U.S. patent application number 10/258013 was filed with the patent office on 2004-02-12 for use of substances with oxytocin activity against climacteric disorders.
Invention is credited to Lundeberg, Thomas, Uvnas-Moberg, Kerstin.
Application Number | 20040029787 10/258013 |
Document ID | / |
Family ID | 20279369 |
Filed Date | 2004-02-12 |
United States Patent
Application |
20040029787 |
Kind Code |
A1 |
Uvnas-Moberg, Kerstin ; et
al. |
February 12, 2004 |
Use of substances with oxytocin activity against climacteric
disorders
Abstract
The present invention relates to the use of substances with
oxytocin activity against climacteric disorders or similar symptoms
due to dysfunction in the ovaries. It also relates to a
pharmacetucial composition comprising at least one substance with
oxytocin activity against climacteric disorders.
Inventors: |
Uvnas-Moberg, Kerstin;
(Danderyd, SE) ; Lundeberg, Thomas; (Lidingo,
SE) |
Correspondence
Address: |
YOUNG & THOMPSON
745 SOUTH 23RD STREET 2ND FLOOR
ARLINGTON
VA
22202
|
Family ID: |
20279369 |
Appl. No.: |
10/258013 |
Filed: |
June 10, 2003 |
PCT Filed: |
April 18, 2001 |
PCT NO: |
PCT/SE01/00854 |
Current U.S.
Class: |
514/11.6 ;
514/10.2; 514/16.7; 514/17.5; 514/21.1 |
Current CPC
Class: |
A61K 38/095 20190101;
A61P 5/24 20180101; A61K 38/095 20190101; A61K 2300/00
20130101 |
Class at
Publication: |
514/9 |
International
Class: |
A61K 038/12 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 18, 2000 |
SE |
0001440-7 |
Claims
1. Use of a substance with oxytocin activity for the preparation of
a pharmaceutical composition against climacteric disorders, such as
weight changes, mood swings, hot flushes, dry and ulcerous
membranes, fissures, and bone loss.
2. Use according to claim 1, characterised in that the substance is
selected from the group consisting of the following compounds:
9 {overscore (.vertline. )}S-R-S{overscore ( .vertline.)}
X.sub.1-X.sub.2-X.sub.3-X.sub.4-Asn- Cys-X.sub.5-X.sub.6-X.sub.7--
NH.sub.2 (SEQ ID NO: 2)
wherein X.sub.1 is selected from the group consisting of Cys and
nothing, X.sub.2 is selected from the group consisting of Tyr, Phe,
and nothing, X.sub.3 is selected from the group consisting of Ile,
Val, Hoph, Phe, Cha, and nothing, X.sub.4 is selected from the
group consisting of Gln, Ser, Thr, Cit, Arg, and Daba, X.sub.5 is
selected from the group consisting of Pro, and nothing. X.sub.6 is
selected from the group consisting of Ile, Leu, nothing, Val, Hos,
Daba, Thr, and Cit, X.sub.7 is selected from the group consisting
of Gly, nothing, and Ala, R is a chemical bond or, in the case
neither of X.sub.1, X.sub.2, X.sub.3 and X.sub.4 represents Cys,
represents nothing.
3. Use according to claim 1-2, characterised in that the substance
is selected from the group consisting of the following compounds:
SEQ ID NO: 1, SEQ D NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:
6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID
NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,
SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.
4. Use according to claim 1-3, characterised that the
pharmaceutical composition comprises substances that increase the
release of oxytocin and/or the number or affinity of oxytocin
receptors, such as oestrogen, or drugs having an
.alpha..sub.2-agonistic effect, such as clonidine.
5. Use according to claim 1-4, characterised in that the substance
is administered in amount of 0.01-100 ng/g body weight of the
patient.
6. Use according to claim 1-5, characterised in that the substance
is administered in amount of 0.1-10 ng/kg body weight of the
patient.
7. Pharmaceutical composition against climacteric disorders,
characterised in that it comprises an effective concentration of at
least one substance with oxytocin activity in mixture or otherwise
together with at least one pharmaceutically acceptable carrier or
excipient, wherein the substance is selected from the group
consisting of the following compounds:
10 {overscore (.vertline. )}S-R-S{overscore ( .vertline.)}
X.sub.1-X.sub.2-X.sub.3-X.sub.4-Asn- Cys-X.sub.5-X.sub.6-X.sub.7-
-NH.sub.2 (SEQ ID NO: 2)
wherein X.sub.1 is selected from the group consisting of Cys and
nothing, X.sub.2 is selected from the group consisting of Tyr, Phe,
and nothing, X.sub.3 is selected from the group consisting of Ile,
Val, Hoph, Phe, Cha, and nothing, X.sub.4 is selected from the
group consisting of Gln, Ser, Thr, Cit, Arg, and Daba, X.sub.5 is
selected from the group consisting of Pro, and nothing, X.sub.6 is
selected from the group consisting of Ile, Leu, nothing, Val, Hos,
Daba, Thr, and Cit, X.sub.7 is selected from the group consisting
of Gly, nothing, and Ala, R is B chemical bond or, in the case
neither of X.sub.1, X.sub.2, X.sub.3 and X.sub.4 represents Cys,
represents nothing.
8. Pharmaceutical composition according to claim 7, characterised
in that the substance is selected from the group consisting of the
following compounds: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ
ID NO: 5, SEQ ED NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,
SEQ ID NO: 10, SEQ ID NO. 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID
NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,
and SEQ ID NO: 19.
9. Pharmaceutical composition according to claims 7-8,
characterised in that it comprises substrates that increase the
release of oxytocin and/or the number or affinity of oxytocin
receptors, such as oestrogen, or drugs having an
.alpha..sub.2-agonistic effect, such as clonidine.
10. Pharmaceutical composition according to claims 7-9,
characterised in that the effective concentration is 4-70% by
weight, preferably 0.1-50% by weight.
Description
[0001] The present invention relates to the use of substances with
oxytocin activity against climacteric disorders or similar symptoms
due to dysfunction in the ovaries. It also relates to a
pharmaceutical composition comprising at least one substance with
oxytocin activity against climacteric disorders.
