U.S. patent application number 15/956625 was filed with the patent office on 2018-11-29 for methods and compositions related to improving properties of pharmacological agents targeting nervous system.
The applicant listed for this patent is University of Utah Research Foundation. Invention is credited to Grzegorz Bulaj, H. Steve White.
Application Number | 20180340012 15/956625 |
Document ID | / |
Family ID | 38256917 |
Filed Date | 2018-11-29 |
United States Patent
Application |
20180340012 |
Kind Code |
A1 |
Bulaj; Grzegorz ; et
al. |
November 29, 2018 |
METHODS AND COMPOSITIONS RELATED TO IMPROVING PROPERTIES OF
PHARMACOLOGICAL AGENTS TARGETING NERVOUS SYSTEM
Abstract
Disclosed are compositions and methods related to improving
pharmacological properties of bioactive compounds targeting nervous
system.
Inventors: |
Bulaj; Grzegorz; (Salt Lake
City, UT) ; White; H. Steve; (Salt Lake City,
UT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Utah Research Foundation |
Salt Lake City |
UT |
US |
|
|
Family ID: |
38256917 |
Appl. No.: |
15/956625 |
Filed: |
April 18, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15699991 |
Sep 8, 2017 |
|
|
|
15956625 |
|
|
|
|
15411905 |
Jan 20, 2017 |
|
|
|
15699991 |
|
|
|
|
14558570 |
Dec 2, 2014 |
9586999 |
|
|
15411905 |
|
|
|
|
13853945 |
Mar 29, 2013 |
8933020 |
|
|
14558570 |
|
|
|
|
12160035 |
Dec 18, 2008 |
8435940 |
|
|
PCT/US2007/000261 |
Jan 5, 2007 |
|
|
|
13853945 |
|
|
|
|
60757047 |
Jan 5, 2006 |
|
|
|
60844024 |
Sep 11, 2006 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 25/08 20180101;
A61P 29/00 20180101; A61P 25/24 20180101; C07K 14/47 20130101; C07K
7/23 20130101; A61P 25/04 20180101; C07K 7/08 20130101; C07K 7/06
20130101; C07K 14/575 20130101; C07K 2319/01 20130101; A61P 35/00
20180101; A61P 25/00 20180101; A61P 25/28 20180101; C07K 14/57545
20130101; C07K 5/1019 20130101; C07K 14/6555 20130101; C07K 14/665
20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; C07K 7/08 20060101 C07K007/08; C07K 14/665 20060101
C07K014/665; C07K 14/655 20060101 C07K014/655; C07K 14/575 20060101
C07K014/575; C07K 7/23 20060101 C07K007/23; C07K 7/06 20060101
C07K007/06; C07K 5/11 20060101 C07K005/11 |
Claims
1. A method of increasing permeability of the blood-brain barrier
for a peptide, the method comprising modifying the peptide to have
increased lipophilic character and increased basicity of the
peptide compared to the unmodified form of the peptide.
2. The method of claim 1, wherein the lipophilic character is
increased by conjugating the peptide to a hydrophobic moiety.
3. The method of claim 2, wherein the hydrophobic moiety is
polyaliphatic chains.
4. The method of claim 1, wherein the lipophilic character is
increased by the substitution of one or more aromatic residues with
a halogenated aromatic amino acid residue.
5. The method of claim 1, wherein the basicity is increased by
introducing homo- and heterooligomers of positively charged amino
acid residues, including, but not limited to Lysine, Arginine,
homo-Lysine, homo-Arginine, Ornitine in L- or D-isomer
configuration; 2,3-Diaminopropioic acid; 2,4-Diaminobutyric
acid.
6. The method of claim 1, wherein the basicity is increased by
conjugation to polyamine-based moieties, such as spermine,
spermidine, polyamidoamine dendrimers or polyamine toxins and
derivatives thereof.
7. The method of claim 1, wherein the peptide can cross the
blood-brain barrier with 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, or 100% more efficiency compared to the unmodified
peptide.
8. The method of claim 1, further comprising increasing
glycosylation of the peptide compared to the unmodified form of the
peptide.
9. The method of claim 1, comprising a first modification relative
to the unmodified peptide that increases the lipophilic character
of the modified neuropeptide when compared to the unmodified
peptide; with the first modification being selected from at least
one of: (a) a hydrophobic moiety conjugated to one or more amino
acid residues of the modified neuropeptide; and (b) substitution of
one or more aromatic amino acid residues with a halogenated
aromatic amino acid residue, and a second modification relative to
the unmodified peptide that increases the basicity of the modified
neuropeptide when compared to the unmodified peptide, with the
second modification being selected from at least one of: (a) an
oligomer of positively charged amino acid residues introduced into
the amino acid sequence of the unmodified peptide, wherein the
oligomer is selected from the group consisting of homooligomers and
heterooligomers comprising Lysine, Arginine, homo-Lysine,
homo-Arginine, L-Ornithine, D-Ornithine, 2,3-Diaminopropioic acid,
and 2,4-Diaminobutyric acid, and (b) a polyamine-based moiety
conjugated to the modified neuropeptide, wherein the
polyamine-based moiety is selected from the group consisting of
spermine, spermidine, polyamidoamine, dendrimers, and polyamine
toxins.
10. An isolated polypeptide modified to have increased permeability
of the blood-brain barrier according to the method of claim 1.
11. The isolated polypeptide of claim 10, wherein the modified
polypeptide is derived from an unmodified peptide selected from
galanin, somatostatin, delta-sleep inducing peptide, neuropeptide
Y, and neurotensin.
12. The isolated polypeptide of claim 10 comprising SEQ ID NO:3,
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, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ
ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:
27, SEQ ID NO: 28, and SEQ ID NO: 29, SEQ ID NO: 50, SEQ ID NO: 56,
SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID
NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67,
SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID
NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77,
SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID
NO: 82, SEQ ID NO: 83, SEQ ID NO: 101, SEQ ID NO: 105, and SEQ ID
NO: 142.
13. A method of treating, preventing, or ameliorating pain or other
neurological disorders comprising administering to a subject in
need thereof an effective amount of a polypeptide modified to have
increased permeability of the blood-brain barrier according to the
method of claim 1.
14. The method of claim 13, wherein the modified polypeptide is
derived from an unmodified peptide selected from galanin,
somatostatin, delta-sleep inducing peptide, neuropeptide Y, and
neurotensin.
15. The method of claim 13, wherein the modified polypeptide is a
galanin analog.
16. The method of claim 13, wherein the modified polypeptide is a
galanin analog selected from at least one of SEQ ID NO: 56, SEQ ID
NO: 66, and SEQ ID NO: 67.
17. The method of claim 13, wherein the pain is caused by one or
more of the following, or the neurological disorder is selected
from one or more of the following: chronic back pain, cancer,
fibromyalgia, postherpetic neuralgia, multiple sclerosis, diabetic
neuropathy, peripheral nerve injury, traumatic mononeuropathy,
complex regional pain syndrome, and spinal cord injury.
18. A method of treating epilepsy, comprising administering to a
subject in need thereof an effective amount of a polypeptide
modified to have increased permeability of the blood-brain barrier
according to the method of claim 1.
19. A method of treating spinal cord injury in a subject,
comprising administering to the subject a polypeptide modified to
have increased permeability of the blood-brain barrier according to
the method of claim 1.
20. A method of treating multiple sclerosis in a subject,
comprising administering to the subject a polypeptide modified to
have increased permeability of the blood-brain barrier according to
the method of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/699,991, filed Sep. 8, 2017, which is continuation of U.S.
application Ser. No. 15/411,905, which was filed Jan. 20, 2017,
which is a continuation of U.S. application Ser. No. 14/558,570,
which was filed Dec. 2, 2014 (U.S. Pat. No. 9,586,999), which is a
continuation of Ser. No. 13/853,945, which was filed Mar. 29, 2013
(U.S. Pat. No. 8,933,020), which is a continuation of U.S.
application Ser. No. 12/160,035 (U.S. Pat. No. 8,435,940), filed
Dec. 18, 2008, which claims the benefit of priority under 35 U.S.C.
.sctn. 371 of International Application No. PCT/US2007/000261,
filed Jan. 5, 2007, which claims the benefit of the filing dates of
U.S. Provisional Application No. 60/757,047, filed Jan. 5, 2006,
and U.S. Provisional Application No. 60/844,024, filed Sep. 11,
2006. The content of these earlier filed applications are hereby
incorporated by reference herein in their entirety.
BACKGROUND OF THE INVENTION
[0002] The blood-brain barrier (BBB) separates the mammalian brain
from the systemic circulation and plays a vital role in the
homeostasis of the central nervous system (CNS). Despite the
continuous progress in understanding transport of peptides through
the blood-brain barrier, their efficient delivery directly into the
CNS has remained a major challenge in developing neuropeptides as
potential therapeutics.
[0003] Epilepsy, for example, is a complex neurological disorder.
Intractable epilepsy is estimated to affect 30% of the patient
population. Despite availability of various antiepileptic drugs
(AEDs), certain types of seizures and epilepsy syndromes respond
with limited success to only a few AEDs. Therefore, there is an
ongoing need to discover and develop new anticonvulsant
therapeutics with improved efficacy and safety profiles. Moreover,
recent discoveries of neurobiological changes that occur prior to
an epileptic seizure have opened an opportunity for the discovery
of new antiepileptogenic compounds, and such antiepileptogenic
agents can include neuropeptides and neurotrophins.
[0004] Neuropeptides and their receptors that have been implicated
in the mechanisms of epileptic seizures include galanin,
neuropeptide Y, somatostatin and opioid peptides. Some of these
neuropeptides, when delivered directly into the central nervous
system (CNS), possess an anticonvulsant activity, but their poor
bioavailability and marginal metabolic stability preclude
development of neuropeptide-based antiepileptic drugs. On the other
hand, advanced peptide engineering has produced many successful
instances of peptide analogs with improved stability or
bioavailability. However, none of the available peptide engineering
techniques have been applied to neuropeptides with anticonvulsant
activity. What is needed in the art are methods and compositions
for improving permeability through the blood-brain barrier.
I. SUMMARY OF THE INVENTION
[0005] In accordance with the purposes of this invention, as
embodied and broadly described herein, this invention, in one
aspect, relates to an isolated polypeptide comprising SEQ ID NO: 3,
an amino acid sequence at least about 90% identical to the amino
acid sequence of SEQ ID NO: 3, or the amino acid sequence of SEQ ID
NO: 3 having one or more conservative amino acid substitutions.
[0006] Also disclosed is an isolated polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO: 2, 4-29, 37-39, 50, 64, 65, 66, 67, 80, 82, and 89, an amino
acid sequence at least about 90% identical to an amino acid
sequence selected from the group consisting of SEQ ID NO: 2, 4-29,
37-39, 50, 64, 65, 66, 67, 80, 82, and 89, or the amino acid
sequence selected from the group consisting of SEQ ID NO: 2, 4-29,
37-39, 50, 64, 65, 66, 67, 80, 82, and 89 having one or more
conservative amino acid substitutions.
[0007] Also disclosed is an isolated polypeptide comprising an
amino acid segment selected from the group consisting of SEQ ID NO:
31-36, an amino acid sequence at least about 90% identical to the
amino acid sequence selected from the group consisting of SEQ ID
NO: 31-36, or the amino acid sequence selected from the group
consisting of SEQ ID NO: 31-36 having one or more conservative
amino acid substitutions.
[0008] Also disclosed is an isolated polypeptide comprising SEQ ID
NO: 40, an amino acid sequence at least about 90% identical to the
amino acid sequence of SEQ ID NO: 40, or the amino acid sequence of
SEQ ID NO: 40 having one or more conservative amino acid
substitutions.
[0009] Also disclosed is an isolated polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO: 105, 106, 107, 108, 109-112, and 113-118, an amino acid
sequence at least about 90% identical to the amino acid sequence
selected from the group consisting of SEQ ID NO: 105, 106, 107,
108, 109-112, and 113-118, or the amino acid sequence selected from
the group consisting of SEQ ID NO: 105, 106, 107, 108, 109-112, and
113-118 having one or more conservative amino acid
substitutions.
[0010] Also disclosed is an isolated polypeptide comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO: 58 and 135-141, an amino acid sequence at least about 90%
identical to the amino acid sequence selected from the group
consisting of SEQ ID NO: 58 and 135-141, or the amino acid sequence
selected from the group consisting of SEQ ID NO: 58 and 135-141
having one or more conservative amino acid substitutions.
[0011] Further disclosed is a composition with increased
permeability of the blood-brain barrier, wherein the composition
comprises a peptide with increased lipophilic character and
increased basicity when compared to the non-altered form of the
peptide.
[0012] Disclosed is a method of increasing permeability of the
blood-brain barrier for a peptide, comprising increasing lipophilic
character and increasing basicity of the peptide compared to the
non-altered form of the peptide.
[0013] Also disclosed is a method of treating epilepsy, comprising
administering to a subject in need thereof an effective amount of a
polypeptide as disclosed herein. Further disclosed is a method of
treating epilepsy, comprising administering to a subject in need
thereof an effective amount of a composition as disclosed
herein.
[0014] Disclosed is a method of treating, preventing, or
ameliorating pain or other neurological disorders comprising
administering to a subject in need thereof an effective amount of a
polypeptide as disclosed herein.
[0015] Also disclosed is a method of treating a subject in need of
a composition that crosses the blood-brain barrier, comprising:
identifying the composition to be used in treatment of the subject;
modifying the composition by increasing lipophilicity and basicity
of the composition; and administering the modified composition to
the subject in need thereof.
[0016] Further disclosed is a method of treating a subject in need
of a composition that crosses the blood-brain barrier, comprising:
identifying the composition to be used in treatment of the subject;
modifying the composition by increasing lipophilicity,
glycosylation, and basicity of the composition; and administering
the modified composition to the subject in need thereof.
[0017] Also disclosed is a method of treating a subject in need of
a composition that crosses the blood-brain barrier, comprising:
identifying the composition to be used in treatment of the subject;
modifying the composition by increasing lipophilicity,
glycosylation, and basicity of the composition; inserting the
modified composition into a vector; and administering the vector to
the subject in need thereof.
[0018] Further disclosed is a method of treating a subject in need
of a composition that crosses the blood-brain barrier, comprising:
identifying the composition to be used in treatment of the subject;
modifying the composition by increasing lipophilicity and basicity
of the composition; inserting the modified composition into a
vector; and administering the vector to the subject in need
thereof.
II. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the invention and together with the description,
serve to explain the principles of the invention.
[0020] FIG. 1 shows the role of neuropeptides in GABA- and
GLU-mediated neurotransmission in SSSE hippocampal neuronal
circuitry. Abbreviations: CA, pyramidal neurons; DYN, dynorphin;
GABA, .gamma.-amino butyric acid; GAL, galanin; GLU, glutamate; NE,
norepinephrine; NPY, neuropeptide Y; SOM, somatostatin; SubsP,
substance P (Wasterlain et al. 2002).
[0021] FIG. 2 shows galanin injected into the hilus before of after
stimulation, shortened the duration of seizures in rats. Upper
plot. Galanin (50 and 500 picomoles) was injected 30 minutes prior
to perforant-path stimulation (PPS). Lower plot. Only injection of
500 picomoles of galanin was effective in reducing the duration of
seizures when injected 30 minutes after the PPS (Mazarati et al.
1998).
[0022] FIG. 3 shows both a nonpeptidic galanin receptor agonist and
galanin increased latency and decreased seizure scores on the
PTZ-induced seizures in mice. Inset summarizes the maximal seizures
score (black, control; open, galnon; grey, galanin). (Saar et al.
2002).
[0023] FIG. 4 shows anticonvulsive activity of somatostatin after
intrahippocampal injection in the self-sustaining status
epilepticus model in rats. (Mazarati and Wasterlain, 2002).
[0024] FIG. 5 shows anticonvulsive activity of dynorphin and
neuropeptide Y after intrahippocampal injection in the
self-sustaining status epilepticus model in rats. Refer to FIG. 4
for the control experiment. (Mazarti and Wasterlain, 2002).
[0025] FIG. 6 shows key factors affecting permeability of peptides
through the blood-brain barrier. Basicity and lipophilicity may
improve the passive transport via diffusion and absorptive-mediated
endocytsis, whereas glycosylation or vectors can also contribute to
the active transport through the blood-brain barrier.
[0026] FIG. 7 shows a general strategy for the development of
neuropeptide analogs with anticonvulsant activity. Two model
neuropeptides were selected to evaluate the technology for
improving their permeability though the blood-brain barrier.
[0027] FIG. 8 shows structural organization of a prototype of an
ideal neuropeptide drug. Stars denote residues of the
pharmacophore. Grey boxes represent backbone and side chain
modifications that increase metabolic stability, basicity and
lipophilicity of the peptide moiety. The BBB/PK-modifier is a
bulky, polymer-based structure containing lipophilic and cationic
modules and endogenous nutrient mimetics. The cationic module
increases absorptive-mediated endocytosis through electrostatic
interactions with membranes. The lipophilic module increases
passive transport through the blood-brain barrier. The active
transport mimetic structure (e.g., hexose or phenylalanine) serves
as a substrate for interactions with inwardly directed nutrient
transporters located in the blood-brain barrier.
[0028] FIG. 9 shows the strategy used to design peptide analogs
that penetrate the BBB. Combinations of two or more distinct
chemical modifications that improve permeability of the analogs
through the BBB are shown.
[0029] FIG. 10 shows a systematic approach to engineering
neuropeptides to improve their permeability through the blood-brain
barrier. Key: DAB, diaminobutyric acid; DAP, diaminopropioic acid;
PEG, polyethylene glycol; Mmt, 4-methyltrityl.
[0030] FIG. 11 shows modular structure of the N-terminal extension
in somatostatin analogs. The modules are coupled during solid-phase
peptide synthesis. The number and order of modules is arbitrary,
and the structural composition of the BBB/PK modulator can be
optimized.
[0031] FIG. 12 shows Gal BBB-2 displays time (inset) and
dose-dependent protection against audiogenic seizures in the Frings
mouse. For the time course mice were treated with 4 mg/kg, i.p. Gal
BBB-2 and tested at various times after administration. For the
dose-response study groups of Frings mice were treated with
increasing doses of Gal BBB-2 and tested one hour after i.p.
administration.
[0032] FIG. 13 shows general experimental strategy of optimizing
GAL-BBB2. First three boxes summarize activity defined in Example
1. Approximately 40 analogs are synthesized and screened in the
competitive binding assay. Only the most potent galanin ligands are
further screened for their potent and long-lasting anticonvulsant
activity, before more detailed pharmacological
characterization.
[0033] FIG. 14 shows structural organization of GAL-BBB2 and
proposed SAR studies. Black boxes illustrate key pharmacophore
residues.
[0034] FIG. 15 shows "backbone prosthesis"--replacement of
non-pharmacophore residues (R2 and R3) with a non-peptide spacer
(5-aminovaleric acid). Other backbone spacers include aminohexanoic
acid or amino-3,6-dioxaoctanoic acid (PEG-spacer).
[0035] FIG. 16 shows GAL-BBB2 (labeled as NAX-5055 in the figure)
(0.52-5 mg/kg) produced a dose-dependent reduction in paw licking
during both the initial acute phase as well as the prolonged
inflammatory phase. In contrast, the un-modified native fragment
Gal 1-16 was found to be inactive following i.p. administration of
a dose 4 times higher than the highest dose of GAL-BBB2 tested
(i.e., 20 mg/kg).
[0036] FIG. 17 shows that 5 mg/kg GAL-BBB2 was found to be
equivalent to a 10 mg/kg dose of gabapentin.
[0037] FIG. 18 shows that GAL-BBB2 (labeled as NAX-5055 in the
figure) displayed a time-dependent increase in the threshold for
mechanical allodynia in the sciatic ligation model of chronic pain.
Furthermore, GAL-BBB2 was equi-potent to morphine and several fold
more potent that gabapentin.
[0038] FIG. 19 shows the structure of GAL-BBB2, also referred to as
NAX 5055.
[0039] FIG. 20 shows NAX 5055 (GAL-BBB2), but not the native
peptide fragment, is active in Frings mouse. Anticonvulsant
efficacy was quantitated at the time to peak effect (i.e., 1 h) in
a dose-response study. The results of this study demonstrated that
GAL-BBB2 displayed a dose-dependent effect against sound-induced
seizures. The calculated median effective dose (i.e., ED.sup.50)
and 95% confidence intervals were obtained from a Probit analysis
of the dose-response data was 3.2 (2.3-6.1) mg/kg. The native
peptide fragment GAL(1-16) was inactive at a dose of 20 mg/kg, i.p.
