Note: Descriptions are shown in the official language in which they were submitted.
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Medicament
FIELD OF THE INVENTION
The present invention relates to a medicament, and in particular to a
pharmaceutical composition. The medicament is considered particularly suited
to treatment of neural disorders, although a number of other disorders may be
treatable with the invention. Aspects of the invention also relate to methods
of
preparation of such a medicament, and to methods of treatment of disorders
using said medicament.
BACKGROUND TO THE INVENTION
PCT publications W003/004049 and W003/064472 describe therapeutic agents
and treatments which are based on a serum composition with many surprising
beneficial effects. The respective content of each of these two texts is
incorporated in full by specific reference. In particular, the reader is
referred to
them for an understanding of how the therapeutic agent can be prepared, and
for the indications which can be treated.
Typically a goat is immunised with H> V-3B viral lysate raised in H9 cells.
The
resulting serum is believed to be active against, among other disorders,
multiple
sclerosis. The reader is further referred in particular to the section on
pages 3
and 4 of W003/004049 headed 'Example of Production of Goat Serum' for
further details of the production of serum. This section is incorporated
herein by
reference. A brief summary is given below.
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Preparation of serum
Approximately 400 cc of blood is taken from a 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 serum. 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 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 is then subjected to diafiltration against a PBS buffer with a
molecular weight cut-off of 10,000 Daltons. at 4 C. After diafiitration the
product is fiitered through a 0.2 micron filter into a sterile container and
adjusted to a protein concentration of 4mg/ml. The solution is put into vials
to
give single doses of Iml, and stored at -22 C prior to use. This product is
referred to herein as the serum composition, the composition, or the product,
while treatment of a patient involves administering the composition to the
patient by an appropriate route (usually subcutaneously).
The use of 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 supernate 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
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composition. Other suitable immunogens are recited on pages 12 and 13 of
W003/064472.
A pyrogenic material (for example, RIBI or Freund's adjuvant) can be used to
promote production of the active component in the serum. Another possible
factor may be exposure of the animal to daylight, with greater daylight hours
(or exposure to daylight equivalent) may increase active component serum
levels.
Uses of the serum composition
The composition is believed to be effective against a number of disorders, in
particular multiple sclerosis. Reference is also made in the previously-
identified
publications to the composition as being useful in the treatment of
inflammatory diseases such as rheumatoid arthritis; optic neuritis; motor
neurone disease; autoimmune diseases; axonal or nerve damage; and cancers.
The composition is also believed to cause a reduction in viral load in HIV
patients, and an increase in CD4+ cells.
Several other diseases which may be treated by the composition are described,
and the reader is referred to these earlier publications for a full
understanding
of the range and nature of conditions which may be treated. In particular, the
contents of W003/004049 and W003/064472 are specifically incorporated
herein by reference.
A non-exhaustive list of disorders against which the serum composition is
believed to be effective, in addition to those mentioned above, includes
cancers, in particular myelomas, melanomas, and lymphomas; cardiovascular
diseases; and neural disorders, both demyelinating and non-demyelinating.
Examples of disorders which may be treated in accordance with the present
invention include cerebrovascular ischaemic disease; Alzheimer's disease;
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Huntingdon's chorea; mixed connective tissue diseases; scieroderma;
anaphylaxis; septic shock; carditis and endocarditis; wound healing; contact
dermatitis; occupational lung diseases; glomerulnephritis; transplant
rejection;
temporal arteritis; vasculitic diseases; hepatitis; and burns. All of these
disorders may have an inflammatory component, but are believed to be
additionally treatable based on the non-demyelinating neural aspect of the
disorder. Further non-demyelinating disorders which may be treated, and which
are considered to have a degenerative component include multiple system
atrophy; epilepsy; muscular dystrophy; schizophrenia; bipolar disorder; and
depression. Other non-demyelinating disorders which may be treated include
channelopathies; myaesthenia 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.
Demyelinating disorders which may be treatable 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.
The composition may be useful in the treatment of autoimmune diseases
including lupus, psoriasis, eczema, thyroiditis, and polymyositis.
The composition is also believed to be effective against inflammatory
conditions.