BACKGROUND OF THE INVENTION
[0002] Oxytocin was one of the first peptide hormones to be
isolated and sequenced. It is a nonapeptide with two cysteine
residues that form a disulfide bridge between positions 1 and 6 and
corresponds to the formula 1
[0003] For a long time the only effects attributed to oxytocin were
its stimulating effects on milk ejection and uterine contractions,
but in the past decades it has been shown tat oxytocin exerts a
wide spectrum of effects within the central nervous system, CNS. It
has been suggested that oxytocin participates in the control of
memory and learning processes and of various types of behaviour
such as feeding, locomotion, as well as maternal and sexual
behaviour. Oxytocin is also suggested to participate in the control
of cardiovascular functions, thermoregulation, and pain threshold
and fluid balance. There is also evidence that oxytocin is involved
in the control of various immunological processes. It has recently
been demonstrated that oxytocin injections cause a lowering of
blood pressure and increased weight gain-long lasting effects after
repetitive administration. As a central stimulating substance
oxytocin plays an important role in the intention between mother
and progeny in mammals. The products, may also be used prophylactic
in young human beings e.g. already in new born babies or young
children to prevent the development of diseases later on in life
which diseases are dependent on stress conditions during the fetal
life. Such conditions may be heart/vessel diseases such as stroke,
heat infarct, hypertension, and diabetes.
[0004] In human body oxytocin is produced in the paraventricular
nucleus, PVN, and the supraoptic nucleus, SON, of the hypothalamus.
It differs by only two amino acids from vasopressin, which is also
produced in these nuclei. The magnocellular oxytocinergic neurones
of the SON and PVN send oxons to the posterior pituitary from which
oxytocin is released into the circulation. Parvocellular neurones
that originate in the PVN project into multiple areas within CNS.
The oxytocin-producing cells are innervated by cholinergic,
catecholaminergic as well as peptidergic neurones. The presence of
oxytocin in different tissues outside the brain, such as the
uterus, ovaries, testis, thymus, adrenal medulla and pancreas has
been demonstrated and oxytocin is suggested to exert local effects
in these organs.
[0005] A parallel secretion of oxytocin into the brain regions and
into the circulation occurs in response to some stimuli such as
suckling, but other stimuli can cause separate activation of
oxytocinergic neurones, terminating in the brain or the
pituitary.
[0006] It has now turned out that oxytocin has a relieving effect
on climacteric disorders.
[0007] There are several oxytocin derivatives, i.e. compounds with
a structure similar to that of oxytocin. The inventors have
preliminary indications that other oxytocin derivatives than
oxytocin could give the effects against climacteric disorders and
disorders of ovarian functions as well as parts of the oxytocin
molecule. No publications describe the use of oxytocin or any other
oxytocin derivatives or parts of the oxytocin molecule to have
effects against climacteric disorders or other types of premature
ovarian dysfunction.
[0008] By the expression "climacteric disorders" we understand
premenopausal (ie before the menopause), perimenopausal (ie during
the menopause) and postmenopausal (ie after the menopause) weight
changes, mood swings, hot flushes (transient redness and a feeling
of being warm), somatic discomfort, dry and ulcerous mucous
membranes, fissures, and bone loss. Such symptoms often occur at
the time of the menopause, ie the cessation of menstruation in the
human female, occurring usually around the age of 50.
[0009] It has now turned out that oxytocin improves the vaginal
mucosal membranes of women with postmenopausal disorders and
improves the mood of such women (Example 1). In an animal model of
menopause, i.e. ovariectomy, oxytocin normalises hormone levels and
moderates weight changes (Example 2), increases motor activity and
relieves somatic discomfort (Example 3), reduces hot flushes
(Example 4), reduces hyperactivity in the sympathetic nervous
system (Example 5), and suppresses bone loss (Example 6). These
Examples indicate that oxytocin or that substances with oxytocin
activity may be used against climacteric disorders and ovarian
dysfunction. The oxytocin derivatives according to the invention
are not only suitable a postmenopausal disorders but also against
premenopausal, premenopausal and ovarian dysfunction.
[0010] The effect of oxytocin can be extended or strengthened by
administration in combination with drugs increasing the release of
oxytocin and/or the number or affinity of receptors, such as
oestrogen, or drugs having an .alpha..sub.2-agonistic effect, such
as clonidine.
SUMMARY OF THE INVENTION
[0011] The present invention relates to the use of substances with
oxytocin activity against climacteric disorders. The invention also
relates to a pharmaceutical composition comprising an effective
concentration of at least one substance with oxytocin activity in
mixture or otherwise together with at least one pharmaceutically
acceptable carrier or excipient. Such a pharmaceutical composition
could be used in order to improve the effects against climacteric
disorders.
DETAILED DESCRIPTION OF THE INVENTION
[0012] One object of the present invention is the use of a
substance with oxytocin activity for the preparation of a
pharmaceutical composition against climacteric disorders, such as
weight changes, mood swings, hot flushes, dry and ulcerous mucous
membranes, fissures, and bone loss.
[0013] It is preferred that the substance is selected from the
group consisting of the following compounds: 2
[0014] wherein
[0015] X.sub.1 is selected from the group consisting of Cys and
nothing,
[0016] X.sub.2 is selected from the group consisting of Tyr, Phe,
and nothing,
[0017] X.sub.3 is selected from the group consisting of Ile, Val,
Hoph, Phe, Cha, and nothing,
[0018] X.sub.4 is selected f the group consisting of Gln, Ser, Thr,
Cit, Arg, and Daba,
[0019] X.sub.5 is selected from the group consisting of Pro, and
nothing,
[0020] X.sub.6 is selected from the group consisting of Ile, Leu,
nothing, Val, Hos, Daba, Thr, and Cit,
[0021] X.sub.7 is selected from the group consisting of Gly,
nothing, and Ala,
[0022] R is a chemical bond or, in the case neither of X.sub.1,
X.sub.2, X.sub.3 and X.sub.4 represents Cys, represents
nothing.
[0023] The cystein disulfide bridge in SEQ ID NO: 2 is only present
when X.sub.1 represents Cys, X.sub.2 represents Tyr or Phe, and
X.sub.3 represents Ile, Val, Hoph, Phe or Cha.
