U.S. patent application number 10/463803 was filed with the patent office on 2004-01-15 for template associated npy y2-receptor agonists.
This patent application is currently assigned to B.M.R.A. Corporation B.V.. Invention is credited to Grouzmann, Eric, Lacroix, Jean-Silvain, Mutter, Manfred.
Application Number | 20040009905 10/463803 |
Document ID | / |
Family ID | 21990766 |
Filed Date | 2004-01-15 |
United States Patent
Application |
20040009905 |
Kind Code |
A1 |
Mutter, Manfred ; et
al. |
January 15, 2004 |
Template associated NPY Y2-receptor agonists
Abstract
The present invention is directed to agonists of neuropeptide Y
(NPY) or PYY that are formed by combining these peptides or a
portion of these peptides with a template that promotes
biologically active folds. Typically, templates consist of cyclized
peptides containing one or more naphthyl ring structures. The
agonists may be used in the treatment of diseases and conditions
known to be responsive to NPY or PYY and, particularly in the
treatment of asthma, rhinitis, and bronchitis.
Inventors: |
Mutter, Manfred; (Vaud,
CH) ; Lacroix, Jean-Silvain; (Geneva, CH) ;
Grouzmann, Eric; (Vaud, CH) |
Correspondence
Address: |
FITCH, EVEN, TABIN & FLANNERY
SUITE 401L
1801 K STREET, NW
WASHINGTON
DC
20006-1201
US
|
Assignee: |
B.M.R.A. Corporation B.V.
|
Family ID: |
21990766 |
Appl. No.: |
10/463803 |
Filed: |
June 18, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10463803 |
Jun 18, 2003 |
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09843824 |
Apr 30, 2001 |
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09843824 |
Apr 30, 2001 |
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09229900 |
Jan 14, 1999 |
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6288029 |
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09229900 |
Jan 14, 1999 |
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09054393 |
Apr 3, 1998 |
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6017879 |
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Current U.S.
Class: |
514/1.7 ;
514/15.1; 514/15.7; 514/16.4; 514/17.8; 514/21.1; 514/3.8;
514/5.1 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
25/08 20180101; A61K 38/00 20130101; C07K 1/1075 20130101; A61P
11/00 20180101; A61P 7/10 20180101; A61P 11/16 20180101; A61P 9/04
20180101; A61P 3/04 20180101; A61P 29/00 20180101; A61P 9/12
20180101; C07K 14/57545 20130101; A61P 11/06 20180101; A61P 9/10
20180101; A61P 25/22 20180101; A61P 37/04 20180101; A61P 25/28
20180101; C07K 7/02 20130101; A61P 43/00 20180101 |
Class at
Publication: |
514/9 ; 514/13;
514/14 |
International
Class: |
A61K 038/12; A61K
038/10; A61K 038/08 |
Claims
What is claimed is:
1. An agonist of neuropeptide Y (NPY) comprising: (a) a template
comprising a cyclized peptide 3 to 10 amino acids in length,
wherein at least two residues in said cyclized peptide are joined
by a naphthyl ring; and (b) at least one linear peptide between 12
and 37 amino acids in length covalently bound to said template,
wherein said linear peptide has a C-terminal sequence selected from
the group consisting of: RHYTNLITRQRY, (SEQ ID NO:3); and
RHYLNLVTRQRY (SEQ ID NO:4); and wherein the C-terminal tyrosine of
said linear peptide is amidated.
2. The agonist of claim 1, wherein said template has the structure:
3and wherein said linear peptide is attached to said template at
either or both of the lysine residues.
3. The agonist of claim 2, wherein said C-terminal sequence of said
linear peptide is preceded at the N terminal end by between 1 and
24 residues of amino acids 1-24 of NPY, said sequence being:
YPSKPDNPGEDAPAEDMARYYSAL, (SEQ ID NO:5).
4. The agonist of claim 3, wherein said linear peptide has the
sequence: YSALRHYINLITRQRY, (SEQ ID NO:6).
5. The agonist of claim 3, wherein said linear peptide further
comprises an aminooxy acetylated glycine at its N terminus.
6. The agonist of claim 3, wherein said linear peptide is bound to
said template by an oxime bond.
7. The agonist of claim 6, wherein said agonist is TASP-V.
8. The agonist of claim 2, wherein said C-terminal sequence of said
linear peptide is preceded at the N terminal end by between 1 and
24 residues of amino acids 1-24 of PYY, said sequence being:
YPIKPEAPGEDASPEELNRYYASL (SEQ ID NO:7).
9. The agonist of claim 8, wherein said linear peptide has the
sequence: YASLRHYLNLVTRQRY (SEQ ID NO:8).
10. The agonist of claim 9, wherein said linear peptide further
comprises an aminooxy acetylated glycine at its N terminus.
11. The agonist of claim 9, wherein said linear peptide is bound to
said template by an oxime bond.
12. The agonist of claim 6, wherein said agonist is TASP-V2.
13. A pharmaceutical composition comprising the agonist of claim
1.
14. A method of reducing airway resistance in a patient suffering
from a bronchial disease or condition, comprising administering a
therapeutic agent selected from the group consisting of: NPY, PYY,
an agonist of NPY or an agonist of PYY.
15. The method of claim 14, wherein said patient is administered
either NPY or PYY.
16. The method of claim 14, wherein said patient is administered
either: a) an agonist that is a peptide comprising the sequence of
amino acids 25-36 of NPY; or b) an agonist that is a peptide
comprising the sequence of amino acids 25-36 of PYY.
17. The method of claim 16, wherein said agonist is either TASP-V
or TASP-V2.
18. The method of claim 14, wherein said therapeutic agent is in a
pharmaceutical composition administered by inhalation and said
therapeutic agent is administered at a dose of between 1 and 100
.mu.g.
19. The method of claim 14, wherein said bronchial disease or
condition is asthma.
20. The method of claim 14, wherein said bronchial disease or
condition is bronchitis.
21. In a method for treating a disease or condition that is
responsive to NPY or PYY, the improvement comprising administering
the agonist of claim 1 at a unit dose of between 1 and 100
.mu.g.
22. The improvement of claim 21, wherein said agonist comprises the
template: 4and wherein the peptide bound to said template: a)
comprises a sequence selected from the group consisting of: amino
acids 25-36 of NPY (SEQ ID NO:3); and amino acids 25-36 of PYY (SEQ
ID NO:4); b) further comprises 0-24 residues a sequence selected
from the group consisting of: amino acids 1-24 of NPY (SEQ ID
NO:5); and amino acids 1-24 of PYY (SEQ ID NO:7).
23. The improvement of claim 22, wherein said agonist is either
TASP-V or TASP-V2.
24. The improvement of claim 21, wherein said disease or condition
is selected from the group consisting of: laryngitis, chronic
rhinosinusitis, oedema, inflammation, anxiety, congestive heart
failure, cardiomyopathy, coronary artery disease, diminished
cardiac vagal activity, hypertension, Alzheimer's Disease,
epilepsy, ischemia, angina, myocardial infarction, AIDS and
diseases characterized by a decreased immune responsiveness.
25. The improvement of claim 21, wherein said agonist is
administered to increase body weight or as an antihistamine.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to a new type of agonist
that interacts preferentially with the neuropeptide Y (NPY) Y2
receptor. The agonist contains one or more peptides with sequences
from the C-terminal end of neuropeptide Y (NPY) or peptide YY (PYY)
bound to a template that promotes the correct folding of these
peptides. In addition, the present invention is directed to methods
for reducing airway resistance in bronchial patients by
administering NPY, PYY, or agonists of these peptides.
BACKGROUND OF THE INVENTION
[0002] Neuropeptide Y (NPY) is an amidated peptide widely
distributed in the central and peripheral nervous systems
(Tatemoto, et al., Nature 296:659-660 (1982); Ekblad, et al.,
Regul. Peptides 8:225-235 (1984)). It is present in all sympathetic
nerves innervating the cardiovascular system and is the most
abundant peptide in the brain and the heart (Tatemoto, et al.,
Nature 296:659-660 (1982)). In addition, NPY is present in
platelets (Ericsson, et al, Proc. Natl. Acad Sci. U.S.A.