(six times the ED50 for GAL-BBB2)
[0040] FIG. 21 shows the chemical structure and schematic for
engineering octreocide.
[0041] FIG. 22 shows that NAX 5055 (GAL-BBB2) is more potent and
more efficacious than the native peptide in 6 Hz (32 mA) test.
[0042] FIG. 23 shows that NAX 5055 (GAL-BBB2) (4 mg/kg, i.p.)
displays a time-dependent anticonvulsant activity in Frings
Mouse.
[0043] FIG. 24 shows that NAX 5055 (GAL-BBB2) displays
dose-dependent protection against audiogenic seizures in the Frings
mouse.
[0044] FIG. 25 shows NAX 5055 (GAL-BBB2) (4 mg/kg) is active in
pharmaco-resistant seizure model.
[0045] FIG. 26 shows NAX 5055 (GAL-BBB2) displays excellent
bioavailability following i.p. and s.c. administration in 6 Hz
seizure test. NAX 5055 was injected either intraperitoneally or
subcuatenously into groups (n=6-8) male CF-1 mice. After 60 min,
individual mice in each group were stimulated (32 mA, 6 Hz, 3 sec
duration) via corneal electrodes. Mice not displaying limbic
seizures were considered protected. Results demonstrate that the
anticonvulsant activity of NAX 5055 is retained following
subcutaneous administration. These findings show that a depot
formulation amenable for subcutaneous delivery of NAX 5055 and/or
other neuroactive peptides can be used.
[0046] FIG. 27 shows NAX 5055 (GAL-BBB2) increases the efficacy and
potency of CMPD X in 6 Hz (44 mA) test. When administered by
itself, CMPD A (levetiracetam) is minimally effective against 6 Hz
limbic seizures at very high doses (i.e., maximum 50% protection at
1000 mg/kg). In contrast, when a minimally effective dose of
NAX5055 (1.5 mg/kg) is administered in combination with CMPDA A
(levetiracetam), efficacy and potency is markedly increased. These
results show that modulation of galanin receptors by NAX 5055 leads
to a synergistic enhancement of the anticonvulsant efficacy of
levetiracetam. When taken together, these findings show that a
combination product that combines NAX 5055 with levetiracetam can
offer therapeutic advantages over even very high doses of
levetiracetam alone.
[0047] FIG. 28 shows NAX 5055 (GAL-BBB2) displays modest protection
in hippocampal kindled rats. In the hippocampal kindled rat model
of partial epilepsy, NAX 5055 decreases the seizure score from 5 to
3. These results show that modulation of galanin receptors by NAX
5055 is useful in preventing secondarily generalized partial
seizures and is consistent with previous intracerebroventricular
studies wherein galanin was directly injected into the brain of
kindled rats. The finding that intraperitoneally administered NAX
5055 is active supports the conclusion that it is gaining access to
the brain following systemic administration.
[0048] FIG. 29 shows the effect of NAX 5055 (GAL-BBB2) on
formalin-induced hyperalgesia. NAX 5055 was administered 60 min
prior to plantar injection of formalin. Time was based on
anticonvulsant time of peak effect.
[0049] FIG. 30 shows somatostatin and Delta sleep-inducing peptide
(DSIP) are both anticonvulsant in 22 mA 6 Hz seizure test. Result
shown in this figure demonstrate that somatostatin and delta
sleep-inducing peptide (DSIP) when administered directly into the
ventricular space of CF-1 mice are effective against 6 Hz (22 mA)
limbic seizures. These results provide the `proof-of-concept` that
modulation of somatostatin and DSIP binding sites in the brain is a
viable approach. They further support the development of
systemically active somatostatin and DSIP neuroactive peptides that
cross the blood-brain-barrier using our proprietary technology.
[0050] FIG. 31 shows the structure of GAL(1-16) analog. Marked are
key pieces of information that were used to design analogs with
increased BBB permeability.
[0051] FIG. 32 shows the effect of twice daily injections of
NAX-5055 (4 mg/kg, i.p.) on the acquisition of mouse corneal
kindling. CF#1 mice were randomized to receive either vehicle (0.9%
saline) or NAX-5055. Mice in the NAX-5055 group received two doses
of NAX-5055 twelve (12 h) and 1 h prior to their first kindling
stimulation. One hour (1 h) prior to each subsequent stimulation,
mice in the NAX-5055 treated group received another dose of
NAX-5055 (4 mg/kg, i.p.). Mice were stimulated twice daily for 16
days. Results are expressed as the mean seizure score per
stimulation. As noted above, the results for the NAX-5055 treated
mice segregated into two populations; i.e., sensitive (green line)
and insensitive (blue line). Saline vs. NAX-5055 sensitive
significantly different at p<0.0002; NAX-5055 sensitive vs.
NAX-5055 insensitive significantly different at p<0.0001. The
results obtained for this study support the claim that
galanin-based peptides such as NAX-5055 possess the ability to
prevent the acquisition of kindling and can be disease-modifying in
patients at risk for the development of epilepsy and other
neurological disorders.
[0052] FIG. 33 shows the effect of twice daily treatment with
NAX-5055 (mg/kg, i.p.) on the rate of corneal kindling. Mice
treated with NAX-5055 (see legend to Figure X for experimental
details) segregated into two treatment groups; i.e., NAX-5055
sensitive and NAX-5055 insensitive.
[0053] Results are expressed as the number of stimulations required
to reach a particular seizure score+/-S.E.M.; i.e., 1 to 5. Mice in
the NAX-5055 sensitive group required two times more stimulations
to reach a Stage 1 seizure and 35-40% more stimulations to reach
Stage 2 and Stage 3 seizures, respectively. Furthermore, none of
the mice in the NAX-5055 sensitive group reached Stage 5 seizures.
One Way ANOVA, p<0.0209; post hoc analysis: saline vs. NAX-5055
insensitive, p>0.05; NAX-5055 sensitive vs. NAX-5055
insensitive, p<0.05. These results support the conclusion that
modified galanin-based neuropeptides possess the ability to modify
the development of kindling acquisition and that they are useful
for the prevention of network hyperexcitability in a patient
population susceptible to developing epilepsy and other
neurological disorders.
[0054] FIG. 34 shows structures of glycosyl groups introduced to
enkephalin analogs (Elmagbari, Egleton et al. 2004).
[0055] FIG. 35 shows that the combination of two distinct chemical
modifications is superior over individual modifications.
Cationization or lipidization alone did not improve penetration of
the 5055 analog as a combination of both.
[0056] FIG. 36 shows examples of lipoamino acids which were used to
improve permeability of peptides through the blood-brain-barrier.
Such lipoamino acids can be combined with chemical modifications
that increase basicity of the target peptide.
[0057] FIG. 37 shows chemical modifications improve metabolic
stability of the neuropeptide analogs. Analogs 5055 (SEQ ID NO: 3)
or 1205-2 (SEQ ID NO: 50) or unmodified analog Gal(1-16) were
incubated in diluted rat serum at 37.degree. C. Remaining amounts
of the peptides were determined by HPLC.
[0058] FIG. 38 shows the different types of neuropathic pain can be
treated, prevented or reversed by neuropeptide analogs that cross
the BBB.
III. DETAILED DESCRIPTION
[0059] The present invention may be understood more readily by
reference to the following detailed description of preferred
embodiments of the invention and the Examples included therein and
to the Figures and their previous and following description.
[0060] Before the present compounds, compositions, articles,
devices, and/or methods are disclosed and described, it is to be
understood that this invention is not limited to specific synthetic
methods, specific recombinant biotechnology methods unless
otherwise specified, or to particular reagents unless otherwise
specified, as such may, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only and is not intended to be
limiting.
A. DEFINITIONS
[0061] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a pharmaceutical carrier" includes mixtures of two or
more such carriers, and the like.
[0062] Ranges may be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that when a value is disclosed that "less than
or equal to" the value, "greater than or equal to the value" and
possible ranges between values are also disclosed, as appropriately
understood by the skilled artisan. For example, if the value "10"
is disclosed then "less than or equal to 10" as well as "greater
than or equal to 10" is also disclosed.
[0063] In this specification and in the claims which follow,
reference will be made to a number of terms which shall be defined
to have the following meanings:
[0064] "Optional" or "optionally" means that the subsequently
described event or circumstance may or may not occur, and that the
description includes instances where said event or circumstance
occurs and instances where it does not.
B. COMPOSITIONS AND METHODS
[0065] 1. The Blood-Brain Barrier
[0066] The blood-brain barrier (BBB) is the specialized system of
capillary endothelial cells that protects the brain from harmful
substances in the blood stream, while supplying the brain with the
required nutrients for proper function. Unlike peripheral
capillaries that allow relatively free exchange of substance
across/between cells, the BBB strictly limits transport into the
brain through both physical (tight junctions) and metabolic
(enzymes) barriers. Thus the BBB is often the rate-limiting factor
in determining permeation of therapeutic drugs into the brain.
[0067] A number of obstacles currently limit the use of many
compounds for use as central nervous system (CNS) therapeutic
agents. First, the brain is equipped with a barrier system. The
brain barrier system has two major components: the choroid plexus
and the blood-brain barrier (BBB). The choroid plexus separates
cerebrospinal fluid (CSF) from blood and the BBB separates brain
ISF from blood.
[0068] Also, the BBB has about 1000 times more surface area than
the choroid plexus and is the primary obstacle to delivery of
therapeutic compounds to the CNS. The BBB acts as a selective
partition, regulating the exchange of substances, including
peptides, between the CNS and the peripheral circulation. The
primary structure of the BBB is the brain capillary endothelial
wall. The tight junctions of brain capillary endothelial cells
prevent circulating compounds from reaching the brain ISF by the
paracellular route. Furthermore, recent work suggests the existence
of a physiological barrier at the level of the basal lamina, in
addition to the barrier provided by the tight junctions. (Kroll et
al., Neurosurgery, Vol. 42, No. 5, p. 1083 (May 1998)). Other
unique characteristics of the BBB include lack of intracellular
fenestrations and pinocytic vesicles and a net negative charge on
the luminal surface of the endothelium.
[0069] The mechanisms by which substances may traverse the BBB may
generally be divided into active and passive transport mechanisms.
Lipophilic molecules readily traverse the BBB by passive transport
or diffusion through the endothelial plasma membranes. Hydrophilic
molecules, such as peptides, typically require an active transport
system to enable them to cross the BBB. Certain larger peptides,
such as insulin, have receptors on the luminal surface of the brain
capillaries which act as active transcytosis systems.
[0070] There are two main mechanisms for transporting peptides
across the blood-brain barrier: (1) simple diffusion through the
membrane, determined primarily by molecular size and lipophilicity,
and (2) active influx, mediated by specific receptors and carriers
located on the surface of the endothelial cells in the blood-brain
barrier, or by non-specific absorption trancytosis (Tamai and
Tsuji, 2000; Pan and Kastin, 2004a; Smith et al., 2004).
[0071] 2. Improving Permeability
[0072] A number of strategies have been tested to improve
permeability of peptides and proteins through the blood-brain
barrier. These can be divided into several categories: (1)
conjugation to a vector or nutrient-active transport that enhances
uptake of the peptide into the CNS; (2) lipidization/halogenation
resulting in increased lipophilicity to enhance simple diffusion,
(3) cationization (increased basicity) to enhance transport through
absorptive transcytosis; and (4) glycosylation or prodrugs that
improve active/passive transport and the pharmacokinetic profiles
of peptides ((Witt et al., 2001) and (Pan and Kastin, 2004a)).
Examples of such studies and major findings are shown in Table 1.
Each reference is herein incorporated in its entirety for its
teaching regarding penetration of the blood-brain barrier.
TABLE-US-00001 TABLE 1 Examples of improving permeability of
peptides through the blood-brain barrier. Strategy Peptides Major
Findings References Halogenation of Enkephalins Chloro-Phe
containing peptides (Weber et al., 1991; aromatic residues elicited
a much greater analgesic Abbruscato et al., effect after
intravenous 1996) administration Conjugation with .alpha.-Conotoxin
4-fold Increase in blood-brain (Blanchfield et al., lipoamino acids
MII barrier permeability 2003) Conjugation with .beta.-Amyloid-
7-fold increase in the blood-brain (Poduslo et al., 1999) polyamine
derived barrier permeability derivatives peptide Conjugation with
Octreotide/ More efficient brain uptake of (Luyken et al., 1994;
DOPA/DTPA somatostatin radiopharmaceuticals for MRI and Kurihara
and derivatives Epidermal treatment of brain tumors Pardridge,
1999) growth factor Glycosylation Enkephalins Adding glycosyl
moiety resulted in (Elmagbari et al., 20-fold increase in systemic
2004) bioavaliability Selective amino- Neurotensin Trp replaced by
Neo-Trp and Ile (Hertel et al., 2001) acid side-chain replaced by
Tert-Leu resulted in an replacements analog that crosses
blood-brain barrier Selective amino- Thyrotropin- His replaced by
pyridinium moiety (Prokai et al., 2004) acid side-chain releasing
increased 2- to 3-fold central replacements hormone activity of TRH
(TRH) Prodrug strategy TRH-like Esterification of Glu residues
(Prokai-Tatrai et al., peptides enhanced analeptic activity 2003)
Prodrug strategy Enkephalin C-terminal Extension with Phe (Greene
et al., 1996) increased half-life and permeability Prodrug strategy
Enkephalin Conjugation with adamantine (Kitagawa et al., moiety
resulted in improved 1997) activity in subcutaneous administration
Vector-mediated Enkephalin SynB3 peptide-based vectors (Rousselle
et al., delivery Dalargin enhanced brain uptake and 2003) analgesic
activity
[0073] Regarding the first item, conjugation, the increased
molecular size of the conjugated peptides does not seem to hamper
permeability. Adding the peptide-based vector SynB1 (MW=2,099) to
the opioid peptide dalargin (MW=726) resulted in an almost
four-fold increase in size, but also in an 18-fold increase in
brain uptake (Rousselle et al., 2003). Similarly, adding
disaccharide moieties to enkephalin analogs increased their
antinociceptve activity up to 21-fold, following intravenous
administration (Elmagbari et al., 2004).
[0074] Two important factors, namely lipophilicity and basicity,
contribute to increased permeability of peptides through the
blood-brain barrier without the need for specific transporters to
or carriers. The lipophilic character of a peptide can be altered
by either conjugation to a hydrophobic moiety (e.g., polyaliphatic
chains), or halogenation of aromatic residues (e.g., chloro-Phe, as
compared to Phe). It has been shown that polyamine-modified
proteins and peptides cross the blood-brain barrier more
efficiently, as compared to unmodified ones (Poduslo and Curran,
1996a; b; Poduslo et al., 1999). It has also been shown (Tamai et
al., 1997) that the increased basicity of small peptides was an
important determinant of transport through the blood-brain barrier
via absorptive-mediated endocytosis (AME).
[0075] In addition to direct modification of the peptides, there
are a few other drug delivery strategies with improved uptake of
drugs into the CNS. These include liposome-, micelle- or
nanoparticle-mediated delivery of peptides through the blood-brain
barrier (Kreuter et al., 2003; Pan and Kastin, 2004b). These novel
drug delivery technologies can also be applicable to
neuropeptide-based compositions disclosed herein and known in the
art.
[0076] There are several peptide-engineering strategies to improve
permeability of neuropeptides through the blood-brain barrier.
These are summarized in FIG. 6. However, none of the studies
mentioned above showed a systematic approach of combining these
strategies to further boost the permeability of the peptides
through the blood-brain barrier. What is disclosed herein is the
application of these peptide-engineering strategies in combination
to known anticonvulsant neuropeptides, such as somatostatin or
galanin, thereby making these peptides anticonvulsant via the
intravenous (i.v.) or subcutaneous (s.c.) route of
administration.
[0077] Disclosed herein are methods and compositions for increasing
permeability through the blood-brain barrier. By "increasing" is
meant a higher percentage of the composition is able to cross the
blood-brain barrier compared with the wild type, non-altered, or
native peptide, or with a control composition. For example, the
rate of increase can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250,
275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650,
700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250,
2500, 2750, 3000, 3500, or 4000 percent when compared with the
control, native, or wild type peptide or composition.
[0078] Specifically disclosed herein is a composition with
increased permeability of the blood-brain barrier, wherein the
composition comprises a peptide with increased lipophilic character
and increased basicity when compared to the non-altered form of the
peptide (FIG. 7). Also disclosed are compositions with increased
permeability of the blood-brain barrier, wherein the composition
comprises a peptide with increased lipophilic character, increased
basicity, and increased glycosylation when compared to the
non-altered form of the peptide.
[0079] Also disclosed herein are methods of increasing permeability
of the blood-brain barrier for a peptide, comprising increasing
lipophilic character and increasing basicity of the peptide
compared to the non-altered form of the peptide. Another method of
increasing permeability of the blood-brain barrier for a peptide,
comprises increasing lipophilic character, increasing basicity, and
increasing glycosylation of the peptide compared to a non-altered
form of the peptide.
[0080] a) Lipophilic Character
[0081] The lipophilic character of the composition can be increased
by conjugating the peptide to a hydrophobic moiety, for example.
Examples of hydrophobic moieties include, but are not limited to,
polyaliphatic chains or aromatic residues. The lipophilic character
can also be increased by increasing halogenation of aromatic
residues or polyaliphatic reagents, such as perfluorohexanoic
acid
[0082] b) Basicity
[0083] The basicity of the composition can be increased by
introducing homo- and heterooligomers of positively charged amino
acid residues, including, but not limited to Lysine, Arginine,
homo-Lysine, homo-Arginine, Ornitine in L- or D-isomer
configuration; 2,3-Diaminopropioic acid; 2,4-Diaminobutyric
acid.
[0084] The basicity can also be increased by conjugation to
polyamine-based moieties, such as spermine, spermidine,
polyamidoamine dendrimers or polyamine toxins and derivatives
thereof.
[0085] c) Glycosylation
[0086] The glycosylation can be introduced by conjugation to
xylose, glucose, galactose, maltose, maltotriose, mannose, lactose,
melibiose or similar saccharides.
[0087] d) Vectors
[0088] Also disclosed are vectors comprising the compositions
disclosed herein. An example of vectors able to cross the BBB can
be found in Toyobuku et al. (J Pharmacol Exp Ther. 2003 April;
305(1):40-7.),
C. METHODS OF TREATMENT
[0089] Disclosed herein are methods of treating specific diseases
and disorders involving the central nervous system, or any
application that involves the need for a compound to cross the
blood-brain barrier. A variety of diseases and disorders can be
treated with the methods and compositions disclosed herein,
including stroke and related ischemic diseases, chronic back pain,
spinal cord injuries, peripheral nerve injuries, traumatic brain
injuries, retinal degeneration, neurodegenerative disorders,
cataracts, antibiotic-induced ototoxicity, Alzheimer's disease,
Amyotrophic Lateral Sclerosis (ALS, Lou Gehrig's disease), epilepsy
(such as generalized, partial, or refractory epilepsy),
Huntington's disease, Parkinson's disease, Multiple Sclerosis,
chronic back pain, fibromyalgia, postherpetic neuralgia, diabetic
neuropathy, traumatic mononeuropathy, complex regional pain
syndrome, adjuvant analgesic, rhizotomy/nerve ablation, preamptive
analgesia/amputations, epileptogenesis/trauma, chemical exposure,
status epilepticus, chemotherapy-induced neuropathy, cancer, opioid
withdrawal, and chronic neuropathic pain. Methods and routes of
administration, dosages, and pharmaceutical compositions are
discussed in more detail below.
[0090] Regarding the use of the compositions disclosed herein to
treat spinal cord injury and multiple sclerosis, the following
references are hereby incorporated in their entirety for their
teaching concerning the treatment of these diseases: Hawes J J,
Narasimhaiah R, Picciotto M R Galanin and galanin-like peptide
modulate neurite outgrowth via protein kinase C-mediated activation
of extracellular signal-related kinase. Eur J Neurosci. 2006 June;
23(11):2937-46. Suarez V, et al, The axotomy-induced neuropeptides
galanin and pituitary adenylate cyclase-activating peptide promote
axonal sprouting of primary afferent and cranial motor neurons Eur
J Neurosci. 2006 September; 24(6):1555-64
[0091] The compositions and methods disclosed herein can also be
useful in preventing anoxic damage, increasing growth hormone
secretion in humans, controlling prolactin release from pituitary
adenomas, prolonging morphine analgesia, as an antidepressant, and
in feeding disorders, for example.
[0092] Also disclosed are methods of treating pain and other
neurological disorders comprising administering to a subject in
need thereof an effective amount of the polypeptides disclosed
herein.