[0024] It is even more preferred that the substance is selected
from the group consisting of the following compounds: 3
[0025] X.sub.1 is Cys, X.sub.2 is Tyr, X.sub.3 is Ile, X.sub.4 is
Glu, X.sub.5 is Pro, X.sub.6 is Leu, and X.sub.7 is Gly in claim 2
and 7 4
[0026] X.sub.1 is Cys, X.sub.2 is Tyr, X.sub.3 is Ile, X.sub.4 is
Gln, X.sub.5 is Pro, X.sub.6 is Ile, and X.sub.7 is Gly in claim 2
and 7 5
[0027] X.sub.1 is Cys, X.sub.2 is Tyr, X.sub.3 is Ile, X.sub.4 is
Ser, X.sub.5 is Pro, X.sub.6 is Ile, and X.sub.7 is Gly in claim 2
and 7 6
[0028] X.sub.1 is Cys, X.sub.2 is Phe, X.sub.3 is Val, X.sub.4 is
Arg, X.sub.5 is Pro, X.sub.6 is Thr, and X.sub.7 is Gly in claim 2
and 7 7
[0029] X.sub.1 is Cys, X.sub.2 is Tyr, X.sub.3 is Ile, X.sub.4 is
Gln, and X.sub.5-X.sub.7 is nothing in claim 2 and 7 8
[0030] X.sub.1 is Cys, X.sub.2 is Tyr, X.sub.3 is Ile, X.sub.4 is
Gln, X.sub.5 is Pro, and X.sub.6-X.sub.7 is nothing in claim 2 and
7 9
[0031] X.sub.1 is Cys, X.sub.2 is Tyr, X.sub.3 is Ile, X.sub.4 is
Gln, X.sub.5 is Pro, X.sub.6 is Leu, and X.sub.7 is nothing in
claim 2 and 7
[0032] Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH.sub.2
[0033] SEQ ID NO: 9
[0034] X.sub.1 is nothing, X.sub.2 is Tyr, X.sub.3 is Ile, X.sub.4
is Gln, X.sub.5 is Pro, X.sub.6 is Leu, and X.sub.7 is Gly in claim
2 and 7
[0035] Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH.sub.2
[0036] SEQ ID NO: 10
[0037] X.sub.1-X.sub.2 is nothing, X.sub.3 is Ile, X.sub.4 is Gln,
X.sub.5 is Pro, X.sub.6 is Leu, and X.sub.7 is Gly in claim 2 and
7
[0038] Gln-Asn-Cys-Pro-Leu-Gly-NH.sub.2
[0039] SEQ ID NO: 11
[0040] X.sub.1-X.sub.3 is nothing, X.sub.4 is Gln, X.sub.5 is Pro,
X.sub.6 is Leu, and X.sub.7 is Gly claim 2 and 7
[0041] Ile-Gla-Asn-Cys-Pro-NH.sub.2
[0042] SEQ ID NO: 12
[0043] X.sub.1-X.sub.2 is nothing, X.sub.3 is Ile, X.sub.4 is Gln,
X.sub.5 is Pro, and X.sub.6-X.sub.7 is nothing in claim 2 and 7
10
[0044] X.sub.1 is Cys, X.sub.2 is Tyr, X.sub.3 is Val, X.sub.4 is
Thr, X.sub.5 is Pro, X.sub.6 is Leu, and X.sub.7 is Gly in claim 2
and 7
1 SEQ ID NO: 14 {overscore (.vertline. )}S{overscore (
)}S{overscore ( .vertline.)}
Cys-Tyr-Hoph-Thr-Asn-Cys-Pro-Val-Gly-NH.sub.2
[0045] X.sub.1 is Cys, X.sub.2 is Tyr, X.sub.3 is Hoph, X.sub.4 is
Thr, X.sub.5 is Pro, X.sub.6 is Val, and X.sub.7 is Gly in claim 2
and 7
2 SEQ ID NO: 15 {overscore (.vertline. )}S{overscore (
)}S{overscore ( .vertline.)}
Cys-Tyr-Phe-Cit-Asn-Cys-Pro-Leu-Gly-NH.sub.2
[0046] X.sub.1 is Cys, X.sub.2 is Tyr, X.sub.3 is Phe, X.sub.4 is
Cit, X.sub.5 is Pro, X.sub.6 is Leu, and X.sub.7 is Gly in claim 2
and 7
3 SEQ ID NO: 16 {overscore (.vertline. )}S{overscore (
)}S{overscore ( .vertline.)}
Cys-Tyr-Cha-Arg-Asn-Cys-Pro-Hos-Ala-NH.sub.2
[0047] X.sub.1 is Cys, X.sub.2 is Tyr, X.sub.3 is Cha, X.sub.4 is
Arg, X.sub.5 is Pro, X.sub.6 is Hos, and X.sub.7 is Ala in claim 2
and 7
4 SEQ ID NO: 17 {overscore (.vertline. )}S{overscore (
)}S{overscore ( .vertline.)}
Cys-Tyr-Val-Dada-Asn-Cys-Pro-Dada-Ala-NH.sub.2
[0048] X.sub.1 is Cys, X.sub.2 is Tyr, X.sub.3 is Val, X.sub.4 is
Daba, X.sub.5 is Pro, X.sub.6 is Cit, and X.sub.7 is Ala in claim 2
and 7
5 SEQ ID NO: 18 {overscore (.vertline. )}S{overscore (
)}S{overscore ( .vertline.)}
Cys-Tyr-Hoph-Dada-Asn-Cys-Pro-Cit-Ala-NH.sub.2
[0049] X.sub.1 is Cys, X.sub.2 is Tyr, X.sub.3 is Hoph, X.sub.4 is
Daba, X.sub.5 is Pro, X.sub.6 is Cit, and X.sub.7 is Ala in claim 2
and 7
6 SEQ ID NO: 19 {overscore (.vertline. )}S{overscore (
)}S{overscore ( .vertline.)}
Cys-Tyr-Phe-Arg-Asn-Cys-Pro-Val-Ala-NH.sub.2
[0050] X.sub.1 is Cys, X.sub.2 is Tyr, X.sub.3 is Phe, X.sub.4 is
Arg, X.sub.5 is Pro, X.sub.6 is Val, and X.sub.7 is Ala in claim 2
and 7,
[0051] wherein Cha stands for cyclohexylalanine, 11
[0052] Hoph stands for homophenylalanine, 12
[0053] Cit stands for citruline, 13
[0054] Daba stands for diaminobutyric acid, and 14
[0055] Hos stands for homoserine. 15
[0056] There are different processes described for the synthetical
production of oxytocin; commercial processes are for instance
described in U.S. Pat. Nos. 2,938,991 and 3,076,797.