84:5587-5591 (1987)), the endothelium (Id.); the adrenal medulla
(Allen, et al., J. Auton. Nerv. Sys. 9:559-566 (1983)); the
pancreas (Jamal, et al., Endocrinology 129:3372-3380 (1991)); the
kidney (Grouzmann, et al., Peptides 15 (8):1377-1382 (1994)); and
the pituitary gland (Gehiert, et al., Peptides 15 (4):651-656
(1994)). Peptide YY (PYY) is a closely related peptide that has
similar biological effects to NPY and which is found primarily in
the gut.
[0003] The biological actions of NPY and peptide YY are mediated by
a number of G-protein coupled receptors termed Y1, Y2, Y3, Y4/PP
and Y5 (Herzog, et al., Proc. Natl. Acad. Sci. U.S.A. 89:5794-5798
(1992)). Of these, the physiological effects associated with the Y1
and Y2 receptors are the best characterized. Exposure to a Y1
agonist causes an increase in blood pressure and potentiates
post-synaptically the action of other vasoactive substances
(Wahlestedt, et al., J. Pharmacol. Exp. Ther. 234:735-741 (1985)).
In contrast, Y2 receptors are mainly located presynaptically and,
upon stimulation, mediate the inhibition of neurotransmitter
release (Westfall, et al., J. Cardiovasc. Pharmacol. 10:716-722
(1987)).
[0004] NPY has a number of biological effects of potential
therapeutic importance. Intranasal administration of NPY reduces
nasal airway resistance and vascular permeability without affecting
submucosal gland secretion (Baraniuk, et al., Am. J. Respir. Cell.
Mol. Biol. 3:165-173 (1990); Baraniuk, et al., J. Appl. Physiol. 73
(5):1867-72 (1992)). In healthy volunteers, intranasal pretreatment
with exogenous NPY markedly reduces vasodilation and nasal
secretion induced by afferent nerve stimulation with capsaicin or
histamine (Lacroix, et al., Br. J. Pharmacol. 118:2079-2084
(1996)). Therapeutic application of NPY in the treatment of
rhinitis has been recently suggested since allergen-evoked nasal
responses in patients are significantly attenuated after local
pretreatment with the peptide (Lacroix, et al., J. Allergy Clin.
Immunol. 98:611-616 (1996)).
[0005] NPY also plays an important role in modulating the
cardiovascular system, behavior, anxiety and the secretion of
certain hormones (Wahlestedt, et al., Annu. Rev. Pharmacol.
Toxicol. 33:309-352 (1993); Michel, Trends PharmacoL Sci.
12:389-394 (1991)). It contributes to the central and peripheral
control of blood pressure, the regulation of feeding behavior,
obesity, diabetes and psychiatric disorders (Walker, et al., Trends
Pharmacol Sci 12:111-115 (1991); Sahu, et al., Trends Endocrinol.
Metab. 4:217-224 (1993); Stanley, et al., Proc. Natl. Acad. Sci.
USA 82:3940-3943 (1985)).
[0006] B. Structure of NPY and PYY
[0007] NPY is derived from the 97 amino acid precursor shown as SEQ
ID NO:1 (Minth, et al., Proc. Natl. Acad. Sci. USA 81:4577-4581
(1984)). Amino acids 29-64 represent the 36 amino acid sequence
which undergoes processing resulting in the addition of an
N-terminal glycine and the amidation of the C-terminal tyrosine.
The complete NPY sequence is needed for binding to the Y1 receptor,
whereas C-terminal fragments are selective for the Y2 receptor
(Ekblad, et al., Regul. Peptides 8:225-235 (1984)). The C-terminal
pentapeptide amide is important for both receptors and probably
represents the binding site (Beck-Sickinger, et al., Eur. J.
Biochem. 225:947-958 (1994)). However, Arg33 and Arg35 may not be
exchanged by L-alanine in the Y1 system, whereas Arg35 and Tyr36
are the most critical residues for the Y2 receptor. NPY fragments
shorter than NPY 27-36 are no longer able to bind to the Y2
receptor.
[0008] Peptide YY also binds to the Y2 receptor. It is 36 amino
acids in length and shares a 70% sequence homology with NPY. Its
sequence is shown as SEQ ID NO:2.
[0009] C. Template Assembled Synthetic Proteins or Peptides
(TASP)
[0010] In order to bypass the folding problem that has typically
been associated with peptide and protein synthesis, a conceptually
different approach to de novo protein design has recently been
taken, the synthesis of template-assembled synthetic proteins or
peptides (TASP). In this approach, topological templates direct
covalently attached peptide blocks to a predetermined
three-dimensional packing arrangement (FIGS. 1-3), thereby
modifying their biological and pharmacokinetic properties ((Mutter,
et al., Helv. Chim. Acta, 71:835-47 (1988); Mutter, Trends Biochem.
Sci., 13:260-5 (1988); Mutter, et al., J. Am. Chem. Soc.
114:1463-1470 (1992); Grouzmann, et al., Eur. J. Biochem. 234:44-49
(1995)). Typically, templates are constrained peptides,
cyclodextrines or polycyclic systems.
[0011] Recently, the TASP concept was used to design a compound
that selectively antagonizes the action of NPY at the Y2 receptor.
A cyclic peptide exhibiting four attachment sites and a naphthyl
derivative was used as template and NPY33-36 segments were attached
by means of an oxime bond (Grouzrnann, et al., J. Biol. Chem. 292
(12):7699-7706 (1997)). This TASP molecule was investigated for
binding to NPY Y1 and Y2 receptors and its antagonistic activity
was established by its ability to prevent the NPY-induced increase
in intracellular calcium.
[0012] D. TASP Agonists of NPY Y2 Receptor Interaction
[0013] It has now been discovered that template assembled synthetic
peptides can produce NPY and PYY agonists that interact
specifically with the Y2 receptor. These compounds may be used in
the treatment of several conditions, including rhinitis. In
addition, it has been discovered that NPY, PYY and agonists of
these peptides may be used in treating bronchial diseases and
related conditions.
SUMMARY OF THE INVENTION
[0014] The present invention is based upon two main discoveries.
The first is that template assembled synthetic peptides can be
produced that are agonists of NPY and PYY. These agonists can be
used to effectively treat rhinitis and a variety of other
physiological conditions. The second discovery is that NPY, PYY and
their agonists reduce bronchial airway resistance. Thus, these
agents may be used in treating bronchitis, asthma and related
conditions.
[0015] In its first aspect, the present invention is directed to an
agonist of NPY comprising a template and one or more peptides
derived from NPY or PYY covalently bound to the template. The
template is a cyclized peptide between 3 and 10 amino acids in
length containing at least two residues that are joined by a
naphthyl ring. At least one, and preferably two, linear peptides
between 12 and 27 amino acids in length are covalently bound to the
template, e.g.,by an oxime bond. The C-terminal sequence of the
bound peptide(s) has either the sequence: RHYINLITRQRY, (SEQ ID
NO:3); or the sequence RHYLNLVTRQRY (SEQ ID NO:4). In either case,
the C-terminal tyrosine should be amidated. In a preferred
embodiment, linear peptides are attached at the lysine residues of
the following template: 1
[0016] The 12 amino acid C-terminal sequences shown above may be
preceded by additional portions of the NPY sequence, up to the full
additional 24 amino acids found in NPY: YPSKPDNPGEDAPAEDMARYYSAL,
(SEQ ID NO:5). For example, SEQ ID NO:3 may be preceded at its
N-terminal end by NPY1-24; NPY2-24; NPY3-24 etc. It is expected
that conservative changes in this 24 amino acid sequence can be
made without affecting activity and, in particular, the "M" at
position 17 can be effectively replaced with L. If agonists
specific for the Y2 receptor are desired, then the full length NPY
peptide should not be used. The most preferred peptide for
attachment to the template has the sequence: YSALRHYINLITRQRY, (SEQ
ID NO:6). The peptides may be preceded at their N-terminal end by a
single aminooxy acetylated glycine. Although these peptides may be
joined to templates by other covalent bonds, oxime bonds are
generally preferred. The most preferred structure is that of TASP-V
as follows: 2
[0017] As an alternative, the linear peptides attached to the TASP
template may be preceded by additional portions of the PYY sequence
up to the full additional 24 amino acids: YPIKPEAPGEDASPEELNRYYASL
(SEQ ID NO:7). In all cases, additions should be made so as to
maintain the correct sequence order of PYY. The most preferred PYY
fragment for attachment to templates is: YASLRHYLNLVTRQRY (SEQ ID
NO:8). The attached peptides may be preceded at their N-terminal
end by an aminooxy acetyl glycine and they are preferably bound to
the template by an oxime bond. When the PYY fragment of SEQ ID NO:8
is used in place of the NPY fragments in TASP-V, a second preferred
agonist is produced. In order to distinguish this second agonist
from TASP-V, it is designated as TASP-V2.
