NZ749158B2 - Antisecretory factor 17 - Google Patents
Antisecretory factor 17 Download PDFInfo
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- NZ749158B2 NZ749158B2 NZ749158A NZ74915817A NZ749158B2 NZ 749158 B2 NZ749158 B2 NZ 749158B2 NZ 749158 A NZ749158 A NZ 749158A NZ 74915817 A NZ74915817 A NZ 74915817A NZ 749158 B2 NZ749158 B2 NZ 749158B2
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/12—Antidiarrhoeals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4703—Inhibitors; Suppressors
Abstract
The present invention relates to a new peptide called Antisecretory Factor (AF) 17 which is an isolated recombinant and/or synthetically produced which has a t1/2 of at least 1.1h. The peptide is e.g. useful for normalizing pathological fluid transport and/or inflammatory reactions in animals and in humans. AF-17 and pharmaceutical compositions of AF-17 can e.g. be used for treating and/ or preventing TBI and/or secondary brain injuries associated with TBI, as well as for treating and/ or preventing acquired brain injuries and to optimize cancer treatment. humans. AF-17 and pharmaceutical compositions of AF-17 can e.g. be used for treating and/ or preventing TBI and/or secondary brain injuries associated with TBI, as well as for treating and/ or preventing acquired brain injuries and to optimize cancer treatment.
Description
Antisecretory Factor 17
Field of invention
Antisecretory factor (AF) is a protein x which inhibits inflammation and regulates
fluid-transport; the AF complex resides in modified proteasomes. Synthetic peptides,
comprising the antidiarrhoeic sequence AF-16, d between the amino acid
positions 35 and 50 on the antisecretory factor (AF) protein sequence, have prior been
terized (WO 97/08202; WO 05/030246). AF16 is known to be rapidly degrading
in plasma. The present invention discloses s major metabolic fate in plasma,
which is a rapid disulfide formation of AF16, resulting in AF-16 sing a cysteine
disulfide at amino acid position 2 (C2) (hereinafter called AF-17). This action, being
reversible, clearly protects AF16 from rapid peptidase degradation. Based on this
surprising insight, the present invention for the first time discloses synthetic peptides,
comprising AF-17, which, when administered to a patient in need f, provide a
substantially prolonged half-life of AF16.
Background of the invention
Antisecretory factor (AF)
Antisecretory factor (AF) is a 41 kDa protein that originally was described to provide
protection against diarrhea es and intestinal inflammation (for a review, see: The
cretory factor: synthesis, anatomical and ar distribution, and biological
action in experimental and clinical studies. Int Rev Cytol, 2001. 210: p. ). The
cretory factor (AF) protein has been sequenced and its cDNA cloned. The
antisecretory activity seems to be mainly exerted by a peptide located between the
amino acid positions 35 and 50 on the antisecretory factor (AF) protein sequence (i.e.
the antidiarrhoeic sequence/consensus sequence) comprising at least 4-16, such as in
particular 4, 6, 7, 8 or 16 amino acids of the antidiarrhoeic sequence. Immunochemical
and immunohistochemical investigations have revealed that the antisecretory factor
(AF) protein is present and may also be synthesized by most tissues and organs in a
body. Synthetic peptides, comprising the arrhoeic/consensus ce or
fragments thereof, have prior been characterized (WO 97/08202; WO 05/030246).
Antisecretory factor (AF) proteins and peptides have previously been disclosed to
normalize pathological fluid transport and/or inflammatory reactions. such as in the
intestine and the choroid plexus in the central nervous system after challenge with
cholera toxin (WO 97/08202). Food and feed with the capacity to either induce
endogenous synthesis of AF or uptake of added AF have ore been suggested to
be useful for the treatment of edema, diarrhea, dehydration and inflammation e.g. in
W0 97/08202. WO 98/21978 discloses the use of products having enzymatic ty
for the production of a food that induces the formation of antisecretory factor (AF)
proteins. W0 00/038535 further ses food products enriched in native
antisecretory factor (AF) proteins as such (NASP).
Antisecretory factor (AF) proteins and fragments thereof have also been shown to
improve the repair of nervous tissue, and proliferation, apoptosis, differentiation, and/or
migration of stem and progenitor cells and cells derived thereof in the treatment of
conditions associated with loss and/or gain of cells (W0 246) and to be equally
effective in the treatment and/or prevention of intraocular hypertension (W0
07/126364), as for the treatment and/or prevention of compartment syndrome (W0
07/126363).
The present inventors have r shown that AF is able to monitor and/or beneficially
affect the structure, distribution and multiple functions of lipid rafts, receptors and/or
caveolae in membranes and thus to be useful for the treatment and/or prevention of
structural disorganization and dysfunction of lipid rafts and/or ae in cell
membranes (W0 07/126365).
The present inventors have further been able to prove that the same antisecretory
factor (AF) protein, as well as peptides and fragments thereof, can intervene in the
biological activation of embrane ns, e.g. NKCC1 h FAK and CAP,
and that it can thus ly regulate the pathological activity of the ion channel in
pathological and/or perturbed cells, effectively izing the intracellular pressure
and transmembrane protein function in said cell, and thus allowing an improved uptake
of drugs used in e.g. cancer therapy ().
The present inventors isolated a protein named AF1 ecretory factor 1) from blood,
and sequenced its encoding gene. Later, AF 1 was shown to be a constituent of the
198 proteasome subunit, and as such named PSMD4, RPN10 or 85a. It was further
shown that bacterial enterotoxins and processed cereals were able to induce an altered
form of antisecretory factor (AF), which inhibited inflammation and fluid ion in the
gut. This modified form of AF was found to bind to the polysaccharide agarose. After
elution with d-methylglucoside, its concentration could be determined by ELISA.
Surprisingly, it was recently demonstrated that proteasomes react with the complement
factors C3 after intake of processed cereals (SPC). This reaction results in re of
usly hidden antisecretory epitopes, and the proteasome/complement complex
formation results in the splitting of CB into its inactive form 03c.
ingly, there are many medical conditions which would benefit from the
administration of AF-16. Unfortunately, it has been shown to have a very short half-life
in the body of the patients once administered.
The present invention for the first time presents an isolated, recombinant, or
synthetically produced protected AF16 metabolite (herein referred to as AF-17), with a
substantially prolonged half-life in the body of the patients once administered.
Summary of the present invention
In one embodiment, the present invention relates to an isolated recombinant and/or
synthetically produced peptide, hereinafter referred to as AF-17 (as shown in
SEQ.|D.NO. 7), or a pharmaceutically active salt thereof, having equivalent functional
activity, which comprises an amino acid sequence as shown in SEQ.ID.NO. 3 )
and a ne disulfide in amino acid position no. 2 of SEQ.|D.NO. 3, said peptide
having antisecretory ty.
The relative stability of AF16 in different species is shown in figure 12. As is for the first
time documented , irrespective of species, AF16 is y disappearing, with an
in vitro half-life (t1/2) less than or equal to 10 min. The present inventors were
furthermore for the first time able to determine the molecular fate of the peptide AF-16
after administration, leading to a thorough understand of the cokinetic basis of
any pharmacological action of AF and to the development of a new tide (AF-17)
with improved in vitro half-life (tug), enabling improved means for monitoring the fate of
the active AF substance after administration to a patient in need thereof and
consequently g to improved means for optimizing dosage regimen of the active
AF and/or AF peptide.
The present ion for the first time identifies AF’s major lite as a cysteine
disulfide of AF16 (AF—17). The rapid disulfide formation of AF16, being reversible,
clearly protects AF16 from rapid peptidase degradation and is a protective function
which enables AF16 to reach its target intact to a much higher degree, and/or which
improves means for monitoring the fate of the active AF substance after stration
to a t in need thereof and consequently leads to an optimized dosage regimen of
the active AF and/or AF peptide.
As revealed herein, the present inventors for the first time synthesize and study the in
1O vitro pharmacokinetic properties of AF-17, proving that AF-17 can be administered
directly to a patient in need thereof and that AF-17 is at least as effective as AF-16 for
normalizing pathological fluid transport and/or inflammatory ons, such as in the
intestine, after challenge with the cholera toxin, as is shown e.g. in experiment 3. Given
that AF-16 has prior been shown to be effective in a vast variety of different diseases
and condition, selected from but not limited to normalizing pathological fluid transport
and/or inflammatory reactions, treating and/or preventing TBI, tumors and/or tumor
related complications, for treating cancer, compartment syndrome, glioblastoma,
diabetes and diarrhea, for zing cellular uptake of a given drug, such as a small
molecular drug, for neuroprotection, as well as for normalizing calveola, and AF-17 is
the naturally occurring metabolite of AF-16, it can be assumed that the herein sed
AF-17 is at least as ive as AF-16 for treating the same diseases and/or conditions
as AF-16 is known to be effective for.
Thus, the present invention ses a new and improved ed recombinant or
synthetic active peptide ting of, comprising, derived from and/or based on the
antisecretory factor (AF) protein (AF-17) and its use(s) in medicine. in particular, the
present invention relates to the use of the herein described AF-17 for treating
Traumatic Brain Injury (TBl).
What is more, as it is well known from extensive studies of the cretory protein
(AF) over many years, the endogenous protein is post-transcriptionally processed into
a plethora of smaller peptides, all with proven similar antisecretory effect, as long as
the core active sequence of the protein (AF-6, shown in SEQ.|D.NO. 2) is intact. It thus
stands to reason, that in in the natural environment, the antisecretory protein (AF) will
be degraded and/or post-transcriptionally processed into smaller peptides sing
AF—6, and that the metabolite of those smaller peptides will in analogy to the observed
fate of AF-16, also be protected at least by a disulfide in the only cysteine of AF-6, i.e.
in amino acid position 1 of AF-6, as shown in SEQ.ID.NO. 2.