[0057] A substance with oxytocin activity refers, whenever
applicable, in addition to oxytocin also to precursors, metabolic
derivatives, oxytocin agonists or analogues displaying the same
properties. It can be shown that a substance has oxytocin activity
by performing tests showing the activity of the actual substance eg
by performing a double blind cross-over randomised protocol as
described in Example 1.
[0058] Annetocin has been isolated from the earthworm, as described
in Oumi T. Ukena K, Matsushima O, Ikeda T, Fujita T, Minakata H,
Nomoto K, Annetocin: an oxytocin-related peptide isolated from the
earthworm, Eisenia foetida, Biochem Biophys Res Commun 1994,
January 14; 198(1); 393-399.
[0059] Other substances with oxytocin activity could also be used,
such as naturally occurring or artificially modified variants,
analogues, and derivatives of oxytocin, mesotocin, isotocin, and
annetocin. Such substances could be obtained by addition,
insertion, elimination, or substitution of at least one amino acid
in these hormones. By a substance with an oxytocin like activity is
also understood precursors, metabolites such as metabolic
derivatives e.g. metabolic degradation products, agonists, or
analogues of the substances mentioned herein displaying the same
properties. Metabolic derivatives or metabolic degradation products
may be oxytocin like peptides e.g. wit nine amino acids such as
oxytocin, mesotocin, isotocin, and annetocin from which one or more
amino acids has been deleted from either the carboxyl terminal end
or the amino terminal end or both the carboxyl terminal and the
amino terminal end, preferably 1-3 amino acids from each terminal.
It could be ascertained that these variants are analogues of
oxytocin, mesotocin, isotocin or annetocin by immunological
methods, e.g. RIA (radioimmunoassay), IRMA (radiometric methods),
RIST (radioimmunosorbent test), and RAST (radioallergosorbent
test). The invention also includes substances having at least 50,
60, 70, 80 and most preferably 90% homology to oxytocin, and
showing oxytocin activity.
[0060] As mentioned above there are indications that subfragments
of the oxytocin molecule could have effects against cimacteric
disorders. Examples of subfragments of the oxytocin molecule are
the following compounds: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,
SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.
[0061] There is also a possibility to create new compounds with
oxytocin activity by means of computer simulation. Methods for
computer simulation are known by a person skilled in the art, e.g.
as described in EP 0660 210 A2. Seven new compounds have been
created by means of computer simulation, namely the following
peptides: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:
16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.
[0062] The invention also relates to the peptides mentioned above
in both D- and L-form, and racemates thereof. Especially the
invention relates to the L-form. By inversion of the peptide
sequence thereof, the D-form could be converted to the L-form. The
effect of the D- and L-forms are the same. These and the peptides
above can be produced by methods known to a person skilled in the
art, e.g. according to Merrifield, P. B., "Solid Phase Synthesis",
Angew. Chemie, 1985, No. 97, p. 801.
[0063] The pharmaceutical compositions according to the invention
may contain substances that extend or strengthen the effects of
oxytocin. Such substances could increase the release of oxytocin
and/or the member or affinity of oxytocin receptors, such as
oestrogen, or drugs having an .alpha..sub.2-agonistic effect, such
as clonidine.
[0064] It is preferred tat a substance with oxytocin activity is
administered in an amount of 0.01-100 ng/kg body weight of the
patient, in particular 0.1-10 ng/kg.
[0065] Another object of the invention is a pharmaceutical
composition against climacteric disorders comprising an effective
concentration of at least one substance with oxytocin activity in
mixture or otherwise together with at least one pharmaceutically
acceptable carrier or excipient. It is preferred that the substance
is selected from the group consisting of compounds with the formula
SEQ ID NO: 2. It is even more preferred that the substance is
selected from the group consisting of the following compounds: SEQ
ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,
SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:
11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ
ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.
[0066] The pharmaceutical compositions according to the invention
may contain substances that extend or strengthen the effects of
oxytocin. Such substances could increase the release of oxytocin
and/or the number or affinity of oxytocin receptors, such as
oestrogen, or drugs having an .alpha..sub.2-agonistic effect such
as clonidine.
[0067] The pharmaceutical compositions are prepared in a manner
known to a person skilled in the pharmaceutical art. The carrier or
the excipient could be a solid, semi-solid or liquid material that
could serve as a vehicle or medium for the active ingredient.
Suitable carriers or excipients are known in the art. The
pharmaceutical composition could be adapted to oral, parenteral,
intravaginal, or topical use and could be administered to the
patient as tablets, capsules, suppositories, solutions, suspensions
or the like.
[0068] The pharmaceutical compositions could be administered
orally, e.g. with an inert diluent or with an edible carrier. They
could be enclosed in gelatine capsules or be compressed to tablets.
For oral therapeutic administration the compounds according to the
invention could be incorporated with excipients and used as
tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers,
chewing gums and the like. These preparations should contain at
least 4% by weight of the compounds according to the invention, the
active ingredient, but could be varied according to the special
form and could, suitably, be 4-70% by weight of the unit. The
amount of the active ingredient that is contained in compositions
is so high that a unit dosage form suitable for administration is
obtained.
[0069] The tablets, pills, capsules, lozenges and the like could
also contain at least one of the following adjuvants: binders such
as microcrystalline cellulose, gum tragacanth or gelatin,
excipients such as starch or lactose, disintegrating agents such as
alginic acid, Primogel, corn starch, and the like, lubricants such
as magnesium stearate or Sterotex, glidants such as colloidal
silica dioxide, and sweetening agents such as saccharose or
saccharin could be added or flavourings such as peppermint, methyl
salicylate or orange flavouring. When the unit dosage form is a
capsule it could contain in addition to the type above a liquid
carrier such as polyethylene glycol or a fatty oil. Other unit
dosage forms could contain other different mars that modify the
physical form of the unit dosage form, e.g. as coatings.
Accordingly, tablets or pills could be coated with sugar, shellac
or other enteric coating agents. A syrup could in addition to the
active ingredient contain saccharose as a sweetening agent and some
preservatives, dyes and flavouring agents. Materials that are used
for preparation of these different compositions should be
pharmaceutically pure and non-toxic in the amounts used.
[0070] For parenteral administration the compounds according to the
invention could be incorporated in a solution or suspension.