The composition is useful in the treatment of all kinds of peripheral
neuropathy
of axonal and demyelinating type, including 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
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to pressure palsies (HNPP) - also called Tomaculous neuropathy; Hereditary
Motor and Sensory Neuropathy with Deafness - Lom (HMSNL); Proximal
Hereditary Motor and Sensory Neuropathy / Neuronopathy (HMSNP); Hereditary
Neuralgic Amyotrophy; Hereditary Sensory and Autonomic Neuropathies
5 (HSANI, 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; Adrerioleukodystrophy; Refsum's disease (HMSN IV); Tangier
Disease; Friedreich's ataxia; Spinal cerebellar ataxia (SCA) all types - SCA1,
SCA2, SCA3, SCA4, SCA5, SCA6, SCA7, SCA8, SCAIO, SCA11, SCA12, SCA13,
SCA14, SCA16; Spinocerebellar Ataxia; Cockayne's syndrome; and Giant axonal
neuropathy.
The composition may also be useful in the treatment of chronic inflammatory
demyelinating polyneuropathy (CIDP), and Guillain-Barre syndrome.
The composition may also have anti-angiogenic properties, caused by the
molecules thrombospondin-1 (TSP-1) and platelet factor-4 (PF-4).
It is believed that the composition may also be effective for treatment of
animals, in particular, but not exclusively, the treatment of canine atopic
dermatitis, canine oral melanoma, and equine pulmonary disorders.
Nature of the serum
Although the serum composition has exhibited many surprising effects, and has
been studied extensively, until now the active component or components of the
serum have not yet been identii"led. This has been disadvantageous, both in
terms of isolating the active component for further study, and in terms of
exploring possible alternative sources of the active component or components.
Further, it has been necessary to administer the treatment to patients as
serum, which necessitates injections, and imposes certain restrictions on the
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handling and processing of the composition. It is believed that the serum has
bioactive components sensitive to protease degradation.
We have now identified a number of potential active components of the serum.
This identification allows the manufacture of novel pharmaceutical
compositions
comprising one or more of the active components in various forms, and the
treatment of one or more of the disorders recited above and in our earlier
patent applications using the active component(s). The identification also
opens
up novel approaches for treating the various disorders based on the active
component(s) and not simply the serum itself.
SUMMARY OF THE INVENTION
The invention resides in a bioactive composition which triggers a molecular
cascade in treated patients.
According to a first aspect of the present invention, there is provided a
pharmaceutical composition comprising a corticotropin releasing factor (CRF)
peptide. CRF is also known as corticotropin releasing hormone (CRH).
CRF is a peptide produced in the hypothalamus, and is believed to be involved
in stress response. Human CRF is described in detail in entry 122560 of OMIM
(online mendelian inheritance in man, accessible through
http=.Ilwww.ncbi.nim.nih.goyL). The nucleotide and amino acid sequence of
human CRF is also known, and has GENBANK accession number BC011031.
Knowledge of the sequence and size data for human CRF will allow the skilled
person to determine the equivalent information for non-human CRF, including
goat CRF.
By "a CRF peptide" is meant any peptide having a corresponding sequence,
structure, or function. It will be apparent to the skilled person that the
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canonical nucleotide and/or amino acid sequences given for human CRF 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; and fragments and derivatives of CRF.
Administration of CRF to a patient is believed to stimulate production of
endogenous CRF, which in turn stimulates production of proopiomelanocortin
(POMC) and its related component peptides.
POMC is a peptide (prohormone) produced in the pituitary gland (as well as a
number of other organs, certain tumours such as melanomas, and normal skin
cells) which is the precursor of a set of corticotrophic hormones which exert
a
number of effects on the host. POMC is the precursor to alpha, beta, and
gamma melanocyte stimulating hormone (MSH); adrenocorticotrophin (ACTH);
beta and gamma lipotropin (LPH); and beta endorphin. All of these hormones
are cleaved from a single large precursor, POMC, and are termed herein "POMC
products".