[0093] The methods and compositions disclosed herein can also be
used in the prevention, amelioration, or treatment of neurological
disorders, such as those disclosed above and known to those of
skill in the art.
[0094] The methods and compositions disclosed herein can be used in
conjunction with other compositions or treatment methods. For
example, the following drugs and classes of drugs can be used in
combination with the compositions disclosed herein for pain,
epilepsy, neuroprotection, and depression, bipolar, other
psychiatric disorders, as well as for any other disease or disorder
treatable by the compositions disclosed herein: opioids and opioid
peptides, morphine, hydroxymorphine, fentanyl, oxycodone, codeine;
capsaicin; as well as antiepileptic drugs as a class including but
not limited to carbamazepine, primidone, gabapentin, pregabalin,
diazepam, felbamate, fluorofelbamate, lamotrigine, lacosamide,
levetiracetam, phenobarbital, phenytoin, fosphenytoin, topiramate,
valproate, vigabatrin, zonisamide, oxcarbazepine, nonsteroidal
anti-inflamatory drugs (NSAIDs), local anesthetics (such as
lidocaine), glutamate receptor antagonists, NMDA antagonists,
alpha-adrenoceptor agonists and antagonists, adenosine,
cannabinoids, NK-1 antagonist (CI-1021), antidepressants
(amitriptyline, desipramine, imipramine, for example), analogs and
derivatives of galanin, somatostatin, delta-sleep inducing peptide,
enkephalins, oxytocin. cholecystikinin, calcitonin, cortistatin,
nociceptin and other neuropeptide-based therapeutics, and pluronic
P85 block copolymer.
[0095] The following drugs and classes of drugs can be used in
combination with the compositions disclosed herein for Alzheimer
disease: amyloid lowering agents, such as Flurizan; galantamine
(Razadyne); rivastigmine (Exelon); donepezil (Aricept); tacrine
(Cognex); memantine (Namenda); and vaccine for Alzheimer's disease.
Also disclosed are methods of treating a subject in need of a
composition that crosses the blood-brain barrier, comprising
identifying the composition to be used in treatment of the subject;
modifying the composition by increasing lipophilicity and basicity
of the composition; and administering the modified composition to
the subject in need thereof.
[0096] By "combination" is meant one or more additional
compositions, in addition to the compositions disclosed herein, can
be administered to the subject. These compositions can ben
[0097] Also disclosed are methods of treating a subject in need of
a composition that crosses the blood-brain barrier, comprising:
identifying the composition to be used in treatment of the subject;
modifying the composition by increasing lipophilicity,
glycosylation, and basicity of the composition; and administering
the modified composition to the subject in need thereof.
[0098] Also disclosed are methods of treating a subject in need of
a composition that crosses the blood-brain barrier, comprising
identifying the composition to be used in treatment of the subject;
modifying the composition by increasing lipophilicity,
glycosylation, and basicity of the composition; inserting the
modified composition into a vector; administering the vector to the
subject in need thereof.
[0099] Also disclosed is a method of treating a subject in need of
a composition that crosses the blood-brain barrier, comprising:
identifying the composition to be used in treatment of the subject;
modifying the composition by increasing lipophilicity and basicity
of the composition; inserting the modified composition into a
vector; and administering the vector to the subject in need
thereof.
[0100] 1. Methods of Using the Compositions as Research Tools
[0101] The disclosed compositions can be used in a variety of ways
as research tools. For example, the disclosed compositions, such as
SEQ ID NOs: 1-55, can be used as reagents to study epilepsy, for
example.
[0102] The compositions can be used, for example, as targets in
combinatorial chemistry protocols or other screening protocols to
isolate molecules that possess desired functional properties, such
as galanin agonists or antagonists or partial agonists.
[0103] The compositions can be used to discover individual and
network interactions between different neuropeptides, other
neurotransmitters, receptors and ion channels in the nervous
system.
[0104] For example, the disclosed compositions can be used to
discover synergistic interactions between galanin receptor
antagonists and drugs that act on molecular targets expressed on
the same neurons. Such positive drug-drug interactions are
beneficial, since they can improve efficacy or/and safety of a
treatment when two drugs are applied in combination.
[0105] The disclosed compositions can be used as discussed herein
as either reagents in micro arrays or as reagents to probe or
analyze existing microarrays. The disclosed compositions can be
used in any known method for isolating or identifying single
nucleotide polymorphisms. The compositions can also be used in any
known method of screening assays, related to chip/micro arrays. The
compositions can also be used in any known way of using the
computer readable embodiments of the disclosed compositions, for
example, to study relatedness or to perform molecular modeling
analysis related to the disclosed compositions.
[0106] 2. Methods of Gene Modification and Gene Disruption
[0107] The disclosed compositions and methods can be used for
targeted gene disruption and modification in any animal that can
undergo these events. For example, a gene producing galanin can be
altered to express a galanin analog with increased permeability of
the blood-brain barrier. Gene modification and gene disruption
refer to the methods, techniques, and compositions that surround
the selective removal or alteration of a gene or stretch of
chromosome in an animal, such as a mammal, in a way that propagates
the modification through the germ line of the mammal. In general, a
cell is transformed with a vector which is designed to homologously
recombine with a region of a particular chromosome contained within
the cell, as for example, described herein. This homologous
recombination event can produce a chromosome which has exogenous
DNA introduced, for example in frame, with the surrounding DNA.
This type of protocol allows for very specific mutations, such as
point mutations, to be introduced into the genome contained within
the cell. Methods for performing this type of homologous
recombination are disclosed herein.
[0108] One of the preferred characteristics of performing
homologous recombination in mammalian cells is that the cells
should be able to be cultured, because the desired recombination
event occurs at a low frequency.
[0109] Once the cell is produced through the methods described
herein, an animal can be produced from this cell through either
stem cell technology or cloning technology. For example, if the
cell into which the nucleic acid was transfected was a stem cell
for the organism, then this cell, after transfection and culturing,
can be used to produce an organism which will contain the gene
modification or disruption in germ line cells, which can then in
turn be used to produce another animal that possesses the gene
modification or disruption in all of its cells. In other methods
for production of an animal containing the gene modification or
disruption in all of its cells, cloning technologies can be used.
These technologies generally take the nucleus of the transfected
cell and either through fusion or replacement fuse the transfected
nucleus with an oocyte which can then be manipulated to produce an
animal. The advantage of procedures that use cloning instead of ES
technology is that cells other than ES cells can be transfected.
For example, a fibroblast cell, which is very easy to culture can
be used as the cell which is transfected and has a gene
modification or disruption event take place, and then cells derived
from this cell can be used to clone a whole animal.
D. COMPOSITIONS
[0110] Disclosed are the components to be used to prepare the
disclosed compositions as well as the compositions themselves and
to be used within the methods disclosed herein. These and other
materials are disclosed herein, and it is understood that when
combinations, subsets, interactions, groups, etc. of these
materials are disclosed that while specific reference to each
various individual and collective combinations and permutation of
these compounds may not be explicitly disclosed, each is
specifically contemplated and described herein. For example, if a
particular galanin analog is disclosed and discussed and a number
of modifications that can be made to a number of molecules
including the variant are discussed, specifically contemplated is
each and every combination and permutation of the galanin analog
and the modifications that are possible unless specifically
indicated to the contrary. Thus, if a class of molecules A, B, and
C are disclosed as well as a class of molecules D, E, and F and an
example of a combination molecule, A-D is disclosed, then even if
each is not individually recited each is individually and
collectively contemplated meaning combinations, A-E, A-F, B-D, B-E,
B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any
subset or combination of these is also disclosed. Thus, for
example, the sub-group of A-E, B-F, and C-E would be considered
disclosed. This concept applies to all aspects of this application
including, but not limited to, steps in methods of making and using
the disclosed compositions. Thus, if there are a variety of
additional steps that can be performed it is understood that each
of these additional steps can be performed with any specific
embodiment or combination of embodiments of the disclosed
methods.
[0111] 1. Compositions Related to Permeability of the Blood-Brain
Barrier
[0112] Spontaneous epileptic seizures result from excessive
discharge in hyperexcitable neurons primarily located in the
hippocampus. The brain controls seizures by balancing inhibitory
mechanisms employing .gamma.-aminobutyric acid (GABA) and
excitation mechanisms mediated by glutamate (Wasterlain et al.,
2002). Neurotransmission is modulated by a number of endogenous
neuropeptides, including neuropeptide Y, galanin,
nociceptin/orphanin FQ and endomorphin-1. FIG. 1 illustrates
possible relationships between glutamate, GABA and neuropeptides in
neuronal circuitry of hippocampus in self-sustained status
epilepticus.
[0113] The role of these neuropeptides has been elucidated using
both pharmacological and genetic (knockout or overexpression)
approaches. For example, Oberto and coworkers (Oberto et al., 2001)
used transgenic mice to characterize the interactions between the
GABAergic system, neuropeptide Y and neuropeptide Y(1) receptors in
the amygdala (also reviewed in (Eva et al., 2004)). Similarly,
Leresche and coworkers (Leresche et al., 2000) showed that
somatostatin inhibited GABA-mediated neurotransmission via a
presynaptic mechanism. Modulation of glutamate release by galanin
in the hippocampus was investigated in two transgenic mouse models:
knockout of galanin (GalKO) and overexpressing (GalOE) mice
(Mazarati et al., 2000). In GalKO and GalOE mice,
depolarization-induced glutamate release was increased and
decreased by centrally administered galanin, respectively,
indicating a role of hippocampal galanin as an anticonvulsant
through the glutamatergic system (Mazarati, 2004).
[0114] At least three neuropeptides and their receptors were shown
to play a role in epileptogenesis: galanin, somatostatin and
neuropeptide Y. Galanin immunoreactivity in the hippocampus is
diminished after limbic status epilepticus. Injection of galanin
into the hippocampal dentate hilus prevented onset of limbic status
epilepticus and stopped status epilepticus. It thus appears that
galanin acts as an endogenous anticonvulsant that inhibits status
epilepticus (Mazarati et al., 1998). Evidence of this was shown by
examining the phenotype of transgenic mice with overexpression of
galanin (Kokaia et al., 2001). In this study, galanin suppressed
kindling epileptogenesis.
[0115] The role of neuropeptides in modulating neurotransmitter
release and seizure control has been recognized as an opportunity
for new therapeutic treatments. As described below, a number of
published studies showed potent anticonvulsant activity of
neuropeptides in animal models.
[0116] a) Neuropeptide Y and Dynorphin
[0117] Neuropeptide Y suppressed epileptiform activity in rat
hippocampal slices in vitro (Klapstein and Colmers, 1997). In
another study (Baraban et al., 1997), mice lacking neuropeptide Y
had uncontrollable seizures in response to kainic acid. Moreover,
93% of knockout mice progressed to death, whereas death was rare in
wild-type animals. Intracerebroventricular neuropeptide Y prevented
death induced by kainic acid administration. Finally, the
anticonvulsant action of neuropeptide Y was demonstrated to be
mediated through the Y5 receptors (Sperk and Herzog, 1997).
[0118] Hippocampal opioid peptides, including dynorphin, have been
implicated in epileptogenesis and epileptic seizures (reviewed by
(Hong, 1992) and (Solbrig and Koob, 2004)). Seizures induced by
either electroconvulsive shocks or amygdala kindling resulted in
the initial release of both enkephalin and dynorphin, but also
caused a long-term decrease in dynorphin (Gall, 1988).
Anticonvulsant activity of dynorphin was shown in the rat model of
self-sustained status epilepticus (Mazarati and Wasterlain, 2002),
as illustrated in FIG. 5.
[0119] As with dynorphin, i.h. injection of neuropeptide Y also
reduced distribution of seizures in the self-sustaining status
epilepticus model (Mazarati and Wasterlain, 2002). NPY administered
into the lateral ventricle appeared to be a potent inhibitor of
kainate-induced seizures (Woldbye et al., 1997). It had been
suggested that the antiepileptic effect was mediated by
neuropeptide Y5 receptors, a finding that was confirmed in a study
with Y5R-deficient mice (Marsh et al., 1999).
[0120] b) Galanin and Analogs Thereof
[0121] Galanin has been recognized as a potential anticonvulsant
agent since the work of Mazarati and coworkers (Mazarati et al.,
1992). When injected directly into the lateral brain ventricle or
hippocampus, galanin decreased the severity of picrotoxin-induced
kindled convulsions in rats. In the animal model of status
epilepticus, perihilar injection of galanin, before or after
perforant path stimulation (PPS), shortened the duration of
seizures (Mazarati et al., 1998). These effects were reversed by
co-application of galanin antagonists. As illustrated in FIG. 2,
doses as low as 50 to 500 picomoles were effective in stopping
established self-sustaining status epilepticus (SSSE).
[0122] One strategy for treating epilepsy is to use
neuropeptide-based therapeutics. As a proof-of-concept, two
non-peptide galanin receptor agonists, galnon and galmic, were
recently shown to possess anticonvulsant and antiepileptic
activities ((Saar et al., 2002) and (Bartfai et al., 2004),
respectively). Both compounds appeared to possess a midrange
micromolar affinity for GalR1 or GalR2 receptors, and exhibited
anticonvulsant activity in animal models of epilepsy when
administered systemically. As shown in FIG. 3, galnon (2 mg/kg,
i.p.) or galanin (0.5 nmoles, i.c.v.) had comparable effects on
both latency and seizure score in the pentylenetetrazo
(PTZ)-induced test in mice (Saar et al., 2002; Ceide et al.,
2004).
[0123] Below is a table showing the affinity of NAX 5055 (GAL-BBB2)
toward galanin receptors:
TABLE-US-00002 TABLE 2 Ligand GalR1 GalR2 Gal(1-16) 8.5 nM 8.3 nM
NAX 5055 ~9 nM ~6 nM Galmic 34,000 nM >100,000 nM Galnon 12,000
nM 24,000 nM
[0124] When injected i.p., galanin was found to reduce the severity
and increased latency for pentylenetertazole-induced seizures in
mice. Intrahippocampal injection of galnon was also demonstrated to
shorten the duration of self-sustained status epilepticus.
Similarly, galmic blocked status epilepticus when injected i.h. or
i.p. Thus, these two galanin agonists are useful anticonvulsants,
and validate galanin receptors as therapeutic targets for
epilepsy.
[0125] Galanin is a 30-amino-acid neuropeptide, but SAR studies
identified that the N-terminal portion is still a highly potent
agonist as compared to the whole-length peptide (Langel and
Bartfai, 1998). A galanin(1-16) analog can be used with the methods
disclosed herein, in which the Gly.sup.1 residue is replaced by
N-methyl-Gly (sarcosine, SAR), as shown below:
TABLE-US-00003 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Sar Trp Thr
Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Ala Val
[0126] N-methylation of Gly.sup.1 protected the peptide from
accelerated proteolytic degradation from the N-terminus, whereas it
did not significantly change its affinity for the galanin receptor
(Rivera Baeza et al., 1994). SAR studies identified the following
residues critical for biological activity: Gly.sup.1, Trp.sup.2,
Asn.sup.5, Tyr.sup.9 and Gly.sup.12 (Land et al., 1991). The same
study identified that the N-terminal extensions caused a loss of
the biological activity. On the other hand, the C-terminal portion
of galanin(1-16) appears to be very robust when it comes to
attaching to larger structures (Pooga et al., 1998). Therefore, the
strategy for design of [Sar.sup.1]galanin analogs is similar to
that used with somatostatin only with regard to amino acid
replacements, but it differs by introducing the extensions at the
C-, rather than at the N-terminus.
[0127] The galanin analog, GAL-BBB2 (SEQ ID NO: 3), exhibited
potent anticonvulsant activity (ED50.about.3 mg/kg) when given i.p.
(Example 2). The smallest galanin analog with the most potent and
long-lasting anticonvulsant activity can be obtained from GAL-BBB2.
Examples of these sequences can be found in SEQ ID NOS: 4-29
(described in detail in Example 2). These peptides can have
increased stability when compared to galanin, for example. Also
disclosed are galanin analogs GAL-BBB3, GAL-BBB4, GAL-BBB5,
GAL-BBB6, GAL-BBB7, and GAL-BBB8 (SEQ ID NOS: 49-54). Each of these
can also poses anticonvulsant activity.
[0128] Limited structure-function relationship studies are carried
out to identify the smallest fragment of the GAL-BBB2 analog that
maintains anticonvulsant activity when administered systemically.
Galanin analogs containing either the C-terminal and central
truncations are synthesized and tested. In addition, limited
structure-function relationship study of the C-terminal motif are
carried out to optimize permeability of the analog through the
blood-brain-barrier. FIG. 14 illustrates the structure of GAL-BBB2
in the context of structure-function studies. Discussed below are
various galanin analogs and methods for their design and synthesis
(see examples 1 and 2).
[0129] c) Somatostatin
[0130] There are many lines of evidence showing that brain
somatostatin plays an important role as an inhibitor of seizures
and epileptogenesis (Vezzani and Hoyer, 1999). Somatostatin is a
major neuropeptide expressed in GABAergic interneurons of the
hippocampus. Moreover, somatostatin release from rat hippocampal
neurons was stimulated by glutamate (Fontana et al., 1996). The
expression of somatostatin and its receptors is significantly
changed after epileptic seizures, and this neuropeptide has also
been postulated to control neuronal excitability during
epileptogenesis (reviewed in (Schwarzer et al., 1996) and (Vezzani
and Hoyer, 1999)). Receptor-subtype-knockout and pharmacological
studies have suggested the involvement of at least four subtypes of
somatostatin receptors (sst1, sst2, sst3 and sst4) in
glutamate-mediated neurotransmission in hippocampus (Pikwo et al.,
1996). The recent study of Csaba and coworkers (Csaba et al., 2004)
provided additional evidence that somatostatin sst2A receptors can
play a key role in epileptogenesis and anticonvulsant activity.
[0131] The most direct evidence of the anticonvulant activity of
somatostatin comes from studying its pharmacological effects on
seizures and epileptogenesis in animal epilepsy models (Vezzani et
al., 1991; Perez et al., 1995; Mazarati and Wasterlain, 2002).
(Mazarati and Wasterlain, 2002). Injection of somatostatin or its
subtype-selective analogs resulted in a reduced number of seizures,
and raised the latency to seizures induced by kainic or quinolonic
acid (Vezzani et al., 1991). Similarly, infusion of RC-160, the
somatostatin sst2-selective agonist decreased the number of animals
with pentylenetetrazol-induced tonic-clonic seizures (Perez et al.,
1995). As illustrated in FIG. 4, the intrahippocampal injection
(i.h.) of somatostatin dramatically decreased the distribution of
seizures in a rat model of self-sustained status epilepticus
(Mazarati and Wasterlain, 2002).
[0132] Somatostatin is a 14-amino-acid hypothalamic peptide with a
single disulfide bridge, originally discovered in 1973 (Brazeau et
al., 1973). The sequence of somatostatin is shown below:
TABLE-US-00004 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Ala Gly Cys Lys Asn
Phe Phe Trp Lys Thr Phe Thr Ser Cys
[0133] Extensive SAR studies have identified five key residues:
Phe.sup.6, Phe.sup.7, Trp.sup.8, Lys.sup.9 and Phe.sup.11, whereas
alanine substitutions of Gly.sup.2, Lys.sup.4, Asn.sup.5,
Thr.sup.10, Thr.sup.12 or Ser.sup.13 did not significantly affect
biological activity (Vale et al., 1975). In addition, the
D-Trp.sup.8-containing analog was shown to be more potent, due to
greater resistance to proteolysis and/or better stabilization of
the active conformation.
[0134] The [D-Trp.sup.8] or [L-Trp.sup.8] somatostatin can be used
as the metabolically stable analog with the methods disclosed
herein. To increase basicity, Thr, Ser or Asn residues can be
systematically replaced with isosterically similar, but positively
charged DAB (diaminobutyric acid) or DAP (diaminopropionic acid)
residues. To increase lipophilicity, a Lys-palmitoyl moiety can be
introduced in place of Lys.sup.4 or Asn.sup.5, and/or Phe residues
can be substituted with halogenated equivalent, chloro-Phe
residues. As summarized in Table 5, nine analogs are synthesized
and assayed for their affinity to somatostatin receptors. The
modifications that do not negatively affect high affinity binding
are combined together. These 2.sup.nd-generation analogs comprise
2-4 combined modifications. These sequences can be found in SEQ ID
NOS: 31-36.