Parenteral administration refers to the administration not trough
the alimentary canal but rather by injection through some other
route, as subcutaneous, intramuscular, intraorbital, intracapsular,
intraspinal, intrasternal, intravenous, intranasal, intrapulmonary,
through the tract, through eye drops, rectal or intravaginal (e.g.
as a suppository, a vagitorium, a cream or an ointment), through
the lactiferous rat in cattle, into an organ such as bone marrow,
etc. Bone marrow may also be treated in vitro. These preparations
could contain at least 0.1% by weight of an active compound
according to the invention but could be varied to be approximately
0.1-50% thereof by weight. The amount of the active ingredient that
is contained in such compositions is so high that a suitable dosage
is obtained.
[0071] The solutions or suspensions could also comprise at least
one of the following adjuvants: sterile diluents such as water for
injection, saline, fixed oils, polyethylene glycols, glycerol,
propylene glycol or other synthetic solvents, antibacterial agents
such as benzyl alcohol or methyl paraben, antioxidants such as
ascorbic acid or sodium bisulfite, chelating agents such as
ethylene diamine tetraacetic acid, buffers such as acetates
citrates or phosphates, and agents for adjustment of the tonicity
such as sodium chloride or dextrose. The parenteral preparation
could be enclosed in ampoules, disposable syringes or multiple
dosage vessels made of glass or plastic.
[0072] For topical administration the compounds according to the
invention could be incorporated in a solution, suspension,
ointment, or gel. These preparations could contain at least 0.1% by
weight of an active compound according to the invention but could
be varied to be approximately 0.1-50% thereof by weight. The amount
of the active ingredient that is contained in such compositions is
so high that a suitable dosage is obtained. The administration
could be facilitated by applying touch, pressure, massage, heat,
warms, or infrared light on the skin, which leads to enhanced skin
permeability. Hirvonen, J., Kalia, Y N, and Gay, R H. Transdermal
delivery of peptides by iontophoresis. Nat Biotechnol December
1996; 14(13): 1710-1713 describes how to enhance the transport of a
drug via the skin using the driving force of an applied electric
field. Preferably, iontophoresis is effected at a slightly basic
pH.
[0073] Other administration forms are inhalation through the lungs,
buccal administration via the mouth, enteral administration via the
small intestine, and local administration with a release,
preferably a slow release, of the active substance eg in the form
of a ring. All these administration forms could be effected by
means known by a person skilled in the art.
[0074] All publications mentioned herein are hereby incorporated by
reference. By the expression "comprising" we understand including
but not limited to. Thus, other non-mentioned substances, additives
or carriers may be present.
SHORT DESCRIPTION OF THE FIGURES
[0075] FIG. 1 is a bar graph showing the plasma concentration of
insulin in ovariectomised rats administered subcutaneously with
saline (NaCl) and oxytocin.
[0076] FIG. 2 is a bar graph showing the plasma concentrations of
corticosterone and growth hormone (GH) in ovariectomised rats
subjected to subcutaneous treatment. Group A refers to rats treated
with oestrogen, progesterone, and oxytocin. Group B refers to rats
treated with oestragen and progesterone. Group C refers to rats
treated with oxytocin. Group D refers to rats treated with
saline.
[0077] FIG. 3 shows the weight change in ovariectomised rats in
response to consecutively given subcutaneous treatments with
oxytocin compared to controls receiving saline.
[0078] FIG. 4 shows the somatic discomfort relieving effect for
ovariectomised rats given intravaginal treatments with oxytocin
compared to controls receiving saline.
[0079] The invention will be illuminated by the following Examples,
which are only intended to illuminate and not restrict the
invention in any way.
EXAMPLES
[0080] Materials and Methods
Animals (Example 2-3)
[0081] Female sexually mature Sprague-Dawley rats weighing 250-300
g were used (B & K Universal Sollentuna, Sweden). The rats were
housed 5 to each cage with free access to food and tap water at
20.+-.2.degree. C. and with a 12 h light/12 h dark cycle for at
least 3 weeks before and throughout the experimental period.
Animals (Example 4-5)
[0082] Virgin adult cycling Sprague-Dawley rats weighing 300-350 g
(Example 4) and 190-210 g (Example 5) and with regular 4-day
oestrous cycles were used (Mollegaard, Denmark). The rats were
housed 4 to each cage with free access to pelleted food and tap
water at 22.degree. C. and with a 12 h light/12 h dark cycle for at
least 1 week before and throughout the experimental period.
Animals (Example 6)
[0083] Four lots of 120-day-old female Wistar rats (mean.+-.SD
initial weight 273.+-.28 g) were used. The rats were randomized
into groups based on their body weight. Rats weighing the most and
those weighing the least were assigned each time alternatively to
one group, so at the end of the randomization process the mean body
weight of each group was comparable. Sample size was calculated in
a pilot study after determining the variability of densitometric
measurements.
[0084] All rats were fed standard chow feed containing 7.1 g/kg of
calcium and 5 g/kg of phosphorus; the energy content of the feed
was 3100 kcal/kg. A control group of 15 rats were not manipulated
but fed the stud diet (control group, mean initial weight 269.+-.31
g). A experimental group of 15 rats was ovariectomized at 120 days
of life and given oxytocin for 10 days 1.0 mg/kg sc. (OVX+Oxy
group, mean initial weight 277.+-.32 g). A group of 15 rats
underwent ovariectomy (OVX group, mean initial weight 282.+-.27 g).
The OVX group did not receive oxytocin and was fed the standard
diet. The ovariectomy was performed as in previous studies [Rico H,
Amo C, Revilla M, Arribas I, Gonzalez-Riola J, Villa L F,
Rodriguez-Puyol M Etidronate versus Clodronate in the prevention of
postovariectomy bone loss. An experimental study in rats. Clin Exp
Rheumatol 1994, 12:301-304]. The manipulated rats were controlled
to observe the development of intestinal function, alterations, and
infection or dehiscence of surgical sutures. All rats received
water ad libitum. The rats were kept for 30 days in the animal
laboratory of department of Physiology and Pharmacology, Karolinska
Institutet Living conditions (12 hours of light and 12 hours of
darkness; mean room temperature 22.degree. C.), habitat, and diet
met current guidelines of the European Union. Weight was measured
with a precision biomedical balance.