Preferably the pharmaceutical composition comprises non-human CRF;
conveniently ungulate CRF; and most preferably goat CRF. It has been
surprisingly identified that goat serum contains CRF, particularly when the
goat
is stimulated by physiological stress, such as bleeding or immunization. This
provides a convenient source for CRF for pharmaceutical compositions of the
present invention. It is also believed that CRF may have a self-sustaining
effect
in the patient, in that administration of an initial amount of CRF leads to
endogenous production of CRF in the patient; thus, an initial administration
of a
low level of CRF may have a significant effect on the patient, including an
increase in the levels of POMC peptides.
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Administration of pharmaceutical compositions of the invention may be
accomplished orally or parenterally. Methods of parenteral delivery include
topical, intra-arterial, intramuscular, subcutaneous, intramedullary,
intrathecal,
intraventricular, intravenous, intraperitoneal, or intranasal administration.
In
addition to the active ingredients, such compositions may comprise suitable
pharmaceutically acceptable carriers comprising excipients and other
components which facilitate processing of the active compounds into
preparations suitable for pharmaceutical administration.
Pharmaceutical compositions for oral administration can be formulated using
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.
Pharmaceutical preparations 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 methylcelfulose, hydroxypropylmethylcellulose,
or sodium carboxymethylcel[u[ose; and gums including arabic and tragacanth;
as well as proteins such as gelatin and coliagen. 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
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added to the tablets or dragee coatings for product identification or to
characterise the quantity of active compound.
Pharmaceutical preparations which can be used orally 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 1=liler 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.
Pharmaceutical formulations for parenteral administration include aqueous
solutions of active compounds. For injection, the pharmaceutical compositions
of the invention may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hanks'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 pharmaceutical compositions of the present invention can be manufactured
substantially in accordance with standard manufacturing procedures known in
the art.
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The composition may also comprise one or more peptide regulatory or releasing
factors, which may induce a cascade of release of further peptides by a
variety
of cells in the patient. Such additional factors are preferably derived from
the
5 same source as the CRF, in particular goat serum. Suitable factors include
aW
HLA, TGF-p, and IL-10, among others.
In preferred embodiments, the composition may comprise one or more of
vasopressin, beta endorphin, and an enkephalin. In certain embodiments, the
10 composition may comprise CRF binding protein, CRF-BP. This binds CRF and
may act as a reservoir for subsequent release of CRF to the patient.
The composition may further comprise a POMC peptide or a POMC product;
certain POMC products may be useful to administer to a patient to stimulate
further production, or to obtain a desired response before endogenous POMC
can be produced.
Human POMC is described in detail in entry 176830 of OMIM (online mendelian
inheritance in man, accessible through http=//www.ncbi.nlm.nih.gov/). The
nucleotide and amino acid sequence of human POMC is also known, and has
GENBANK accession number BC065832. Human POMC gives rise to a
glycosylated protein precursor having a molecular weight of 31 kDa.
By "a POMC peptide" is meant any peptide having a corresponding sequence,
structure, or function. It will be apparent to the skilled person that the
canonical nucleotide and/or amino acid sequences given for human POMC in
the GENBANK entry referenced above may be varied to a certain degree
without affecting the structure or function of the peptide. In particular,
allelic
variants and functional mutants are included within this definition. Mutants
may
include conservative amino acid substitutions. "A POMC peptide" as used herein
refers to any peptide acting as a precursor to at least one form of MSH, ACTH,
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at least one form of LPH, p endorphin, met-enkephalin and leu-enkephalin; and
preferably all of a, p, and y MSH; ACTH; p and y LPH; and p endorphin, met-
enkephalin and leu-enkephalin
Although research has been carried out into the pharmaceutical potential of
certain of the individual products of the POMC peptide, it is believed that
administration of POMC itself has not been determined to have any medical
use. It is possible that POMC itself is inactive, and must be cleaved into its
products before having an activity.
Preferably the pharmaceutical composition comprises non-human POMC;
conveniently ungulate POMC; and most preferably goat POMC. Although POMC
is produced in the pituitary gland, and so would not be expected to be present
in serum, at least at signihcant levels, it has been surprisingly identified
that
goat serum contains POMC, POMC-related peptides, and molecules associated
with the POMC cascade, particularly when the goat is stimulated by
physiological stress, such as bleeding or immunization. This provides a
convenient source for POMC for pharmaceutical compositions of the present
invention. It is also believed that POMC may have a self-sustaining effect in
the
patient, in that administration of an initial amount of POMC leads to
endogenous production of POMC in the patient; thus, an initial administration
of
a low level of POMC may have a significant effect on the patient.
