Formulation Comprising A Stabilized Complex Of Corticotropin Releasing Hormone And Alpha-2 Macroglobulin

Abstract

The present invention relates to a CRH formulation having improved stability/efficacy. The improved CRH formulation is particularly suitable for treatment of various disorders. The invention also relates to a method of producing the CRH formulation, and to methods of treatment using said CRH formulation.

Claims

1. A method of manufacturing a stabilised complex of corticotropin releasing hormone (CRH) and alpha-2 macroglobulin, the method comprising: (a) providing hyperimmune serum from an ungulate that has been immunised 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) aliquoting said nano-filtered serum into a vial, wherein said nano-filtered serum is in a form for administration to a patient and comprises the stabilised complex of CRH and alpha-2 macroglobulin; wherein the serum remains unfrozen throughout steps (b) to (d).

2. The method of claim 1, wherein the serum is frozen after step (d) and thawed prior to patient administration, though with the proviso that said serum is not subjected to a subsequent freezing step prior to said patient administration.

3. The method of claim 1, wherein the nanofiltration step is carried out using a 35 nanometre filter, for example using a 35 nanometre hollow fibre filter.

4. The method of claim 1, further comprising the step of adjusting the final protein concentration to 4-5 milligrams protein per millilitre.

5. The method of claim 4, wherein the hyperimmune serum is only frozen after said adjusting of the final protein concentration.

6. The method of claim 1, wherein exposure to ambient temperature is strictly minimised at all stages of the method.

7. A formulation comprising a stabilised complex of CRH and alpha-2 macroglobulin, wherein the formulation is produced by the method of claim 1.

8. The formulation of claim 7, comprising more than 50,000 pg/ml of alpha-2 macroglobulin.

9. The formulation of claim 8, comprising between 100,000 pg/ml and 150,000 pg/ml of alpha-2 macroglobulin.

10. The formulation of claim 7, wherein the formulation when administered to a patient results in an in vivo CRH expression concentration that is greater after 36 hours than after 48 hours from the time of administration.

11. The formulation of claim 8, comprising 80-120 pg/ml of CRH.

12. The formulation of claim 11, comprising 90-110 pg/ml of CRH.

13. The formulation of claim 8, wherein the CRH is non-human.

14. The formulation of claim 8, further comprising one or more stabilisers.

15. (canceled)

16. The formulation of claim 8, further comprising a pro-opiomelanocortin (POMC) peptide.

17. The formulation of claim 16, wherein the POMC peptide is non-human.

18. The formulation of claim 16, comprising at least 140 pmol/L of POMC peptide.

19. The formulation of claim 8, further comprising one or more of vasopressin, ACTH, MSH such as alpha-MSH, -MSH, and -MSH, LPH such as -LPH and -LPH, -endorphin, enkephalin such as met-enkephalin and leu-enkephalin, CLIP, and Lipotrophin-gamma.

20. The formulation of claim 8, further comprising CRH binding protein (CRH-BP).

21. (canceled)

22. A method of treatment for a disease 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; the method comprising administering the formulation of claim 8 to a patient in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0076] These and other aspects of the present invention will now be described by way of example only with reference to the accompanying drawings, in which:

[0077] FIGS. 1A, 1B, 1C, 2A, 2B, and 2C show in vivo change in CRH concentrations in pg/ml (FIG. 1A to 1C) and in vivo change in CRH binding protein concentrations (FIG. 2A to 2C) in mice treated with either the composition of the invention Aimspro (1A, 2A), nave serumthough otherwise prepared by exactly the same manufacture protocol as the present invention Nave serum (1B, 2B), or CRH formulation prepared by the present applicant's prior art manufacture protocol Prior art CRH formulation (1C, 2C);

[0078] FIGS. 3 and 4 show mass spectrometry analyses of patient sera before and after treatment with the stabilised complex of the invention;

[0079] FIGS. 5 and 6 show in vivo activity of the stabilised complex of the invention following treatment;

[0080] FIGS. 7, 8, 9, and 10 show evidence for a switch in inflammatory profile of patients following treatment with the stabilised complex of the invention;

[0081] FIG. 11 shows levels of CRH in human serum and the stabilised complex of the invention;

[0082] FIG. 12 shows a summary statistical table of absolute CRH levels and changes relative to baseline (picograms per millilitre) in patients with diffuse systemic sclerosis, measured at baseline and at 26 weeks, following a twice-weekly treatment with either the composition of the invention or placebo (controls);

[0083] FIG. 13 shows a plot of the difference between the baseline (pre-treatment) level of CRH and the final level of CRH in patients with diffuse systemic sclerosis, measured at baseline and at 26 weeks, following a twice-weekly treatment with either the composition of the invention or placebo;

[0084] FIG. 14 shows a summary statistical table of absolute CRH levels (nanograms per millilitre) in patients with secondary progressive multiple sclerosis, measured at baseline and at 4 weeks following a twice-weekly treatment with either the composition of the invention or placebo (controls).