[0085] Ovariectomy
[0086] Ovariectomy means removal of an ovary or ovaries and can eg
be effected according to Merchentaler, I., Funkhouser, J. M.,
Carver, J. N, Lundeen, S. G., Ghosh, K, Winneker, R. C. The effect
of estrogens and antiestrogens in a rat model for hot flush.
Maturitas, Nov 16, 1998; 30(3): 307-316.
Statistics (Example 1-3 and 6)
[0087] The results are presented as means.+-.SD. Statistical
analysis was performed by means of a one-way ANOVA, followed by
Dunnett's t-test for post hoc comparisons. .sup.nap>0.05;
*p<0.05; **p<0.01.
Statistics (Example 4-5)
[0088] Statistical analyses were carried out using the SPSS 8.0
software. NGF concentrations in the pituitary gland, the
hypothalamus, the hippocampus, the ovary, and the adrenal glands
were analysed and the groups compared using analyses of variance
(ANOVA) followed by multiple comparison procedures Bonferroni
test).
[0089] All results are presented as mean.+-.standard error of mean
(SEM). A p-value less than 0.05 is considered significant. The 95%
confidence interval (CI) was given when p<0.05.
Example 1
Intravaginal Oxytocin Administration to Postmenopausal Women
[0090] Seven women at an age of 60-70 years, otherwise completely
healthy, with expressed postmenopausal disorders in the form of
white, thin, atrophic, easy-bleeding and ulcerous vaginal mucous
membranes were included in the study. They were treated with
oxytocin 1 mg/ml mixed with 1 ml cellulose gel intravaginally
driving five consecutive days,
[0091] The treatment was effected by a gynaecologist who inspected
the mucous membranes. After a treatment lasting 2-3 days, the
mucous membranes had improved in that they looked like those in a
female woman. They were all perfused with blood and all ulcers had
disappeared.
[0092] At the same time, it was observed that the mood of the women
was improved. They seemed obviously happy and reported that they
felt happy, and many of them had resumed their sex lives.
[0093] Atrophic mucous membranes have formerly been treated with
oestrogen cream or gel with oestradiol or oestrone as an active
substance. Then, a certain improvement is observed after a
treatment of approximately 2 weeks, but the improvement is not as
complete as after the preliminary observations with the oxytocin
gel. Furthermore, the improvement is often preceded by a period
with local itch and irritation. No mood improvement has been
observed after a local oestrogen treatment.
[0094] Besides, a further experiment was conducted, wherein 20
women, all of them at two years into menopause and otherwise
healthy. None of them underwent hormone replacement therapy. All
patients had objective and subjective symptoms of vaginal rash,
pain and a feeling of dryness.
[0095] Ten patents were treated with oxytocin 1 mg/ml mixed with 1
ml close gel intravaginally during five consecutive days, whereas
the remaining ten patients were treated with 1 ml cellulose gel
intravaginally dog five consecutive days as control. Before
treatment with either control or oxytocin gel we took a biopsy,
this procedure was repeated after 5 days of treatment. The protocol
was a double blind protocol, ie the microscopic evaluations were
performed by the same experienced pathologist before the code was
broken.
[0096] None of the women receiving control showed improvement in
the microscopic evaluation, and they did not express subjective
relief in the interviews. The women with oxytocin treatment
expressed relief and the histology showed normalization of the
epithelium (except in the cases where the mucous epithelium was
normal before treatment) and also no or a lesser degree of
inflammation.
Example 2
Subcutaneous Oxytocin Administration to Rats
[0097] In an experimental animal study, eight ovariectomised rats
were treated with oxytocin. The ovaries were removed in order to
create a menopause-like situation. After a mouth, an endocrine
study was made. The "menopausal" rats get a changed hormone
balance. Their insulin levels and, to a certain extent their blood
sugar levels rise, possibly in consequence of increased insulin
resistance. Their cortisol and growth hormone levels decrease,
which expresses an inability to mobilise calories for activity. An
oxytocin treatment during five days (1 mg/kg given subcutaneously
during five days) restored the hormone balance. The insulin levels
were normalised ie lowered in oxytocin treated rats compared to
control rats treated with NaCl (see FIG. 1), as well as the blood
sugar levels.
[0098] In another experimental animal study, the effect of oxytocin
(1 mg/kg given subcutaneously during five days) combined with
hormone treatment was investigated. All treatment groups A-D each
contained eight ovariectomised rats. The corticosterone levels were
significantly higher in rats treated with oxytocin only than in
rats treated with oestrogen+progesterone. The GH levels were
significantly higher in rats treated with
oestrogen+progesterone+oxytocin, oestrogen+progesterone, or
oxytocin than in saline treated rats (see FIG. 2). Analogously to
the insulin levels, the corticosterone and GH levels were
normalised (but increased) by oxytocin treatment.
[0099] A rat having its ovaries removed also gains weight compared
to untreated rats. Rats treated with oxytocin for 10 days do not
gain weight as much despite of the fact that they do not eat less
(see FIG. 3).
Example 3
Intravaginal Oxytocin Administration to Ovariectomised Rats
[0100] The ovaries of seven female rats were removed. After 2
weeks, the rats were treated with oxytocin gel given intravaginally
(conc. 1 mg/ml). Two days after completed treatment, the rats given
oxytocin showed some behaviour changes reflecting an increased
motor activity such as forward locomotion compared to rats only
treated with gel without oxytocin. This indicates that it is
possible to create central oxytocin effects by giving oxytocin
intravaginally, even in rats. Most probably, there is an activation
of nerves present in the vaginal mucous membrane.
[0101] The following Table I shows the forward locomotion of
ovariectomised rats (rats R1-R7) after intravaginal oxytocin
administration compared to saline administered control rats (rats
B1-B7). By forward locomotion is meant successive interruptions of
photocells in the lower rows when the animal is moving in the same
direction, i.e. initially, the location of the animal is defined by
the photocell being interrupted, the next interruption indicates
that the animal is moving, and successive interruptions are
registered as locomotion, as long as the animal moves along the
same vector (for further details, see Carter, S. Oxytocin and
sexual behavior. Neurosci. Biobehav. Rev. 16:131-144; 1992). The
total experimental time is 30 minutes per rat, and the values in
table I show the forward locomotion for the first 15 minutes, for
the whole experimental time, and for the last 15 minutes.