It is believed that, on administration of POMC and its associated molecules to
a
subject, the peptide is proteolysed to provide one or more of the products of
POMC in a readily available form to the subject; there is also the induction
of a
molecular cascade which stimulates the hypotha[amo-pituitary-adrenal axis
(HPA). This would be consistent with previously observed effects of the
unpurihed goat serum; for example, a rapid 'buzz' effect on sublingual
administration may be due to proteolysis of POMC to release p endorphin,
which can then be absorbed through the mucous membranes. In addition,
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alpha MSH is known to have an effect on TL-10 and TGF-p production, which
results in an anti-inflammatory effect, consistent with that which has been
observed with goat serum. Alpha MSH is also known to inhibit the release of
pro-inflammatory cytokines.
The composition may also have anti-inflammatory properties. We believe there
is a passive transfer of anti-inflammatory response from the goat or other
animal used to produce the serum to the patient. This is a consequence of the
puriflcation process used to prepare the composition, in which a variety of
active factors are retained in the serum. The composition may also comprise
additional active components which provide an anti-inflammatory effect.
As mentioned above, it is believed that an initial administration of POMC
(optionally together with CRF and/or vasopressin) may stimulate native
production of POMC and its regulatory peptides. A further aspect of the
present
invention therefore provides a method of stimulating POMC production in a
patient, comprising administering exogenous POMC to the patient. The
exogenous POMC is preferably non-human, and more preferably goat POMC.
Conveniently, administration is subcutaneous; this gives a subcutaneous depot
of active composition for subsequent slow release into the patient's system.
A further aspect of the present invention provides a pharmaceutical
composition
comprising a POMC peptide.
According to a further aspect of the invention, there is provided a
pharmaceutical composition comprising two or more of alpha, beta, and gamma
melanocyte stimulating hormone (MSH); adrenocorticotrophin (ACTH); beta and
gamma lipotropin (LPH); and beta endorphin. Given the likely proteolysis of
POMC on administration, it may be possible to achieve similar effects by
administration of two or more of the individual hormones derived from POMC.
The pharmaceutical composition may provide the recited hormones as
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individual peptides, or as one or more precursor molecules (for example,
partial
breakdown products of POMC). Preferably three, four, five, six, or seven of
the
hormones are included in the pharmaceutical composition which (optionally
together with CRF) induce a cascade for continued production of such
molecules. The various components may be provided in combination with one
or more carrier molecules which bind one or more of the components, and so
act as a depot or reservoir for release of the component. A carrier molecule
may also be used in combination with POMC and its related peptides.
According to a further aspect of the present invention, there is provided a
method of treatment for a disease selected from multiple sclerosis; rheumatoid
arthritis; optic neuritis; motor neurone disease; autoimmune diseases
including
lupus, psoriasis, eczema, thyroiditis, and polymyositis; axonal or nerve
damage;
cancers, in particular myelomas, melanomas, and lymphomas; neural disorders,
both demyelinating and non-demyelinating; inflammatory conditions; obesity;
nerve conduction disorders; and sexual dysfunction, in particular erectile
dysfunction; the method comprising administering CRF to a patient in need
thereof. Alternatively, the method may comprise administering POMC.
The optimal dosage of the treatment has not yet been determined; however it
may be appropriate to administer the treatment in a dosage of between 0.01
and 10 mg/kg to the subject; more preferably between 0.01 and 5 mg/kg,
between 0.025 and 2 mg/kg, and most preferably between 0.05 and 1 mg/kg.
In preliminary studies, a serum product has been administered to patients with
a total protein concentration of 4 mg/mi.
The precise dosage to be administered may be varied depending on such
factors as the age, sex and weight of the patient, the method and formulation
of administration, as well as the nature and severity of the disorder to be
treated. Other factors such as diet, time of administration, condition of the
patient, drug combinations, and reaction sensitivity may be taken into
account.