The present invention thus further relates to an isolated recombinant and/or
synthetically produced peptide, or a pharmaceutically active salt f, having
equivalent functional activity, said peptide corresponding to a fragment of the
antisecretory factor (AF) protein as shown in SEQ.ID.NO. 1 (AF) and/or a homologue
f, having equivalent activity, wherein said peptide at least comprises an amino
acid sequence as shown in SEQ.ID.NO. 2 (AF—6) and a cysteine disulfide in amino acid
position no. 1 of SEQ.ID.NO. 2, said peptide having antisecretory activity and an
improved in vitro half-life (tug) compared to a peptide consisting of an identical nt
of the antisecretory factor (AF) protein shown in SEQ.ID.NO. 1 (AF) and/or a
homologue thereof, wherein said peptide does not comprise a cysteine disulfide in
amino acid position no. 1 of SEQ.ID.NO. 2.
In a tly preferred embodiment, the isolated recombinant and/or synthetically
produced peptide of the present invention comprises an amino acid sequence as
shown in SEQ.ID.NO. 3 (AF-16) and a cysteine ide in amino acid position no. 2 of
.NO. 3.
An isolated recombinant and/or synthetically produced peptide according to the present
invention is 6-25 amino acids long, ably 7-17, 6—16, 7-20, 7—17, 16-25, 16-20, 17-
, 17—25, such as 6, 7, 8, 9, 16 or 17 amino acids long. In certain embodiments, it is at
least 6, 7, 16, or 17 amino acids long and/or at the most 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long.
An isolated recombinant and/or synthetically produced peptide according to the present
invention typically comprises the amino acid sequence (VC(C)HSKTRSNPENNVGL),
as shown in SEQ.ID.NO.7, or the amino acid ce (C(C)HSKTR) as shown in
SEQ.ID.NO.8, or the amino acid sequence (VC(C)HSKTR) as shown in SEQ.ID.NO.9.
An isolated inant and/or synthetically produced peptide according to the t
invention typically consists of the amino acids (VC(C)HSKTRSNPENNVGL) as shown
in SEQ.ID.NO.7, or the amino acid sequence SKTR) as shown in SEQ.ID.NO.8,
or the amino acid sequence (VC(C)HSKTR) as shown in SEQ.ID.NO.9.
An isolated recombinant and/or synthetically produced peptide according to the present
invention typically has a t1/2 of at least 0.2h, preferably of at least 0.25h, 0.3h, 0.4h,
0.5h, 1h, or 1.5h, 1.9h, 2.0h, 2.5h, such as a t1/2 of at least 1.8h.
The t invention further relates to an isolated recombinant and/or synthetically
produced peptide according to the present ion for use in medicine, such as for
use in treating and/or ting diseases and ions selected from the list
consisting of ogical fluid transport, infections, inflammations, inflammatory
ons, TBI, TBI related conditions, , tumor related complications, cancer,
compartment syndrome, glioblastoma, diabetes, and diarrhea. The present invention
further relates to an isolated recombinant and/or synthetically produced peptide
according to the present invention for optimizing cellular uptake of an active nce,
for neuroprotection and/or for normalizing la.
In one embodiment, a pharmaceutical and/or cosmetic composition is envisioned,
comprising an isolated recombinant and/or synthetically produced peptide according to
the present invention and a suitable pharmaceutical carrier.
A pharmaceutical and/or ic composition according to the present invention is
intended for use in ng and/or preventing diseases and conditions selected from
the list consisting of pathological fluid transport, infections, inflammations, inflammatory
reactions, TBI, TBI related conditions, tumors, tumor related cations, cancer,
compartment me, glioblastoma, diabetes, and diarrhea. The present invention
further relates to a pharmaceutical and/or cosmetic composition according to the
t invention for optimizing cellular uptake of an active substance, for
neuroprotection and/or for normalizing calveola.
Also related to is a method of normalizing pathological fluid transport and/or
inflammatory reaction in patient in need thereof, and/or a method for optimizing cellular
3O uptake of an active nce, for neuroprotection and/or for normalizing calveola
and/or for treating and/or preventing diseases and conditions selected from the list
consisting of pathological fluid transport, infections, inflammations, inflammatory
reactions, TBI, TBI related conditions, tumors, tumor related cations, cancer,
compartment syndrome, astoma, diabetes, and diarrhea, comprising
administering to an animal or a human being in need thereof an effective amount of an
isolated inant and/or synthetically produced peptide according to the present
invention or a pharmaceutical composition comprising said e.
The present invention in on relates to an dy against a peptide having
essentially the amino acid sequence shown in SEQ.|D.NO. 7, 8 or 9, as well as to its
use in detecting said protein or homologues or fragments thereof in organisms, such as
animals, including ians and humans.
Also provided herein is the use of a nucleic acid coding for a peptide having essentially
the amino acid sequence shown in any of SEQ. ID. NO.1- 9, for producing a
corresponding peptide, wherein said peptide comprises at least one cysteine disulfide
in at least amino acid (aa) position no.36 ofSE.|D.NO. 1, in aa position no. 1 of
SE.ID.NO. 2, in aa position no. 2 of SE.|D.NO. 3, and/or in aa position no. 2 of
SE.|D.NO. 4.
Figure legends
Figure 1
Oxidation of cysteine. A: Sulfenic acid, B: Sulfinic acid, C: Sulfonic acid
Figure 2
N—ethyl maleimide analog of AF16 (AF16-NEM)
Figure 3
Human plasma and buffer stability of AF 16 (A) and AF16-NEM (B)
Figure 4
1O LC-MS detected degradation products of AF16 in human plasma
Figure 5
AF16 degradation in the presence of Caco-2 cells. +Pl indicates se inhibitor
Figure 6
Metabolite formation cs of AF16 degradation in the presence of Caco-2 cells. Blue
diamond AF16. Red square AF16 + inhibitor cocktail
Figure 7
Metabolite ion kinetics of AF16 degradation in the presence of Caco—2 cells. Blue
diamond AF16. Red square AF16 + inhibitor cocktail
Figure 8
Metabolite formation kinetics of AF16 degradation in the presence of Caco-2 cells. Blue
diamond AF16. Red square AF16 + inhibitor cocktail
Figure 9
Degradation of AF16 in the ce of Caco-2 cells. Suggested pathways of
determined structures
Figure 10
Relative stability of AF16 with different precipitation methods over 20h at 10°C
Figure 10 shows the results of repeated injection of the same sample three times over
20h. It is clear that TCA and MeCN shows good nt stability over time. A slight
disappearance is noted with ZnSO4 at 20h. A significant loss over time is shown with
MeOH. It is likely that AF16 is stable with MeOH but the loss stem from peptide
precipitation since it is known that peptides may have limited solubility in alcoholic
mixtures.
Figure 11
ve stability of AF16 with different precipitation methods over 20h at 10°C
Figure 11 shows a m of the data ed above in comparison to each other in
terms of MS-intensity (ion counts). TCA precipitation showed the strongest signal and
was set as the reference. It is clear that the nonorganic methods fall behind, most likely
due to co-eluting suppressing ions. The two methods with best stability (TCA and
MeCN) show over 500-fold ence in sensitivity, thus for the ued studies, TCA
was chosen to be used throughout the study.
Figure 12
Relative ity of AF16 with different precipitation methods over 20h at 10°C
qum13
Kinetics of AF16 and the identified metabolites in human (top) and rat (bottom) plasma
Figure 14
Enhanced resolution scan of the 5923/5944 metabolite
Figure 15
Kinetics of AF16 and the identified metabolites using ZnSO4 as plasma precipitant and
+/- DTT. A: human + DTT, B: human — DTT, C: rat + DTT and D: rat - DTT. AF16 is
showninquckde
Figure 16
Relative formation of major identified products in human plasma
Figure 17
Relative formation of major identified products in rat plasma
Figure 18
Sequence listing
Definitions and abbreviations
Abbreviations
IFP: interstitial fluid pressure;
PBS: phosphate buffered saline;
AF: antisecretory factor, Full-length AF protein (as shown in SEQ.|D.NO. 1)
AF-6: a hexa peptide CHSKTR (as shown in SEQ.|D.NO. 2);
AF-7: a peptide composed of the amino acids C(C)HSKTR (as shown in SEQ.|D.NO.
AF-16: a peptide composed of the amino acids VCHSKTRSNPENNVGL (as shown in
SEQ.ID.NO. 3);
AF-17: a peptide composed of the amino acids VC(C)HSKTRSNPENNVGL (as shown
in .NO. 7);
AF-8: a septa e VCHSKTR (as shown in .NO. 4);
AF-9: a peptide composed of the amino acids VC(C)HSKTR (as shown in SEQ.ID.NO.
9);
Octa peptide IVCHSKTR (as shown in SEQ.|D.NO. 5);
Penta peptide HSKTR (as shown in .NO. 6);
SPC: Specially Processed Cereals;
RTT: Method for measuring a standardized secretion response in rat small intestine, as
published in SE 9000028-2 (publication number 466331) for ing content of AF
(ASP);
AF: Antisecretory Factor;
ELISA: -linked immunosorbent assay;
PBS: phosphate buffered saline;
AP: alkaline atase;
BSA: bovine serum albumin;
mAb: monoclonal antibody;
LC-MS/MS: nanoflow liquid chromatography-tandem mass spectrometry;
PAGE: rylamide gel electrophoresis.