[0018] All of the peptide agonists described above may be
incorporated into a pharmaceutical composition and administered to
a patient for the purpose of treating diseases or conditions that
respond to NPY or PYY. In general, the agonists should be
administered to patients in a dosage range of about 1 to 100 .mu.g.
Any route of delivery is consistent with the present invention but
non-oral routes will typically be used to avoid possible
destruction of agents in the gut.
[0019] In another aspect, the present invention is directed to a
method of reducing airway resistance in a patient suffering from a
bronchial disease or condition by administering NPY, PYY, or an NPY
or PYY agonist, preferably an agonist specific for the Y2 receptor.
When either NPY or PYY is used in treatments, the amidated, full
length form of the peptide should be used. When a Y2 agonist is
used, it should be a peptide containing, at a minimum, the sequence
of amino acids 25-36 of NPY or PYY but not the full 36 amino acids.
The most preferred agonists are TASP-V and TASP-V2.
[0020] Bronchial conditions or diseases are preferably treated by
administering therapeutic agent in a pharmaceutical composition
delivered by inhalation. A unit dose should provide a patient with
between about 1 and 100 .mu.g of active agent. Among the bronchial
diseases and conditions that may be treated using this procedure
are asthma and bronchitis.
[0021] In another aspect, the invention is directed to an
improvement in methods for treating diseases or conditions
responsive to NPY or PYY. This is accomplished by administering any
of the TASP-type agonists described above, preferably one specific
for the Y2 receptor, at a unit dose of between 1 and 100 .mu.g. By
"TASP-type agonists" we mean agonists in which peptides are
covalently bound to a template such as those described herein. The
preferred template is that shown above and the linear peptide
attached to this template should contain the sequence of amino
acids 25-36 of NPY or PYY. The linear peptide may also contain any
portion of the additional contiguous amino acids which make up the
sequence of intact NPY or PYY. The most preferred agonists are,
again, TASP-V and TASP-V2. The NPY or PYY agonist may be used as an
antihistamine, to increase body weight, or to treat rhinitis,
asthma or bronchitis. Other diseases and conditions that may be
treated are laryngitis, mucovisidose, chronic rhinosinusitis,
oedema, inflammation, anxiety, congestive heart failure,
cardiomyopathy, coronary artery disease, diminished cardiac vagal
activity, hypertension, Alzheimer's Disease, epilepsy, ischemia,
angina, myocardial infarction and diseases characterized by
decreased immune responsiveness such as AIDS.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1: The concept of template-assembled synthetic proteins
(TASP) for the construction of functional protein mimetics.
Topological templates (e.g. cyclic peptides, see FIG. 2) induce
folding or spatial rearrangement of covalently attached peptide
blocks (e.g. fragments of bioactive compounds) into predetermined
packing arrangements. The enforced spatial proximity as well as the
induction of specific conformations of the template assembled
peptides may result in enhanced bioactivity and specificity and
alter the pharmacokinetics in a characteristic way. The symbols
denote chemoselectively reactive groups as outlined in FIG. 3.
[0023] FIG. 2: A variety of molecules may serve as templates, e.g.
cyclic peptides, monosaccharides, steroids, cyclodextrs,
calixarenes or porphyrins. The structural requirement is an
appropriate spatial orientation of selectively addressable
attachment sites, e.g. orthogonally protected amino groups as
schematically shown (center).
[0024] FIG. 3: Recently introduced chemoselective ligation methods
allow for the condensation of completely unprotected peptide
fragments (P) to correspondingly functionalized templates in
aqueous solution.
[0025] FIG. 4: Reactions leading to the production of turn mimics
(1a, 2a and 3a) for incorporation into TASP templates (from Ernest
et al., Helv. Chim. Acta 76:1539-1563(1993), seepage 1541).
[0026] FIG. 5: Synthesis of TASP-V. Abbreviations: Lys=lysine;
Gly=glycine; DIEA=diisopropylethylamine; oxime-bond=template-Lys
(.sup..epsilon.-N--CO--CH.dbd.NO--CH.sub.2CO-peptide;
Fmoc=fluorenylmethoxycarbonyl;
PyBOP=benzotriazole-1-yl-oxy-tris-pyrrolid- ino-phosphonium
hexafluorophosphate; Boc-AO-OSu=t-butyloxycarbonyl-aminoox-
yacetyl-N-hydroxy succinic ester; T=threonine; R=arginine;
Q=glutamine; Y=tyrosine; Pmc=pentamethyl chroman sulphonyl;
G=glycine; L=leucine; N=asparagine; H=histidine; I=isoleucine;
S=serine; A=alanine.
[0027] FIG. 6: Representative concentration response curves of the
displacement of .sup.125I-NPY by selective peptides for the Y1
receptor in SK-N-MC cells. Four experiments were performed with
each analogue. The percentage inhibition of .sup.125I-NPY binding
to the receptor, which is caused by the increasing concentrations
of competitors, is shown on the y axis. High affinity binding to
the Y1 receptor on SK-N-MC cells was found for NPY (.diamond.) and
Leu31, Pro34 NPY (.DELTA.), whereas poor affinity was observed with
NPY13-36(.largecircle.) and TASP-V (.box-solid.).
[0028] FIG. 7: Representative concentration response curves of the
displacement of .sup.125I-NPY by selective peptides for the Y2
receptor in LN319 cells. Four experiments were performed with each
analog. The percentage inhibition of .sup.125I-NPY binding to the
receptor, which is caused by the increasing concentrations of
competitors, is shown on the y axis. High affinity binding to the
Y2 receptor on LN319 cells was found for NPY (.diamond.), NPY13-36
(.largecircle.) and TASP-V (.box-solid.). Leu31, Pro34 NPY
(.DELTA.) exhibited poor affinity binding.
[0029] FIG. 8: Prejunctional activity of TASP-V measured in rat and
expressed as the maximum percent inhibition of the increase in
pulse interval (.DELTA.PI) evoked by stimulation of the vagus nerve
following injection of TASP-V.
[0030] FIG. 9: Prejunctional activity of TASP-V measured in rat and
expressed as the time to half recovery of this effect (T50).
[0031] FIG. 10: Postjunctional activity of TASP-V measured in rat
as the peak response following injection of the peptide
(.DELTA.BP)
[0032] FIG. 1: Postjunctional activity of TASP-V measured in rat as
the duration of this response (BP duration)
[0033] FIG. 12 (panels a and b): Time course variations of
subjective (panel a) and objective (panel b) nasal airway
resistance (NAR, measured by a visual analogue scale graded from 0
to 5 and anterior rhinomanometry, respectively) in the homolateral
nostril following an intranasal application of histamine (1 mg in
200 .mu.g of saline) and after pretreatment with TASP-V or placebo
(saline spray). Pretreatment with TASP-V significantly limits the
subjective and objective increase of nasal airway resistance.
