26. The method or formulation according to any one of the preceding claims, wherein sucrose is added to the nano-filtered serum or formulation (preferably to the formulation), preferably wherein sucrose is added to a final concentration of about 1- 1000 nM, more preferably about 10-500 nM.

27. The method or formulation according to any one of the preceding claims, wherein the formulation comprises sucrose, preferably wherein the formulation comprises sucrose at a final concentration of about 1-1000 nM, more preferably about 10-500 nM.

28. A formulation according to any one of claims 6-27 for use in treating one or more disorder(s) selected from the group consisting of: an inflammatory disorder such as rheumatoid arthritis; optic neuritis; motor neuron disease; autoimmune diseases including lupus, psoriasis, eczema, thyroiditis, and polymyositis; 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; systemic sclerosis (SSc); Parkinson’s disease; Huntingdon’s chorea; mixed connective tissue diseases; scleroderma; anaphylaxis; septic shock; carditis and endocarditis; wound healing; contact dermatitis; occupational lung diseases; obesity; nerve conduction disorders; sexual dysfunction, in particular erectile dysfunction ; 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, CMT1B, 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, SCA11, SCA12, SCA13, SCA14, SCA16; Spinocerebellar Ataxia; Cockayne’s syndrome; Giant axonal neuropathy; chronic inflammatory demyelinating polyneuropathy (Cl DP); and Guillain- Barre syndrome.

29. A method of treating one or more disorder(s), the method comprising administering a formulation according to any one of claims 6-27 to a subject, wherein the one or more disorder(s) are selected from the group consisting of: an inflammatory disorder such as rheumatoid arthritis; optic neuritis; motor neuron disease; autoimmune diseases including lupus, psoriasis, eczema, thyroiditis, and polymyositis; 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; systemic sclerosis (SSc); Parkinson’s disease; Huntingdon’s chorea; mixed connective tissue diseases; scleroderma; anaphylaxis; septic shock; carditis and endocarditis; wound healing; contact dermatitis; occupational lung diseases; obesity; nerve conduction disorders; sexual dysfunction, in particular erectile dysfunction ; 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, CMT1B, 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, SCA11, SCA12, SCA13, SCA14, SCA16; Spinocerebellar Ataxia; Cockayne’s syndrome; Giant axonal neuropathy; chronic inflammatory demyelinating polyneuropathy (Cl DP); and Guillain- Barre syndrome.

30. Use of a formulation according to any one of claims 6-27 in the manufacture of a medicament for treating one or more disorder(s) selected from the group consisting of: an inflammatory disorder such as rheumatoid arthritis; optic neuritis; motor neuron disease; autoimmune diseases including lupus, psoriasis, eczema, thyroiditis, and polymyositis; 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; systemic sclerosis (SSc); Parkinson’s disease; Huntingdon’s chorea; mixed connective tissue diseases; scleroderma; anaphylaxis; septic shock; carditis and endocarditis; wound healing; contact dermatitis; occupational lung diseases; obesity; nerve conduction disorders; sexual dysfunction, in particular erectile dysfunction ; 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, CMT1B, 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, SCA11, SCA12, SCA13, SCA14, SCA16; Spinocerebellar Ataxia; Cockayne’s syndrome; Giant axonal neuropathy; chronic inflammatory demyelinating polyneuropathy (Cl DP); and Guillain-Barre syndrome.

31. The formulation for use according to claim 28, the method according to claim 29 or the use according to claim 30, wherein the disorder is one or more selected from Alzheimer’s disease; systemic sclerosis (SSc); multiple sclerosis; rheumatoid arthritis; optic neuritis; motor neuron disease; hepatitis, in particular hepatitis C; 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; Parkinson’s disease; inflammatory conditions; obesity; nerve conduction disorders; and sexual dysfunction, in particular erectile dysfunction, preferably wherein the disorder is one or more selected from Alzheimer's disease, vasculitis, systemic sclerosis, inflammatory bowel disease, arthritis, diabetes mellitus, multiple sclerosis, and obesity.