HSV1: herpes simplex virus-1
TBl: Traumatic Brain Injury
Definitions
Proteins are biological macromolecules constituted by amino acid residues linked
together by peptide bonds. Proteins, as linear polymers of amino acids, are also called
polypeptides. Typically, ns have 50—800 amino acid residues and hence have
molecular weights in the range of from about 6,000 to about several hundred thousand
Dalton or more. Small proteins are called peptides, ptides, or oligopeptides. The
terms “protein", “polypeptide", “oligopeptide” and “peptide” may be used
interchangeably in the present context. es can have very few amino acid
residues, such as between 2-50 amino acid residues (aa).
The term “antisecretory” refers in the present t to inhibiting or decreasing
ion and/or fluid transfer. In the present context, the terms an “Antisecretory
factor protein”, “antisecretory factor (AF) protein”, “AF- protein”, AF, or a gue,
derivative or fragment thereof, may be used interchangeably with the term
ecretory factors” or “antisecretory factor ns” as defined in WO 97/08202,
and refer to an antisecretory factor (AF) protein or a peptide or a homologue, derivative
and/or fragment thereof having antisecretory and/or equivalent functional and/or
analogue activity, or to a modification thereof not altering the function of the
ptide. Hence, it is to be understood that an “antisecretory factorI! “
, antisecretory
factor protein”, “antisecretory peptide”, “antisecretory fragment”, or an “antisecretory
factor (AF) protein” in the present context, also can refer to a derivative, homologue or
fragment thereof. These terms may all be used interchangeably in the context of the
present invention. Furthermore, in the present context, the term “antisecretory factor”
may be abbreviated “AF". Antisecretory factor (AF) protein in the present context also
refers to a protein with antisecretory properties as previously defined in W097/08202
and WO 00/38535. Antisecretory factors have also been disclosed eg. in WO
05/030246.
SPC© is a medical food sing specially processed cereals (SPC).
A “medical food”, in the present context, refers to a food, a feed or food supplement,
or a food for special y use, which has been prepared with an cretory factor
(AF) n, or alternatively, has the capability to induce synthesis and/or activation of
endogenous AF. Said food may be any suitable food, in fluid or solid form, such as a
liquid or a powder, or any other suitable foodstuff. Examples of such matter may be
found in WC 0038535 or WO 91/09536.
m© Also intended by the term antisecretory factor are native antisecretory
factors (NASP) which can be provided in egg yolk with a high content of antisecretory
2017/068111
factors (NASP), as e.g. disclosed in SE 900028-2 and WO 35, and as further
described below.
ed ption of the invention
The t invention for the first time identifies AF's major metabolite as a cysteine
disulfide of AF16 (AF-17). The rapid disulfide formation of AF16, being ible,
clearly protects AF16 from rapid peptidase degradation and is a protective function
which s AF16 to reach its target intact to a much higher degree, and improves
means for monitoring the fate of the active AF substance after administration to a
patient in need thereof and consequently leads to an optimized dosage regimen of the
active AF substance, full-length AF, a fragment of AF and/or AF peptide. As is for the
first time documented herein, disclosed is a new synthetically produced or isolated
recombinant AF-peptide (AF-17) with improved in vitro half-life (tug), enabling improved
means for monitoring the fate of the active AF substance after administration to a
patient in need thereof and consequently leading to improved means for optimizing
dosage regimen of the active AF and/or AF peptide.
In one embodiment, the present invention relates to an isolated recombinant and/or
tically produced peptide hereinafter called AF-17 (as shown in SEQ.ID.NO. 7), or
a pharmaceutically active salt thereof, having equivalent functional activity, which
ses an amino acid sequence as shown in SEQ.|D.NO. 3 ) and a cysteine
disulfide in amino acid position no. 2 of SEQ.lD.NO. 3, said peptide having
antisecretory activity.
In another embodiment, the present invention relates to an isolated recombinant and/or
synthetically produced peptide, hereinafter ed to as AF-7 (as shown in
SEQ.|D.NO. 8), or a pharmaceutically active salt thereof, having equivalent functional
activity, which comprises an amino acid sequence as shown in .NO. 2 (AF-6)
and a cysteine disulfide in amino acid position no. 1 of SEQ.ID.NO. 3, said peptide
having antisecretory activity.
In yet another embodiment, the present invention relates to an isolated inant
and/or synthetically produced peptide, hereinafter referred to as AF-9 (as shown in
SEQ.|D.NO. 9), or a ceutically active salt thereof, having equivalent functional
activity, which comprises an amino acid sequence as shown in SEQ.lD.NO. 4 (AF-8)
and a cysteine disulfide in amino acid position no. 2 of SEQ.lD.NO. 4, said peptide
having antisecretory activity.
As revealed herein, the present inventors for the first time disclose a peptide according
to the present invention which is at least as effective as AF-16 for izing
pathological fluid transport and/or inflammatory reactions, such as in the intestine, after
challenge with the a toxin, as is shown eg. in experiment 3. A peptide ing
to the present invention is effective in a vast variety of different diseases and ion,
selected from but not limited to normalizing pathological fluid transport and/or
1O inflammatory reactions, treating and/or preventing TBl, tumors and/or tumor related
complications, for ng , compartment syndrome, glioblastoma, diabetes and
ea, for optimizing ar uptake of a given drug, for neuroprotection, as well as
for normalizing calveola.
Thus, the present invention discloses a new and improved isolated recombinant or
synthetic active peptide consisting of, comprising, derived from and/or based on the
antisecretory factor (AF) n (AF-17) and its use(s) in medicine. In particular, the
present invention relates to the use of one or a ation of the herein described AF-
17, AF-7, and/or AF-9 for treating and/or preventing diseases and conditions ed
from the list consisting of ogical fluid transport, infections, inflammations,
inflammatory reactions, TBl, TBl related conditions, tumors, tumor related
complications, cancer, compartment syndrome, glioblastoma, diabetes, and diarrhea,
or for optimizing cellular uptake of an active substance, for neuroprotection and/or for
normalizing calveola.
The present invention discloses a new and improved isolated recombinant or synthetic
active e consisting of, comprising, derived from and/or based on the
antisecretory factor (AF) protein (AF-17) and in particular a new peptide called
Antisecretory Factor (AF) 17. The peptide is eg. used for normalizing pathological fluid
transport and/or inflammatory reactions in animals including humans. AF-17 can further
be used for detection, as feed additive for growing animals and as antidiarrheal
and drug against diseases involving edema, dehydration and/or inflammation.
The cretory factor
The cretory factor is a class of proteins that occurs naturally in the body. The
human antisecretory factor AF protein is a 41 kDa n, comprising 382-288 amino
acids when isolated from the pituitary gland. The active site according to the present
invention can be localized to the protein in a region close to the N-terminal of the
protein, in particular localized to amino acids 1-163 of SEQ lD NO 1, more specifically
to amino acid positions 35 - 50 on the antisecretory factor (AF) protein sequence. The
ical effect of AF is exerted by any peptide or polypeptide comprising at least 6
amino acids, as shown in SEQ.|D.NO. 2 (AF-6), of said consensus sequence, or
comprising a modification thereof not altering the biological function of the polypeptide
and/or peptide.
The t inventors have shown that the antisecretory factor is to some extent
gous with the protein 85a, and an10, which tutes a subunit of a
constituent prevailing in all cells, the 26 S proteasome, more specifically in the 19 S/PA
700 cap. In the present invention, antisecretory factor (AF) proteins are defined as a
class of homologue proteins having the same functional properties. Antisecretory factor
is also highly similar to angiocidin, another protein isoform known to bind to
thrombospondin—1 and associated with cancer progression.
Homologues, tives and fragments of antisecretory factor (AF) proteins and/or
peptides according to the present ion all have analogous biological activity.
Homologues, derivatives and fragments, in the t context, comprise at least 6
amino acids (as shown in SEQ.|D.NO. 2) corresponding to those of a naturally
occurring antisecretory factor (AF) protein, which may be further modified by changing
one or more amino acids in order to optimize the antisecretory factor’s biological
activity, without altering the essential biological function of the ptide and/or
peptide.
By a derivative is in the present context intended a n having equivalent activity
and/or a functional equivalent activity to an antisecretory factor as defined herein, being
derived from another substance either directly or by modification or partial substitution,
wherein one or more amino acids have been substituted by another amino acid, which
amino acid can be a modified or an unnatural amino acid. For example, the
antisecretory factor derivatives according to the invention may comprise an N terminal
and/or a C al protecting group. One example of an N terminal protecting group
includes acetyl. One example of a C terminal protecting group includes amide.
Furthermore, any amino acid sequence being at least 70% identical, such as being at
least 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% identical with the amino acid sequence of an antisecretory factor
(AF) protein, peptide, gue, derivative and/or fragment according to the
invention, is also considered to be inside the scope of the present ion.
By proteins, homologues, derivatives, peptides and/or fragment thereof having an
amino acid sequence at least, for example 95% identical to a reference amino acid
sequence, is intended that the amino acid ce of eg. the e is identical to
the reference ce, except that the amino acid sequence may include up to 5
point mutations per each 100 amino acids of the reference amino acid sequence. In
other words, to obtain a polypeptide having an amino acid sequence at least 95%
identical to a reference amino acid sequence, up to 5% of the amino acids in the
reference sequence may be deleted or substituted with r amino acid, or a
number of amino acids up to 5% of the total amino acids in the reference sequence
may be inserted into the reference sequence. These mutations of the reference
sequence may occur at the amino or y terminal positions of the reference amino
acid ce or anywhere between those terminal positions, interspersed either
individually among amino acids in the reference sequence or in one or more
contiguous groups within the reference sequence.
In the present invention, a local algorithm program is best suited to determine identity.