Maximum effect is obtained 15 minutes after the pretreatment
(n=11). *p<0.05, **p<0.01 (one-way analysis of variance
ANOVA).
[0034] FIG. 13: Time course variations of the minimal cross
sectional area (MCSA, measured by acoustic rhinometry) in the
homolateral nostril following an intranasal application of
histamine (1 mg in 200 .mu.g of saline) and after pretreatment with
TASP-V or placebo (saline spray). Pretreatment with TASP-V
significantly limits the decrease in cross sectional surface
following the histamine challenge. Maximum effect is obtained 15
minutes after the pretreatment (n=11). *p<0.05, **p<0.01
(one-way analysis of variance ANOVA).
[0035] FIG. 14: Effect of an intranasal spray of histamine (1 mg in
200 .mu.g of saline) on the homolateral nasal airway resistance
(NAR) measured by anterior rhinomanometry (n=11) and following
pretreatment with placebo or TASP-V. Pretreatment with TASP-V
significantly limits the increase of nasal airway resistance
(n=11). *p<0.05 (one-way analysis of variance ANOVA).
[0036] FIG. 15: Effects of an intranasal spray of histamine (1 mg
in 200 .mu.g of saline) on the homolateral minimal cross sectional
area (MCSA) measured by acoustic rhinometry and following
pretreatment with placebo or TASP-V. Pretreatment with TASP-V
significantly limits the decrease of the nasal cross section
surface (n=11). *p<0.05 (one-way analysis of variance
ANOVA).
DETAILED DESCRIPTION OF THE INVENTION
[0037] In the following description, reference will be made to
various methodologies well known to those skilled in the art of
chemistry and medicine. Such methodologies are described in
standard reference works setting forth the general principles of
these disciplines.
[0038] I. Synthesis of TASP NPY Y2 Agonists
[0039] A. Making of Template
[0040] The procedures used in the making of TASP templates in
general, and, in particular, in making the template used in TASP-V,
have been fully described in the literature (Ernest, et al., Helv.
Chim. Acta 76:1539-1563; Ernest, et al., Tetrahedron Lett.
31:4011-4014 (1990)). The basic approach is to introduce an
artificial turn-inducing mimic into a cyclized peptide chain so as
to constrain it into a semi-rigid spatial arrangement. The three
most commonly used turn mimics are:
8-amino-5,6,7,8-tetrahydronaphth-2-oic acid;
8-(aminomethyl)-5,6,7,8-tetr- ahydronaphth-2-oic acid; and
8-(aminomethyl)naphth-2-oic acid. Each of these may be synthesized
from commercially available 4-phenylbutanoic acid. The basic
reaction scheme used for producing each of these peptide mimics is
shown in FIG. 4.
[0041] Prior to their incorporation into peptide chains, reactive
groups in the turn mimics are transformed into their N-Boc and
N-Fmoc derivatives. Free carboxyl groups on the blocked mimic are
then reacted with the N-terminal amino acid in a peptide to form an
amide bond. The C-terminal end of the peptide is reacted with
deprotected NH groups on the mimic to form a cyclic structure. Many
variations of these reactions may be performed depending upon the
particular template desired. The peptides used in forming templates
will typically be between 3 and 10 units long with the most
preferred structure consisting of a glycine residue flanked at
either end by one or two lysine amino acids. A typical template
formed in this manner is shown as structure I in FIG. 5.
[0042] Reactive side chain groups of the amino acids in the
cyclized template form the site of attachment for one or more
linear peptides derived from NPY or PYY. In the case of template I
the epsilon amino groups of lysine may be derivatized with a group
facilitating attachment of peptides. For example, as shown in FIG.
5, the epsilon amino groups may be reacted with glyoxylic acid
1,1-diethylacetalsuccinimide ester to form the diethylacetal
derivative. After hydrolysis, the resulting aldehyde functions may
then be reacted with linear NPY or PYY derived peptides to form a
covalent oxime bond. Other covalent bonds that may be formed are
shown in FIG. 3 and include hydrazones, amides, thioethers,
thioesters, thiazolidines and oxazolidines. A complete description
of reactions and the purification of reaction products may be found
in the Ernest references cited above as well as in Example 1
below.
[0043] B. Synthesis of NPY Linear Peptides
[0044] Any method can be used for synthesizing the linear peptides
to be attached to templates. Typically peptides have been made
using solid phase synthesis techniques (Stewart, et al., Solid
Phase Peptide Synthesis, 2nd ed., (1984); Fields, et al., Int. J.
Peptide Protein Res. 35:161-214 (1990)), or by fragment
condensation involving the coupling of peptide segments in solution
(Lloyd-Williams, et al., Int. J. Peptides Protein Res. 37:58-60
(1990); Ernest, et al., Tetrahedron Lett. 31:4015-4018 (1990)).
Although solid phase synthesis has been optimized so that proteins
of about 100 amino acids in length can be made, the accumulation of
side products over many coupling steps may render the purification
of the target product both laborious and time consuming.
Condensation strategies have the advantage that synthesis and
purification of peptide segments up to about 30 residues in length
is straightforward but they are limited by the poor solubility of
fully protected peptide segments in aqueous solution and the
tendency of alpha-carboxy-activated peptides to racemize. Many of
the difficulties associated with these synthesis methods can be
circumvented by using recently developed chemoselective ligation
methods (Rose, et al., Bioconj. Chem. 7:552-556 (1996); Liu, et
al., J. Am. Chem. Soc. 116:4149-4153 (1994); Dawson, et al., J. Am.
Chem. Soc. 115:7263-7266 (1993); Kemp, et al., J. Org. Chem.
58:2216-2222 (1993)). These methods allow for the condensation of
completely unprotected peptide fragments in aqueous medium (FIG.
3).
[0045] In choosing appropriate linear peptides for attachment to
templates, the structure of NPY or PYY should serve as a guide.
C-terminal residues 25-36 must be present in the linear peptide but
longer segments, up to and including the full NPY or PYY sequences,
may be used. In addition, conservative amino acid substitutions may
be introduced into the sequence. For example, it is expected that a
hydrophobic residue in the NPY sequence may generally be
substituted with another hydrophobic amino acid without
substantially affecting activity. In order to determine whether a
particular substitution is acceptable, a linear peptide-template
compound may be tested for its ability to bind to NPY receptors and
activate cAMP using the procedures described below in Examples 3
and 4.
[0046] C. Formation of TASP Agonists
[0047] The preferred method for attaching linear peptides to
templates is by means of the chemoselective ligation procedures
described above. In particular, peptide blocks and templates
exhibiting chemoselective addressable functional groups (e.g.,
aminooxy and aldehyde groups) are prepared by standard methods and,
after cleavage of side chain protecting groups, these are reacted
to produce bioactive TASP molecules. For example, fragments derived
from NPY of variable chain length and sequence (e.g., NPY 2-36, NPY
21-36, NPY 25-36) can be selectively attached to templates
according to the strategy outlined in FIG. 5. Similar strategies
have been effectively employed for producing a variety of other
TASP molecules (see e.g., Grouzmann, et al., Eur. J. Biochem.
234:44-49(1995); Tuchscherer, et al., Protein Sci. 1:1377-1386
(1992); Futaki, et al., Tetrahedron Lett. 38:6237-6240 (1997);
Grove, et al., J. Am. Chem. Soc. 115:1100-1115 (1993)). Additional
guidance concerning appropriate methods that can be employed are
set forth in Example 1 using TASP-V as a model.
[0048] Once appropriate TASP compounds have been formed, they may
be purified using standard procedures in peptide chemistry. One
procedure that has been found effective is to purify compounds by
reverse-phase HPLC using linear gradients of acetonitrile (see
Example 1).