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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, preferably by
subcutaneous injection, although alternative routes which may be used include
intramuscular or intralesional injection, oral, aerosol, parenteral, or
topical.
The treatment is preferably 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
1s or parenteral administration.
The invention also provides the use of CRF in the preparation of a medicament
for the treatment of one or more of the diseases recited above. Also provided
is
the use of POMC in the preparation of a medicament for the treatment of one
or more of the diseases recited above. The CRF or the POMC may be isolated,
purifled CRF or POMC, although it is preferred that they are administered in
combination with the various other components as discussed above. In
particular, bioactive carrier proteins and vasopressin may be used.
According to a further aspect of the present invention, there is provided a
method of producing CRF, the method comprising the steps of obtaining a
blood sample from a goat; separating the serum from the remaining blood
components; and purifying the serum by precipitation of solids.
The precipitate may further be resuspended in a physiologically acceptable
buffer; for example, PBS buffer. The resuspended precipitate may further be
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purified by dialysis or diai=litration; for example, with a molecular weight
cut-off
of 50,000 Da, preferably 40,000 Da, and more preferably 31,000 Da.
The precipitate may undergo further purification to isolate CRF; for example,
5 antibody or other affinity puriflcation. CRF may be bound by antibodies
raised
against CRF, or by use of CRF-BP.
Separation of the serum may be achieved by centrifugation.
10 The serum may further be purihed by viral flitration; it is preferred that
any
purii=lcation method used does not inactivate or remove any of the bioactive
components of the serum, which may include components other than CRF /
POMC.
15 Precipitation may be carried out by ammonium sulphate precipitation, or by
caprylic acid purification. Other suitable precipitating agents may be used.
Preferably the goat is an immunized goat. It is believed that immunization of
the goat stimulates the production of CRF and vasopressin, such that it is
present in the serum in higher levels. The method may also comprise the step
of immunizing a goat. Alternatively, the goat may be subject to physiological
stress, for example, bleeding.
It is also believed that, in some circumstances, the use of an immunogen may
not be necessary, and that useful product may be obtained from a non-
immunised animal (that is, one which has not been pre-immunised with a
specific immunogen). It is accepted that a normal goat may well have been
previously exposed to environmental immunogens, and such goats may be used
in preparation of the compositions of the present invention. The present
invention is therefore intended also to encompass pharmaceutical compositions
comprising serum obtained from a non-immunised goat. The invention also
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extends to uses of such compositions or such serum in the treatment of, or in
the preparation of medicaments for the treatment of, the various diseases or
disorders recited above in the section headed "uses of the serum composition".
The invention still further extends to the preparation of POMC and/or CRF from
serum obtained from a non-immunised goat.
BRIEF DESCRIPTION OF THE DRAWINGS
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:
Figures I. to 4 show mass spectrometry analyses of tryptic digests of serum
components;
Figures 5 to 7 show mass spectrometry analyses of patient sera before and
after treatment with the composition;
Figures 8 and 9 show analyses of induction of POMC peptides by treatment with
the composition;
Figures 10 to 14 show evidence for a switch in inflammatory profile of
patients
following treatment with the composition;
Figure 15 shows levels of vasopressin in human serum and the composition;
Figure 16 shows levels of CRF in human serum and the composition; and
Figure 17 shows a summary diagram of the proposed elements of the
composition and their method of action.
DETAILED DESCRIPTION OF THE INVENTION
Preparation of serum composition
Approximately 400 cc of blood is taken from a 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 serum. The blood is then centrifuged to separate the
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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 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)
sufticient to redissolve the precipitate.
The solution is then subjected to diafiltration against a PBS buffer with a
1o molecular weight cut-off of 10,000 Daltons. at 4 C. After diafiltration the
product is flltered through a 0.2 micron filter into a sterile container and
adjusted to a protein concentration of 4mg/ml. The solution is put into vials
to
give single doses of lml, and stored at -22 C prior to use.
Discussion
The effects of the serum have been previously described, while determination
of the active components has not previously been effected.