Local algorithm programs, (such as Smith Waterman) compare a subsequence in one
sequence with a subsequence in a second sequence, and find the combination of sub-
sequences and the ent of those quences, which yields the highest overall
similarity score. Internal gaps, if allowed, are penalized. Local algorithms work well for
comparing two multi domain proteins, which have a single domain, orjust a binding site
in common.
Methods to determine identity and similarity are codified in publicly available programs.
Preferred computer program methods to determine identity and similarity between two
ces e, but are not limited to, the GCG program package eux, J et al
(1994)) BLASTP, BLASTN, and FASTA (Altschul, SF. et al (1990)). The BLASTX
program is publicly available from NCBI and other sources (BLAST Manual, Altschul,
S.F. et al, Altschul, S.F. et al (1990)). Each sequence analysis program has a default
scoring matrix and default gap penalties. In general, a molecular biologist would be
expected to use the default settings established by the software m used.
The cretory factor (AF) proteins or a peptide or a gue, derivative and/or
nt thereof having equivalent activity as defined herein, can comprise 6 amino
acids or more, such as 6-16 amino acids, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19 or 20 amino acids or more. In other preferred embodiments, the
antisecretory factor ts of 7, 9 or 17 amino acids. In certain embodiments, the
antisecretory factor (AF) protein, a homologue, derivative, peptide and/or fragment
thereof, according to the present invention, ts of 6, 7, 8, 9, 15, 16 or 17 amino
acids.
In a presently preferred ment, an isolated recombinant and/or synthetic peptide
AF-17 (as shown in SEQ.ID.NO. 7), or a pharmaceutically active salt thereof, having
equivalent functional activity, comprises an amino acid sequence as shown in
SEQ.ID.NO. 3 (AF-16) and a cysteine disulfide in amino acid position no, 2 of
SEQ.ID.NO. 3, said peptide having antisecretory activity.
In another preferred embodiment, an isolated recombinant and/or synthetic peptide, or
a pharmaceutically active salt f, having equivalent functional activity, at least
comprises an amino acid sequence as shown in SEQ.|D.NO. 2 (AF-6) and a cysteine
disulfide in amino acid position no. 1 of SEQ.ID.NO. 2, said peptide having
antisecretory activity.
In yet another preferred embodiment, an isolated recombinant and/or synthetic peptide,
or a ceutically active salt thereof, having equivalent functional activity, at least
ses an amino acid sequence as shown in SEQ.ID.NO. 4 (AF-8) and a cysteine
disulfide in amino acid position no. 2 of SEQ.|D.NO. 4, said e having
antisecretory activity.
The cretory factor (AF) protein, a homologue, derivative, peptide and/or fragment
thereof, according to the present invention, can be produced in vivo or in vitro, e.g.
recombinantly, synthetically and/or chemically synthesized, and/or isolated from a
lly occurring source of antisecretory factors, such as from pig pituitary glands or
bird’s eggs. After production, the antisecretory factor (AF) protein, homologue,
derivative, e and/or fragment thereof, according to the present invention, may be
further processed, such as by chemical or enzymatic cleavage to smaller antisecretory
active nts and/or by modification of amino acids and/or by addition of a ne
in amino acid position no. 1 of SEQ.lD.NO, 2, alternatively in amino acid position no. 2
of SEQ.lD.NO.3, via a ide link in the cysteine in the peptide.
It is presently not possible to obtain antisecretory factor (AF)—protein in pure form by
purification. It is however possible to produce a biologically active cretory factor
protein recombinantly or synthetically, as previously disclosed in WO 97/08202 and
WO 05/030246. WO 97/08202 also discloses the production of biologically active
fragments of this protein of 7—80 amino acids.
In the present context, the antisecretory factor (AF) protein, a gue, derivative,
peptide and/or fragment thereof, according to the present invention is either a natural
metabolite of antisecretory factor (AF) n produced in a mammal (excluding
humans), or recombinantly produced and ally chemically modified, or a
synthetically produced peptide.
The antisecretory factor (AF) n, a homologue, tive, peptide and/or fragment
thereof, according to the present invention, may further comprise an N terminal and/or
a C terminal protecting group. One example of an N terminal protecting group es
acetyl. One example of a C terminal protecting group includes amide.
In a preferred ment of the present invention the antisecretory factor (AF)
protein, a homologue, derivative, peptide and/or fragment thereof, according to the
t ion is selected among SEQ ID NO 7-9, i.e. VC(C)HSKTRSNPENNVGL
(SEQ ID NO 7, in this context also called AF-17), C(C)HSKTR (SEQ ID NO 8, in this
context also called AF—7), VC(C)HSKTR (SEQ ID NO 9 in this context also called AF-
9), using the common one letter abbreviations for amino acids. As specified in the
accompanying sequence listing, some of the amino acids in the above-specified
sequences may be replaced by other amino acids.
Also intended by the present invention is the combination of two or more of any of the
peptides according to SEQ ID NO 7-9.
In yet another embodiment, the invention relates to the use of a pharmaceutical
composition as disclosed herein, which comprises two or more the antisecretory factor
(AF) protein, a homologue, derivative, peptide and/or fragment thereof, according to
the present invention.
Disulfide
In the present context, a disulfide refers to a functional group with the general structure
R—S—S—R. The linkage is also called an SS-bond or a disulfide bridge and is usually
derived by the coupling of two thiol groups. In formal terms, the connection is a
fide, in analogy to its congener, peroxide (R—O—O—R).
Cysteine (abbreviated as Cys or C) is a semi-essential proteinogenic amino acid with
the a HOZCCH(NH2)CHZSH>. It is encoded by the codons UGU and UGC. The
thiol side chain in Cys often participates in enzymatic reactions, as a nucleophile. The
thiol is susceptible to oxidization to give the ide derivative ne, which serves
an important structural role in many proteins.
Cysteine residues are among the most chemically involved amino acids, participating
typically in redox chemical reactions but also as a nucleophile against reactive
electrophiles such as reactive oxygen species or lically ed
xenobiotics/drugs. AF16 contains one cysteine residue in on 2 of SEQ.ID.NO.2.
AF-17, or any other disulfide comprising peptide according to the present application,
such as but not limited to AF—7 and AF-9, is (are) thus ted against both oxidation
of the sulfur, from reacting with other cysteines (disulfide formation), ng with
electrophiles and is thus more resilient against proteolytic activity.
Synthesis
Disulfide bonds are usually formed from the oxidation of sulfhydryl (—SH) groups.
A variety of oxidants promote this reaction including air and hydrogen peroxide. Such
reactions are thought to proceed via sulfenic acid intermediates. In the laboratory,
iodine in the presence of base is commonly ed to oxidize thiols to disulfides.
Alternatively, disulfide bonds in proteins are often formed by disulfide exchange.
Such reactions are mediated by enzymes in some cases and in other cases are under
brium control, especially in the ce of a catalytic amount of base.
Many specialized s have been developed for forming disulfides, for applications
in organic synthesis and can be employed for producing the synthetic AF-17 or AF-7 or
AF-9 or AF based AF-disulfide peptides according to the present invention.
AF16 shows a high degree of sensitivity s plasma as shown in figure 3A. The
kinetics of degradation tes an in vitro half-life (t1/2) of 0.4h. AF-17 is anticipated
to have an almost 5-fold higher stability, t1/2 = 1.8h. AF16 is highly sensitive for
enzymatic and chemical degradation in systemic circulation. AF—17 is more resilient
towards tic or al reactions as the ne moiety is ed.
Quantitative pilot experiments of AF16 during Caco-2 cell permeability experiments
clearly showed a rapid disappearance of the peptide (not shown). It is well known that
in the ine, and as such at the apical side of Caco-2 cells, brush-border peptidases
exist. The kinetics of degradation of AF16 is very rapid as described with a t1/2 of 8
min.
AF16 and the isotopically labelled peptide in similar quantity were incubated in plasma
of rat and human as described in experiment 1. For analysis of fullscan MS data, a
metabolite identification software from Sciex, Lightsight was used, which compares the
incubated MS response to the quality control (QC) sample and assigns apparent peaks
as metabolites with specific mass over charge (m/z) values. The largest metabolite
peak areas were rank ordered and in some cases verified by MS/MS fragmentation.
Skyline methodology was used to predict ntation of the identified peptides, but
also aided in creating sensitive MRM methods so that low s could be monitored.
Table 6 lists the identified products and their relative amount at 30 min incubation. MS
sensitivity, may differ and thus individual percentages may change. Upon reviewing the
s, it was clear that based on the ve area of the identified peaks one
metabolite was much larger than the other, designated M1 in table 6. However the
3O mass pair identified (m/z 625/630) did not correspond to any catabolic products
(expected proteolytic peptide bond cleavage). This pair is now identified as the cysteine
disulfide of AF-16 (AF-17).
Medical treatments
TBI
Traumatic brain injury (TBI) is a complex injury with a broad spectrum of symptoms and
disabilities. tic brain injury (TBI) is also known as intracranial injury, it occurs
when an external force traumatically injures the brain. TBI can be classified based on
severity, mechanism (closed or penetrating head injury), or other features (e.g.,
occurring in a ic location or over a widespread area). In the present context, TBI
is also meant to include head , i.e. it can involve damage to structures other than
the brain, such as the scalp and skull.
TBI is a major cause of death and disability worldwide, especially in children and young
adults. Causes include falls, vehicle accidents, and ce. Brain trauma can occur
as a consequence of a focal impact upon the head, by a sudden
ration/deceleration within the cranium or by a complex combination of both
movement and sudden . In addition to the damage caused at the moment of
injury, brain trauma causes secondary injury, a variety of events that take place in the
s and days following the injury. These processes, which include alterations in
cerebral blood flow and the pressure within the skull, contribute substantially to the
damage from the initial injury.