[0049] D. Testing of Compounds for Activity
[0050] The compounds synthesized by the methods described above may
be assayed to determine the extent to which they mimic the effects
of NPY or PYY. Radioreceptor binding assays such as those described
in Example 3 may be employed to determine whether the compound
selectively binds to the Y1 or Y2 receptor. This is accomplished by
using cell lines that exclusively produce either Y2 (LN319 cells)
or Y1 (SK-N-MC cells). Cyclic AMP assays may be performed in
conjunction with binding assays in order to determine whether
compounds interacting with receptors are acting as agonists or
antagonists of NPY.
[0051] Alternatively, any biological assay that has been employed
to demonstrate a measurable effect of NPY or PYY may be used in
screening TASP compounds for activity. For example, the effect of
compounds on rhinitis and bronchospasm may be determined directly
using procedures such as those set forth in Example 6.
[0052] II. Therapeutic Methods Employing NPY, PYY or Their
Agonists
[0053] TASP compounds synthesized by the methods described above
may be used in treating any disease or condition that responds to
NPY or PYY. Because agonists in which linear peptides having less
than the full length NPY or PYY sequence interact preferentially
with the Y2 receptor, it is expected that these compounds will
produce therapeutically desirable effects with fewer undesired side
effects. In the case of bronchial diseases and conditions, either
agonists, NPY or PYY may be administered for the purpose of
reducing airway resistance.
[0054] The total daily dosage of agonist, NPY or PYY administered
to a patient should be at least the amount required to minimize,
reduce or eliminate one or more of the symptoms associated with the
disease or condition treated. For example, in the case of rhinitis,
sufficient drug should be administered to reduce rhinorrhea and/or
alter airway resistance. Ordinarily, a unit dose should contain
between 1 and 100 .mu.g of active agent with the optimal daily dose
being determined by methods well known in the art. Dosages may be
provided either in a single or multiple daily regimen.
[0055] The present invention is not limited to any particular
dosage form or route of administration. Although inhalation will
generally be most convenient and is preferred in the treatment of
rhinitis and bronchial conditions, parenteral, transdermal,
sublingual, peroral, nasal, rectal, vaginal, auricular, implantable
or other routes of administration may be used as well. Therapeutic
agents may be administered in a substantially purified form or as
part of a pharmaceutical composition containing one or more
excipients, flavoring agents, or other active ingredients.
Preparations may be solid or liquid or take any of the
pharmaceutical forms presently used in human medicine, e.g.,
tablets, powders, solutions, creams, ointments, suspensions, gel
capsules, granules, suppositories, transdermal compositions or
injectable preparations.
[0056] The active agents may be incorporated into dosage forms in
conjunction with the vehicles that are commonly employed in
pharmaceutical preparations, e.g., talc, gum arabic, lactose,
starch, magnesium stearate, cocoa butter, aqueous or non-aqueous
solvents, oils, paraffin derivatives, glycols, etc. Methods for
preparing appropriate formulations are well known in the art (see,
e.g., Remington's Pharmaceutical Sciences, 16th ed., A. Oslo. ed.,
Easton Pa. (1980)).
[0057] In order to determine the effect of an administered
composition on a particular disease or condition, patients should
be evaluated on a regular basis over an extended period of time. In
some cases, it may take several weeks for the full therapeutic
effect of a treatment to become apparent. In other cases, e.g., in
the treatment of bronchitis, asthma, and rhinitis, agonists such as
TASP-V should produce relief within an hour after administration.
Since agonists specific for the Y2 receptor should not produce a
rebound effect on airway resistance, these may be administered
repeatedly by the patient as desired. The preferred route for these
conditions is by inhalation and, as indicated above, each unit dose
administered should contain between about 1 and 100 .mu.g of active
agent.
EXAMPLES
[0058] A new Y2-receptor agonist, TASP-V (FIG. 5) was synthesized
and characterized with respect to its effect on the functional
response to subsequent histamine challenge. Studies on the
modulation of histamine-evoked bronchial and nasal responses by
local pretreatment with TASP-V were performed in pigs and in
humans. The results demonstrate that:
[0059] 1. Intranasal pretreatment with TASP-V reduces nasal
obstruction induced by histamine.
[0060] 2. Intrabronchic pretreatment with TASP-V reduces
bronchoconstriction evoked by histamine challenge.
Example 1
Synthesis of the Y2-Receptor Agonist TASP-V
[0061] Based on molecular dynamics simulations derived from the
crystal structure of avian pancreatic polypeptide, the 3D structure
of NPY was proposed to be a polyproline-type II helix for residues
1-8, followed by a .beta.-turn at positions 9-14. An amphipatic
.alpha.-helical segment 15-32 is stabilized by hydrophobic
interactions with the polyproline helix and a C-terminal turn
structure is adopted by residues 33-36.
[0062] The C-terminal part of NPY is essential for receptor binding
and biological activity. It is believed that the N-terminal segment
1-4 stabilizes the C-terminal .alpha.-helical structure 25-36. The
antiparallel PP-fold is of structural importance for the receptor
binding of NPY, and its main function is to present the combined C-
and N-terminal segments of the molecule to the receptor. As the
tetrapeptide NPY33-36 per se does not bind to the receptor, it must
be assumed that factors like conformational stabilization by
N-terminal extension are of particular importance.
[0063] The preparation of the cyclic template (I in FIG. 5) has
been described previously (Ernest, et al., Tetrahedron Lett.,
31:4011-4014 (1990); Ernest, et al., Helv. Chim. Acta 76:1539-1563
(1993)). The synthesis of the fully protected neuropeptide Y
analogue, NPY21-36 amide (IV in FIG. 5) was performed on Rink amide
resin using an Fmoc strategy (Fields, et al., Int. J. Peptide
Protein Res. 35:161-214 (1990)). All amino acids were used as
N.sup..alpha.-protected derivatives. Side-chain protecting groups
were Tyr(t-Bu), Arg(Pmc), Gln (Trt), Thr(t-Bu), Asn(Trt) and
Ser(t-Bu). Peptides were constructed semi-automatically according
to the published cycle (Akaji, et al., Chem. Pharm. Bull.
37:2661-2664 (1989)) consisting of (i) a 20 min deprotection of
Fmoc with 25% piperidine/DMF (N,N-dimethylformamide) and (ii) a 45
min coupling of the Fmoc amino acid derivatives (1.5 equiv.) by the
aid of PyBOP (benzotriazol-1-yl-oxy-tris(pyrrolidino)-phosphonium
hexafluorophosphate) (1.7 equiv.) and DIEA
(N,N-diisopropylethylamine) (3.5 equiv.) in DMF
(N,N-dimethylformamide). The completeness of each coupling was
confirmed by the Kaiser test (Kaiser, et al., Anal. Biochem.
34:595-598 (1970)). Washing cycles after coupling and deprotection
consisted of successive treatments with DMF and dichloromethane.
After the peptide sequence was assembled, the functional residue
for chemoselective ligation was introduced onto the N-terminus
using Boc-aminooxyacetyl-OSu-ester.
[0064] For cleavage from the resin and partial deprotection of the
fully side-chain protected peptide, the dried resin (800 mg) was
treated with 10% trifluoroacetic acid-dichloromethane (5.times.15
ml). After each treatment, the resin was separated by filtration,
and the filtrates were concentrated in vacuo.
[0065] In order to completely deprotect the peptide, the residue
prepared as described above was retreated for 90 min with
trifluoroacetic acid-dichloromethane (9:1, 15 ml) in the presence
of triisopropyl-silane/water (1:1, 0.5 ml), and cold diethyl ether
was then added to precipitate the product. The crude
octadecapeptide amide was collected by centrifugation and
lyophilized from water-acetonitrile (1:1) to afford a colorless
hygroscopic solid (300 mg).