EXAMPLES

[0085] CRH and products containing CRH are very sensitive to proteolytic degradation and suffer from the above-mentioned poor effective half-life following in vivo administration. We show here that the stabilized complex of the invention has improved in vivo stability and, in view of said enhanced biological half-life, is able to demonstrate a greater therapeutic efficacy.

Example 1: Manufacture of the Stabilised Complex of the Invention

[0086] Hyperimmune ungulate serum is centrifuged to separate any unwanted components, and the method carried out as a continuous process, avoiding any freezing or thawing step(s) prior to final aliquoting. This avoids any aggregation and loss of the CRH component from the formulation.

[0087] In more detail, a serum composition comprising CRH was stored at 2 to 8 degrees C. (and not frozen) and was diluted at a ratio of 1:2 parts cold PBS, and supersaturated ammonium sulphate was added slowly with constant agitation until a ratio of 47:53 of ammonium sulphate:PBS was reached. This was carried out on a cold tray and the resulting solution was maintained at this temperature for 30 to 60 minutes with constant agitation.

[0088] The serum solution was then centrifuged in a Beckman J6M/E centrifuge at 3500 rpm for 45 minutes at 4 degrees C. The supernatant was removed and discarded. The precipitated solid material was re-suspended in cold 50% saturated ammonium sulphate:PBS solution and re-centrifuged at 3500 rpm at 4 degrees C. for 45 minutes. The supernatant was again discarded and the precipitated solid material re-suspended in ice cold PBS buffer. This solution (the serum component) was then subjected to diafiltration at 4 degrees C. against PBS with a molecular weight cut-off of 10,000 Daltons.

[0089] A two-stage filtration step was then carried out, whereby the solution was filtered through a 0.2 micrometre filter and was then passed through a 35 nanometre hollow fibre filter. Following the filtration step, the solution was adjusted to a protein concentration of between 4 to 5 milligrams per millilitre with ice-cold PBS.

[0090] Small batches of the solution (1.1 millilitres) containing the stabilised composition of the invention were put into vials in an isolator. 1 millilitre single doses were thus obtained and stored at 15 to 25 degrees C. prior to use.

[0091] All steps were carried out as a continuous process in the cold except nanofiltration and filling into vials, which were both conducted so as to minimise exposure to ambient temperature at all times. Cold trays were used whenever possible. Following treatment of the serum with PBS and supersaturated ammonium sulphate, freezing was avoided until the final filling stage and subsequent storage.

Example 2: The Stabilised Complex Provides a Persistent, Elevated Concentration of CRH In Vivo

[0092] The stabilised complex of the invention has been compared with prior art formulations as previously disclosed by applicant.

[0093] Applicant has disclosed the same basic manufacture protocols in WO 2003/004049, WO 2003/064472, WO 2005/056053, WO 2005/097183, WO 2006/021814, and WO 2007/077465.

[0094] In this Example, male mice, C57BL/6, 25 gm were divided into three groups: one group was administered the stabilised complex of the invention (Aimspro); another group was administered a nave caprine serum (i.e. from a goat that had not been immunised) but which had been otherwise prepared by exactly the same manufacture (including 35 nanometre filtration step) method of the present invention (Nave serum); and the third group was administered a composition comprising a CRH formulation prepared by Applicant's prior art basic manufacture protocol (Prior art CRH formulation).

[0095] Samples of approximately 250 l of whole blood (retro-orbital) were collected via the retroorbital plexus using a microcapillary (EDTA) tube at intervals of 15 minutes, half an hour, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours and 48 hours from initial administration. Li Heparin-whole blood was centrifuged at 4 degrees C. for 10 minutes at a RCF of 1000 and stored at 80 degrees C. until CRH determination was carried out by ELISA.

[0096] The average CRH concentration was calculated in respect of each population and the results are shown in FIG. 1A through 1C.

[0097] It can be seen from FIG. 1A that the composition of the invention (labelled Aimspro) provided a steady, sustained increase in CRH concentration within the population over time. In particular, CRH concentration was still increasing at the 48-hour point.