[0135] Next, the N-terminal extensions are introduced to
[D-Trp.sup.8] or [L-Trp.sup.8] somatostatin. These extensions
(BBB/PK modulators, as shown in FIG. 8) serve a dual purpose: (1)
to improve permeability through the blood-brain barrier by both
passive and active mechanisms, and (2) to improve pharmacokinetic
properties of neuropeptide drugs by adding a bulky moiety that
reduces clearance and improves resistance to proteolytic
degradation. Since such "BBB/PK modulators" are a new concept,
several combinations of a few structural modules are used that
constitute extensions. Table 6 provides information about the
structure and function of the proposed modules.
[0136] Also disclosed for use with the compositions and methods
disclosed herein is octreotide. For example, SEQ ID NO: 40
discloses an octreotide molecule. FIG. 21 shows the chemical
structure and schematic for engineering octreocide. Octreotide is a
somatostatin analog that more selective toward sst2 subtype of
somatostatin receptors (there are 5 known subtypes). Somatostatin
has been shown to be involved in epilepsy and epileptogenesis. The
following reference is incorporated in its entirety for its
teaching concerning octreotide, somatostatin, and epilepsy: Vezzani
A and Hoyer D, Eur J Neurosci, 1999, vol 11, pp3767-3776.
Similarly, a role of somatostatin in nociception was shown in
Chapman V and Dicjkenson A H, Neuropeptides 1992, vol 23, 147-152,
herein incorporated by reference in its entirety for its teaching
concerning somatostatin, octreotide, and nociception. A role of
somatostatin in a development of Alzheimer disease has recently
described (Saito T et al, Nature Medicine, 2005, vol 11, p.
434-439, herein incorporated by reference in its entirety for its
teaching concerning octreotide, somatostatin, and Alzheimer
disease. Discussed below are methods for designing and synthesizing
somatostatin analogs (see Example 1).
[0137] d) Delta Sleep Inducing Peptide
[0138] Delta Sleep Inducing Peptide (DSIP) is an anticonvulsant
neuropeptide (Schoenenberger 1984; Kovalzon and Strekalova 2006).
DSIP shares some structural similarity with dermorphin, opioid
agonist. DSIP was effective in suppressing seizures in the
metaphit-induced epilepsy model. Moreover, it has been shown that
DSIP potentiated anticonvulsant activity of valproate in the same
epilepsy model (Hrncic, Stanojlovic et al. 2006). In addition, this
peptide was shown to modulate interactions between enkephalins with
opioid receptors, resulting in analgesic effects of DSIP (Nakamura,
Nakashima et al. 1986). Neuroprotective activity of DSIP was shown
in a model of toxic cerebral oedema. DSIP and some analogs were
reported to penetrate the BBB (Kastin, Nissen et al. 1981; Kastin,
Banks et al. 1982).
[0139] 2. Compositions with Increased Permeability of the
Blood-Brain Barrier
[0140] The compositions disclosed herein have shown increased
permeability of the blood-brain barrier. As described herein,
disclosed is set of neuropeptide analogs that are designed and
synthesized to test their ability to bind with high affinity to
their respective receptors. This set includes approximately ten
analogs per neuropeptide. High-affinity analogs are further tested
for their ability to penetrate the blood-brain barrier. Results
from 1st-generation analogs are followed by the synthesis and
evaluation of 2nd- and, subsequently, 3rd-generation analogs. The
most promising analogs are selected (high-affinity ligands with
enhanced permeability through the blood-brain barrier) to confirm
their agonist activity in functional assays. A subset of these
analogs (potent agonists with enhanced permeability through the
blood-brain barrier) are then pharmacologically tested in vivo.
[0141] To become a drug, a neuropeptide analog should possess
several important features, including: (1) high potency and
selectivity, (2) metabolic stability, (3) relatively long half-life
and reduced clearance from systemic circulation, and (4) increased
permeability through the blood-brain barrier. Most neuropeptides
exhibit high potency and selectivity. Metabolic stability is often
introduced by peptide backbone modifications and/or replacements of
susceptible residues with residues that are not recognized by
proteolytic enzymes. An increase in half-life and decrease in
elimination rate can be efficiently achieved by conjugating a
polymer-based moiety to a peptide (e.g., PEGylation). Greater
permeability through the blood-brain barrier can be introduced by
increase in lipophilicity or cationization, as well as by adding
prodrug, nutrient transport mimetic or glycosylation. The structure
of an ideal drug neuropeptide is schematically shown in FIG. 8.
[0142] As illustrated in FIG. 8, a new concept in neuropeptide
engineering is the "BBB/PK modulator." The BBB/PK modulator
comprises a polymer-based bulky moiety with lipophilic, cationic
and transport mimetic modules; this modulator serves a dual
purpose, enhancement of the permeability through the blood-brain
barrier, and improvement of the pharmacokinetic properties. The
cationic and lipophilic modules promote interactions with
negatively charged membrane surfaces, and improve the diffusion
through the membranes, respectively. The function of the active
transport mimetic structure is to increase the specificity of
neuropeptide uptake into the brain by enhancing interactions with
specific nutrient transporters located on the surface of the brain
endothelial cells. The structural framework comprising all of these
modules can also improve pharmacokinetic properties of the peptide,
mimicking/replacing the role of the commonly used PEG moiety. These
bulky moieties are tested as the N- or C-terminal extensions of the
model neuropeptides, and more versatile positions of attachment
within the neuropeptide structure are also disclosed herein.
[0143] The following strategy was used to design neuropeptide
analogs with enhanced blood-brain barrier penetrability: begin with
metabolically-stable analogs, if available. Identify additional AA
positions in the analogs amenable to side chain replacements.
Identify positions at the N- and C-termini amenable to introduction
of bulky moieties. Increase lipophilicity and basicity of analogs
by side-chain replacements. Introduce the extension to a peptide
analog that will further increase its lipophilicity and basicity,
while improving the pharmacokinetic properties (BBB/PK modulator).
Include a nutrient mimetic structure at the extension to improve
specificity of the blood-brain barrier penetration. Combine the
analogs with side-chain modifications with the extension moiety
(BBB/PK modulator).
[0144] A key to the successful design of such analogs is the
correct combination of the above-mentioned modifications. To
achieve this goal, a systematic approach in designing and
evaluating individual sets of modifications and their optimal
combinations can be taken. The general strategy is schematically
illustrated in FIG. 10. The modification of amino acids as
disclosed herein can be introduced during solid-phase peptide
synthesis using an automated peptide synthesizer. All non-natural
amino acids or conjugated structures are as commercially available
Fmoc-protected derivatives.
[0145] It is understood that when variants are referred to, the
variants designate specific properties dependent on the specific
substitutions denoted, however, other substitutions, deletions,
and/or insertions, for example, conservative substitutions,
insertions, and/or deletions at positions other than the
specifically denoted positions are also contemplated provided the
variants retain the disclosed activities.
[0146] Disclosed are analogs of galanin that have desireable
properties, such as increased permeability of the blood brain
barrier. Also disclosed are analogs of somatostatin that have
increased permeability of the blood-brain barrier. As defined
above, by "increasing" or "increased" is meant a higher percentage
of the composition is able to cross the blood-brain barrier
compared with the wild type, non-altered, or native peptide, or
with a control composition. For example, the rate of increase can
be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,
375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850,
900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000,
3500, or 4000 percent when compared with the control, native, or
wild type peptide or composition.
[0147] It is also understood that each individual analog discussed
in the tables in the Examples also has a base permeability which
can be determined from the disclosed activities of the composition.
It is understood that these percentages of increased activity can
be calculated from a base permeability of a wild type, native, or
control peptide obtained at any time which provides data in the
analytical range of the assay, unless otherwise indicated.
[0148] Disclosed are substitutions, deletions, modifications,
additions, and extensions to the known, or wild type, peptide, as
disclosed in Examples 1 and 2. For example, in Table 7, disclosed
are N-terminal extensions for somatostatin. The extensions
disclosed herein can be used with a native, wild type, or known
peptide, or can be used in combination with an analog of a known
peptide. For example, side chain modifications can be made to the
known peptide, and then combined with an extension as disclosed
herein.
[0149] Also disclosed are amino acid substitutions and additions,
wherein the substitution or addition is of a non-naturally
occurring substance. Examples include, but are not limited to,
sarcosine, diaminobutyric acid (DAB), diaminopropionic acid (DAP),
Lys-palmityoyl, Chloro-phe, aminohexanoic acid (AHX),
perfluorohexanoic acid (PerFHX), 8-amino-3,6,-dioxaoctanic acid,
and oligo-Lys, tert-leucine,
[0150] Further disclosed are replacements of amino acid residues
with a "backbone prothesesis", such as a non-peptidic spacer.
Examples include, but are not limited to, aminovaleric or
aminohexanoic acid. This can result in a minimization of the
overall molecular size without significant change of spacing
between key residues. The spacer can replace 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, or 50
residues, for example.
[0151] Also disclosed herein are variations of amino acids wherein
their conformation has been changed. For example, disclosed herein
are D-Lys, D-Trp, and L-Trp.
[0152] Disclosed are analogs of known compounds, such as galanin
and somatostatin, having at least 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, or 95% identity (for example) to the
parent sequence (such as galanin or somatostatin) and wherein the
analog comprises at least one, at least two, at least three, at
least 4, at least 5, or at least 6 of any of the substitutions,
deletions, additions, or extensions disclosed herein.
[0153] 3. Sequence Similarities
[0154] It is understood that as discussed herein the use of the
terms homology and identity mean the same thing as similarity.
Thus, for example, if the use of the word homology is used between
two non-natural sequences it is understood that this is not
necessarily indicating an evolutionary relationship between these
two sequences, but rather is looking at the similarity or
relatedness between their nucleic acid sequences. Many of the
methods for determining homology between two evolutionarily related
molecules are routinely applied to any two or more nucleic acids or
proteins for the purpose of measuring sequence similarity
regardless of whether they are evolutionarily related or not.
[0155] In general, it is understood that one way to define any
known variants and derivatives or those that might arise, of the
disclosed genes and proteins herein, is through defining the
variants and derivatives in terms of homology to specific known
sequences. This identity of particular sequences disclosed herein
is also discussed elsewhere herein. In general, variants of genes
and proteins herein disclosed typically have at least, about 40,
50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or
99 percent homology to the stated sequence or the native sequence.
Those of skill in the art readily understand how to determine the
homology of two proteins or nucleic acids, such as genes. For
example, the homology can be calculated after aligning the two
sequences so that the homology is at its highest level.
[0156] Another way of calculating homology can be performed by
published algorithms. Optimal alignment of sequences for comparison
may be conducted by the local homology algorithm of Smith and
Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment
algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by
the search for similarity method of Pearson and Lipman, Proc. Natl.
Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations
of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by inspection.
[0157] The same types of homology can be obtained for nucleic acids
by for example the algorithms disclosed in Zuker, M. Science
244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA
86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306,
1989 which are herein incorporated by reference for at least
material related to nucleic acid alignment. It is understood that
any of the methods typically can be used and that in certain
instances the results of these various methods may differ, but the
skilled artisan understands if identity is found with at least one
of these methods, the sequences would be said to have the stated
identity, and be disclosed herein.
[0158] For example, as used herein, a sequence recited as having a
particular percent homology to another sequence refers to sequences
that have the recited homology as calculated by any one or more of
the calculation methods described above. For example, a first
sequence has 80 percent homology, as defined herein, to a second
sequence if the first sequence is calculated to have 80 percent
homology to the second sequence using the Zuker calculation method
even if the first sequence does not have 80 percent homology to the
second sequence as calculated by any of the other calculation
methods. As another example, a first sequence has 80 percent
homology, as defined herein, to a second sequence if the first
sequence is calculated to have 80 percent homology to the second
sequence using both the Zuker calculation method and the Pearson
and Lipman calculation method even if the first sequence does not
have 80 percent homology to the second sequence as calculated by
the Smith and Waterman calculation method, the Needleman and Wunsch
calculation method, the Jaeger calculation methods, or any of the
other calculation methods. As yet another example, a first sequence
has 80 percent homology, as defined herein, to a second sequence if
the first sequence is calculated to have 80 percent homology to the
second sequence using each of calculation methods (although, in
practice, the different calculation methods will often result in
different calculated homology percentages).
[0159] 4. Nucleic Acids
[0160] There are a variety of molecules disclosed herein peptides,
such as various galanin and somatostatin analogs. It is understood
that these peptide based molecules can be encoded by a number of
nucleic acids, including for example the nucleic acids that encode,
for example, SEQ ID NOS 1-55, and it is understood that for
example, when a vector is expressed in a cell, that the expressed
mRNA will typically be made up of A, C, G, and U.
[0161] a) Sequences
[0162] There are a variety of sequences related to, for example,
galanin, which can be found at, for example, Genbank database which
can be accessed at www.pubmed.gov. These sequences and others are
herein incorporated by reference in their entireties as well as for
individual subsequences contained therein.
[0163] One particular sequence set forth in SEQ ID NO: 3 is used
herein, as an example, to exemplify the disclosed compositions and
methods. It is understood that the description related to this
sequence is applicable to any sequence related to a galanin analog
unless specifically indicated otherwise. Those of skill in the art
understand how to resolve sequence discrepancies and differences
and to adjust the compositions and methods relating to a particular
sequence to other related sequences (i.e. sequences of galanin
analogs). Primers and/or probes can be designed for any
galanin-related nucleic acid sequence, for example, given the
information disclosed herein and known in the art.
[0164] 5. Delivery of the Compositions to Cells (Vectors)
[0165] There are a number of compositions and methods which can be
used to deliver nucleic acids or peptides to cells, either in vitro
or in vivo. The vectors disclosed herein can be used in multiple
ways. In one example, the vectors disclosed herein can be used to
deliver nucleic acids encoding the peptides disclosed herein to
cells and subjects. Vectors can also be used with peptides to
facilitate the crossing of the blood-brain barrier, as discussed
above.
[0166] Methods and compositions relating to vectors can largely be
broken down into two classes: viral based delivery systems and
non-viral based delivery systems. For example, nucleic acids and
peptides can be delivered through a number of direct delivery
systems such as, electroporation, lipofection, calcium phosphate
precipitation, plasmids, viral vectors, viral nucleic acids, phage
nucleic acids, phages, cosmids, or via transfer of genetic material
in cells or carriers such as cationic liposomes. Appropriate means
for transfection, including viral vectors, chemical transfectants,
or physico-mechanical methods such as electroporation and direct
diffusion of DNA, are described by, for example, Wolff, J. A., et
al., Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352,
815-818, (1991). Such methods are well known in the art and readily
adaptable for use with the compositions and methods described
herein. In certain cases, the methods will be modified to
specifically function with large DNA molecules. Further, these
methods can be used to target certain diseases and cell populations
by using the targeting characteristics of the carrier. For the
purpose of further improvement of delivering the compositions
across blood-brain barrier, the TAT protein transduction domain can
be used (Dietz G P and Bahr M, Mol Cell Neurosci, 2004, vol 27, p.
85-131).
[0167] As used herein, plasmid or viral vectors are agents that
transport the disclosed nucleic acids or peptides, such as those
related to galanin and somatostatin analogs, into the cell without
degradation. In some embodiments the delivery systems are derived
from either a virus or a retrovirus. Viral vectors are, for
example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia
virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis and
other RNA viruses, including these viruses with the HIV backbone.
Also preferred are any viral families which share the properties of
these viruses which make them suitable for use as vectors.
Retroviruses include Murine Maloney Leukemia virus, MMLV, and
retroviruses that express the desirable properties of MMLV as a
vector. Retroviral vectors are able to carry a larger genetic
payload, i.e., a transgene or marker gene, than other viral
vectors, and for this reason are a commonly used vector. However,
they are not as useful in non-proliferating cells. Adenovirus
vectors are relatively stable and easy to work with, have high
titers, and can be delivered in aerosol formulation, and can
transfect non-dividing cells. Pox viral vectors are large and have
several sites for inserting genes, they are thermostable and can be
stored at room temperature. A preferred embodiment is a viral
vector which has been engineered so as to suppress the immune
response of the host organism, elicited by the viral antigens.
Preferred vectors of this type will carry coding regions for
Interleukin 8 or 10.
[0168] Viral vectors can have higher transaction abilities than
chemical or physical methods to introduce genes into cells.
Typically, viral vectors contain, nonstructural early genes,
structural late genes, an RNA polymerase III transcript, inverted
terminal repeats necessary for replication and encapsidation, and
promoters to control the transcription and replication of the viral
genome. When engineered as vectors, viruses typically have one or
more of the early genes removed and a gene or gene/promotor
cassette is inserted into the viral genome in place of the removed
viral DNA. Constructs of this type can carry up to about 8 kb of
foreign genetic material. The necessary functions of the removed
early genes are typically supplied by cell lines which have been
engineered to express the gene products of the early genes in
trans.
[0169] (1) Retroviral Vectors
[0170] A retrovirus is an animal virus belonging to the virus
family of Retroviridae, including any types, subfamilies, genus, or
tropisms. Retroviral vectors, in general, are described by Verma,
I. M., Retroviral vectors for gene transfer. In Microbiology-1985,
American Society for Microbiology, pp. 229-232, Washington, (1985),
which is incorporated by reference herein. Examples of methods for
using retroviral vectors for gene therapy are described in U.S.
Pat. Nos. 4,868,116 and 4,980,286; PCT applications WO 90/02806 and
WO 89/07136; and Mulligan, (Science 260:926-932 (1993)); the
teachings of which are incorporated herein by reference.
[0171] A retrovirus is essentially a package which has packed into
it nucleic acid cargo. The nucleic acid cargo carries with it a
packaging signal, which ensures that the replicated daughter
molecules will be efficiently packaged within the package coat. In
addition to the package signal, there are a number of molecules
which are needed in cis, for the replication, and packaging of the
replicated virus. Typically a retroviral genome, contains the gag,
pol, and env genes which are involved in the making of the protein
coat. It is the gag, pol, and env genes which are typically
replaced by the foreign DNA that it is to be transferred to the
target cell. Retrovirus vectors typically contain a packaging
signal for incorporation into the package coat, a sequence which
signals the start of the gag transcription unit, elements necessary
for reverse transcription, including a primer binding site to bind
the tRNA primer of reverse transcription, terminal repeat sequences
that guide the switch of RNA strands during DNA synthesis, a purine
rich sequence 5' to the 3' LTR that serve as the priming site for
the synthesis of the second strand of DNA synthesis, and specific
sequences near the ends of the LTRs that enable the insertion of
the DNA state of the retrovirus to insert into the host genome. The
removal of the gag, pol, and env genes allows for about 8 kb of
foreign sequence to be inserted into the viral genome, become
reverse transcribed, and upon replication be packaged into a new
retroviral particle. This amount of nucleic acid is sufficient for
the delivery of a one to many genes depending on the size of each
transcript. It is preferable to include either positive or negative
selectable markers along with other genes in the insert.
[0172] Since the replication machinery and packaging proteins in
most retroviral vectors have been removed (gag, pol, and env), the
vectors are typically generated by placing them into a packaging
cell line. A packaging cell line is a cell line which has been
transfected or transformed with a retrovirus that contains the
replication and packaging machinery, but lacks any packaging
signal. When the vector carrying the DNA of choice is transfected
into these cell lines, the vector containing the gene of interest
is replicated and packaged into new retroviral particles, by the
machinery provided in cis by the helper cell. The genomes for the
machinery are not packaged because they lack the necessary
signals.
[0173] (2) Adenoviral Vectors
[0174] The construction of replication-defective adenoviruses has
been described (Berkner et al., J. Virology 61:1213-1220 (1987);
Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et
al., J. Virology 57:267-274 (1986); Davidson et al., J. Virology
61:1226-1239 (1987); Zhang "Generation and identification of
recombinant adenovirus by liposome-mediated transfection and PCR
analysis" BioTechniques 15:868-872 (1993)). The benefit of the use
of these viruses as vectors is that they are limited in the extent
to which they can spread to other cell types, since they can
replicate within an initial infected cell, but are unable to form
new infectious viral particles. Recombinant adenoviruses have been
shown to achieve high efficiency gene transfer after direct, in
vivo delivery to airway epithelium, hepatocytes, vascular
endothelium, CNS parenchyma and a number of other tissue sites
(Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin.
Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092
(1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle,
Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem.
267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993);
Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation
Research 73:1201-1207 (1993); Bout, Human Gene Therapy 5:3-10
(1994); Zabner, Cell 75:207-216 (1993); Caillaud, Eur. J.
Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen. Virology
74:501-507 (1993)). Recombinant adenoviruses achieve gene
transduction by binding to specific cell surface receptors, after
which the virus is internalized by receptor-mediated endocytosis,
in the same manner as wild type or replication-defective adenovirus
(Chardonnet and Dales, Virology 40:462-477 (1970); Brown and
Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J.
Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650-655
(1984); Seth, et al., Mol. Cell. Biol. 4:1528-1533 (1984); Varga et
al., J. Virology 65:6061-6070 (1991); Wickham et al., Cell
73:309-319 (1993)).
[0175] A viral vector can be one based on an adenovirus which has
had the E1 gene removed and these virons are generated in a cell
line such as the human 293 cell line. In another preferred
embodiment both the E1 and E3 genes are removed from the adenovirus
genome.
[0176] (3) Adeno-Asscociated Viral Vectors
[0177] Another type of viral vector is based on an adeno-associated
virus (AAV). This defective parvovirus is a preferred vector
because it can infect many cell types and is nonpathogenic to
humans. AAV type vectors can transport about 4 to 5 kb and wild
type AAV is known to stably insert into chromosome 19. Vectors
which contain this site specific integration property are
preferred. An especially preferred embodiment of this type of
vector is the P4.1 C vector produced by Avigen, San Francisco,
Calif., which can contain the herpes simplex virus thymidine kinase
gene, HSV-tk, and/or a marker gene, such as the gene encoding the
green fluorescent protein, GFP.
[0178] In another type of AAV virus, the AAV contains a pair of
inverted terminal repeats (ITRs) which flank at least one cassette
containing a promoter which directs cell-specific expression
operably linked to a heterologous gene. Heterologous in this
context refers to any nucleotide sequence or gene which is not
native to the AAV or B19 parvovirus.
[0179] Typically the AAV and B19 coding regions have been deleted,
resulting in a safe, noncytotoxic vector. The AAV ITRs, or
modifications thereof, confer infectivity and site-specific
integration, but not cytotoxicity, and the promoter directs
cell-specific expression. U.S. Pat. No. 6,261,834 is herein
incorporated by reference for material related to the AAV
vector.
[0180] The vectors of the present invention thus provide DNA
molecules which are capable of integration into a mammalian
chromosome without substantial toxicity.
[0181] The inserted genes in viral and retroviral usually contain
promoters, and/or enhancers to help control the expression of the
desired gene product. A promoter is generally a sequence or
sequences of DNA that function when in a relatively fixed location
in regard to the transcription start site. A promoter contains core
elements required for basic interaction of RNA polymerase and
transcription factors, and may contain upstream elements and
response elements.
[0182] (4) Large Payload Viral Vectors
[0183] Molecular genetic experiments with large human herpesviruses
have provided a means whereby large heterologous DNA fragments can
be cloned, propagated and established in cells permissive for
infection with herpesviruses (Sun et al., Nature genetics 8: 33-41,
1994; Cotter and Robertson, Curr Opin Mol Ther 5: 633-644, 1999).
These large DNA viruses (herpes simplex virus (HSV) and
Epstein-Barr virus (EBV), have the potential to deliver fragments
of human heterologous DNA>150 kb to specific cells. EBV
recombinants can maintain large pieces of DNA in the infected
B-cells as episomal DNA. Individual clones carried human genomic
inserts up to 330 kb appeared genetically stable. The maintenance
of these episomes requires a specific EBV nuclear protein, EBNA1,
constitutively expressed during infection with EBV. Additionally,
these vectors can be used for transfection, where large amounts of
protein can be generated transiently in vitro. Herpesvirus amplicon
systems are also being used to package pieces of DNA>220 kb and
to infect cells that can stably maintain DNA as episomes.
[0184] Other useful systems include, for example, replicating and
host-restricted non-replicating vaccinia virus vectors.
[0185] b) Non-Nucleic Acid Based Systems
[0186] The disclosed compositions, such as nucleic acids encoding
galanin analogs, can be delivered to the target cells in a variety
of ways. For example, the compositions can be delivered through
electroporation, or through lipofection, or through calcium
phosphate precipitation. The delivery mechanism chosen will depend
in part on the type of cell targeted and whether the delivery is
occurring for example in vivo or in vitro.
[0187] Thus, the compositions can comprise, in addition to the
disclosed variants or vectors for example, lipids such as
liposomes, such as cationic liposomes (e.g., DOTMA, DOPE,
DC-cholesterol) or anionic liposomes. Liposomes can further
comprise proteins to facilitate targeting a particular cell, if
desired. Administration of a composition comprising a compound and
a cationic liposome can be administered to the blood afferent to a
target organ or inhaled into the respiratory tract to target cells
of the respiratory tract. Regarding liposomes, see, e.g., Brigham
et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Felgner et
al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No.
4,897,355. Furthermore, the compound can be administered as a
component of a microcapsule that can be targeted to specific cell
types, such as macrophages, or where the diffusion of the compound
or delivery of the compound from the microcapsule is designed for a
specific rate or dosage.
[0188] In the methods described above which include the
administration and uptake of exogenous DNA into the cells of a
subject (i.e., gene transduction or transfection), delivery of the
compositions to cells can be via a variety of mechanisms. As one
example, delivery can be via a liposome, using commercially
available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE
(GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc.
Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison,
Wis.), as well as other liposomes developed according to procedures
standard in the art. In addition, the nucleic acid or vector of
this invention can be delivered in vivo by electroporation, the
technology for which is available from Genetronics, Inc. (San
Diego, Calif.) as well as by means of a SONOPORATION machine (ImaRx
Pharmaceutical Corp., Tucson, Ariz.).
[0189] The materials may be in solution, suspension (for example,
incorporated into microparticles, liposomes, or cells). These may
be targeted to a particular cell type via antibodies, receptors, or
receptor ligands. The following references are examples of the use
of this technology to target specific proteins to tumor tissue
(Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe,
K. D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J.
Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem.,
4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother.,
35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews,
129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol,
42:2062-2065, (1991)). These techniques can be used for a variety
of other specific cell types. Vehicles such as "stealth" and other
antibody conjugated liposomes (including lipid mediated drug
targeting to colonic carcinoma), receptor mediated targeting of DNA
through cell specific ligands, lymphocyte directed tumor targeting,
and highly specific therapeutic retroviral targeting of murine
glioma cells in vivo. The following references are examples of the
use of this technology to target specific proteins to tumor tissue
(Hughes et al., Cancer Research, 49:6214-6220, (1989); and
Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187,
(1992)). In general, receptors are involved in pathways of
endocytosis, either constitutive or ligand induced. These receptors
cluster in clathrin-coated pits, enter the cell via clathrin-coated
vesicles, pass through an acidified endosome in which the receptors
are sorted, and then either recycle to the cell surface, become
stored intracellularly, or are degraded in lysosomes. The
internalization pathways serve a variety of functions, such as
nutrient uptake, removal of activated proteins, clearance of
macromolecules, opportunistic entry of viruses and toxins,
dissociation and degradation of ligand, and receptor-level
regulation. Many receptors follow more than one intracellular
pathway, depending on the cell type, receptor concentration, type
of ligand, ligand valency, and ligand concentration. Molecular and
cellular mechanisms of receptor-mediated endocytosis has been
reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409
(1991)).
[0190] Nucleic acids that are delivered to cells which are to be
integrated into the host cell genome, typically contain integration
sequences. These sequences are often viral related sequences,
particularly when viral based systems are used. These viral
intergration systems can also be incorporated into nucleic acids
which are to be delivered using a non-nucleic acid based system of
deliver, such as a liposome, so that the nucleic acid contained in
the delivery system can be come integrated into the host
genome.
[0191] Other general techniques for integration into the host
genome include, for example, systems designed to promote homologous
recombination with the host genome. These systems typically rely on
sequence flanking the nucleic acid to be expressed that has enough
homology with a target sequence within the host cell genome that
recombination between the vector nucleic acid and the target
nucleic acid takes place, causing the delivered nucleic acid to be
integrated into the host genome. These systems and the methods
necessary to promote homologous recombination are known to those of
skill in the art.
[0192] c) In Vivo/Ex Vivo
[0193] As described above, the compositions can be administered in
a pharmaceutically acceptable carrier and can be delivered to the
subjects cells in vivo and/or ex vivo by a variety of mechanisms
well known in the art (e.g., uptake of naked DNA, liposome fusion,
intramuscular injection of DNA via a gene gun, endocytosis and the
like).
[0194] If ex vivo methods are employed, cells or tissues can be
removed and maintained outside the body according to standard
protocols well known in the art. The compositions can be introduced
into the cells via any gene transfer mechanism, such as, for
example, calcium phosphate mediated gene delivery, electroporation,
microinjection or proteoliposomes. The transduced cells can then be
infused (e.g., in a pharmaceutically acceptable carrier) or
homotopically transplanted back into the subject per standard
methods for the cell or tissue type. Standard methods are known for
transplantation or infusion of various cells into a subject.
[0195] 6. Expression Systems
[0196] The nucleic acids that are delivered to cells typically
contain expression controlling systems. For example, the inserted
genes in viral and retroviral systems usually contain promoters,
and/or enhancers to help control the expression of the desired gene
product. A promoter is generally a sequence or sequences of DNA
that function when in a relatively fixed location in regard to the
transcription start site. A promoter contains core elements
required for basic interaction of RNA polymerase and transcription
factors, and may contain upstream elements and response
elements.
[0197] a) Viral Promoters and Enhancers
[0198] Preferred promoters controlling transcription from vectors
in mammalian host cells may be obtained from various sources, for
example, the genomes of viruses such as: polyoma, Simian Virus 40
(SV40), adenovirus, retroviruses, hepatitis-B virus and most
preferably cytomegalovirus, or from heterologous mammalian
promoters, e.g. beta actin promoter. The early and late promoters
of the SV40 virus are conveniently obtained as an SV40 restriction
fragment which also contains the SV40 viral origin of replication
(Fiers et al., Nature, 273: 113 (1978)). The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment (Greenway, P. J. et al., Gene 18:
355-360 (1982)). Of course, promoters from the host cell or related
species also are useful herein.
[0199] Enhancer generally refers to a sequence of DNA that
functions at no fixed distance from the transcription start site
and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci.
78: 993 (1981)) or 3' (Lusky, M. L., et al., Mol. Cell Bio. 3: 1108
(1983)) to the transcription unit. Furthermore, enhancers can be
within an intron (Banerji, J. L. et al., Cell 33: 729 (1983)) as
well as within the coding sequence itself (Osborne, T. F., et al.,
Mol. Cell Bio. 4: 1293 (1984)). They are usually between 10 and 300
bp in length, and they function in cis. Enhancers function to
increase transcription from nearby promoters. Enhancers also often
contain response elements that mediate the regulation of
transcription. Promoters can also contain response elements that
mediate the regulation of transcription. Enhancers often determine
the regulation of expression of a gene. While many enhancer
sequences are now known from mammalian genes (globin, elastase,
albumin, -fetoprotein and insulin), typically one will use an
enhancer from a eukaryotic cell virus for general expression.
Preferred examples are the SV40 enhancer on the late side of the
replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers.
[0200] The promotor and/or enhancer may be specifically activated
either by light or specific chemical events which trigger their
function. Systems can be regulated by reagents such as tetracycline
and dexamethasone. There are also ways to enhance viral vector gene
expression by exposure to irradiation, such as gamma irradiation,
or alkylating chemotherapy drugs.
[0201] In certain embodiments the promoter and/or enhancer region
can act as a constitutive promoter and/or enhancer to maximize
expression of the region of the transcription unit to be
transcribed. In certain constructs the promoter and/or enhancer
region be active in all eukaryotic cell types, even if it is only
expressed in a particular type of cell at a particular time. A
preferred promoter of this type is the CMV promoter (650 bases).
Other preferred promoters are SV40 promoters, cytomegalovirus (full
length promoter), and retroviral vector LTF.
[0202] It has been shown that all specific regulatory elements can
be cloned and used to construct expression vectors that are
selectively expressed in specific cell types such as melanoma
cells. The glial fibrillary acetic protein (GFAP) promoter has been
used to selectively express genes in cells of glial origin.
[0203] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human or nucleated cells) may also
contain sequences necessary for the termination of transcription
which may affect mRNA expression. These regions are transcribed as
polyadenylated segments in the untranslated portion of the mRNA
encoding tissue factor protein. The 3' untranslated regions also
include transcription termination sites. It is preferred that the
transcription unit also contain a polyadenylation region. One
benefit of this region is that it increases the likelihood that the
transcribed unit will be processed and transported like mRNA. The
identification and use of polyadenylation signals in expression
constructs is well established. It is preferred that homologous
polyadenylation signals be used in the transgene constructs. In
certain transcription units, the polyadenylation region is derived
from the SV40 early polyadenylation signal and consists of about
400 bases. It is also preferred that the transcribed units contain
other standard sequences alone or in combination with the above
sequences improve expression from, or stability of, the
construct.
[0204] b) Markers
[0205] The viral vectors can include nucleic acid sequence encoding
a marker product. This marker product is used to determine if the
gene has been delivered to the cell and once delivered is being
expressed. Preferred marker genes are the E. Coli lacZ gene, which
encodes B-galactosidase, and green fluorescent protein.
[0206] In some embodiments the marker may be a selectable marker.
Examples of suitable selectable markers for mammalian cells are
dihydrofolate reductase (DHFR), thymidine kinase, neomycin,
neomycin analog G418, hydromycin, and puromycin. When such
selectable markers are successfully transferred into a mammalian
host cell, the transformed mammalian host cell can survive if
placed under selective pressure. There are two widely used distinct
categories of selective regimes. The first category is based on a
cell's metabolism and the use of a mutant cell line which lacks the
ability to grow independent of a supplemented media. Two examples
are: CHO DHFR-cells and mouse LTK-cells. These cells lack the
ability to grow without the addition of such nutrients as thymidine
or hypoxanthine. Because these cells lack certain genes necessary
for a complete nucleotide synthesis pathway, they cannot survive
unless the missing nucleotides are provided in a supplemented
media. An alternative to supplementing the media is to introduce an
intact DHFR or TK gene into cells lacking the respective genes,
thus altering their growth requirements. Individual cells which
were not transformed with the DHFR or TK gene will not be capable
of survival in non-supplemented media.
[0207] The second category is dominant selection which refers to a
selection scheme used in any cell type and does not require the use
of a mutant cell line. These schemes typically use a drug to arrest
growth of a host cell. Those cells which have a novel gene would
express a protein conveying drug resistance and would survive the
selection. Examples of such dominant selection use the drugs
neomycin, (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327
(1982)), mycophenolic acid, (Mulligan, R. C. and Berg, P. Science
209: 1422 (1980)) or hygromycin, (Sugden, B. et al., Mol. Cell.
Biol. 5: 410-413 (1985)). The three examples employ bacterial genes
under eukaryotic control to convey resistance to the appropriate
drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or
hygromycin, respectively. Others include the neomycin analog G418
and puramycin.
[0208] 7. Peptides
[0209] a) Protein Variants
[0210] As discussed herein there are numerous variants of a peptide
that are known and herein contemplated. In addition, to the
disclosed functional variants and analogs related to the positions
disclosed herein, there are known functional naturally occurring
variants at positions other than those disclosed herein, which also
function as desired. Protein variants and derivatives are well
understood to those of skill in the art and can involve amino acid
sequence modifications or functional fragments. For example, amino
acid sequence modifications typically fall into one or more of
three classes: substitutional, insertional or deletional variants.
Insertions include amino and/or carboxyl terminal fusions as well
as intrasequence insertions of single or multiple amino acid
residues. Insertions ordinarily will be smaller insertions than
those of amino or carboxyl terminal fusions, for example, on the
order of one to four residues. Immunogenic fusion protein
derivatives, such as those described in the examples, are made by
fusing a polypeptide sufficiently large to confer immunogenicity to
the target sequence by cross-linking in vitro or by recombinant
cell culture transformed with DNA encoding the fusion. Deletions
are characterized by the removal of one or more amino acid residues
from the protein sequence. Typically, no more than about from 2 to
6 residues are deleted at any one site within the protein molecule.
These variants ordinarily are prepared by site specific mutagenesis
of nucleotides in the DNA encoding the protein, thereby producing
DNA encoding the variant, and thereafter expressing the DNA in
recombinant cell culture. Techniques for making substitution
mutations at predetermined sites in DNA having a known sequence are
well known, for example M13 primer mutagenesis and PCR mutagenesis.
Amino acid substitutions are typically of single residues, but can
occur at a number of different locations at once; insertions
usually will be on the order of about from 1 to 10 amino acid
residues; and deletions will range about from 1 to 30 residues.
Deletions or insertions preferably are made in adjacent pairs, i.e.
a deletion of 2 residues or insertion of 2 residues. Substitutions,
deletions, insertions or any combination thereof may be combined to
arrive at a final construct. The mutations must not place the
sequence out of reading frame and preferably will not create
complementary regions that could produce secondary mRNA structure.
Substitutional variants are those in which at least one residue has
been removed and a different residue inserted in its place. Such
substitutions generally are made in accordance with the following
Tables 3 and 4 and are referred to as conservative
substitutions.
TABLE-US-00005 TABLE 3 Amino Acid Abbreviations Amino Acid
Abbreviations alanine Ala A allosoleucine AIle arginine Arg R
asparagine Asn N aspartic acid Asp D cysteine Cys C glutamic acid
Glu E glutamine Gln Q glycine Gly G histidine His H isolelucine Ile
I leucine Leu L lysine Lys K phenylalanine Phe F proline Pro P
pyroglutamic acid pGlu serine Ser S threonine Thr T tyrosine Tyr Y
tryptophan Trp W valine Val V
TABLE-US-00006 TABLE 4 Amino Acid Substitutions Exemplary
Conservative Substitutions, Original Residue others are known in
the art. Ala ser Arg lys, gln, his Asn gln; his Asp glu Cys ser Gln
asn, lys Glu asp Gly Ala His asn; gln Ile leu; val Leu ile; val Lys
arg; gln; his Met Leu; ile Phe met; leu; tyr Ser thr, asn Thr ser,
gln Trp tyr Tyr trp; phe Val ile; leu
[0211] Substantial changes in function or immunological identity
are made by selecting substitutions that are less conservative than
those in Table 3, i.e., selecting residues that differ more
significantly in their effect on maintaining (a) the structure of
the polypeptide backbone in the area of the substitution, for
example as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site or (c) the bulk
of the side chain. The substitutions which in general are expected
to produce the greatest changes in the protein properties will be
those in which (a) a hydrophilic residue, e.g. seryl or threonyl,
is substituted for (or by) a hydrophobic residue, e.g. leucyl,
isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline
is substituted for (or by) any other residue; (c) a residue having
an electropositive side chain, e.g., lysyl, arginyl, or histidyl,
is substituted for (or by) an electronegative residue, e.g.,
glutamyl or aspartyl; or (d) a residue having a bulky side chain,
e.g., phenylalanine, is substituted for (or by) one not having a
side chain, e.g., glycine, in this case, (e) by increasing the
number of sites for sulfation and/or glycosylation.
[0212] For example, the replacement of one amino acid residue with
another that is biologically and/or chemically similar is known to
those skilled in the art as a conservative substitution. For
example, a conservative substitution would be replacing one
hydrophobic residue for another, or one polar residue for another.
The substitutions include combinations such as, for example, Gly,
Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and
Phe, Tyr. Such conservatively substituted variations of each
explicitly disclosed sequence are included within the mosaic
polypeptides provided herein.
[0213] Substitutional or deletional mutagenesis can be employed to
insert or disable sites for N-glycosylation (Asn-X-Thr/Ser) or
O-glycosylation (Ser or Thr). Deletions or substitutions of
cysteine or methionine (for example in "neutrophil-resistant"
proteins due to generation of oxidants by neutrophils) or other
labile residues also may be desirable. Deletions or substitutions
of potential proteolysis sites, e.g. Arg, may be accomplished for
example by deleting one of the basic residues or substituting one
by glutaminyl or histidyl residues.
[0214] Certain post-translational derivatizations are the result of
the action of recombinant host cells on the expressed polypeptide.
Glutaminyl and asparaginyl residues are frequently
post-translationally deamidated to the corresponding glutamyl and
asparyl residues. Alternatively, these residues are deamidated
under mildly acidic conditions. Other post-translational
modifications include hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of amines in the epsilon-amino group of lysine,
arginine, and histidine side chains (T. E. Creighton, Proteins:
Structure and Molecular Properties, W.H. Freeman & Co., San
Francisco pp 79-86 [19831]), acetylation of the N-terminal amine
and, in some instances, amidation of the C-terminal carboxyl.