[0066] After lyophilization, the crude product was purified by
preparative reverse-phase HPLC on a Vydac 218 TP54 column (5 .mu.m,
C18, 25 mm.times.250 mm) using a linear gradient from 20% to 60% of
0.9% trifluoroacetic acid in acetonitrile over a period of 30
minutes at a flow rate of 18.0 ml/min. The eluate was monitored by
measuring the UV absorption at 214 nm, and appropriate fractions
were lyophilized. The overall yield was 200 mg (45%). The partial
sequence of neuropeptide Y, NPY21-36 (IV in FIG. 5), was
characterized by electronspray mass spectrometry and amino acid
analysis.
[0067] For the effective synthesis of the TASP-V (FIG. 5),
chemoselective ligation methods were applied. Oxime bond formation
(Nyanguile, et al., Lett. Peptide Sci. 1 (1):9-16 (1994); Liu, et
al., J. Am. Chem. Soc. 116:4149-4153 (1994); Dawson, et al., J. Am.
Chem. Soc. 115:7263-7266 (1993)) was used to attach the aminooxy
acetyl-containing peptide fragments IV to the cyclic peptide
template III. I contains two lysine residues (acting as attachment
sites) and the .beta.-turn mimic, 8-aminomethyl-naphth-2-oic acid
(Ernest, et al., Tetrahedron Lett., 31:4011-4014 (1990); Ernest, et
al., Helv. Chim. Acta 76:1539-1563 (1993)).
[0068] The .epsilon.-amino groups of lysine were converted to
diethylacetal functions by reaction with glyoxylic acid
1,1-diethylacetalsuccinimide ester. The derivatization reaction was
monitored by analytical reverse-phase HPLC. Interestingly, two
intermediates were observed during the reaction at t.sub.R=18.70
min and 18.90 min, which could be identified as the isomeric
templates containing one derivatized lysine residue. After 4 h, the
template molecule II was purified by preparative reverse-phase HPLC
(60% yield) and characterized by electronspray mass
spectrometry.
[0069] Hydrolysis of the diethylacetal in II was performed by
repeated treatment of II (FIG. 5) with IN
HCl--CH.sub.3COOH--H.sub.2O (2:1:2) for 1 h, followed by
evaporation under reduced pressure. The reaction is apparently
sluggish due to the deactivating effect of the adjacent carbonyl
group, and indeed use of trifluoroacetic acid proved to be
ineffective. However satisfactory results were obtained with 1N
hydrochloric acid. Complete hydrolysis as judged by analytical
reverse-phase HPLC was achieved after six treatments to yield III
(80%). The byproduct IIa was identified as the analogue carrying an
aldehydic function on only one of the lysine residues.
[0070] The ligation reaction proceeded as follows. The template
di-aldehyde III was dissolved in 1M sodium acetate, and the pH was
adjusted to 4.5 with acetic acid. A 1.2-fold excess (with respect
to the aldehyde groups) of NPY21-36, (IV), in 1M sodium acetate was
added, and the mixture was stirred at room temperature. After 3 h,
the condensation reaction was checked by analytical reverse-phase
HPLC. Two major products were observed and characterized by ESI-MS.
After 15 h, the condensation reaction was complete and the crude
product was purified directly by preparative reverse-phase HPLC
(45% yield). The isolated TASP-V was characterized by
electronspray-mass spectrometry and amino acid analysis.
[0071] After each step in the reaction scheme, the product formed
was purified before proceeding on to the next step. The
.alpha.-helix NPY amide (structure IV) and derivatized templates II
and III were purified on a Vydac 218TP1022 (5 .mu.m, C18,
22.times.250 mm) column, using a buffer gradient of B (buffer A:
0.9% trifluoroacetic acid in water; buffer B: 0.9% trifluoroacetic
acid in acetonitrile), at 18 ml/min over 30 min, monitoring at 214
nm. The .alpha.-helix NPY amide was purified using a gradient of
20-60% buffer B and eluted at 19.8 minutes. Derivatized templates
II and III were purified using a gradient of 0-100% buffer B and
eluted at 21.9 minutes and 14.8 minutes respectively. TASP-V was
purified on the same type of column, using a buffer gradient of
10-50-80% buffer B at 18 ml/min over 40 min, again monitoring at
214 nm. TASP-V eluted at 25.7 min.
Example 2
Conformational Properties
[0072] The conformational properties of TASP-V were studied in
solution mainly by circular dichroism (CD) spectroscopy under
various experimental conditions. In order to detect effects of
template attachment on secondary and tertiary structure formation,
the corresponding single (not template attached) peptide IV was
also studied.
[0073] CD spectra were recorded on a Jobin Yvon Dichrograph Mark VI
calibrated with D(+)-10-camphorsulfonic acid. All measurements were
performed at 295K using quartz cells with a path length of 0.1 cm
and each spectrum was the average of three repeated scans between
185 nm to 250 nm, with an integration time of Is for 0.8 nm steps.
The spectra were corrected by substraction of the background
solvent spectrum obtained under identical experimental conditions
and smoothed for clarity of display. CD intensities are expressed
as mean residue ellipticities (deg cm2 dmol-1), calculated by
dividing the total molar ellipticities by the number of amino acids
in the peptide.
[0074] The CD curve of TASP-V displays the typical features of
peptides in an .alpha.-helical conformation, i.e., strong negative
Cotton effects at 222 nm (.theta..sub.M=31000 deg cm2 dmol-1) and
210 nm (.theta..sub.M=34000 deg cm2 dmol-1), a zero-crossover at
202 nm, and a strong positive Cotton effect at 194 nm. While the
single helical peptide IV exhibits weak helicity in TFE (<30%),
the attachment of IV to the cyclic template III results in a
dramatic increase in secondary structure content (>80%),
indicating a strong secondary structure inducing effect of the
template.
Example 3
Selectivity of TASP-V for the NPY Y2 Receptor
[0075] A. Methods
[0076] Cell Culture
[0077] SK-N-MC cells that exclusively express the NPY Y1 receptor,
were derived from a human neuroblastoma and were cultured according
to the American Type cell culture recommendations (Rockville, USA).
LN319 cells that express exclusively the NPY Y2 receptor were
obtained from a human glioblastoma and grown in Dulbecco's modified
Eagle's medium supplemented with 5% fetal calf serum, glutamine,
100 IU of penicillin, and 100 .mu.g/ml of streptomycin in a 5%
CO.sub.2/95% air incubator at 37.degree. C. Tissue culture media
were purchased from Life Technologies (Basel, Switzerland) and
fetal calf serum was obtained from Seromed (Berlin, Germany). 70%
confluent cells were washed with PBS and harvested using 0.15%
Trypsin containing 0.4 mM EDTA. Cells were further diluted 1/3 and
plated onto either 60 mm cell culture dishes (Nunc, Denmark) or
12-mm glass coverslips (Huber and Co, AG, Reinach, Switzerland).
Media were changed every 3 days.
[0078] Y1 Radioreceptor Binding Assay
[0079] Binding of iodinated NPY (Amersham, Buckingamshire, UK, 74
Tbq/mmol) was performed by incubation at 37.degree. C. for 1 hour
in Eagle's minimum essential medium containing 0.5% BSA, 4 mM
MgCl.sub.2 and 10 mM Hepes. Various peptide dilutions were
incubated with SK-N-MC cells that exclusively express Y1 receptors.
Cells were then washed three times with buffer and lysed in 1%
Nonidet P40 (Fluka, Neu-Ulm, Germany), 8M urea, 3M acetic acid.
Non-specific binding was estimated by carrying out binding
reactions in the presence of 1 .mu.M unlabeled NPY. Displacement
curves were obtained by incubation of various concentrations of
competitive peptides together with a non-saturating dose of
iodinated NPY. At the end of the incubation period, cells were
washed and lysed. Bound radioactivity was determined by gamma
counting. Half maximal inhibition of the binding, obtained with
.sup.125I-NPY, is given as the IC.sub.50. Each point represents the
mean .+-. of at least 4 experiments.