[0098] By contrast, the nave serum (Nave serumFIG. 1B) and the prior art CRH formulation (Prior art serum formulationFIG. 1C) did not provide a steady expression profile and the level of CRH at the 48-hour point is clearly falling back to initial levels.

[0099] By way of comparison, the level of CRH binding protein (CRH-BP) was also measured and the average result was calculated for each population over time. The results are shown in FIG. 2A through 2C. It can be seen that the expression profile is similar in each population.

[0100] The table below provides a comparison of the composition of the stabilised complex of the present invention (Aimspro) against the composition of the prior art CRH formulation (Prior art CRH formulation).

TABLE-US-00001 COMPONENT Aimspro Prior art CRH formulation CRH-BP (pg/ml) 41.7 98.84 a2M (pg/ml) 122,014 35,738 CRH half-life (hours) 10.2 1

Example 3: Treatment Induces Protein/Peptide Expression in Patients' Sera

[0101] Mass spectrometry of patients' sera was carried out before and after treatment with the stabilised complex of the invention. The spectra from 2 kDa to 10 kDa are compared, as it is this molecular weight range which is associated with the bioactive peptides of interest discussed above. The results of serum analysis in a first patient are shown pre-treatment (FIG. 3A) and post-treatment (FIG. 3B). The results of serum analysis in a second patient are shown pre-treatment (FIG. 3C) and post-treatment (FIG. 3D). A comparison of the profiles in the pre- and post-treatment sera of both patients reveals clear differences in the peptide expression in the 2 kDa to 6 kDa region. For ease of comparison an overlapping view of the profiles is also provided (FIG. 3E). A comparative peptide/protein expression in post-treatment sera of six treated patients is also provided (FIG. 4A to FIG. 4F). When compared to the pre-treatment expression of the two patients in FIG. 3A and FIG. 3C, it can be seen in from FIGS. 4A to 4F that each patient shows increased levels of induced peptide/protein expression particularly in the 4 kD region.

Example 3: Evidence for the In Vivo Activity of the Stabilised Complex of the Invention

[0102] FIG. 5 shows comparative levels of ACTH in the sera of patients before and after receiving treatment with the stabilised complex of the invention. This is also compared with levels of ACTH in the sera of healthy volunteers. Sera were diluted 1:100 and quantified by an ELISA of sera compared with the product. Data are the mean of three determinations+/standard errors. Post treatment n=5; pre treatment n=3; normal human sera n=5. The data show that treatment with the stabilised complex of the invention increases ACTH levels. FIG. 6 shows comparative levels of endorphin in the serum of patients before and after receiving treatment with the stabilised complex of the invention. This is compared with levels of endorphin in the sera of healthy volunteers. Sera were diluted 1:100 and quantified by an ELISA of sera compared with the product. Data are the mean of three determinations+/standard errors. The data show that treatment increases 13 endorphin levels.

Example 4: The Stabilised Complex of the Invention Leads to a Change from Pro-Inflammatory TH-1 Profile to an Anti-Inflammatory TH-2 Cytokine Profile in Treated Patients

[0103] FIG. 7 shows the levels of TGF- in the serum of two groups of patients (group 1, shown in FIG. 7A; and group 2, shown in FIG. 7B) before and after treatment with the stabilised complex of the invention. The two groups of patients (n=3 for each group) show differing responses with respect to the concentrations of TGF- produced, but all patients showed an increase in serum levels in response to treatment (pre sera=patients' serum levels before treatment; post 2nd and post 5th=after the 2nd and 5th administration). The data show that treatment with the stabilised complex of the invention induces increased concentration of the anti-inflammatory cytokine TGF-. FIG. 8 shows the levels of IL-4 in the serum of one group of patients before (pre-sera) and after treatment with the stabilised complex of the invention. It can be seen that after treatment (post 2nd), the levels of IL-4 are significantly increased in the patients' sera (n=5). However, following the 5th administration, the levels of IL-4 had dropped in all patients, but nevertheless remained higher than they had been pre-treatment. IL-4 is known to down-regulate the production of the pro-inflammatory cytokines from TH-I cells. It may be that the consistent changes in concentration seen in all patients are consistent with IL-4's role in the switch from TH-1 to TH-2. FIG. 9 shows the levels of IL-6 in the serum of one group of patients before and after treatment. It can be seen that after treatment (post 2nd and post 5th) the levels of IL-6 are reduced in the patients' sera (n=4). FIG. 10 shows the levels of IFN- in the serum of one group of patients before and after treatment. It can be seen that after treatment (post 2nd and post 5th) the levels of IFN- are reduced in the patients' sera.