7 TABLE I Minutes 1-15 Minutes 16-30 Minutes 1-30 Control B1 23.9
24.6 24.2 B2 24.7 15.9 22.1 B3 27.4 22.5 25.5 B4 24.5 9.5 22.6 B5
31.3 16.8 26.7 B6 30.6 22.5 27.6 B7 27.3 16.5 24.3 AVERAGE 27.10
18.33 24.71 SD 2.97 5.22 2.03 Oxytocin R1 30.7 20.3 27.7 R2 27.8
19.8 25.3 R3 28.8 17.7 25.1 R4 28.6 23.2 26.3 R5 32.2 21.6 28.7 R6
32.3 15.3 27.7 R7 31 23 27.6 AVERAGE 30.20 20.13 26.91 SD 1.81 2.86
1.36 T-test 0.04* 0,44 ns 0.03*
[0102] Besides increased motor activity, oxytocin also gives rise
to relief of somatic discomfort. One way of applying somatic
discomfort to an organism is to subject a part of it to a high
temperature. This relief effect of oxytocin was investigated in a
tail-flick test. In this test the tails of ovariectomised rats were
immersed in water at 51.degree. C. With reference to FIG. 4, the
latency time from the immersion of the tails to the withdrawal
thereof were measured for rats treated with oxytocin
(.tangle-solidup.) and rats treated with saline (.quadrature.). For
each group of rats, the test sessions were accomplished three times
with a period of 15 minutes between the sessions. As seen from FIG.
4, an intravaginal administration of oxytocin increases the latency
time. A longer latency time means that the rat could stand the
somatic discomfort better such as increased temperature ta it
otherwise would do.
Example 4
Effects of Subcutaneous Treatment with Oxytocin on Hot Flushes in
Rats
[0103] In this study, ovariectomised rats (300-350 g) were treated
with oxytocin for 8 days subcutaneously. Rats were addicted to
morphine by implanting a morphine pellet (75 mg each)
subcutaneously on days 3 and 5 of treatment. On the last day of
treatment, an infrared thermistor was placed above the tail of each
animal and morphine addiction was withdrawn by naloxone injection
(1.0 mg/kg, subcutaneously). Temperature measurements were taken
for 1 h under ketamine (80 mg/kg. intramuscularly) anaesthesia. In
general, vehicle treated rats showed a 5-6.degree. C. elevation of
their tail skin temperature with the peak occurring about 15 ml
after naloxone injection. Repeated oxytocin treatment 1.0 mg/kg
subcutaneous oxytocin or 0.1 m g subcutaneous
17-alpha-ethinyloestradiol (EA) significantly inhibited the
temperature increase (flashes).
Example 5
Effects of Oxytocin on Ovarian Morphology in Rats with
Experimentally-Induced Polycystic Ovaries
[0104] Introduction
[0105] The pathogenesis of polycystic ovary syndrome (PCOS) has
been attributed to dysfunction in oestrogen synthesis and/or
oestrogen receptors in the ovaries. The experimentally induced rat
PCO model produced by a single injection of oestradiol valerate
(EV) has similarities to human PCOS, and both are associated with
altered concentrations of oestrogen and hyperactivity in the
sympathetic nervous system.
[0106] The main findings in the present study were that
significantly higher concentrations of NGF were found in the
ovaries and the adrenal glands in the rat PCO model than in the
control rats, which were only injected with the vehicle (oil or
NaCl) and that oxytocin treatments in PCO rat resulted in
significantly lower concentrations of NGF in the ovaries compared
to non-oxy-treated PCO rats. The result in the present study
provide support for the theory that oxytocin inhibits hyperactivity
in the sympathetic nervous system by compensating for the effects
of oestrogen, possibly through the direct or indirect activation of
oestrogen receptors including the orphanin ones.
[0107] Polycystic ovary syndrome (PCOS), one of the most common
causes of an ovulation in women of reproductive age, is a complex
endocrine and metabolic disorder (Franks S. Polycystic ovary
syndrome. Arch Dis Child, 1997; 77(1): 99-90).
[0108] Women with PCOS have an increased risk of endometrial
cancer, hypertension, and type II diabetes, and they need some kind
of long-standing treatment (Dahlgren E, Janson P O, Johansson S,
Lapidus L, Lindstedt G. Tengborn L, Hemostatic and metabolic
variables in women with polycystic ovary syndrome [see comments].
Fertil Steril, 1994; 61(3): 455-460). Traditional pharmacological
treatment, including oestrogen, for ovulation induction is
effective but side effects such as superovulation are quite
common.
[0109] All rats (totally 59) received a single intramuscular
injection either of oestradiol valerate (EV), oil, or 0.15 M NaCl
(Kabi Pharmacia AB, Sweden) and were anaesthetised with enflurane.
Twenty rats re injected with 4 mg EV in 0.2 ml oil/rat (ten rats in
the control group and ten in the oxytocin treated). They were
decapitated after 30 days to elucidate when the ovaries display
characteristic features of well-defined PCO (Brawer et al., 1978,
Supra; Brawer et al., 1986, supra). The oxytocin treated group
received subcutaneous oxytocin 1.0 mg/kg subcutaneously every third
day during the experimental period.
[0110] Ovarian Morphology
[0111] The ovaries in the EV control group (4 mg EV in 0.2 ml
oil/rat), displayed the characteristic features of well-defined PCO
in rats. The ovaries in the oxytocin treated group showed
significantly less changes.
[0112] The findings of the present study show that oxytocin
treatment reduces the hormonal changes seen in
experimentally-induced PCO.
Example 6
Effects of Oxytocin on Bone Loss
[0113] Introduction
[0114] There is a close relationship between bone mass and age in
women. Osteoporosis related decrease in bone mass occurs after the
age of 40 years. The rate of bone loss is accelerated in the
decade, which follows the menopause. Interestingly, there is also a
relationship between age and fracture rate. The fracture rate,
initially low, increases rapidly in women over the age of 65 years.
Also, there is a relationship between bone mass and fracture rate
suggesting that the bone which is lost during the initial phases of
aging is not essential to the integrity of the skeleton.
Nevertheless, the decreases in bone mass with age in women would be
expected to result in corresponding increases in mechanical strain
on the remaining bone.