Analysis of serum composition
A sample of the composition was size fractionated on a gel, and a Western blot
performed using antibodies to p endorphin. A strong signal was detected,
indicating the presence of p endorphin, although the apparent molecular weight
was approximately 31 kDa, far larger than the expected size of (3 endorphin.
This suggested that p endorphin was present in the sample as part of a larger
peptide; the size being consistent with that of POMC.
We have also carried out mass spectrometry on the composition, and have
detected at least two POMC-derived peptides, p endorphin and corticotrophin-
related molecules. CRH-BP (corticotropin releasing hormone binding protein)
has also been identified.
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Fiaures 1 to 4
POMC peptides and CRF-BP have been identii=led in the product by
Thermofinnegan LCQ mass spectrometry. CRF mainly regulates the synthesis
and secretion of ACTH in the anterior pituitary. The administration of POMC
and/or its component peptides in addition to CRF and CRF-BP is thought to
initiate a cascade effect thus enhancing the production of systemic and
sustained elevated concentrations of POMC peptides. CRF-BP has the ability to
act as a reservoir for CRF.
Figures I to 4 show the hits obtained from mass spectrometry analysis of
tryptic digests from the product separated from contaminating proteins by SDS-
PAGE. As mentioned above, some of these molecules are inducers and
regulators of the POMC cascade. Further investigation using more focused
analysis (e.g. peptide fractionation, immunoprecipitation and concentration)
will
reveal more of the peptides present. Figure 1 indicates the presence of a
POMC-derived corticotropin, Figure 2 that of CRF-BP, Figure 3 that of
proenkephalin A, and Figure 4 that of proenkephalin B. The presence of CRF-BP
suggests that the product contains some CRF, while POMC and related peptides
are also clearly present.
We have also investigated the effects of treatment with the serum composition
on patients' own sera. These effects are described below.
Treatment induces protein/peptide expression in patients' sera
Figure 5 shows mass spectrometry of patients' sera before and after treatment.
The spectra from 2 to 10 kD are compared. This molecular weight range is
associated with the bioactive peptides of interest. Clear differences in the
peptide expression in the 2 to 6 kD region can be seen by comparing the
profiles in the pre and post treatment sera. For ease of comparison an
overlapping view of the profiies is also provided.
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Figure 6 shows comparative peptide / protein expression in six treated
patients.
Each patient shows increased levels of induced peptide / protein expression
particularly in the 4 kD region.
Figure 7a shows the mass spectrometry profiles of unprocessed goat serum
before vaccination (pre-immune profile, top panel), unprocessed serum 53 days
post-immunisation, and the processed product. It can be seen that in the lower
two panels the profile of the serum is significantly different to that of the
pre-
immune profile, indicative of the induction of protein expression. The
profiles
present here represent the active product, and a specific immunisation / bleed
protocol has been shown to be useful in the induction of this serum profile.
An
overlapping view of the profiles is shown (Figure 7b).
Evidence for the induction of PDMC e tides
Figure 8 shows comparative levels of ACTH in the sera of patients before and
after receiving treatment. This is also compared with levels of ACTH in serum
from healthy volunteers and in the product administered to patients. Sera were
diluted 1:100 and quantihed 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 increases ACTH levels.
Figure 9 compares levels of R endorphin in the serum of treated patients with
that in the sera of the same patients before treatment. This is compared with
levels in the sera of healthy volunteers and in the product. 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 p endorphin levels.
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Evidence for a switch from a pro-inflammatory TH-1 profile to an anti-
inflammatory TH-2 c~,tokine profile in treated patients
Figure 10 shows the levels of TGi=-P in the serum of two groups of patients
before and after treatment. The two groups of patients (n=3 for each group)
5 show differing responses with respect to the concentrations of TGF-D
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 induces
increased concentration of the anti-inflammatory cytokine TGF-p.
Figure 11 shows the levels of IL-4 in the serum of one group of patients
before
(pre-sera) and after treatment. It can be seen that after treatment (post
znd),
the levels of IL-4 are signil=lcantly increased in the patients' sera (n=5).