[0215] Disulfide bonds are covalent interactions between the thiol
group of two cysteine molecules. Through an oxidative reaction, the
hydrogen atoms are removed from the thiol groups allowing the
formation of a disulfide bridge; the resulting bonded cysteines are
termed cystine. Disulfide bonds fall into to categories class I and
class II. It is a class II bond which serves to stabilize the three
dimensional structure of a protein by linking cysteines within a
chain. A class I disulfide bond results when these interactions
occur between separate chains. The formation of class I disulfide
bonds can aid in the formation of dimeric proteins, an important
feature which is often necessary for receptors to provide proper
receptor-ligand interactions. Amino acid substitutions may be made
at sites where cysteine residues occur; typically, conservative
substitutions do not alter cysteine residues involved in disulfide
bonds. Such substitutions may have the effect of changing protein
folding or altering multimer interactions if the substituted
residue is involved in disulfide bonds. It can be determined which
cysteines are involved in disulfide bonds.
[0216] It is understood that the description of conservative
mutations and homology can be combined together in any combination,
such as embodiments that have at least 70% homology to a particular
sequence wherein the variants are conservative mutations.
[0217] As this specification discusses various proteins and protein
sequences it is understood that the nucleic acids that can encode
those protein sequences are also disclosed. This would include all
degenerate sequences related to a specific protein sequence, i.e.
all nucleic acids having a sequence that encodes one particular
protein sequence as well as all nucleic acids, including degenerate
nucleic acids, encoding the disclosed variants and derivatives of
the protein sequences. Thus, while each particular nucleic acid
sequence may not be written out herein, it is understood that each
and every sequence is in fact disclosed and described herein
through the disclosed protein sequence. It is also understood that
while no amino acid sequence indicates what particular DNA sequence
encodes that protein within an organism, where particular variants
of a disclosed protein are disclosed herein, the known nucleic acid
sequence that encodes that protein in the particular organism from
which that protein arises is also known and herein disclosed and
described.
[0218] Also disclosed are fragments of the disclosed proteins and
variants. Typically these fragments will retain at least one of the
functions described herein, such as increased permeability of the
blood-brain barrier. However, it is understood that fragments that
do not retain this activity, for example, can still be used to, for
example, generate antibodies. It is also understood that that there
are a variety of different functional activities held by galanin,
for example. These activities can be related but are not
necessarily required. Those of skill understand how to manipulate
functional domains of the disclosed analogs by, for example,
altering a region contributing to a particular function. Analogs
having specific functional sites removed or altered are disclosed
in Examples 1 and 2.
[0219] 8. Antibodies
[0220] Antibodies as disclosed herein can be useful in identifying
analogs with a desired function. As used herein, the term
"antibody" encompasses, but is not limited to, whole immunoglobulin
(i.e., an intact antibody) of any class. Native antibodies are
usually heterotetrameric glycoproteins, composed of two identical
light (L) chains and two identical heavy (H) chains. Typically,
each light chain is linked to a heavy chain by one covalent
disulfide bond, while the number of disulfide linkages varies
between the heavy chains of different immunoglobulin isotypes. Each
heavy and light chain also has regularly spaced intrachain
disulfide bridges. Each heavy chain has at one end a variable
domain (V(H)) followed by a number of constant domains. Each light
chain has a variable domain at one end (V(L)) and a constant domain
at its other end; the constant domain of the light chain is aligned
with the first constant domain of the heavy chain, and the light
chain variable domain is aligned with the variable domain of the
heavy chain. Particular amino acid residues are believed to form an
interface between the light and heavy chain variable domains. The
light chains of antibodies from any vertebrate species can be
assigned to one of two clearly distinct types, called kappa (k) and
lambda (l), based on the amino acid sequences of their constant
domains. Depending on the amino acid sequence of the constant
domain of their heavy chains, immunoglobulins can be assigned to
different classes. There are five major classes of human
immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these
may be further divided into subclasses (isotypes), e.g., IgG-1,
IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled in the art
would recognize the comparable classes for mouse. The heavy chain
constant domains that correspond to the different classes of
immunoglobulins are called alpha, delta, epsilon, gamma, and mu,
respectively.
[0221] The term "variable" is used herein to describe certain
portions of the variable domains that differ in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not usually evenly distributed through the variable
domains of antibodies. It is typically concentrated in three
segments called complementarity determining regions (CDRs) or
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of the
variable domains are called the framework (FR). The variable
domains of native heavy and light chains each comprise four FR
regions, largely adopting a b-sheet configuration, connected by
three CDRs, which form loops connecting, and in some cases forming
part of, the b-sheet structure. The CDRs in each chain are held
together in close proximity by the FR regions and, with the CDRs
from the other chain, contribute to the formation of the antigen
binding site of antibodies (see Kabat E. A. et al., "Sequences of
Proteins of Immunological Interest," National Institutes of Health,
Bethesda, Md. (1987)). The constant domains are not involved
directly in binding an antibody to an antigen, but exhibit various
effector functions, such as participation of the antibody in
antibody-dependent cellular toxicity.
[0222] As used herein, the term "antibody or fragments thereof"
encompasses chimeric antibodies and hybrid antibodies, with dual or
multiple antigen or epitope specificities, and fragments, such as
F(ab')2, Fab', Fab and the like, including hybrid fragments. Thus,
fragments of the antibodies that retain the ability to bind their
specific antigens are provided. For example, fragments of
antibodies which maintain increased permeability are included
within the meaning of the term "antibody or fragment thereof" Such
antibodies and fragments can be made by techniques known in the art
and can be screened for specificity and activity according to the
methods set forth in the general methods for producing antibodies
and screening antibodies for specificity and activity (See Harlow
and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor
Publications, New York, (1988)).
[0223] Also included within the meaning of "antibody or fragments
thereof" are conjugates of antibody fragments and antigen binding
proteins (single chain antibodies) as described, for example, in
U.S. Pat. No. 4,704,692, the contents of which are hereby
incorporated by reference.
[0224] 9. Pharmaceutical Carriers/Delivery of Pharamceutical
Products
[0225] As described above, the compositions, such as galanin
analogs, can also be administered in vivo in a pharmaceutically
acceptable carrier. By "pharmaceutically acceptable" is meant a
material that is not biologically or otherwise undesirable, i.e.,
the material may be administered to a subject, along with the
nucleic acid or vector, without causing any undesirable biological
effects or interacting in a deleterious manner with any of the
other components of the pharmaceutical composition in which it is
contained. The carrier would naturally be selected to minimize any
degradation of the active ingredient and to minimize any adverse
side effects in the subject, as would be well known to one of skill
in the art.
[0226] The compositions may be administered orally, parenterally
(e.g., intravenously), by intramuscular injection, by
intraperitoneal injection, transdermally, extracorporeally,
topically or the like, and topical intranasal administration or
administration by inhalant can be used. The exact amount of the
compositions required will vary from subject to subject, depending
on the species, age, weight and general condition of the subject
the particular nucleic acid or vector used, its mode of
administration and the like. Thus, it is not possible to specify an
exact amount for every composition. However, an appropriate amount
can be determined by one of ordinary skill in the art using only
routine experimentation given the teachings herein.
[0227] Parenteral administration of the composition, if used, is
generally characterized by injection. Injectables can be prepared
in conventional forms, either as liquid solutions or suspensions,
solid forms suitable for solution of suspension in liquid prior to
injection, or as emulsions. A more recently revised approach for
parenteral administration involves use of a slow release or
sustained release system such that a constant dosage is maintained.
See, e.g., U.S. Pat. No. 3,610,795, which is incorporated by
reference herein.
[0228] The materials may be in solution, suspension (for example,
incorporated into microparticles, liposomes, or cells). These may
be targeted to a particular cell type via antibodies, receptors, or
receptor ligands. The following references are examples of the use
of this technology to target specific proteins to tumor tissue
(Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe,
K. D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J.
Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem.,
4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother.,
35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews,
129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol,
42:2062-2065, (1991)). Vehicles such as "stealth" and other
antibody conjugated liposomes (including lipid mediated drug
targeting to colonic carcinoma), receptor mediated targeting of DNA
through cell specific ligands, lymphocyte directed tumor targeting,
and highly specific therapeutic retroviral targeting of murine
glioma cells in vivo. The following references are examples of the
use of this technology to target specific proteins to tumor tissue
(Hughes et al., Cancer Research, 49:6214-6220, (1989); and
Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187,
(1992)). In general, receptors are involved in pathways of
endocytosis, either constitutive or ligand induced. These receptors
cluster in clathrin-coated pits, enter the cell via clathrin-coated
vesicles, pass through an acidified endosome in which the receptors
are sorted, and then either recycle to the cell surface, become
stored intracellularly, or are degraded in lysosomes. The
internalization pathways serve a variety of functions, such as
nutrient uptake, removal of activated proteins, clearance of
macromolecules, opportunistic entry of viruses and toxins,
dissociation and degradation of ligand, and receptor-level
regulation. Many receptors follow more than one intracellular
pathway, depending on the cell type, receptor concentration, type
of ligand, ligand valency, and ligand concentration. Molecular and
cellular mechanisms of receptor-mediated endocytosis has been
reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409
(1991)).
[0229] a) Pharmaceutically Acceptable Carriers
[0230] The compositions, including galanin analogs, can be used
therapeutically in combination with a pharmaceutically acceptable
carrier.
[0231] Pharmaceutical carriers are known to those skilled in the
art. These most typically would be standard carriers for
administration of drugs to humans, including solutions such as
sterile water, saline, and buffered solutions at physiological pH.
The compositions can be administered intramuscularly or
subcutaneously. Other compounds will be administered according to
standard procedures used by those skilled in the art.
[0232] Pharmaceutical compositions may include carriers,
thickeners, diluents, buffers, preservatives, surface active agents
and the like in addition to the molecule of choice. Pharmaceutical
compositions may also include one or more active ingredients such
as antimicrobial agents, antiinflammatory agents, anesthetics, and
the like.
[0233] The pharmaceutical composition may be administered in a
number of ways depending on whether local or systemic treatment is
desired, and on the area to be treated. Administration may be
topically (including ophthalmically, vaginally, rectally,
intranasally), orally, by inhalation, or parenterally, for example
by intravenous drip, subcutaneous, intraperitoneal or intramuscular
injection. The disclosed compositions, such as galanin analogs, can
be administered intravenously, intraperitoneally, intramuscularly,
subcutaneously, intracavity, or transdermally.
[0234] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives may also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like.
[0235] Formulations for topical administration may include
ointments, lotions, creams, gels, drops, suppositories, sprays,
liquids and powders. Conventional pharmaceutical carriers, aqueous,
powder or oily bases, thickeners and the like may be necessary or
desirable.
[0236] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets, or tablets. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids or binders may be desirable.
[0237] Some of the compositions may potentially be administered as
a pharmaceutically acceptable acid- or base-addition salt, formed
by reaction with inorganic acids such as hydrochloric acid,
hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,
sulfuric acid, and phosphoric acid, and organic acids such as
formic acid, acetic acid, propionic acid, glycolic acid, lactic
acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, and fumaric acid, or by reaction with an inorganic
base such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide, and organic bases such as mono-, di-, trialkyl and aryl
amines and substituted ethanolamines.
[0238] b) Therapeutic Uses
[0239] The dosage ranges for the administration of the compositions
are those large enough to produce the desired effect in which the
symptom's of the disorder are effected. The dosage should not be so
large as to cause adverse side effects, such as unwanted
cross-reactions, anaphylactic reactions, and the like. Generally,
the dosage will vary with the age, condition, sex and extent of the
disease in the patient and can be determined by one of skill in the
art. The dosage can be adjusted by the individual physician in the
event of any counterindications. Dosage can vary, and can be
administered in one or more dose administrations daily, for one or
several days.
[0240] 10. Chips and Micro Arrays
[0241] Disclosed are chips where at least one address is the
sequences or part of the sequences set forth in any of the nucleic
acid sequences disclosed herein. Also disclosed are chips where at
least one address is the sequences or portion of sequences set
forth in any of the peptide sequences disclosed herein.
[0242] Also disclosed are chips where at least one address is a
variant of the sequences or part of the sequences set forth in any
of the nucleic acid sequences disclosed herein. Also disclosed are
chips where at least one address is a variant of the sequences or
portion of sequences set forth in any of the peptide sequences
disclosed herein.
[0243] Also disclosed are chips where at least one address is the
sequences or part of the sequences set forth in any of the nucleic
acid sequences disclosed herein wherein the sequence includes at
least one of the variant sequences disclosed herein. Also disclosed
are chips where at least one address is the sequences or portion of
sequences set forth in any of the peptide sequences disclosed
herein, wherein the peptide sequence comprises at least one of the
galanin analog disclosed herein.
[0244] Also disclosed are chips where at least one address is the
sequences or part of the sequences set forth in any of the nucleic
acid sequences disclosed herein wherein the sequence includes at
least one of the variant sequences within the region defined
herein. Also disclosed are chips where at least one address is the
sequences or portion of sequences set forth in any of the peptide
sequences disclosed herein, wherein the peptide sequence comprises
at least one of the substitutions, additions, mutations, or
deletions disclosed herein.
[0245] 11. Computer Readable Mediums
[0246] It is understood that the disclosed nucleic acids and
proteins can be represented as a sequence consisting of the
nucleotides of amino acids. There are a variety of ways to display
these sequences, for example the nucleotide guanosine can be
represented by G or g. Likewise the amino acid valine can be
represented by Val or V. Those of skill in the art understand how
to display and express any nucleic acid or protein sequence in any
of the variety of ways that exist, each of which is considered
herein disclosed. Specifically contemplated herein is the display
of these sequences on computer readable mediums, such as,
commercially available floppy disks, tapes, chips, hard drives,
compact disks, and video disks, or other computer readable mediums.
Also disclosed are the binary code representations of the disclosed
sequences. Those of skill in the art understand what computer
readable mediums are. Thus, computer readable mediums on which the
nucleic acids or protein sequences are recorded, stored, or saved
are disclosed.
[0247] Disclosed are computer readable mediums comprising the
sequences and information regarding the sequences set forth
herein.
[0248] 12. Kits
[0249] Disclosed herein are kits that are drawn to reagents that
can be used in practicing the methods disclosed herein. The kits
can include any reagent or combination of reagent discussed herein
or that would be understood to be required or beneficial in the
practice of the disclosed methods. For example, the kits could
include amino acids to perform the substitutions discussed in
certain embodiments of the methods, as well as instructions.
[0250] 13. Compositions with Similar Functions
[0251] It is understood that the compositions disclosed herein have
certain functions, such as increased permeability of the
blood-brain barrier. Disclosed herein are certain structural
requirements for performing the disclosed functions, and it is
understood that there are a variety of structures which can perform
the same function which are related to the disclosed structures,
and that these structures will ultimately achieve the same
result.
E. METHODS OF MAKING THE COMPOSITIONS
[0252] The compositions disclosed herein and the compositions
necessary to perform the disclosed methods can be made using any
method known to those of skill in the art for that particular
reagent or compound unless otherwise specifically noted. It is
understood that general molecular biology techniques, such as those
disclosed in Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989) are available for making the disclosed
molecules and practicing the disclosed methods unless otherwise
noted.
[0253] Specifically disclosed herein is a method of making a
composition with increased permeability of the blood brain barrier,
comprising making a composition with increased permeability of the
blood-brain barrier, wherein the composition comprises a peptide
with increased lipophilic character and increased basicity when
compared to the non-altered form of the peptide. In one example,
the lipophilic character can be increased by conjugating the
peptide to a hydrophobic moiety, such as polyaliphatic chains. The
lipophilic character can also be increased by increasing
halogenation of aromatic residues. The basicity can be increased by
introducing homo- and heterooligomers of positively charged amino
acid residues, including, but not limited to Lysine, Arginine,
homo-Lysine, homo-Arginine, Ornitine in L- or D-isomer
configuration; 2,3-Diaminopropioic acid; 2,4-Diaminobutyric acid.
In another example, the basicity can be increased by conjugation to
polyamine-based moieties, such as spermine, spermidine,
polyamidoamine dendrimers or polyamine toxins and derivatives
thereof. The peptide can cross the blood-brain barrier with 1%, 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more
efficiency compared to the non-altered peptide. The peptide can
also have increased glycosylation when compared to the non-altered
form of the peptide. The peptide can comprise a spacer. The spacer
can be selected from the group consisting of: Gly, Ahx, Gly-Ahx, or
PEG-020c.
[0254] 1. Nucleic Acid Synthesis
[0255] For example, the nucleic acids, such as, the
oligonucleotides to be used as primers can be made using standard
chemical synthesis methods or can be produced using enzymatic
methods or any other known method. Such methods can range from
standard enzymatic digestion followed by nucleotide fragment
isolation (see for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) to purely
synthetic methods, for example, by the cyanoethyl phosphoramidite
method using a Milligen or Beckman System 1Plus DNA synthesizer
(for example, Model 8700 automated synthesizer of
Milligen-Biosearch, Burlington, Mass. or ABI Model 380B). Synthetic
methods useful for making oligonucleotides are also described by
Ikuta et al., Ann. Rev. Biochem. 53:323-356 (1984),
(phosphotriester and phosphite-triester methods), and Narang et
al., Methods Enzymol., 65:610-620 (1980), (phosphotriester method).
(Peptide nucleic acid molecules) can be made using known methods
such as those described by Nielsen et al., Bioconjug. Chem. 5:3-7
(1994).
[0256] 2. Peptide Synthesis
[0257] One method of producing the disclosed proteins is to link
two or more peptides or polypeptides together by protein chemistry
techniques. For example, peptides or polypeptides can be chemically
synthesized using currently available laboratory equipment using
either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc
(tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc.,
Foster City, Calif.). One skilled in the art can readily appreciate
that a peptide or polypeptide corresponding to the disclosed
proteins, for example, can be synthesized by standard chemical
reactions. For example, a peptide or polypeptide can be synthesized
and not cleaved from its synthesis resin whereas the other fragment
of a peptide or protein can be synthesized and subsequently cleaved
from the resin, thereby exposing a terminal group which is
functionally blocked on the other fragment. By peptide condensation
reactions, these two fragments can be covalently joined via a
peptide bond at their carboxyl and amino termini, respectively, to
form a protein, or fragment thereof (Grant G A (1992) Synthetic
Peptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky
M and Trost B., Ed. (1993) Principles of Peptide Synthesis.
Springer-Verlag Inc., NY (which is herein incorporated by reference
at least for material related to peptide synthesis). Alternatively,
the peptide or polypeptide is independently synthesized in vivo as
described herein. Once isolated, these independent peptides or
polypeptides may be linked to form a peptide or fragment thereof
via similar peptide condensation reactions.
[0258] For example, enzymatic ligation of cloned or synthetic
peptide segments allow relatively short peptide fragments to be
joined to produce larger peptide fragments, polypeptides or whole
protein domains (Abrahmsen L et al., Biochemistry, 30:4151 (1991)).
Alternatively, native chemical ligation of synthetic peptides can
be utilized to synthetically construct large peptides or
polypeptides from shorter peptide fragments. This method consists
of a two step chemical reaction (Dawson et al. Synthesis of
Proteins by Native Chemical Ligation. Science, 266:776-779 (1994)).
The first step is the chemoselective reaction of an unprotected
synthetic peptide--thioester with another unprotected peptide
segment containing an amino-terminal Cys residue to give a
thioester-linked intermediate as the initial covalent product.
Without a change in the reaction conditions, this intermediate
undergoes spontaneous, rapid intramolecular reaction to form a
native peptide bond at the ligation site (Baggiolini M et al.
(1992) FEBS Lett. 307:97-101; Clark-Lewis I et al., J. Biol. Chem.,
269:16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128
(1991); Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).
[0259] Alternatively, unprotected peptide segments are chemically
linked where the bond formed between the peptide segments as a
result of the chemical ligation is an unnatural (non-peptide) bond
(Schnolzer, M et al. Science, 256:221 (1992)). This technique has
been used to synthesize analogs of protein domains as well as large
amounts of relatively pure proteins with full biological activity
(deLisle Milton R C et al., Techniques in Protein Chemistry IV.
Academic Press, New York, pp. 257-267 (1992)).
[0260] 3. Process for Making the Compositions
[0261] Disclosed are processes for making the compositions as well
as making the intermediates leading to the compositions. For
example, disclosed are proteins in SEQ ID NOs: 1-55. There are a
variety of methods that can be used for making these compositions,
such as synthetic chemical methods and standard molecular biology
methods. It is understood that the methods of making these and the
other disclosed compositions are specifically disclosed.
[0262] Disclosed are proteins produced by the process comprising
linking in an operative way a nucleic acid encoding a galanin
analog comprising the sequence set forth in SEQ ID NO: 3 and a
sequence controlling the expression of the nucleic acid.