[0080] Y2 Radioreceptor Binding Assay
[0081] A human glioblastoma cell line, LN319, was used for Y2
binding studies (Greber, et al., Br. J. Pharmacol. 113:737-740
(1994)). Prior to performing binding assays, adhered LN319 cells
were washed extensively with phosphate buffered saline. The cells
were then harvested in 50 mM Hepes, pH 7.4, containing 145 mM NaCl,
2.5 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 0.1% (w/v) bovine serum
albumin, 0.25 mg/ml bacitracin and 0.025 mg/ml aprotinin. After
centrifugation at 600 g for 15 minutes at a temperature of
4.degree. C., the pellet was resuspended in the harvesting buffer.
Binding was performed in 50 mM Tris, pH 7.5, that contained 100 mM
NaCl, 4 mM MnCl.sub.2, 1 mM EGTA, 0.1% BSA, 0.25 mg/ml bacitracin
and PMSF 0.07 mg/ml. Incubation proceeded at room temperature for
45 minutes. Bound radioactivity was determined after separating the
unbound fraction by centrifugation.
[0082] B. Results
[0083] As described above, the SK-N-MC and LN319 cells express Y1
and Y2 receptor subtypes, respectively. For competitive binding
studies, in addition to native NPY, peptides were used with
differential selectivity for Y1 and Y2. Leu31- and
Pro34-substituted NPY has been shown to be a Y1 agonist (Schwartz,
et al., Ann. N.Y Acad. Sci. 611:35-47 (1990)), whereas NPY13-36 has
been reported to bind preferentially to the Y2 receptor
subtype.
[0084] FIGS. 6 and 7 depict the results of binding experiments
obtained with the two cell lines. SK-N-MC cells (FIG. 6) bind NPY
and Leu31, Pro34 NPY equally-well as shown by the similar
competition displacement curves. In contrast, NPY13-36 binding was
2000 fold less. The template did not bind to SK-N-MC cells
(IC.sub.50>10 .mu.M) and TASP-V shows only a poor affinity for
the Y1 receptor (IC.sub.50=2 .mu.M).
[0085] The LN319 cells (FIG. 7) exhibited a comparably high
affinity for NPY and NPY13-36 with an IC.sub.50 of 0.085 and 0.126
nM, respectively. In contrast, Leu31, Pro34 NPY bound poorly to the
Y2 receptor. The template exhibited no affinity for LN319 cells
(IC.sub.50>10 .mu.M) but good binding to the Y2 receptor was
obtained with TASP-V (IC.sub.50=0.379 nM).
Example 4
Determination of cAMP
[0086] Six-well plates, containing confluent LN319 cell cultures,
were washed and incubated at 37.degree. C. for 1 hour in Eagle's
minimum essential medium containing 0.5% BSA, 4 mM MgCl.sub.2, 10
mM Hepes, 100 .mu.M papaverin and 2.5 .mu.M forskolin and one of
the peptides to be tested in varying dilutions. Cells were washed
once in sodium phosphate buffer (100 mM pH 7.5) and lysed with 0.75
ml of 0.1 M HCl. After centrifugation, the supernatant was
recovered and lyophilized. cAMP concentration was measured by a RIA
using a commercially available kit (Amersham).
[0087] It was found that NPY inhibits forskolin-stimulated cAMP
accumulation in LN319 cells with an IC.sub.50 of 2.5 nM and that
TASP-V has an IC.sub.50 of 3.4 nM. These data indicate that TASP-V
acts as a full agonist at the Y2 receptor.
Example 5
Rat In Vivo Assay
[0088] A. Methods
[0089] Experiments were carried out in adult female Wistar rats
weighing 230-280 g and anaesthetised with sodium pentobarbitone
(Nembutal, Boehringer-Ingleheim; 60 mg/kg, intraperitoneal).
Intravenous supplements of pentobarbitone was given to maintain a
surgical plane of anaesthesia. The trachea was cannulated and
attached to a positive pressure rodent ventilator (Ugo Basile
6025). The left femoral artery was cannulated for continuous
recording of arterial blood pressure via a Gould-Statham
physiological pressure transducer (P23XL) which was connected to
one channel of a pen recorder (Graphtec WR7400). Temperature was
continuously monitored via a rectal probe (Digitron Model 1808) and
kept in the range 34.+-.1.degree. C. An electrocardiogram was
recorded through sub-cutaneous needle electrodes and displayed on a
storage oscilloscope. The electrocardiogram was used to obtain a
beat-by-beat pulse interval (PI-time between successive heart
beats) after processing with Neurolog modules (Digitimer, England
NL200,304,600). Triggering was checked with a counter. PI was
preferred to heart rate because of the linear relation between PI
and frequency of vagal stimulation.
[0090] Both vagus nerves were cut high in the neck. This was done
to eliminate vagally mediated reflex effects on the heart which
occur when blood pressure is increased by NPY. The cardiac end of
the right vagus nerve was stimulated every 30 seconds with a 6
second train of supramaximal stimuli (2 Hz, Ims, 7v) using an
isolated, square wave stimulator (Grass Instruments SD9). The
frequency was chosen to increase pulse interval by approximately
100 ms, a submaximal effect of this variable. The left ferroral
vein was cannulated for administration of NPY (Novabiochem, Human
NPY 1-36) and TASP-V (diluted in saline), as well as further doses
of anaesthetic. To indicate prejunctional activity two parameters
were measured; the maximum percent inhibition of the increase in
pulse interval (.DELTA.PI) evoked by stimulation of the vagus nerve
following injection of the peptide and the time to half recovery of
this effect (T50). For an indication of postjunctional activity,
the pressor action was measured as the peak response following
injection of the peptide (ABP) and the duration of this response
(BP duration).
[0091] B. Results
[0092] As shown in FIGS. 8 and 9, TASP-V was found to have no
postjunctional activity since the pressor action measured as the
peak response following injection of the peptide (ABP) and the
duration of this response (BP duration) was only significant with
NPY that activates both the NPY Y1 and Y2 receptors. In contrast,
TASP-V exhibits a prejunctional activity similar to that observed
with NPY as depicted in FIGS. 10 and 11 by the API and the T50.
Example 6
Effect of TASP-V on Histamine-Induced Rhinitis and Bronchospasm in
Minipigs
[0093] A. Methods
[0094] Animals
[0095] Experiments were performed on 16 domestic pigs of both sexes
(body weight 20-30 kg). The animals were premedicated with atropine
(0.05 mg/kg) and ketamine (Ketalar, ParkeDavis, USA, 20 mg/kg i.m.)
and anaesthetized with thiopentone (5 mg/kg i.v.). A tracheostomy
was performed and artificial ventilation was started using a volume
regulated ventilator (type Siemens 900). During surgery, continuous
i.v. infusion of Ringer's solution, thiopentone (15 mg/kg/h) and
pancuronium bromide (0.25 mg/kg/h) was performed.
[0096] Surgical Procedure
[0097] Catheters were placed in the femoral artery for systemic
blood pressure and heart rate monitoring and in the femoral vein
for thiopentone and fluid administration (300 ml/h). Surgical
preparation of the maxillary artery similar to that described in a
previous report (Lacroix, et al., Acta Physiol. Scand. 132:83-90
(1988)) was performed. Nasal arterial blood flow was monitored with
a Transonic flow probe (RB 143) placed around the sphenopalatine
artery and connected to a T 202 ultrasonic blood flowmeter
(Transonic System Inc., Ithaca, N.Y., USA).
[0098] Administration of Histamine
[0099] In early experiments (n=7), tachyphylaxis to histamine was
investigated by 3 successive nasal and bronchial histamine
challenges at 30 minute intervals. Histamine (5 mg in 1 ml of
saline, Sigma, St. Louis, USA) was sprayed with a hand held
nebulizer in the left nostril under controlled conditions, and
repeated 15 minutes after intranasal spray of TASP-V (85 .mu.g in 1
ml of NaCl 0.9%).