However, following the 5th administration, the levels of IL-4 had dropped in
all
patients, but remained higher than they had been pre-treatment. IL-4 is known
to downregulate the production of the pro-inflammatory cytokines from TH-1
cells. It may be that the consistent changes in concentration seen in all
patients
is consistent with IL-4's role in the TH-1 to TH-2 switch.
Figure 12 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 5
th)
the levels of IL-6 are reduced in the patients' sera (n=4).
Figure 13 shows the levels of IFN-y in the serum of one group of patients
before and after treatment. It can be seen that after treatment (post 2nd and
post 5t") the levels of IFN-y are reduced in the patients' sera.
Figure 14 shows that treatment of human peripheral blood cells (PBMCs)
induces the production of the anti-inflammatory cytokine IL-iQ in the monocyte
sub population. T and B lymphocytes and monocytes were separated from
PBMCs obtained from human volunteers. All cell types were treated with
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equivalent doses of product for 16h, and their supernatants assayed for IL-10
content using ELISA. It can be seen that IL.-10 levels produced by the T cell
population were unaffected by treatment and that only a small increase in IL-
10
was induced in the B cells. However, a significant elevation of IL-10
concentration was induced in the monocytes population by the treatment. All
determinations were made in triplicate +/- standard deviations. These data are
representative of at least three separate experiments.
Evidence for vasopressin and CRF induction
Figure 15 shows the comparative levels of vasopressin in the product, control
patients and patients treated with the product and pre-treatment. The figure
shows that there is no significant difference between any of the serum groups,
however the product contains significant levels of vasopressin, sufficient to
elicit
a response in the patients. It is known that vasopressin acts synergistically
with
CRF to release POMC. All determinations were made in triplicate +/- standard
deviations. These data are representative of at least 3 separate experiments.
Patients pre-treatment n=3; treated patients n= 6.
Figure 16 shows the increased presence of CRF in the product compared with
the placebo and the increase in the treated patients compared with the non-
treated individuals; the latter is evidence for the induction of CRF in the
patients in response to treatment. 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.
Summary and conclusions
Although preliminary, the evidence to date is therefore consistent with the
major active component being CRF acting in concert with other components,
which is thought to induce POMC production. There is also evidence that POMC
itself and POMC-derived peptides may be used as a treatment. This suggests
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new pharmaceutical compositions and uses for CRF and POMC, as well as
indicating additional disorders which may be treatable using CRF and POMC.
We have also provided a convenient method of producing CRF and PDMC from
goats.
The data so far suggests that the product not only contains CRF, POMC
peptides and anti-inflammatory cytokines (iL-10 and TGF-(3) but also induces
the expression and release of CRF and hence POMC peptidds in the patient,
which then transform the patients' immunological profile from a TH-1 pro-
inflammatory profle to a predominantly TH-2 anti-inflammatory profile.
Other observations on the composition effects are consistent with the active
component being CRF which leads to POMC production. For example, effects on
leukocyte adherence may be attributable to beta endorphin. The serum product
increases IL-10 production by human PBMC; alpha MSH affects IL-10
production. Effects on nerve conduction and neuroprotective effects may be
ascribed to ACTH and vasopressin; effects on appetite may be due to alpha
MSH. The product itself also contains significant levels of IL-10 and TGF-p
(data
not shown).
Alpha MSH has potent anti-inflammatory effects in all major forms of
inflammation and it antagonises the effects of pro-inflammatory cytokines such
as TNFa and ILi-(3. Cross talk exists between the cytokine systems and the
POMC system which has been observed in patients treated with the composition
to result in the reduction of pro-inflammatory cytokines and the establishment
(retained over the course of treatment) of a TH-2 anti-inflammatory cytokine
profile including elevated levels of IL-Ip and TGF-p. We have also identified
increased levels of ILI.-P in the serum product.
The serum product has previously been shown to be very sensitive to
proteolytic degradation; this is consistent with the theory that the POMC is
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proteolysed to give individual hormones on administration, but that further
degradation destroys activity. In particular, alpha MSH is believed to have
significantly reduced activity if a terminal tripeptide sequence is removed;
again, this is consistent with the active component including POMC. The
product itself is unstable by nature as its active components are short-lived,
but
exhibit powerful effects.