TBI can cause a host of physical, cognitive, social, nal, and behavioral effects,
and outcome can range from complete recovery to permanent disability or death.
In the present context, the following terms and definitions refer to the ent injuries
relating to/of TBI, all of which are treatable by administering an isolated recombinant
and/or synthetically produced peptide, or a pharmaceutically active salt thereof, having
lent functional activity, said peptide corresponding to a fragment of the
antisecretory factor (AF) protein as shown in SEQ.ID.NO. 1 (AF) and/or a gue
thereof having equivalent activity, wherein said peptide at least comprises an amino
acid ce as shown in .NO. 2 (AF-6) and a cysteine disulfide in amino acid
position no. 1 of SEQ.ID.NO. 2, such as an amino acid sequence as shown in
SEQ.ID.NO. 3 (AF-16) and a cysteine disulfide in amino acid position no. 2 of
SEQ.ID.NO. 3., to a patient in need thereof: Closed Head Injury, Open Head Injury,
Diffuse Axonal Injury, Contusion, Penetrating Trauma, and Secondary Injury.
The presently disclosed isolated recombinant and/or synthetically produced peptide, or
a pharmaceutically active salt thereof, having equivalent functional activity, said peptide
corresponding to a nt of the antisecretory factor (AF) protein as shown in
SEQ.lD.NO. 1 (AF) and/or a homologue thereof having equivalent activity, wherein said
peptide at least comprises an amino acid sequence as shown in SEQ.lD.NO. 2 (AF-6)
and a cysteine disulfide in amino acid position no. 1 of SEQ.lD.NO. 2, such as an
amino acid sequence as shown in SEQ.|D.NO. 3 ) and a cysteine disulfide in
amino acid position no. 2 of SEQ.|D.NO. 3, is particularly useful for treating and/or
preventing ary TBI, such as swelling and release of chemicals that promote
inflammation and cell injury or death. This causes ng in the brain which may
increase the intracranial pressure and t the cerebrospinal fluid from draining out
of the skull. This causes further increase in pressure and brain damage. If this is not
controlled or prevented the brain can herniate (push through) the base of the skull and
cause respiratory failure and death. The prevention of this ary injury is the focus
of the acute medical care after injury.
Thus the present invention in a presently preferred embodiment relates to the use of an
isolated recombinant and/or synthetically produced peptide according to the present
invention for the manufacturing of a pharmaceutical composition for treating and/or
preventing secondary TBI injury. The present invention equally relates to the use of an
isolated recombinant and/or synthetically produced peptide according to the present
invention for treating and/or preventing secondary TBI , to an ed
recombinant and/or synthetically produced peptide according to the present invention
for use in treating and/or preventing secondary TBI injury, as well as to a method of
treating and/or preventing secondary TBI injury by administering to a patient in need
f an isolated recombinant and/or synthetically produced peptide according to the
t invention in an amount sufficient to treat and/or cure said patient and/or to
prevent symptoms of TBI injury.
Secondary TBl lnjury es:
a Intracranial hemorrhage (bleeding inside the skull)
. Brain swelling
. Increased ranial pressure (pressure inside the skull)
. Brain damage associated with lack of oxygen
» Infection inside the skull, common with ating trauma
a Chemical changes leading to cell death
. sed fluid inside the skull (hydrocephalus)
Acquired Brain Injury-
Acquired Brain Injuries are es other than congenital, birth trauma, hereditary or
degenerative. This includes traumatic brain . In the aumatic types of
acquired brain injury, the brain is usually diffusely injured. These injuries are usually
not included in traumatic brain injury but the symptoms span the same spectrum.
Common causes are anoxia and hypoxia. These are lack of oxygen to the brain and
insufficient oxygen to the brain. They can occur because of mechanical problems with
breathing, with cardiac arrest or bleeding. Drugs and poisoning can also cause
acquired traumatic brain injury. Carbon monoxide poisoning is an example of
poisoning that may cause brain .
The presently disclosed isolated recombinant and/or synthetically produced peptide, or
a pharmaceutically active salt thereof, having equivalent functional activity, said peptide
corresponding to a fragment of the antisecretory factor (AF) protein as shown in
SEQ.|D.NO. 1 (AF) and/or a homologue thereof having equivalent activity, wherein said
peptide at least comprises an amino acid sequence as shown in SEQ.|D.NO. 2 (AF-6)
and a cysteine disulfide in amino acid position no. 1 of SEQ.ID.NO. 2, such as an
amino acid sequence as shown in SEQ.ID.NO. 3 (AF-16) and a cysteine disulfide in
amino acid position no. 2 of SEQ.ID.NO. 3, is equally useful for treating and/or
preventing Acquired Brain Injury.
Thus the present ion in a tly preferred embodiment relates to the use of an
isolated recombinant and/or synthetically produced peptide according to the present
invention for the manufacturing of a pharmaceutical composition for ng and/or
preventing Acquired Brain Injury. The present invention equally relates to the use of an
isolated recombinant and/or tically ed peptide according to the present
invention for treating and/or preventing Acquired Brain Injury, to an isolated
recombinant and/or synthetically produced peptide according to the present ion
for use in treating and/or ting Acquired Brain Injury, as well as to a method of
ng and/or preventing Acquired Brain Injury by administering an isolated
recombinant and/or synthetically produced peptide according to the present invention
in an amount sufficient to a patient in need thereof.
Cancer
In one embodiment, the present invention relates to a method for treating cancer, such
as, but not limited to, glioblastoma, characterized by administering an isolated
inant and/or synthetically ed e, or a pharmaceutically active salt
thereof, having equivalent functional activity, said peptide corresponding to a fragment
of the antisecretory factor (AF) protein as shown in SEQ.ID.NO. 1 (AF) and/or a
homologue thereof having equivalent activity, wherein said peptide at least comprises
an amino acid sequence as shown in SEQ.|D.NO. 2 (AF-6) and a cysteine disulfide in
amino acid position no. 1 of SEQ.|D.NO. 2, such as an amino acid ce as shown
in SEQ.lD.NO. 3 (AF-16) and a cysteine disulfide in amino acid on no. 2 of
SEQ.|D.NO. 3., to a patient in need thereof. Said method can in one embodiment of the
present invention be used to facilitate an optimized drug uptake and delivery of a
further pharmaceutical substance.
Said method for treating a mammalian suffering from cancer, such as, but not limited
to, glioblastoma, can in a presently preferred embodiment comprise feeding a food,
food stuff and/or food supplement to said patient and thereby inducing endogenous
production of AF for facilitating an optimized drug uptake and ry of a further
pharmaceutical substance.
Said pharmaceutical substance and/or formulation is in the t context ed
from the group consisting of anticancer drug, antitumor drug, radiation therapy,
immunological substances and/or cells and antibiotic substance, a drug targeting
posttraumatic injury, a drug targeting neurodegeneration, and a drug against
inflammatory conditions. Said r pharmaceutical substance can be in the form of
nano particles and/or formulations thereof in the treatment of , such as, but not
limited to, glioblastoma (a GBM tumor).
Hereinafter, the embodiments of the t invention will be described in detail. It is to
be noted that the embodiments individually disclosed below are es of the
isolated or tic peptide and the intended use of the peptide. The present ion
is not limited to these examples.
3O Compartment Syndrome
Furthermore, the present invention in one embodiment relates to the use of an isolated
recombinant and/or synthetically produced peptide according to the present invention
for the manufacturing of a pharmaceutical composition for treating and/or preventing
tment Syndrome. The present invention equally relates to the use of an
isolated inant and/or synthetically produced peptide according to the t
invention for treating and/or preventing Compartment Syndrome, to an isolated
recombinant and/or synthetically produced peptide according to the t invention
for use in treating and/or preventing Compartment me, as well as to a method of
treating and/or preventing Acquired Brain Injury by stering an isolated
recombinant and/or synthetically produced peptide according to the present invention
in an amount sufficient to a patient in need thereof.
In one embodiment, the present invention relates to a method for treating and/or
preventing Compartment Syndrome or symptoms thereof, characterized by
stering an isolated recombinant and/or synthetically produced e, or a
pharmaceutically active salt thereof, having equivalent functional activity, said peptide
corresponding to a fragment of the antisecretory factor (AF) protein as shown in
SEQ.ID.NO. 1 (AF) and/or a homologue thereof having equivalent activity, wherein said
peptide at least comprises an amino acid sequence as shown in .NO. 2 (AF-6)
and a cysteine disulfide in amino acid position no. 1 of SEQ.ID.NO. 2, such as an
amino acid sequence as shown in SEQ.lD.NO. 3 (AF—16) and a cysteine disulfide in
amino acid position no. 2 of SEQ.ID.NO. 3., to a patient in need thereof. Said method
can in one embodiment of the present invention be used to tate an optimized drug
uptake and delivery of a r pharmaceutical substance.
Experimental section
Example 1
The effect of the peptide fragment of antisecretory factor protein (AF 1 6) is fairly well
studied in various pharmacological setups by several groups. The majority of studies
have focused on the pharmacologic/physiologic effect of the n or peptide. There
are however fewer reports that deal with the pharmacokinetic viewpoint of AF16. Thus,
all the testing in various matrices has assumed that AF, or presumably the added
peptide fragment f, is the molecular species that is causing the . Previous
studies in other labs have in fact indicated that AF or AF16 is sensitive to degradation
1O in eg. human or rodent plasma but no detailed igation have been performed.
This experiment summarizes efforts in understanding the in vitro pharmacokinetics and
the molecular fate of the AF16 peptide in both human pooled plasma but also in
presence of Caco-2 cells, which is commonly used in AF16 pharmacological studies.