[0263] Also disclosed are proteins produced by the process
comprising linking in an operative way a nucleic acid molecule
encoding a galanin analog comprising a sequence having 80% identity
to a sequence set forth in SEQ ID NO: 3, and a sequence controlling
the expression of the nucleic acid.
[0264] Disclosed are cells produced by the process of transforming
the cell with any of the disclosed nucleic acids. Disclosed are
cells produced by the process of transforming the cell with any of
the non-naturally occurring disclosed nucleic acids.
[0265] Disclosed are any of the disclosed peptides produced by the
process of expressing any of the disclosed nucleic acids. Disclosed
are any of the non-naturally occurring disclosed peptides produced
by the process of expressing any of the disclosed nucleic acids.
Disclosed are any of the disclosed peptides produced by the process
of expressing any of the non-naturally disclosed nucleic acids.
[0266] Disclosed are animals produced by the process of
transfecting a cell within the animal with any of the nucleic acid
molecules disclosed herein. Disclosed are animals produced by the
process of transfecting a cell within the animal any of the nucleic
acid molecules disclosed herein, wherein the animal is a mammal.
Also disclosed are animals produced by the process of transfecting
a cell within the animal any of the nucleic acid molecules
disclosed herein, wherein the mammal is mouse, rat, rabbit, cow,
sheep, pig, or primate.
[0267] Also disclosed are animals produced by the process of adding
to the animal any of the cells disclosed herein.
[0268] It is understood that another way of producing the proteins
would be to use rabbit expression systems, such as those types of
systems produced by Bioprotein Technologies. The disclosed
molecules can be produced using these types of vectors and
production systems. For example, these types of systems are
disclosed EPO Patent Application No 92 401 635.5, U.S. Pat. No.
5,965,788) and on a gene insulator (EPO Patent Application No 00
403 658.8), and information can be found at www.bioprotein.com.
[0269] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains. The references disclosed are also
individually and specifically incorporated by reference herein for
the material contained in them that is discussed in the sentence in
which the reference is relied upon.
[0270] It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
embodiments of the invention will be apparent to those skilled in
the art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following
claims.
F. EXAMPLES
[0271] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary of the invention and are not
intended to limit the scope of what the inventors regard as their
invention. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.), but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric.
1. Example 1: Systemically-Active Anticonvulsant Galanin Analog
[0272] To obtain proof-of-concept results that anticonvulsant
neuropeptides can be engineered to enhance their penetration across
the blood-brain barrier, two model neuropeptides were selected:
somatostatin and galanin. As described previously, both of these
neuropeptides possess anticonvulsant activity.
[0273] The general experimental strategy is illustrated in FIG. 7.
A set of neuropeptide analogs (the 1st generation) are designed and
synthesized to test their ability to bind with high affinity to
their respective receptors. This set includes approximately ten
analogs per neuropeptide. High-affinity analogs are further tested
for their ability to penetrate the blood-brain barrier. Results
from 1st-generation analogs are followed by the synthesis and
evaluation of 2nd- and, subsequently, 3rd-generation analogs. The
most promising analogs are selected (high-affinity ligands with
enhanced permeability through the blood-brain barrier) to confirm
their agonist activity in functional assays. A subset of these
analogs (potent agonists with enhanced permeability through the
blood-brain barrier) are then pharmacologically tested in vivo.
[0274] To become a drug, a neuropeptide analog should possess
several important features, including: (1) high potency and
selectivity, (2) metabolic stability, (3) relatively long half-life
and reduced clearance from systemic circulation, and (4) increased
permeability through the blood-brain barrier. Most neuropeptides
exhibit high potency and selectivity. Metabolic stability is often
introduced by peptide backbone modifications and/or replacements of
susceptible residues with residues that are not recognized by
proteolytic enzymes. An increase in half-life and decrease in
elimination rate can be efficiently achieved by conjugating a
polymer-based moiety to a peptide (e.g., PEGylation). Greater
permeability through the blood-brain barrier can be introduced by
increase in lipophilicity or cationization, as well as by adding
prodrug, nutrient transport mimetic or glycosylation. The structure
of an ideal drug neuropeptide is schematically shown in FIG. 8.
[0275] As illustrated in FIG. 8, a new concept in neuropeptide
engineering is introduced: the "BBB/PK modulator." The BBB/PK
modulator comprises a polymer-based bulky moiety with lipophilic,
cationic and transport mimetic modules; this modulator serves a
dual purpose, enhancement of the permeability through the
blood-brain barrier, and improvement of the pharmacokinetic
properties. The cationic and lipophilic modules promote
interactions with negatively charged membrane surfaces, and improve
the diffusion through the membranes, respectively. The function of
the active transport mimetic structure is to increase the
specificity of neuropeptide uptake into the brain by enhancing
interactions with specific nutrient transporters located on the
surface of the brain endothelial cells. The structural framework
comprising all of these modules can also improve pharmacokinetic
properties of the peptide, mimicking/replacing the role of the
commonly used PEG moiety. These bulky moieties are tested as the N-
or C-terminal extensions of the model neuropeptides, and more
versatile positions of attachment within the neuropeptide structure
are also disclosed herein.
[0276] The following strategy was used to design neuropeptide
analogs with enhanced blood-brain barrier penetrability: begin with
metabolically-stable analogs, if available. Identify additional AA
positions in the analogs amenable to side chain replacements.
Identify positions at the N- and C-termini amenable to introduction
of bulky moieties. Increase lipophilicity and basicity of analogs
by side-chain replacements. Introduce the extension to a peptide
analog that will further increase its lipophilicity and basicity,
while improving the pharmacokinetic properties (BBB/PK modulator).
Include a nutrient mimetic structure at the extension to improve
specificity of the blood-brain barrier penetration. Combine the
analogs with side-chain modifications with the extension moiety
(BBB/PK modulator).
[0277] A key to the successful design of such analogs is the
correct combination of the above-mentioned modifications. To
achieve this goal, a systematic approach in designing and
evaluating individual sets of modifications and their optimal
combinations can be taken. The general strategy is schematically
illustrated in FIG. 10. The modification of amino acids as
disclosed herein can be introduced during solid-phase peptide
synthesis using an automated peptide synthesizer. All non-natural
amino acids or conjugated structures are as commercially available
Fmoc-protected derivatives.
[0278] Somatostatin is a 14-amino-acid hypothalamic peptide with a
single disulfide bridge, originally discovered in 1973 (Brazeau et
al., 1973). The sequence of somatostatin is shown below:
TABLE-US-00007 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Ala Gly Cys Lys Asn
Phe Phe Trp Lys Thr Phe Thr Ser Cys
[0279] Extensive SAR studies have identified five key residues:
Phe.sup.6, Phe.sup.7, Trp.sup.8, Lys.sup.9 and Phe.sup.11, whereas
alanine substitutions of Gly.sup.2, Lys.sup.4, Asn.sup.5,
Thr.sup.10Thr.sup.12 or Ser.sup.13 did not significantly affect
biological activity (Vale et al., 1975). In addition, the
D-Trp.sup.8-containing analog was shown to be more potent, due to
greater resistance to proteolysis and/or better stabilization of
the active conformation.
[0280] The [D-Trp.sup.8] somatostatin can be used as the
metabolically stable analog with the methods disclosed herein. To
increase basicity, Thr, Ser or Asn residues can be systematically
replaced with isosterically similar, but positively charged DAB
(diaminobutyric acid) or DAP (diaminopropionic acid) residues. To
increase lipophilicity, a Lys-palmitoyl moiety can be introduced in
place of Lys.sup.4 or Asn.sup.5, and/or Phe residues can be
substituted with halogenated equivalent, chloro-Phe residues. As
summarized in Table 5, nine analogs are synthesized and assayed for
their affinity to somatostatin receptors. The modifications that do
not negatively affect high affinity binding are combined together.
These 2.sup.nd-generation analogs comprise 2-4 combined
modifications.
[0281] Next, the N-terminal extensions are introduced to
[D-Trp.sup.8]somatostatin. These extensions (BBB/PK modulators, as
shown in FIG. 8) serve a dual purpose: (1) to improve permeability
through the blood-brain barrier by both passive and active
mechanisms, and (2) to improve pharmacokinetic properties of
neuropeptide drugs by adding a bulky moiety that reduces clearance
and improves resistance to proteolytic degradation. Since such
"BBB/PK modulators" are a new concept, several combinations of a
few structural modules are used that constitute extensions. Table 6
provides information about the structure and function of the
proposed modules.
TABLE-US-00008 TABLE 5 Summary of side chain replacements in
somatostatin proposed in this study. 1 2 3 4 5 6 7 8 9 10 11 12 13
14 Ala Gly Cys Lys Asn Phe Phe D- Lys Thr Phe Thr Ser Cys Trp Ala
Ala DAB DAP DAP DAP Lys- Lys- palm palm Cl-Phe Cl-Phe Cl-Phe
[0282] DAB, diaminobutyric acid; DAP, diaminopropionic acid;
Lys-palm, Lys-palmitoyl; Cl-Phe, chloro-Phe.
TABLE-US-00009 TABLE 6 Summary of structural and functional
properties of modules used to synthesize BBB/PK modulator. Module
Structure Function/Comments AHX Aminohexanoic acid Increase
lipophilicity in the middle of the extension to improve passive
penetration through membranes. No additional hydrogen bond
donors/acceptors are introduced. PerFHX Perfluorohexanoic acid
Increase lipophilicity by capping N-terminus with extremely
hydrophobic "tail". This is a very efficient strategy to increase
hydrophobilicity without significant increase in the molecular size
of the extension. PEG-spacer 8-amino-3,6- Increase length/size of
the extension using a PEG-based dioxaoctanic acid spacer: this
should result in improved pharmacokinetic properties of the
analogs. Phe Phe-[D-Phe] Mimetic of Phe as substrate recognition
for active transport of nutrients; alternatively, a glycosyl moiety
may also be introduced. Oligo-(Lys) Lys-(D-Lys)-Lys-(D- Increase of
basicity of the extension: this should Lys)-Lys-(D-Lys)-Lys enhance
electrostatic interactions with the membranes.
[0283] The modules can be introduced during solid-phase synthesis
as extensions to the Ala.sup.1 residue of
[D-Trp.sup.8]somatostatin. Table 7 and FIG. 11 summarize the
extensions.
TABLE-US-00010 TABLE 7 Summary of N-terminal extensions in
somatostatin analogs (for abbreviations used, refer to Table 6).
Analog # Module 3 Module 2 Module 1 Analog EXT1 AHX-AHX
[D-Trp.sup.8]SOM EXT2 PerFHX [D-Trp.sup.8]SOM EXT3 PEG-spacer
[D-Trp.sup.8]SOM EXT4 AHX-AHX PEG-spacer [D-Trp.sup.8]SOM EXT5
PerFHX PEG-spacer [D-Trp.sup.8]SOM EXT6 Oligo-(Lys) PEG-spacer
[D-Trp.sup.8]SOM EXT7 Phe AHX-AHX [D-Trp.sup.8]SOM EXT8 Phe AHX
[D-Trp.sup.8]SOM EXT9 AHX-AHX Oligo-(Lys) PEG-spacer
[D-Trp.sup.8]SOM EXT10 Phe Oligo-(Lys) PEG-spacer
[D-Trp.sup.8]SOM
[0284] Initially, ten analogs are synthesized and evaluated for
their binding properties to the somatostatin receptors.
High-affinity analogs are further evaluated for their permeability
properties through the model blood-brain barrier permeability
assay.
[0285] Once the optimal extensions are selected, they can be
attached to somatostatin analogs already containing optimized
side-chain replacements (see Table 5). Since it is difficult to
predict the best combination of "extension analogs" with
"side-chain replacement analogs", a matrix approach is utilized,
wherein each selected analog is synthesized with each selected
extension. 9-12 analogs can be achieved in this round. Such
3.sup.rd-generation analogs can be tested in all three in vitro
assays: (1) binding to the somatostatin receptors, (2) agonist
activity, and (3) permeability through the model blood-brain
barrier. A limited number of the most promising analogs can be
selected for pharmacological testing in the in vivo mouse epilepsy
models.
TABLE-US-00011 TABLE 8 Matrix-approach in designing somatostatin
analogs with the attached N-terminal BBB/PK modulators. Here, three
selected extension structures are combined with three selected
"side-chain replacement" analogs. EXT-A EXT-B EXT-C Analog 1 Analog
2 Analog 3
[0286] An approach similar to that described above for somatostatin
can be undertaken with galanin and its analogs. Galanin is a
30-amino-acid neuropeptide, but SAR studies identified that the
N-terminal portion is still a highly potent agonist as compared to
the whole-length peptide (Langel and Bartfai, 1998). A
galanin(1-16) analog can be used with the methods disclosed herein,
in which the Gly.sup.1 residue is replaced by N-methyl-Gly
(sarcosine, SAR), as shown below:
TABLE-US-00012 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Sar Trp Thr
Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Ala Val
[0287] N-methylation of Gly.sup.1 protected the peptide from
accelerated proteolytic degradation from the N-terminus, whereas it
did not significantly change its affinity for the galanin receptor
(Rivera Baeza et al., 1994). SAR studies identified the following
residues critical for biological activity: Gly.sup.1, Trp.sup.2,
Asn.sup.5, Tyr.sup.9 and Gly.sup.12 (Land et al., 1991). The same
study identified that the N-terminal extensions caused a loss of
the biological activity. On the other hand, the C-terminal portion
of galanin(1-16) appears to be very robust when it comes to
attaching to larger structures (Pooga et al., 1998). Therefore, the
strategy for design of [Sar.sup.1]galanin analogs is similar to
that used with somatostatin only with regard to amino acid
replacements, but it differs by introducing the extensions at the
C-, rather than at the N-terminus. Table 9 summarizes galanin
analogs with amino acid replacements.
TABLE-US-00013 TABLE 9 Replacements of individual residues in
[Sar1]galanin(1-16). For amino acid coding, refer to Table 5. 1 2 3
4 5 6 7 8 9 10 11 12 13 14 15 16 Sar Trp Thr Leu Asn Ser Ala Gly
Tyr Leu Leu Gly Pro His Ala Val DAP DAP DAP DAB DAP DAB Lys- Lys-
Lys- palm palm palm Cl- Tyr
[0288] The C-terminal extensions can be identical to those shown in
Table 7, but introduced at position His.sup.14. (Sar.sup.1)galanin
with the Lys.sup.14(Mmt) residue (side chain protected with a
4-methoxytrityl group) is then synthesized. After coupling
sarcosine, the peptide resin can be treated with 1% TFA in
dichloromethane for 30 minutes. The side-chain amino group of the
Lys.sup.14 residue can be deprotected, followed by coupling of the
extension modules. The design of the 3.sup.rd-generation analogs
with combined side chain replacements and the C-terminal extensions
are identical to that described for the somatostatin analogs.
[0289] Chemical Synthesis of Neuropeptides.
[0290] The peptides are synthesized using Fmoc-based solid-phase
peptide synthesis protocols and an automated peptide synthesizer.
Coupling methods and removal of the peptides from solid support are
performed as described by Chan and White (Chan and White, 2000).
The peptides can be removed from solid support by treatment with
reagent K. Following wash and precipitation, the analogs are
purified using preparative reversed-phase HPLC separations. The
disulfide bridge in the somatostatin analogs can be formed by
incubating purified peptide with 2% DMSO, 30% acetic acid in water,
pH 7.0, as described (Chen et al., 2000). At least several
milligrams of each peptide analog can be produced by this
method.
[0291] To evaluate permeability of neuropeptide analogs though the
blood-brain barrier, pION's PAMPA method can be employed. In this
method, a filter with an immobilized artificial membrane is placed
between two compartments: a donor and an acceptor. The analogs can
be placed in the donor compartment. After the appropriate time
interval, the donor and acceptor compartments are quantified using
UV spectroscopy.
[0292] Somatostatin and galanin binding assays (non-selective) are
performed by Novascreen Biosciences Corporation. Rat forebrain
membranes and radiolabeled parent neuropeptides are used in these
assays. The analogs are tested at a single (1 .mu.M) concentration
to distinguish between high- and low-affinity analogs. The agonist
activity of selected somatostatin and galanin analogs can be
further tested using functional assays provided by MDS-Pharma
Services. The analogs can be tested at a single concentration (1
.mu.M).
[0293] Anticonvulsant Testing.
[0294] Anticonvulsant activity can be established in the Frings
AGS-susceptible mouse model of reflex epilepsy. The AGS-susceptible
mouse is the ideal acute seizure model for initial proof-of-concept
studies because it is non-discriminatory, and effectively detects a
wide variety of CNS active compounds (White et al., 1992). Peptides
found to be active in the Frings mouse can be evaluated for their
ability to block seizures induced by maximal electroshock (MES) and
subcutaneously (s.c.) administered pentylenetetrazol (PTZ). These
two tests measure the ability of an investigational antiepileptic
compound to prevent seizure spread and elevate seizure threshold,
respectively (White et al., 2002). Once a modified peptide has been
demonstrated to be active in one or more of these three seizure
tests, complete dose-response studies are conducted at the
previously determined time of peak effect following i.v.
administration. Results from these proof-of-concept studies are
then compared to efficacy studies conducted following
intracerebroventricular (i.c.v.) administration. A leftward shift
in the i.p. dose-response curve can be observed as greater
penetration of the blood-brain barrier is achieved. Collectively,
the results obtained from these three seizure tests provide
substantial data supporting the approach to make small peptides
more accessible to the brain following systemic administration. The
details of each individual seizure test are outlined below.
[0295] Administration of Neuropeptide Analogs.
[0296] Each of the modified neuroactive peptides are administered
intracerebroventricularly (i.c.v.) in 5 .mu.l artificial
cerebrospinal fluid via a 10 .mu.l Hamilton syringe or
intraveneously (i.v.) in 0.5% methylcellulose in a volume of 0.01
ml/g body weight.
[0297] Audiogenic Seizures.
[0298] The ability of individual modified peptides to prevent
seizures induced by sound in the AGS-susceptible Frings mouse model
can be assessed at the time of peak effect (White et al., 1992).
For this test, individual mice are placed into a plexiglass
cylinder (diameter, 15 cm; height, 18 cm) fitted with an audio
transducer (Model AS-ZC; FET Research and Development, Salt Lake
City, Utah), and exposed to a sound stimulus of 110 decibels (11
KHz) delivered for 20 seconds. Sound-induced seizures are
characterized by wild running followed by loss of righting reflex
with forelimb and hindlimb tonic extension. Mice not displaying
hindlimb tonic extension are considered protected.
[0299] MES Test.
[0300] For the MES test, a drop of anesthetic/electrolyte solution
(0.5% tetracaine hydrochloride in 0.9% saline) can be applied to
the eyes of each animal prior to placement of the corneal
electrodes. The electrical stimulus in the mouse MES test is 50 mA
delivered for 0.2 sec by an apparatus similar to that originally
described by Woodbury and Davenport (Woodbury and Davenport, 1952).
Abolition of the hindleg tonic extensor component of the seizure is
used as the endpoint.
[0301] Minimal Toxicity Tests.
[0302] Minimal toxicity can be identified in mice by the rotarod
procedure (Dunham and Miya, 1957). When a mouse is placed on a
1-inch knurled rod that rotates at a speed of 6 r.p.m., the animal
can maintain its equilibrium for long periods of time. The animal
can be considered toxic if it falls off this rotating rod three
times during a 1-minute period.
[0303] Determination of Median Effective (ED.sub.50) or Toxic Dose
(TD.sub.50).
[0304] All quantitative in vivo anticonvulsant/toxicity studies are
conducted at the previously determined TPE. Groups of at least
eight mice are tested with various doses of the peptide until at
least two points have been established between the limits of 100%
protection or minimal toxicity, and 0% protection or minimal
toxicity. The dose of drug required to produce the desired endpoint
in 50% of animals (ED.sub.50 or TD.sub.50) in each test, the 95%
confidence interval, the slope of the regression line, and the
S.E.M. of the slope is then calculated by a computer program based
on the method described by Finney (Finney, 1971).