We have also conducted experiments which suggest that the serum modulates
nitric oxide production by leukocytes; this is consistent with effects of beta
endorphin. We also believe that the serum inhibits PHA-induced PBMC
proliferation, suggesting an explanation for the serum's immunomodulatory
effects. We have also seen a reduced response of PBMCs in the presence of the
product to LPS-induced stimulation and mixed lymphocyte reactions (data not
shown).
The product may also induce tyrosine phosphorylation in human brain microglial
cells, and has been shown by Western blotting to modulate the NFicB pathway
(data not shown). NFxB is known to regulate the transcription of genes
involved in the regulation of pro-inflammatory cytokines, hence the inhibition
of
NFicB would act to reduce the pro-inflammatory cytokine response in
autoimmune disease and reduce inflammatory responses. Further experiments
to investigate this are underway.
Receptors (MCR3 and MCR4) for some POMC peptides are found in the retinal
ganglion cells that form the optic nerve and may be stimulated by POMC
peptides produced after treatment. This may account for some of the rapid
improvements in vision experienced by MS patients with optic neuritis which
have previously been described. It is known that ACTH triggers the
corticosteroid pathway which can exert effects in as little as 20 to 30
minutes.
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Preliminary data suggests that the concentrations of the peptides in the
product
may be insufficient to elicit therapeutic responses in patients after dilution
in
the blood volume of the patient. However, the product could act locally (as it
is
injected in a subcutaneous bolus) to induce a biochemical cascade which
triggers the synthesis and release of the bioactive peptides in the treated
patients. It is now known that any medical treatments that interfere with the
product, for example by competing for receptors or blocking molecules in the
HPA should be avoided.
In support of this hypothesis mass spectrometry of the product has identified
additional molecules some of which are involved in the induction and
regulation
of the corticotropin system; namely CRH binding protein and leu-enkephalin,
corticotropin-lipotropin precursor and pro-enkephalin A precursors (see
Figures
I to 4). In addition, and perhaps more importantly, we have discovered that
two of the major POMC peptides are upregulated significantly in treated
patients' sera compared with levels before treatment, and also compared with
levels from healthy control volunteers. This finding, together with
immunological data, suggests that the treatment induces the expression and
release of POMC peptides in the patient, which then transforms the patients'
immunological profile from a TH-1 pro-inflammatory proflfe to a TH-2 anti-
inflammatory pro1=<le. The further elucidation of the cascade mechanism in the
patients is currently under investigation.
It should be noted that although the product is anti-inflammatory in nature it
does not completely inhibit the inflammatory response. Our data suggest that
the product induces a shift from the unfavourable TH-1 cytokine profile seen
in
auto-immune diseases to a more favourable balanced cytokine level. This may
appear initially after treatment as a rapid anti-inflammatory TH-2 shift as
the
TH-1 network is turned off. Later on after treatment the TH-1 network operates
albeit at a lower level.
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The reported effects of the serum product on tumours leads us to consider the
possibility of anti-angiogenic effects of the serum. In this regard, the
proteins
thrombospondin-1 (TSP-1) and platelet factor 4 (PF-4) have been identifled in
the product by mass spectrometry of tryptic digests from SDS PAGE gels.
5 Computer database searches using Biowork Browser for peptide identification
yielded strong matches across several species including Homo sapiens.
Although precise quantification of the TSP-1 and PF-4 protein content of the
product has not yet been established, the visible nature of the protein bands
on
SDS PAGE gels indicates that the proteins are present in biologically
significant
10 (upper nanogram) quantities.
A summary of the hypothesised components of the product, and the method of
action, is shown in Figure 17. The product is thought to contain CRF, with
some
levels of CRF-BP, beta endorphin, vasopressin, and enkephalins. CRF induces
15 production of further CRF in the patient, as do beta endorphin and the
enkephalins. Endogenous CRF causes production of POMC, which gives rise to
among others ACTH, alpha MSH, and beta endorphin. This last product acts in a
feedback loop, with low levels stimulating further CRF release, while high
levels
inhibit CRF release. This whole CRF / POMC cascade is thought to induce an
20 immunological switch in the patient, which could explain the surprising
beneficial effects seen in a variety of conditions.