Materials and Methods
Materials
Solid material of AF16 and the stable isotopically labeled (SIL) AF16 IS was provided
by Jan Bruhn at Lantmannen. According to information provided by Ewa Johansson at
enska Hospital the peptide is approximately 70% in , other ents
being 4 x trifluoroacetic acid (TFA) and n x H20. However for sake of simplicity
all concentrations regarding the peptide are considered to be 100%. This assumption
does not influence the s presented herein since they are on a relative basis. All
other chemicals and consumables were from common commercial sources.
Alkylation and oxidation of AF16
To test for oxidation of AF16 a 50 % hydrogen peroxide (H202) solution was used. 2 pl
H202 was added (35 mM final cone.) to a 1 ml on of 15 pM AF16 in 0.1 M
ammonium bicarbonate. ately after mixing, the solution was injected (direct
infusion) to the mass spectrometer (MS) to look for ion products. Only the
oxidative end-product was detected, the sulfonic acid, of which a sensitive multiple
on method (MRM) was created (table 1). In a simultaneous experiment, adding a
surplus of dithiothreitol (DTT), showed that DTT protected against the oxidation.
The N-ethyl maleimide (NEM) alkylated variant of AF16 (AF16-NEM) was produced by
mixing 10 ul 0.1 M NEM with 250 pl 2 mg/ml AF16 in PBS in a HPLC glass vial yielding
imately a 1 mM on. The sample was left standing, sealed, at ambient
temperature for 30 min before MS analysis. After 30 min less than 5% of AF16 was
remaining and a sensitive MRM method of AF16-NEM was created (table 1).
AF16 stability in human plasma and Caco-2 cells
Test of AF16 and AF16-NEM stability in plasma was med using pooled human
plasma (4 donors, two female + two male) in HPLC glass vials at 37°C (20 uM
incubation conc.). Four different matrices were tested: Plasma: isotonic 67 mM
potassium phosphate pH 7.4 (KP) (1:1), plasmazKP + 0.1 mM DTT. KP and KP + 0.1
mM DTT. Incubation times were 0, 1.5 and 3 h. At the indicated times a 50 pl aliquot
was ted and quenched/preicpitated in 150 pl ice-cold methanol and frozen until
analysis. Relative quantification was performed UPLC -MS/MS.
Investigation of the fate of AF16 in the presence of Caco-2 cells was performed on
three separate occasions. Firstly, a quantitative measurement was performed using
MRM methodology (table 1) over 0, 15, 30 and 60 min. Here, AF16 or its possible
sulfonic acid metabolite was red both in the apical and in the basolateral
compartment (not shown). The second experiment was a qualitative ination of
the fate of AF16 during a 60 min incubation. 50 uM AF16 in HBSS buffer was
incubated at the apical side of the Caco-2 cell monolayer and 100 pl aliquots was
extracted at 5, 10, 20, 30 and 60 min. The sample was placed on a 96-well plate
containing 100 pl MeCN/ H20. The plate was sealed and frozen until can UPLC-
MS analysis (see below and table 2). The third ment was a mirror of the second
and the only difference was that a HBSS solution of AF16:AF 16 IS (1:1) was used, to
aid in the structure elucidation.
Analytical procedures
All s from the different assays were analyzed by UPLC-MS/MS. The following
system was used, a Waters XEVO TQ triple-quadrupole mass spectrometer
(electrospray ionization, ESl) coupled to a Waters Acquity UPLC (Waters Corp). For
chromatographic separation a general gradient was used (1% mobile phase B to 50%
over 3 min total run) on a C18 BEH 1.7um column 2x100mm (Waters Corp). Mobile
phase A consisted of 0.05% TFA and mobile phase B 100% acetonitrile. The flow rate
was 0.5 ml/min. 5 uL of the sample were ed and run with the mass spectrometric
settings ed in table 1. In the full—scan MS analysis the same chromatographic
settings was used except that 10 pl was injected (full loop). The MS was set to scan for
ions in the 100-400 or 400-700 m/z window using the cone voltage in table 1. For
daughter scan is of selected parent ions in table 2 the collision energy was
stepped between 10, 20 and 40 V. The parent ion, suggested charge and ion
time of AF16 and found metabolites are listed in table 2.
Table 1. MRM MS specific settings used for detection. Product ions in bold used
for quantitative is.
‘*"’ m/z t) ml:
(product) (V) energy (V)
586.0 784.2/734.7/ 26 16/20/18
791.2/741.6/ 25 16/20/18
7 7
AF16 sulfonic 601.9 758.5/808.1/ 18/14/14
acid 836.7
627.7 797.2/846.8/
531.8
Table 2. Molecular ion mlz values and chromatographic retention time found in
incubations of AF16 with Caco-2 cells.
2-19
, , ,
777777777
D479 —-
s
Alkylation and oxidation of AF16
Cysteine residues are among the most chemically involved amino acids, participating
typically in redox chemical reactions but also as a nucleophile against reactive
electrophiles such as reactive oxygen species or metabolically modified
xenobiotics/drugs. AF16 contains one cysteine e so it was important to test if
sensitivity towards oxidation or other modification occurs in vitro. At first, oxidation of
AF16 with hydrogen de was performed in order see if it was possible to create
analytical MS methods of the possible sulfenic, ic and sulfonic acid derivatives
(figure 1). Hydrogen peroxide was proven to be a too strong oxidizing reagent to allow
detection of the lower order species and thus only method for the sulfonic acid analog
was d.
To further explore the role of the cysteine in subsequent experiments an alkylated
variant was created using the S-alkylating reagent N-ethyl ide (NEM) (figure 2).
NEM modified AF16 is thus protected against both oxidation of the sulfur, from reacting
with other cysteines (disulfide formation), reacting with electrophiles and presumably
more resilient against proteolytic activity, being an unnatural amino acid.
AF16 ity in human plasma.
AF16, the stable isotope labeled (SlL) AF16 and the alkylated analog AF16-NEM were
ted at 1pM in human plasma: 0.1M potassium phosphate pH 7.4 (KP) (1:1),
human plasma:KP (1:1) + 1mM DTT, KP or KP + 1mM DTT over 3h at 37°C to
investigate stability. KP was included to e buffer capacity to the plasma and to
not confuse the s with pH related effects. The results are shown in figure 3.
AF16 show a high degree of ivity towards plasma as shown in figure 3A. The
kinetics of degradation indicates an in vitro half-life (t1/2) of 0.4h. Inclusion of DTT
seems to have a protective effect which increases the t1/2 to 1.1 h. Interestingly the
unnatural AF16 analog (figure SB) has an almost 5-fold higher stability, t1/2 = 1.8h.
Also here DTT imposes a protective effect, t1/2 = 4.2h. These results indicate AF16 to
be highly sensitive for enzymatic and chemical degradation in systemic circulation. it
also shows that AF16 is more resilient towards enzymatic or chemical reactions if the
cysteine moiety is ed. The effect of DTT with both compounds is more difficult to
understand but most likely this reflects that DTT protects against general oxidation
reactions, such as formation of carbonyl products, eg. threonine, lysine, arginine and
e, all present in AF16. Full-scan LC-MS analysis of the 3h incubations of AF16,
SlL-AF16 and AF16-NEM revealed a few interesting results, see figure 4. Two distinct
degradation products were found, cleavage at the 5th e bond (lysine and
threonine) and at the 10th bond (proline and glutamate) yielding the theoretical
fragments VCHSK—TRSNP, TRSNP-ENNVGL and ENNVGL. The fragments TRSNP-
ENNVGL and ENNVGL were detected by LC-MS but not VCHSK-TRSNP. Presumably
the cted fragment underwent further degradation during the incubation.
Interestingly, in the incubation with AF16-NEM, no apparent lysis occurred at
position five, indicating that proteases acting towards the N-terminus are excluded, e.g.
aminopeptidases.
AF16 stability with Caco-2 cells
Quantitative pilot ments of AF1 6 during Caco-Z cell permeability experiments
clearly showed a rapid disappearance of the e (not shown). It is well known that
in the ine, and as such at the apical side of Caco-2 cells, border peptidases
exist. To further investigate this, a separate ment was designed. Caco-2 cells
were exposed at the apical side to 50 uM AF16, with or without a protease inhibitor
cocktail, and were sampled over 1h. The results on AF16 degradation are shown in
figure 5.
The kinetics of degradation of AF16 is very rapid as described with a t1/2 of 8 min. The
inhibitor il significantly slows down the degradation (t1 /2 = 57 min) but not
completely indicating complicated kinetics. LC-MS analysis was pursued in order to
understand the molecular fate of AF16. In the incubations with or without inhibitors
l new metabolites were detected in comparison to the plasma stability
experiment. However this can depend on the higher incubation concentration used.
Figure 6-8 show the formation kinetics of the metabolites M1-M9 over the experimental
time and figure 9 shows the tentatively determined structures to these products.
Table 3 shows the relative amounts of the individual molecular species, :t inhibitor
cocktail, after 30 and 60 min, respectively. The ntly major metabolites (>10 %)
are indicated in bold numbers. It is however important mentioning when doing such
comparisons as in table 3 that we assume that all the individual ions have similar MS
sensitivity. All the identified products relate to cleavages at specific e bonds. The
suggested amino acid compositions are shown in table 4.
In absence of the inhibitor cocktail, three clear metabolites dominate after 30 min
incubation, M1, M3 and M6. After 60 min tion both M3 and M6 decline, whereas
M1 continues to increase linearly (figure 6A/C and 70). Looking at the suggested
pathways in figure 9, it is reasonable to assume that further N-terminal peptide
cleavage of M3 results in M1. Without knowing the structure of M6 and its relatively
rapid decline after 20 min, this may also indicate that M6 contributes to the formation of
M1. The other metabolites are suggested to be further degradation products of M1 but
could of course be formed in a more direct manner as well, by a slower pathway.