[0100] In other experiments, histamine (10 mg in 3 ml of saline)
was aerosolized for 3 minutes in the trachea with a nebulisator
(Acorn 2, ref. 124010, Marquest Medical, Col., USA) fixed to the
inspiratory division of the ventilation tube and supplemented by
oxygen (4 I/min) under controlled conditions. This procedure was
repeated 15 minutes after intratracheal nebulization of 200
.mu.g-400 .mu.g of TASP-V in 3 ml of saline.
[0101] Measurements of Parameters
[0102] In all animals, the bronchial and nasal vascular responses
to histamine challenge were recorded under controlled conditions
before and after pretreatment with TASP-V. By use of a six channel
pen trace recorder (Gould Electronics) the following parameters
were recorded simultaneously:
[0103] 1. Heart rate and systemic arterial blood pressure using the
femoral artery catheter connected to a pressure transducer.
[0104] 2. Pulmonary airway resistance and compliance. Airway
pressure was measured from a catheter positioned at the tip of the
endotracheal tube. Transpulmonary pressure was determined by a
differential pressure transducer (Hewlett-Packard 267B) taking the
difference between tracheal and oesophagus pressure. Tidal volume
was determined by integration of the respiratory flow signal
measured with a pneumotachograph (Gould Godard, model 17212) by
means of a heated Fleisch flow transducer #2 connected to the
endotracheal tube. Transpulmonary pressure, tidal volume and flow
were continuously recorded on a 4-channel recorder
(Hewlett-Packard, 7754 GB). Total airflow resistance across the
lungs (raw) was determined by dividing the difference in
transpulmonary pressure by inspiratory plus expiratory flow at
mid-tidal volume. Dynamic pulmonary compliance (Cdyn) was obtained
by dividing tidal volume by the difference in transpulmonary
pressure at points of zero flow. Respiratory parameters were
averaged for five successive tidal volumes.
[0105] 3. Sphenopalatine arterial blood flow using an Ultrasonic
flow meter probe (see above).
[0106] B. Results
[0107] The initial triple challenge with nasal histamine resulted
in a reproducible 10.+-.4% increase of the sphenopalatine blood
flow, and a 10.+-.4% decrease in vascular resistance. No
tachyphylactic phenomenon could be elicited in the bronchi. There
was a reproducible 100.+-.27% increase in airway resistance and
38.+-.10% reduction of dynamic lung compliance. No cardiac effect
following the challenge, either by the nasal or bronchial route,
was recorded. The intranasal or intrabronchial administration of
TASP-V did not induce any change in heart rate or mean arterial
blood pressure.
[0108] Following pretreatment with TASP-V, the increase in
sphenopalatine blood flow after histamine challenge was
statistically reduced by 50.+-.5.5%, with a maximum effect after 45
minutes. The reduction of vascular resistance induced by histamine
was not significantly modified after TASP-V pretreatment.
[0109] Two different doses of TASP-V were tested. After
pretreatment with 200 .mu.g, the increase of airway resistance
following the histamine challenge was attenuated by 15.+-.10%, with
a maximum effect observed after 90 minutes (p<0.05). The dynamic
lung compliance reduction was also significantly reduced after 45
minutes). A stronger effect was observed when 400 .mu.g dose was
used, with an attenuation of 50.+-.45%, and the same kinetics
(p<0.05).
Example 7
Effect of TASP-V on Histamine-Induced Rhinitis in Healthy
Volunteers
[0110] Eleven healthy volunteers, 6 males and 5 females, aged from
23 to 48, underwent a daily study designed on a randomized, double
blind, cross-over basis. Exclusion criteria included abnormal nasal
mucosa; anatomical nasal obstruction (such as obstructing nasal
polyps); gross anatomical nasal deformity (such as markedly
deviated septum); and use of any nasal treatment, such as
vasoconstrictors or corticosteroids, in the preceding 30 days.
[0111] Pretreatment
[0112] TASP-V (diluted in saline) and the placebo (saline) were
prepared before each experiment by a technician not involved in the
study. Each patient underwent a local pretreatment with saline or
TASP-V (85 .mu.g in a total of 200 .mu.l of NaCl 0.9%). One
substance was applied and tested over a period of 2 hours, the
other substance being applied and tested over the same period
following a break of 3 hours between the 2 tests. In order to
minimize the risk of bias produced by the cross-over, the peptide
was tested in one nostril while the placebo was used in the
contralateral one, with a randomized and double blind
allocation.
[0113] Stimulation
[0114] 15 minutes after pretreatment with 85 .mu.g of TASP-V or
placebo, intranasal application of histamine (1 mg. in 200 .mu.l of
saline, Sigma, St. Louis, USA) was performed in the same
nostril.
[0115] Measurement of Parameters
[0116] The parameters listed below were measured prior to the
TASP-V pretreatment (T-30', T-15'), then once 15 minutes after it.
Following the histamine challenge, the same parameters were
repeatedly measured at 15 minute intervals during 1 hour (T15, 30,
45 and 60). These were as follows:
[0117] 1. Symptoms: A visual analogue scale, graded from 0 to 5
(where 0 represented the absence of symptom and 5 severe intensity
of symptoms) was used to assess the degree of subjective nasal
obstruction and rhinorrhea.
[0118] 2. Nasal secretions and sneezings: Nasal secretions produced
during the 15 minutes after the histamine challenge were collected
by nose-blowing in a pre-weighted tissue. The number of sneezes was
also recorded.
[0119] 3. Rhinomanometry: Nasal airway resistance (NAR) was
recorded in each nostril by anterior rhinomanometry (Rhinotest MP
441, EVG Elektronic, Vertriebs, Germany). Mean resistance values
for each nostril were obtained after 10 normal breaths and were
calculated at a pressure of 150 Pa.
[0120] 4. Acoustic rhinometry: The minimal cross section area
(MCSA) of the nasal airways opposed to the respiratory flow was
evaluated using an acoustic rhinometer (Rhinoclak, Germany), the
patient being seated in an ENT chair with the head fixed on the
same position during each MCSA recording.
[0121] Statistical Analysis
[0122] Data are given as means.+-.SEM. Statistical differences in
symptom scores, NAR, and nasal secretions were estimated using a
paired Student t-test analysis and one-way analysis of variance
(ANOVA) followed by a Dunnett comparison.
[0123] B. Results
[0124] The intranasal spray of TASP-V did not produce any
subjective local irritation, sneezing or increase in rhinorrhea. No
significant modification of the subjective nasal resistance was
recorded (FIG. 12a). Rhinomanometry and acoustic rhinometry did not
demonstrate any significant change of nasal airway resistance or
MCSA (FIGS. 12b and 13).
[0125] In all patients, histamine challenge following placebo
induced rapid onset of itching, sneezing, rhinorrhea and objective
and subjective nasal obstruction (FIGS. 12-15). The maximum
obstructive effect was observed 15 minutes following the histamine
challenge. No statistical residual effect remained after 60
minutes.
[0126] Pretreatment with TASP-V did not significantly reduce
subjective or objective rhinorrhea or the number of sneezes
following histamine challenge. However, the increase of nasal
airway resistance induced by the allergen challenge, expressed in
percent of the initial value (mean of T01 and T02), was
significantly reduced by the TASP-V pretreatment (FIGS. 12b, 14).
The maximum effect was observed after 15 and 30 minutes. Similarly,
the histamine-induced reduction of MCSA was significantly
attenuated by TASP-V pretreatment with a maximum effect at 15 and
30 minutes (FIGS. 13, 15). Though non-significant, the subjective
homolateral nasal obstruction produced by the histamine challenge
appeared to be reduced after TASP-V pretreatment, again with a
maximum effect after 15 and 30 minutes (FIG. 12a). None of the
patients reported any side effects during the 24 hours following
the experiment.
[0127] All references cited herein are fully incorporated by
reference. Having now fully described the invention, it will be
understood by those of skill in the art that the invention may be
performed within a wide and equivalent range of conditions,
parameters and the like, without affecting the spirit or scope of
the invention or any embodiment thereof.
Sequence CWU 1
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