[0115] Osteoclasts are large multinuclear cells associated with the
absorption and removal of bone and are, accordingly, implicated in
bone loss. An object of the invention is, therefore, to counteract
the action of osteoclasts. As shown in the present Example,
administration of oxytocin suppresses bone loss in rats and, hence,
counteracts the action of osteoclasts.
[0116] Experimental Procedure
[0117] Densitometric and Morphometric Studies. After 30 days, all
rats were anesthetized and killed by exsanguination from the
abdominal aorta after being anesthetized with 4 mg/100 g body
weight of sodium pentothal. The femur and 5th lumbar vertebra of
rat were dissected and placed in an 85.degree. C. water bath to
remove soft tissue. Femoral length and vertebral height were
measured with a caliper and bones were weighed on a precision
balance. The BMC and BMD of the whole right femur and 5th lumbar
vertebra were measured using a desitometer equipped with a special
program for studying small Gals and samples. The instrument was
calibrated daily. Because of the influence of weight on bone mass
in rate [Rico H, Amo C, Revilla M, Arribas I, Gonzalez-Riola J,
Villa L F, Rodriguez-Puyol M Etidronate versus Clodronate in the
prevention of postovariectomy bone loss. An experimental study in
rats. Clin Exp Rheumatol 1994, 12:301-304] and human beings [Rico
H, Revilla M, Villa L F, Alvarez de Buergo M A, Ruiz-Contreras D
Determinants of total and regional bone mineral content and density
in postpubertal normal women. Metabolism 1994, 43 :263-266], BMC
was corrected for final body weight (BMC/BW). The BMC and BMD of
the femurs and 5th lumbar vertebra were measured separately.
[0118] Results
[0119] The values obtained for each parameter in every group of
rats are shown in Table II.
[0120] In Table II:
[0121] F-BMC means femur bone mineral content,
[0122] F-BMD means femur bone mineral density,
[0123] V-BMC means 5.sup.th lumbar vertebral bone mineral
content,
[0124] V-BNMD means 5.sup.th lumbar vertebral bone mineral
density,
[0125] OVX means ovariectomized rats, and
[0126] OVX+Oxy means oxytocin-treated ovariectomized rats.
8TABLE II Values for each parameter in three groups of rats
Controls OVX OVX + Oxy Initial weight (g) 269 .+-. 31 282 .+-. 27
277 .+-. 32 Final weight (g) 312 .+-. 22 331 .+-. 25 334 .+-. 31
Femur (mm) 34.6 .+-. 0.5 32.8 .+-. 1.4 35.5 .+-. 1.3 Femur (mg) 729
.+-. 54 669 .+-. 74 712 .+-. 42 F-BMC (mg) 399 .+-. 36 282 .+-. 42
335 .+-. 37 F-BMD (mg/cm.sup.2) 145 .+-. 16 117 .+-. 17 131 .+-. 18
F-BMC/BW (mg/g) 1.38 .+-. 0.05 0.93 .+-. 0.08 1.12 .+-. 0.09
Vertebra (mm) 7.2 .+-. 0.4 6.4 .+-. 0.9 6.9 .+-. 0.6 Vertebra (mg)
269 .+-. 19 215 .+-. 36 273 .+-. 32 V-BMC (mg) 126 .+-. 17 88 .+-.
22 110 .+-. 16 V-BMD (mg/cm.sup.2) 121 .+-. 14 104 .+-. 11 107 .+-.
13
[0127] Statistical analyses (P<0.05) using ANOVA shows that
there were no differences in the initial body weight, and the final
body weight was significantly greater in the OVX versus controls
(P<0.05). Femur length did not differ significantly but femur
weight was significantly lower in the OVX versus controls
(P<0.05) and in OVX versus OVX-oxy (P<0.05). Femoral BMC was
significantly lower in the OVX group than in the other groups (all
P<0.05), and femoral BMD was significantly lower in the OVX than
in the other groups (all P<0.05). Femoral BMC/BW differed in all
the groups (all P<0.05). The fifth lumbar vertebra length did
not differ between groups. Vertebral weight was less in the OVX
group than in the other groups (all P<0.05).
CONCLUSION
[0128] It seems that oxytocin treatment for 10 days suppresses bone
loss in ovariectomized rats.
Sequence CWU 1
1
19 1 9 PRT Artificial Sequence DISULFID (1)..(6) C-term amidated 1
Cys Tyr Ile Gln Asn Cys Pro Leu Gly 1 5 2 9 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 2 Xaa Xaa Xaa
Xaa Asn Cys Xaa Xaa Xaa 1 5 3 9 PRT Artificial Sequence Description
of Artificial Sequence Synthetic peptide 3 Cys Tyr Ile Gln Asn Cys
Pro Ile Gly 1 5 4 9 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 4 Cys Tyr Ile Ser Asn Cys Pro
Ile Gly 1 5 5 9 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 5 Cys Phe Val Arg Asn Cys Pro Thr Gly 1
5 6 6 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 6 Cys Tyr Ile Gln Asn Cys 1 5 7 7 PRT Artificial
Sequence Description of Artificial Sequence Synthetic peptide 7 Cys
Tyr Ile Gln Asn Cys Pro 1 5 8 8 PRT Artificial Sequence Description
of Artificial Sequence Synthetic peptide 8 Cys Tyr Ile Gln Asn Cys
Pro Leu 1 5 9 8 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 9 Tyr Ile Gln Asn Cys Pro Leu Gly 1 5 10
7 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 10 Ile Gln Asn Cys Pro Leu Gly 1 5 11 6 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 11 Gln Asn Cys Pro Leu Gly 1 5 12 5 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 12 Ile Gln Asn
Cys Pro 1 5 13 9 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 13 Cys Tyr Val Thr Asn Cys Pro Leu Gly 1
5 14 9 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 14 Cys Tyr Xaa Thr Asn Cys Pro Val Gly 1 5 15 9
PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 15 Cys Tyr Phe Xaa Asn Cys Pro Leu Gly 1 5 16 9
PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 16 Cys Tyr Xaa Arg Asn Cys Pro Xaa Ala 1 5 17 9
PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 17 Cys Tyr Val Xaa Asn Cys Pro Xaa Ala 1 5 18 9
PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 18 Cys Tyr Xaa Xaa Asn Cys Pro Xaa Ala 1 5 19 9
PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 19 Cys Tyr Phe Arg Asn Cys Pro Val Ala 1 5
* * * * *