Table 3. Tentative s (%) of the respective molecular species at 30 or 60
min incubation with Caco-2 cells. (+I- peptidase inhibitor cocktail)
1O The inhibitor cocktail consisted of three inhibitors of protelytic enzymes, Bestatin
(aminopeptidase), Diprotin A (dipeptidylpeptidase IV) and Captopril (angiotensin
converting enzyme, a carboxypeptidase). It is interesting to see that due to inhibition of
these enzymes, the formation of M1, M3 and M6 is effectively minimized. Moreover,
the theoretical product M7, shown in figure 4, 9 and table 4, is formed in a relatively
linear manner and seems to be stabilized by the inhibitors. This is in agreement with
inhibition of N-terminal acting proteases. Figure 8A suggests M7 to be formed very
y initially without inhibitors but is likely efficiently further metabolized. Although
relatively small, the major metabolites M8 and M9 have not been identified as of yet. It
is quite possible that these two metabolites stem from more complicated chemistry
than peptide bond cleavage as mentioned above. However, in order to try to
understand the structure of the unknown lites M6, M8 and M9 and to verify the
already structurally ed, an additional ment was performed using the SlL
labeled AF16. From this experiment, one could however not t any more
information part from the fact that M6 contains a SlL amino acid and M8 and M9 do
not. This r needs to be further explored.
Table 4. Suggested amino acid composition of tentatively identified peptides.
m/z (03) Amino acid number Amino acid
com osition
. .
, ,
AF16 586 1-16 VCHSK TRSNP ENNVG
TRSNF‘WENNVG L
11-16 ENNVG L
HSK TRSNP ENNVG L
sNPENNve,-
,,,,,,,
Permeability of either AF16 or any of the detected metabolites was also red at
the basolateral side after 60 min incubation time with the Caco-2 cells. No lites
were however found in this experiment except for M8, which gave a distinct peak in the
ted tion.
Conclusions and future studies
A number of important in vitro experiments have been performed in order to further
tand the in vitro pharmacokinetics of AF 16.
AF16 most likely adsorbs to e.g. polystyrene surfaces. However, the described
countermeasures did not show an effect and it is difficult to interpret how severe it is. At
higher concentration, > 1 uM, the effect is not apparent and thus in our incubations the
effect is most likely minor.
The current studies have shown that AF16 e degrades to several peptide
products in the presence of both human plasma and Caco-2 cells.
The degradation is very rapid in both matrices and indicates a similar metabolic
pathway, yielding M1 as a major, linearly formed and apparent stable product
(cleavage at the 5th peptide bond (lysine and ine)).
The involvement of brush border peptidases is proven by a strong effect of the inhibitor
cocktail, which increases the in vitro tm from 8 to 55 min. The change in metabolic
pattern in the presence of protease inhibitors is interesting but the currently unknown
metabolites (M8, M9) are most likely not formed to a significant degree in the absence
of inhibitors.
Protection of the cysteine at position 2 with N-ethyl maleimide greatly stabilizes the
peptide in human plasma. Most likely due to removal of activity of aminopeptidases.
2017/068111
Taking these s together strongly suggests that the in vitro pharmacokinetics of
AF16 is complex and carefully needs to be balanced and interpreted to the
pharmacologic effect that’s been observed under various ions. It is apparent that
AF16 is rapidly disappearing in vitro after a short incubation time with Caco-2 cells.
Further, a few metabolites are formed very rapidly at a rate that parallels AF16
disappearance. It is quite possible that AF16 and the metabolites act against the same
target with similar efficiency this will be proven in a functional assay.
Example 2
AF16: plasma in vitro stability and metabolic fate
Introduction
This experiment izes the latest findings on interspecies (human, mouse, rat
and dog) plasma kinetics and the qualitative catabolism/metabolism of AF16 in human
and rat plasma.
Materials and Methods
Materials
Solid al of AF16 and the stable isotopically labeled (SIL) AF16 IS was provided
by Lantmannen AB. According to information provided by Ewa Johansson at
enska Hospital, the peptide is approximately 70% in , other components
being 4 x trifluoroacetic acid (TFA) and unknown x H20. However, for the sake of
simplicity, all concentrations regarding the peptide are considered to be 100%. This
assumption does not influence the results presented herein, since they are compared
on a relative basis. Human pooled plasma (4 donors, 2 male and 2 female,
nonsmokers) were obtained from the academic hospital. Animal plasma was from
Novakemi AB. All other chemicals and ables were from common commercial
sources.
Test of plasma precipient
To optimize the MS sensitivity of AF16, the most common protein precipitation agents
with apparent similar efficiency were tested. The plasma matrix consisted of 1:2
(plasma: isotonic potassium phosphate buffer) (pH 7.4) and was precipitated in HPLC
glass vials with either: 1:3 a acetonitrile (MeCN)), 1:4 a: methanol
(MeOH)), 1:3 (plasma: zinc sulfate : 5 M NaOH) (10% WM) and 1:3 (plasma:
trichloroacetic acid (TCA) (10% w/v)) (Polson et al, 2003). The sample was spiked with
a 10 uM (final tration) 1:2 mixture of AF16 and the isotopic labelled AF16
(AFIS). The samples were sealed and centrifuged at 3500 rpm. After centrifugation, the
supernatants were analyzed by UHPLC-MS/MS (see below). The identical samples
were injected three times at different time points to probe the peptide stability with a
given precipitant in the auto sampler at 10°C.
AF16 stability, kinetics and metabolite identification in human and rat plasma
All the incubations of the plasma samples utilized a mixture of AF16 (stock solution
1mM in MeCN:H20) and AFIS (stock solution 0.9mM in MeCNzHZO) 1:2, this to aid in
structural interpretation of metabolic or lic products. The compound mixture was
always pipetted to the bottom of the vial prior to the addition of any other solvent. The
stability in plasma was performed using pooled human pooled plasma, Wistar Rat
plasma, CD-1 mouse plasma and Beagle dog plasma, in sealed HPLC glass vials at
37°C (10 uM incubation conc.). Incubation times were in l between 0 (QC
sample) and 2h. At each time point an aliquot was taken and reaction was stopped with
the selected precipitant incl. dithiothreitol (DTT) (1mM final conc.). Relative
quantification (compared to QC) was med using MS/MS (see below).
lite fication was performed using le on monitoring (MRM,
Skyline predicted MRM)), Lightsight software (Sciex) enhanced MS scan (EMS),
enhanced product ion scan (EPI) and enhanced resolution scan (ERS).
Analytical procedures
All samples from the different assays were analyzed by UHPLC-MS/MS with utilization
of the linear ion trap for the EMS, ERS and EPI scan. The following system was used;
Sciex QTRAP 6500 triple-quadrupole mass ometer (electrospray ionization, ESI)
with a linear ion trap coupled to an Agilent 1290 UHPLC. For chromatographic
separation a general gradient was used (0% mobile phase B to 90% over 5-15 min
total run) on a C18 HSS T3 1.8um column 2x50mm (Waters Corp.) Mobile phase A
consisted of 0.05% TFA/0.05% formic acid and mobile phase B 100% acetonitrile
0.05% TFA/0.05% formic acid. The flow rate was 0.5 ml/min. 10 uL of the sample were
injected and run with the mass spectrometric settings reported in table 5.
Table 5. MRM MS specific settings used for detection of AF16 and lites.
Compound ESI m/z m/z Declusterin Collisio
(+/ t (product, g in
potential energy
(V) (V)
586.0 734.7
AF16 IS
CHSKTRSNPENNVGL+3b12+2heavy 554.6 687.8 73.8
SNPENNVGL+2b6|ight + 472.2 656.3 71.4 26.7
SNPENNVGL+2b6heavy + 475.2 662.3 65.5 24.9
RSNPENNVGL+2b7|ight + 550.3 812.4 65.5 23.9
RSNPENNVGL+2b7heavy + 553.3 818.4 65.5 23.9
TRSNPENNVGL+2b8|ight + 600.8 913.4 71.2 28.7
TRSNPENNVGL+2b8heavy 603.8 919.4 74.9
NPENNVGL+2b5Iight 428.7 569.2 74.9
GL+2b5heavy + 431.7 575.2 74.9
7 7'
ENNVGL+1b3|ight + 645.3 358.1 62.4 23.3
3 7
SKTRSNPENNVGL+2b6heavy + 711.4 674.4 78.2 28.1
_HSKTRSNPENNVGL+2b7Iight 776.9 811.4 82.8
HSKTRSNPENNVGL+2b7heavy + 779.9 811.4 82.8 34.4
VC[SCC]HSKTRSNPENNVGL+3b13+2|ight + 625.3 793.8 87.8 36.8
VC[SCC]HSKTRSNPENNVGL+3b13+2hea
+ 630.0 800.9 76.7 30.6
C[SCC]HSKTRSNPENNVGL+3b13+2light + 592.3 744.4 75 35
C[SCC]HSKTRSNPENNVGL+3b13+2heavy + 594.4 747.4 75 35
Results and Discussion
Impact of plasma protein itate on AF16 MS-sensitivity and UHPLC
chromatography
Due to the complexity of the plasma matrix, it was ant to test how AF16
chromatography and mass spectrometry (MS) sensitivity responds to different protein
precipitation methods. The chosen precipitants are commonly applied and have been
shown to be of r efficiency. The results are shown in s 10 and 11.
Figure 10 shows the results of repeated injection of the same sample three times over
20h. It is clear that TCA and MeCN show good apparent stability over time. A slight
disappearance is noted with ZnSO4 at 20h. A significant loss over time is shown with
MeOH. It is likely that AF16 is stable with MeOH but the loss stems from peptide
precipitation since it is known that peptides may have limited solubility in alcoholic
es. r tests are to be performed.