Example 5: The Stabilised Complex of the Invention Leads to Induction of CRH

[0104] FIG. 11 shows firstly, the increased presence of CRH in the stabilised complex of the invention by comparison with a placebo (human albumin at a concentration of 4.5 mg/ml); and secondly, the increase in CRH expression in patients treated with the stabilised complex of the invention by comparison with the non-treated individuals. This latter is evidence for the induction of CRH in patients in response to treatment with the stabilised complex of the invention.

[0105] All determinations were made in triplicate+/standard deviations, These data are representative of at least 3 separate experiments. Control individuals n=4; treated patients n=13. The data indicate that the stabilised complex of the invention induces the expression and release of CRH and hence POMC peptides in the patient, which then transform the patients' immunological profile from a TH-1 pro-inflammatory profile to a predominantly TH-2 anti-inflammatory profile.

Example 6: The Stabilised Complex of the Invention Leads to an Increase in CRH Levels in Patients with Diffuse Systemic Sclerosis

[0106] A double-blind placebo control trial was carried out on patients with diffuse systemic sclerosis. The absolute levels of CRH (in micrograms per millilitre) were measured prior to treatment (baseline) and at 26 weeks following treatment. Treatment was carried out in a double-blind placebo control trial, with one group of subjects receiving the stabilised complex of the invention and the other (control) group receiving a placebo (human albumin at a concentration of 4.5 mg/ml). The patients received the treatment twice-weekly

[0107] FIG. 12 shows a three-fold increase (paired T-test, p<0.043) in CRH levels of the patients treated with the stabilised complex of the invention by comparison with those receiving the placebo.

[0108] FIG. 13 shows a plot of the results over the 26 weeks. The difference between the baseline (pre-treatment) level of CRH and the final level of CRH for each patient is shown graphically. The change in average level of CRH between the baseline and week 26 (shown by a bar) is significantly greater for the stabilised complex of the invention than for the placebo (P=0.04).

Example 7: The Stabilised Complex of the Invention Leads to an Increase in CRH Levels in Patients with Progressive Multiple Sclerosis

[0109] A double-blind placebo control trial was carried out on patients with progressive multiple sclerosis. The absolute levels of CRH (in micrograms per millilitre) were measured prior to treatment (baseline) and at 4 weeks following treatment. Treatment was carried out in a double-blind placebo cross-over trial, with each group of patients receiving a twice-weekly treatment of either the stabilised complex of the invention or a placebo (human albumin at a concentration of 4.5 mg/ml) for four weeks, followed by a 6 week washout period, following which those patients previously receiving the placebo were administered the stabilised complex of the invention twice-weekly for 4 weeks; and those patients previously receiving the stabilised complex of the invention were administered the placebo twice-weekly for 4 weeks.

[0110] FIG. 14 shows that for both groups, there was a significant difference (paired T-test, p<0.0023) in levels of CRH following administration of the stabilised complex of the invention, by comparison with the level of CRH following administration of the placebo.

Example 8: The Stabilised Complex of the Invention can be Used in Treating Hepatitis C

[0111] The subject was a 20 year old female patient with a lifelong history of infection with the Hepatitis C virus (HCV), sub-group type 2b.

[0112] The subject had previously been treated with interferon (24 month-long regime, unsuccessful) and with pegylated interferon alpha-2a and ribavirin (18-month long regime, unsuccessful). Prior to this study, the subject had received no intervention for the preceding 3 years.

[0113] Tests confirmed the presence of hepatitis C using HCV antibody enzyme immunoassay and quantitative PCR (1.5*10.sup.6 IU/ml). No jaundice was observed (bilirubin levels of 10 mol/litre) and transaminits was detected at a mildly elevated level (aparatate transaminase AST 33 IU/litre, alanine transaminase 58 IU/litre). Further questioning of the subject revealed clinical signs and symptoms including lethargy, weight loss, malaise, insomnia and fatigue.

[0114] A 1 ml aliquot of the stabilised complex of the invention (4.5 mg/ml concentration) was prepared as described above. This was delivered by intramuscular route on an every other day treatment regimen for an initial six month course.

[0115] At the end of this six months, HCV quantitative PCR showed a drop to 150,000 IU/ml. The dosing regimen was then increased to a daily delivery of the stabilised complex of the invention for a further six month course.

[0116] Subsequent HCV titres were measured at 90,000 IU/ml, falling to 8,000 IU/ml and then (at the end of the further six months) a negative HCV titre, according to quantitative PCT. Normal liver function tests were also observed at the end of the further six month course.