Figure 11 shows a diagram of the data acquired above in comparison to each other in
terms of MS-intensity (ion counts). TCA itation showed the strongest signal and
was set as the reference. it is clear that the nonorganic methods fall behind, most likely
due to co-eluting suppressing ions. The two methods with best stability (TCA and
MeCN) show over 500-fold difference in ivity, thus for the ued s, TCA
was chosen to be used throughout the study.
Plasma stability of AF16
The relative stability of AF16 in different species is shown in figure 12. Irrespective of
species, AF16 is rapidly disappearing with an in vitro half-life (t1,2) less than or equal to
min. Thus, it is of utmost importance to determine the molecular fate of the e
in order to understand the pharmacokinetic basis of any pharmacological action.
Molecular fate of AF16 in human and rat plasma
AF16 and the isotopically labelled peptide in similar quantity were incubated in plasma
of rat and human as described above. For analysis of full scan MS data, the metabolite
identification software from Sciex, Lightsight, was used, which compares the incubated
MS response to the quality control (QC) sample and assigns apparent peaks as
metabolites with specific mass over charge (m/z) values. The software is currently not
optimal to use with multi-charged nds such as peptides so the te of
metabolite detection is fairly high and manual assessment of each found peak had to
be performed. The largest metabolite peak areas were then rank ordered and in some
cases verified by MS/MS fragmentation. Skyline methodology was used to predict
fragmentation of the identified peptides, but also aided in creating sensitive MRM
methods so that low amounts could be monitored.
Table 6 lists the identified products and their relative amount at 30 min incubation. It
must however be emphasized that this ison assumes that each product has the
same MS sensitivity, which may differ and thus individual percentages may change.
Upon reviewing the s it was quite clear that based on the relative area of the
fied peaks one metabolite was much larger than the others, designated M1 in
table 6. However, the mass pair identified (m/z 625/630) did not correspond to any
lic products (expected lytic peptide bond cleavage). This pair is now
identified as the ne disulfide of AF16.
Table 6. Relative area % of identified metabolites in human and rat plasma.
°/o of QC area at 30 min incubation
time
e sequence Human Rat
VCHSKTRSNPENNVGL
CHSKTRSNPENNVGL
HSKTRSNPENNVGL
SKTRSNPENNVGL
TRSNPENNVGL
RSNPENNVGL
NVGL
NPENNVGL
ENNVGL
Figure 13 shows the relative kinetics of the identified metabolites and it is apparent that
the cysteine disulfide M1 is formed similarly, in both rate and amount, in human and rat
plasma, indicating that rat may be a good model for cological studies. The
greatest disparity between human and rat is the formation of M9, which is formed
linearly over time in human but to a very low extent in rat. One explanation to this could
be that human plasma lytic activity of the smaller fragments is higher than in the
rat plasma. One metabolite (M10) with an m/z pair of 5923/5944 (not shown in table
6/figure 13) remained puzzling and was first understood after MS/MS studies and ERS
mode scan (figure 14). ERS showed that it was triple d on the 13C isotope
pattern (0.3 Da step between isotopic peaks) and agreed with a M2 disulfide product.
M2 is formed in apparent low amounts but this formation could be obscured by an
efficient cysteine oxidation, forming the disulfide. Alternatively M10 is formed from M1
but that is less likely. Simple cleavage of the N-terminal Valine is quite surprising
(forming M2) since most N-terminal acting peptidases out two amino acids at a time
and it is, to our knowledge, rare that a peptidase would act so close to the disulfide and
form M10.
To further verify that indeed these products, M1 and M10, were disulfides, a reaction
mixture was incubated with the cysteine selective alkylating agent N-ethyl maleimide
which confirmed no reaction with these metabolites (not shown).
The kinetics shown in figure 13 was surprising given the fact that DTT was used in the
quenching solution with TCA, although it is likely that the ng capability of DTT is
lowered in acidic conditions. It was therefore tested if the same pattern occurred when
using a neutral precipitant ZnSO4 (without NaOH). This precipitation method is not as
efficient but would hopefully give more information on the formation of the major
disulfide products. The results are shown in figure 15.
From figure 15 and ing to figure 13 it is clear that no large difference is
indicated and it is also very sing how resilient the M1/M1O ide is against
DTT reduction.
sions and future studies
A number of important in vitro experiments have been med in order to further
understand the in vitro pharmacokinetics of AF16 and how it may translate to the in
vivo situation.
1. Trichloroacetic acid has been fied as the optimal choice for removing the
plasma proteins in the tion mixture and maintaining a good MS sensitivity
and chromatography.
2. AF16 is known to be rapidly degrading in plasma from earlier studies. This
investigation further validates this but also shows that the rate is similar
between different s.
3. The apparent major metabolic fate in plasma, of both rat and human, is shown
to be rapid disulfide formation of AF16. This action, being reversible, clearly
protects AF16 from rapid peptidase degradation, which has been shown earlier
with N-ethyl ide stabilization which is less reversible. This can be viewed
as a protective function which enables AF16 to reach its target intact to a much
higher degree.
4. The M1/M10 disulfide is surprisingly resilient against DTT reduction.
Example 3
M1 (AF-17) is synthesized to be able to accurately fy in vitrofin vivo but also to
study the in vitro pharmacokinetic properties in l and to be used in
pharmacological studies.
Biological activity of AF-17 - The antisecretory activity was measured in a rat intestinal
loop model previously described (Lange, S. (1982) FEMS Microbiol. Lett. 15, 239-242).
A l loop was challenged with 3 pg of cholera toxin. Different doses of synthetically
produced AF-17, AF-16 or control (= no peptide, only buffer (XY)) was injected either
intravenously or intramuscular, before nge with cholera toxin. The weight of the
accumulated fluid in the intestinal loop (mg/cm (mg/ml» was recorded after five hours.
Each AF preparation was tested in at least six rats. Fisher's PLSD was used for
statistical analysis of the data.
Biological activity of AF-17 - The biological ty of the AF-17 was tested in a rat
model. The capacity of the AF-17 to inhibit intestinal fluid secretion when injected
intravenously or intramuscular 20-30 sec before intestinal challenge with cholera toxin
is shown in table 7. In control animals injected with buffer only, the cholera toxin
caused a pronounced secretion, 390 mg fluid per cm intestine.
AF-17 caused dose-dependent inhibition of the a ion which was
significantly different from the response to the buffer (p < 0.001, n=6).
Table 7
AF-17 Mean i SEM Significance
Administration
mode
Control 390:5
10pg intravenously 16619 ’ Vs control,
1 p<0.001
1pg intravenously 190:1? Vs control,
p<0.001
20pg intramuscular 152114 Vs control,
I p<0.001
Table 8
‘ AF—16 Mean i SEM N Significance
Administration
mode
l 413.51: (?)
0.1ug intravenously 1683-L (?) ‘ 8 Vs control,
p<0.001
‘ 0.1 pg subcutan 2985: (?) 1 8 Vs control,
p<0.05
1pg subcutan 161i (?) 8 Vs control,
p<0.001
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Claims (8)
- Claims 1. An isolated inant and/or synthetically produced peptide, or a pharmaceutically active salt thereof, having equivalent functional activity, said peptide corresponding to a fragment of the antisecretory factor (AF) protein as shown in SEQ.ID.NO. 1 (AF) and/or a homologue f having equivalent activity, wherein said peptide at least comprises an amino acid sequence as shown in SEQ.ID.NO. 2 (AF-6) and a cysteine disulfide in amino acid (aa) position no. 1 of SEQ.ID.NO. 2, said peptide having antisecretory activity.
- 2. An isolated recombinant and/or synthetically produced peptide according to claim 1, wherein said peptide comprises an amino acid sequence as shown in SEQ.ID.NO. 3 (AF-16) and a cysteine disulfide in amino acid position no. 2 of SEQ.ID.NO. 3.
- 3. An isolated recombinant and/or synthetically produced e according to claim 1, wherein said peptide comprises an amino acid sequence as shown in .NO. 4 (AF-8) and a cysteine disulfide in amino acid position no. 2 of SEQ.ID.NO. 4.
- 4. An isolated inant and/or tically produced peptide according to any one of claims 1-3, which is 6-25 amino acids long, preferably 7-17, 7-16, 7-20, 8-17, 8-20, 17-25, 17-20, such as 7, 8, 16 or 17 amino acids long.
- 5. An isolated recombinant and/or synthetically produced peptide according to any one of claims 1-3, which is at least 6, 7, 8, 16, or 17 amino acids long and at the most 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long.
- 6. An isolated recombinant and/or synthetically produced peptide according to any one of the preceding , comprising the amino acids as shown in SEQ.ID.NO. 7, or the amino acid ce: as shown in .NO. 8, or the amino acid sequence as shown in SEQ.ID.NO. 9.
- 7. An isolated recombinant and/or synthetically produced peptide according to any one of the preceding claims, consisting of the amino acid sequence as shown in SEQ.ID.NO.7, or the amino acid sequence as shown in SEQ.ID.NO. 8, or the amino acid sequence as shown in .NO. 9.
- 8. An isolated recombinant and/or synthetically produced peptide according to any one of the preceding claims, which has a t
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE1651074-5 | 2016-07-18 | ||
SE1651074 | 2016-07-18 | ||
PCT/EP2017/068111 WO2018015379A1 (en) | 2016-07-18 | 2017-07-18 | Antisecretory factor 17 |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ749158A NZ749158A (en) | 2021-06-25 |
NZ749158B2 true NZ749158B2 (en) | 2021-09-28 |
Family
ID=
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