[0305] Analogs of somatostatin can be found in Table 10 (all
analogs have a disulfide bridge formed between two cysteine
residues):
TABLE-US-00014 TABLE 10 SOM-BBB1 (NN3APG)(AHX)AGCKNFFWKTFTSC (SEQ
ID NO: 41) SOM-BBB2 (NN3APG)(AHX)AGCKNFF(.sub.DW)KT(Cl-Phe)T(Dap)C
(SEQ ID NO: 42) SOM-BBB3
W(AHX)KKCKNFF(.sub.DW)KT(Cl-Phe)(Dab)(Dab)C (SEQ ID NO: 43)
SOM-BBB21 KK(Lys-P)K(AHX)(.sub.DF)CF(.sub.DW)KTC-Thr(ol) (SEQ ID
NO: 44) SOM-BBB22
KKK(Lys-P)K(AHX)(AHX)(.sub.DF)CF(.sub.DW)KTC-Thr(ol) (SEQ ID NO:
45) SOM-BBB23 (Lys-P)KK(Lys-P)K(AHX)(.sub.DF)CF(.sub.DW)KTC-Thr(ol)
(SEQ ID NO: 46) SOM-BBB24
KK(Lys-P)K(AHX)KK(Lys-P)K(AHX)(.sub.DF)CF(.sub.DW)KTC-Thr(ol) (SEQ
ID NO: 47) SOM-BBB25
(PFHA)K(.sub.DK)K(ACPA)KK(Lys-P)K(AHX)(.sub.DF)CF(.sub.DW)KTC-Th-
r(ol) (SEQ ID NO: 48)
[0306] In the above table, (AHX) is aminohexanoic acid, (Dab) is
diaminobutyric acid, (Dap) is diaminopropionic acid, (Tle) is
tert-Leucine, (Cl-Phe) is 4-chlorophenylalanine (NN3APG) is
N,N-bis(3-aminopropyl)glycine, (AHX) is aminohexanoic acid, (Lys-P)
is Lys-palmitoyl, Thr(ol) is Threoninol, DK, DF, DW denotes
D-isomer, PFHA is 2H, 2H, 3H, 3H-perfluoroheptanoic acid, and ACPA
is 8-aminocaprylic acid.
[0307] There are also other examples of analogs that can be used
other than galanin and somatostatin. For example, analogs of
Delta-sleep inducing peptide (DSIP) follow:
[0308] DSIP-BBB8: (AHX)GGWAGGDASGE (SEQ ID NO: 55). Additional DSIP
peptides can be found in Table 31.
2. Example 2: Anticonvulsant Galanin Analogs
[0309] Galanin is a 30-amino-acid neuropeptide, with the the
N-terminal portion being a highly potent agonist as compared to the
whole-length peptide (Langel and Bartfai, 1998). A truncated
galanin (1-16) analog (below) was used to introduce modifications
that enhance its permeability through the blood-brain-barrier.
TABLE-US-00015 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Gly Trp Thr
Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Ala Val
[0310] The following residues critical for biological activity were
identified: Gly.sup.1, Trp.sup.2, Asn.sup.5, Tyr.sup.9 and
Gly.sup.12 (Land et al., 1991). The N-terminal extensions or
truncations caused a loss of the biological activity. On the other
hand, the C-terminal portion of galanin (1-16) is very robust when
it comes to either truncations or attaching larger structures
(Pooga et al., 1998).
[0311] Based on available structure-activity relationship data, two
peptide-based galanin analogs were designed, chemically
synthesized, and tested. The structures of both analogs (GAL-BBB1
and GAL-BBB2) are provided below:
TABLE-US-00016 GAL-BBB1:
Sar-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-
Pro-His-(Lys-palm)-Tle-NH.sub.2 GAL-BBB2:
Sar-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-
Pro-Lys-Lys-(Lys-palm)-Lys-NH.sub.2
[0312] where Sar is sarcosine, Tle is tert-Leucine and Lys-palm is
lysine residue coupled with palmityoyl moiety via epsilon amino
group and --NH2 denotes amidation at the C-terminus. The peptides
were synthesized on solid support using the standard Fmoc chemistry
and purified by HPLC. The purified analogs were then tested in the
Frings audiogenic-seizure susceptible mouse model of epilepsy.
Results from this study were compared to the native galanin peptide
fragment (1-16).
[0313] Other analogs include the following:
TABLE-US-00017 GAL-BBB3: (SEQ ID NO: 49) WTLNSAGYLLGPKKXK-NH2
GAL-BBB4: (SEQ ID NO: 50)
Sar-WTLNSAGYLLGP(D-Lys)(D-Lys)X(D-Lys)-NH2 GAL-BBB5: (SEQ ID NO:
51) Sar-WTLNSAGYLLGPRRXR-NH2 GAL-BBB6: (SEQ ID NO: 52)
Sar-WTLNSAGYLLGPHHXH-NH2 GAL-BBB7: (SEQ ID NO: 53)
Sar-WTLNSAGYLLKKKKXK-NH2 GAL-BBB8: (SEQ ID NO: 54)
Sar-WTLNSAGYLLKKXK-NH2
[0314] where Sar is sarcosine, and X is Lys-palmitoyl residue.
[0315] The two modified galanin analogs (GAL-BBB1 and GAL-BBB2)
were administered i.p. to a group of Frings audiogenic seizure
susceptible mice in a dose of 4 mg/kg. At various times after
administration (i.e., 15, 30, 60, 120, and 240 min) each mouse was
placed into a cylindrical test chamber fitted with an audio
transducer and challenged with a high-intensity sound stimulus (110
dB, 11 KHz for 20 sec). Animals not displaying tonic hind-limb
extension were considered protected. As summarized in FIG. 12, the
results obtained from this study demonstrated that GAL-BBB2
displays a time-dependent anticonvulsant effect that was rapid in
onset (within 30 min) and moderate in duration (between two and
four hours). In contrast, the other modified galanin analog
GAL-BBB1 was not active at any time point tested, even at a higher
dose of 12 mg/kg. In a subsequent study, anticonvulsant efficacy
was quantitated at the time to peak effect (i.e., 1 h) in a
dose-response study. The results of this study demonstrated that
GAL-BBB2 displayed a dose-dependent effect against sound-induced
seizures. The calculated median effective dose (i.e., ED50) and 95%
confidence intervals were obtained from a Probit analysis of the
dose-response data was 3.2 (2.3-6.1) mg/kg. The native peptide
fragment GAL(1-16) was inactive at a dose of 20 mg/kg, i.p. (six
times the ED50 for GAL-BBB2) (FIG. 20).
[0316] The galanin analog, GAL-BBB2, exhibited potent
anticonvulsant activity (ED50-3 mg/kg) when given i.p. This
proof-of-concept analog represents the prototype on which more
"drug-like" analogs are designed. The smallest galanin analog with
the most potent and long-lasting anticonvulsant activity can
therefore be obtained. This requires a two-step approach: (1)
define the smallest fragment of GAL-BBB2 analog that maintains the
anticonvulsant activity: this will include terminal and central
truncations, (2) optimize the C-terminal structural motif that will
further improve BBB permeability of the analog. The synthesized
analogs are first screened in the galanin competitive binding
assay. Those analogs that displace galanin at concentrations 1
.mu.M or lower are further screened for anticonvulsant activity
using an audiogenic-seizure mouse model of epilepsy. Those analogs
that exhibit long-lasting anticonvulsant activity at a single dose
(i.e., 2 mg/kg, when given i.p.), are further evaluated in more
pharmacological assays. The general experimental strategy is
summarized in FIG. 13. FIGS. 23 and 24 shows time-dependent
anticonvulsant activity in Frings Mouse and dose-dependent
protection against audiogenic seizures in the Frings mouse,
respectively, when given GAL-BBB2.
[0317] Limited structure-function relationship studies are carried
out to identify the minimal fragment of the GAL-BBB2 analog that
maintains anticonvulsant activity when administered systemically.
Galanin analogs containing either the C-terminal and central
truncations are synthesized and tested. In addition, limited
structure-function relationship study of the C-terminal motif are
carried out to optimize permeability of the analog through the
blood-brain-barrier. FIG. 14 illustrates the structure of GAL-BBB2
in the context of structure-function studies.
[0318] In order to generate truncated analogs of GAL-BBB2, four
consecutive deletions are from the Pro13 to Leu10 residues
(summarized in Table 11). Since Tyr9 is critical to the galanin
activity, further C-terminal truncations can result in a complete
loss of biological activity (reference Land et al, Int J Pept Prot
1991). In each truncated analog, the C-terminal "-Lys-Lys-LysP-Lys"
is retained for improved permeability through the BBB.
TABLE-US-00018 TABLE 11 Structure of GAL-BBB2 and truncated
analogs. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Sar Trp Thr Leu
Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Lys-P Lys Sar Trp Thr
Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Lys Lys-P Lys Sar Trp Thr
Leu Asn Ser Ala Gly Tyr Leu Leu Lys Lys Lys-P Lys Sar Trp Thr Leu
Asn Ser Ala Gly Tyr Leu Lys Lys Lys-P Lys Sar Trp Thr Leu Asn Ser
Ala Gly Tyr Lys Lys Lys-P Lys
[0319] To minimize GAL-BBB2 analog, central truncations between key
residues are introduced. This is an alternative strategy to design
active peptide analogs with a simplified structure, without
compromising the length of a given peptide. Backbone replacement
(i.e., "backbone prosthesis") can be achieved by substituting two
or more consecutive "non-key" residues with a non-peptide spacer,
for example aminovarelic or aminohexanoic acid ("backbone spacer").
This concept is better illustrated in FIG. 15.
[0320] In the case of the GAL-BBB2 analog, three parts of the
peptide are probed by systematic replacements of residues with a
backbone spacer (Table 11): between Trp2 and Asn5, between Asn5 and
Tyr9 and between Tyr9 and the C-terminal motif. Approximately 14 of
such analogs are synthesized and tested for binding to the galanin
receptor. If some analogs maintain the anticonvulsant activity, two
or more spacers in different positions can be introduced (see the
example in Table 12).
TABLE-US-00019 TABLE 12 "Backbone-prosthesis walk" in the GAL-BBBB2
analog. Replacement of two residues at a time with non-peptidic
backbone spacer, such as aminovaleric or aminohexanoic acids
results in a minimization of the overall molecular size without
significant change of a spacing between the key pharmacophore
residues. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Sar Trp Thr Leu
Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Lys-P Lys Sar Trp
spacer Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Lys-P Lys Sar
Trp Thr Leu Asn spacer Gly Tyr Leu Leu Gly Pro Lys Lys Lys-P Lys
Sar Trp Thr Leu Asn Ser spacer Tyr Leu Leu Gly Pro Lys Lys Lys-P
Lys Sar Trp Thr Leu Asn Ser Ala Gly Tyr spacer Gly Pro Lys Lys
Lys-P Lys Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu spacer Pro Lys
Lys Lys-P Lys Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu spacer
Lys Lys Lys-P Lys Sar Trp Thr Leu Asn Spacer Tyr Leu Leu Gly Pro
Lys Lys Lys-P Lys Optional: Sar Trp spacer Asn Spacer Tyr Leu Leu
Gly Pro Lys Lys Lys-P Lys
[0321] Next, the C-terminal structural motif,
"-Lys-Lys-LysP-Lys-NH2", can be optimized to improve the BBB
permeability. The initial compound, GAL-BBB2 can be optimized by
introduction of the following structural changes, summarized in
Table 13. Replacements of Lys residues with homo-Lys, D-Lys or
diaminobutyric acid probes efficiency of the BBB permeability of
positively charged residues with varying lipophilic nature of their
side chains. Replacement of Lys-palmitoyl moiety in the position 16
with 2-amino-tetradecanoic acid or 3,3-diphenylalanine determines
how flexible is this position to other hydrophobic residues that
can also enhance the BBB permeability.
TABLE-US-00020 TABLE 13 Modification of the C-terminal motif that
enhances permeability of the galanin analogs through the BBB. 1 2 3
4 5 6 7 8 9 10 11 12 13 14 15 16 17 Sar Trp Thr Leu Asn Ser Ala Gly
Tyr Leu Leu Gly Pro Lys Lys Lys-P Lys Sar Trp Thr Leu Asn Ser Ala
Gly Tyr Leu Leu Gly Lys Lys Lys Lys-P Lys Sar Trp Thr Leu Asn Ser
Ala Gly Tyr Leu Leu Lys Lys Lys Lys Lys-P Lys Sar Trp Thr Leu Asn
Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Lys Lys Sar Trp Thr Leu Asn
Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys-P Lys Lys Sar Trp Thr Leu
Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys-P Lys Lys Lys Sar Trp Thr
Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro D-Lys D-Lys Lys-P D-Lys Sar
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro h-Lys h-Lys Lys-P
h-Lys Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro DAB DAB
Lys-P DAB Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys
Lys TDA Lys Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys
Lys DPA Lys
[0322] TDA is 2-amino-tetradecanoic acid, DAB is diaminoobyturic
acid, D-Lys is D-isomer of Lys and h-Lys is homo-Lys, DPA is
3,3-diphenylalanine
TABLE-US-00021 Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro
Lys Lys Lys-P Lys X
[0323] In addition to the above list of analogs, two analogs with a
lipophilic, non-peptidic extension at the C-terminus can be
produced, as shown below:
[0324] where X denotes: 12-amino-dodecanoic acid or
2-amino-tetradecanoic acid.
[0325] As illustrated in FIG. 13, each synthesized and purified
analog is tested for its binding properties to galanin receptors.
Only those analogs that displace full-length galanin at
concentration 1 .mu.M or lower are studied further. Galanin binding
assays can be performed by Novascreen Biosciences Corporation, for
example. Rat forebrain membranes and radiolabeled parent
neuropeptides can be used in these assays. The analogs can be
tested at a single (1 .mu.M) concentration to distinguish between
high- and low-affinity analogs. The agonist activity of selected
galanin analogs can be further tested using functional assays
provided by MDS-Pharma Services. For example, the analogs can be
tested at a single concentration (1 .mu.M).
[0326] The below table summarizes various SAR analogs, and the
percentage of protection afforded at 1, 2 and 4 hours by a dose of
4 mg/kg, i.p.
TABLE-US-00022 TABLE 14 % Protection at 1, 2 and 4 hours afforded
by a dose of 4 mg/kg, NAX Structure i.p. Gal(1-16) GWTLNSAGYLLGPHAV
(SEQ ID NO: Not active 1) 5055 (Sar)WTLNSAGYLLGPKK(Lys-P)K 100%,
100%, 0% (0.8 mg/kg) (SEQ ID NO: 56) Subtype selectivity 1205-1
WTLNSAGYLLGPKK(Lys-P)K (SEQ ID 50%, 50%, 0%, * (5.7 mg/kg) NO: 57)
"KKKpK" motif 1205-2 (Sar)WTLNSAGYLLGPDKDK(Lys- 100%, 50%, 75% (1.2
mg/kg) P)DK (SEQ ID NO: 50) 1205-3 (Sar)WTLNSAGYLLGPRR(Lys-P)R
100%, 75%, 0% (SEQ ID NO: 59) 1205-4 (Sar)WTLNSAGYLLKKKK(Lys-P)K
75%, 100%, 66%, * (SEQ ID NO: 60) 1105-2 (Sar)WTLNSAGYLLGPKKKK 30%,
0%, 0% (3.7 mg/kg) (SEQ ID NO: 61) Truncations 1205-5
(Sar)WTLNSAGYLLKK(Lys-P)K 100%, 25%, 0% (2.8 mg/kg) (SEQ ID NO: 62)
306-3 (Sar)WTLNSAGYKK(Lys-P)K 75%, 25%, 0% (2.7 mg/kg) (SEQ ID NO:
63) Backbone spacers 306-2 (Sar)WTLNSAGYLLGP(Ahx)KK(Lys- 100%, 75%,
0%, * P)K (SEQ ID NO: 64) 306-4 (Sar)WTLNSAGY(Ahx)KK(Lys-P)K 50%,
0%, 0% (2.95 mg/kg) (SEQ ID NO: 65)
[0327] Anticonvulsant activity can first be established in the
Frings AGS-susceptible mouse model of reflex epilepsy. The
AGS-susceptible mouse is the ideal acute seizure model because it
is non-discriminatory, and effectively detects a wide variety of
CNS active compounds (White et al., 1992). Peptides found to be
active in the Frings mouse can be evaluated for their ability to
block seizures induced by maximal electroshock (MES) and
subcutaneously (s.c.) administered pentylenetetrazol (PTZ). These
two tests measure the ability of an investigational antiepileptic
compound to prevent seizure spread and elevate seizure threshold,
respectively (White et al., 2002). Once a modified peptide has been
demonstrated to be active in one or more of these three seizure
tests, complete dose-response studies are conducted at the
previously determined time of peak effect following i.p.
administration. Results from these proof-of-concept studies are
then be compared to efficacy studies conducted following
intracerebroventricular (i.c.v.) administration. A leftward shift
in the i.p. dose-response curve can be observed as greater
penetration of the blood-brain barrier is achieved. Collectively,
the results obtained from these three seizure tests provides
substantial data supporting the approach to make small peptides
more accessible to the brain following systemic administration. The
details of each individual seizure test are outlined below.
[0328] Administration of Neuropeptide Analogs.
[0329] Each of the modified galanin analogs are administered
intracerebroventricularly (i.c.v.) in 5 .mu.l artificial
cerebrospinal fluid via a 10 .mu.l Hamilton syringe or
intraperitoneally (i.p.) in 0.5% methylcellulose in a volume of
0.01 ml/g body weight.
[0330] Audiogenic Seizures.
[0331] The ability of individual modified analogs to prevent
seizures induced by sound in the AGS-susceptible Frings mouse model
can be assessed at the time of peak effect (White et al., 1992).
For this test, individual mice are placed into a plexiglass
cylinder (diameter, 15 cm; height, 18 cm) fitted with an audio
transducer (Model AS-ZC; FET Research and Development, Salt Lake
City, Utah), and exposed to a sound stimulus of 110 decibels (11
KHz) delivered for 20 seconds. Sound-induced seizures are
characterized by wild running followed by loss of righting reflex
with forelimb and hindlimb tonic extension. Mice not displaying
hindlimb tonic extension are considered protected.
[0332] MES Test.
[0333] For the MES test, a drop of anesthetic/electrolyte solution
(0.5% tetracaine hydrochloride in 0.9% saline) is applied to the
eyes of each animal prior to placement of the corneal electrodes.
The electrical stimulus in the mouse MES test is 50 mA delivered
for 0.2 sec by an apparatus similar to that originally described by
Woodbury and Davenport (Woodbury and Davenport, 1952). Abolition of
the hindleg tonic extensor component of the seizure is used as the
endpoint.
[0334] s.c. PTZ Test.
[0335] For the s.c. PTZ test, a dose of 85 mg/kg PTZ is s.c. into a
loose fold of skin on the back of each mouse. Mice will be placed
into individual plexiglas observation boxes and observed for 30
minutes for the presence of a minimal clonic seizure. Mice not
displaying clonic seizure activity will be considered
protected.
[0336] Minimal Toxicity Tests.
[0337] Minimal toxicity will be identified in mice by the rotarod
procedure (Dunham and Miya, 1957). When a mouse is placed on a
1-inch knurled rod that rotates at a speed of 6 r.p.m., the animal
can maintain its equilibrium for long periods of time. The animal
is considered toxic if it falls off this rotating rod three times
during a 1-minute period.
[0338] Determination of Median Effective (ED.sub.50) or Toxic Dose
(TD.sub.50).
[0339] All quantitative in vivo anticonvulsant/toxicity studies re
conducted at the previously determined TPE. Groups of at least
eight mice are tested with various doses of the peptide until at
least two points have been established between the limits of 100%
protection or minimal toxicity, and 0% protection or minimal
toxicity. The dose of drug required to produce the desired endpoint
in 50% of animals (ED.sub.50 or TD.sub.50) in each test, the 95%
confidence interval, the slope of the regression line, and the
S.E.M. of the slope are calculated by a computer program based on
the method described by Finney (Finney, 1971).
3. Example 3: GAL-BBB2 Possesses Potent Pain Relief
[0340] a) Formalin Test
[0341] An injection of 0.5% formalin is made into the planter
region of a mouse right hind paw. This elicits a distinct biphasic
behavioral profile characterized by the mouse licking the affected
paw. Immediately following the injection the mouse licks the paw
for about 10 minutes. This is phase 1 (acute) and is followed by a
brief latent period where there is little behavioral activity. A
more prolonged period of about 20 to 30 minutes of paw licking
ensues which constitutes phase 2 (inflammatory).
[0342] Prior to the administration of the active peptide, drug or
vehicle each mouse undergoes a 15-minute conditioning period in one
of several 6'' tall plexiglass observation tubes (4'' diameter)
that are placed in front of a mirror. Following the conditioning
period, mice were treated i.p. with to either GAL-BBB2, the
inactive native fragment Gal 1-16, or gabapentin then returned to
its home tube. One hour after treatment, formalin was injected
sub-dermally (20 .mu.l; 27 gauge needle) into the plantar surface
of the right hind foot. The bevel of the needle is placed facing
down toward the skin surface. Following the injection of the
formalin each animal is observed for first 2 minutes of each 5
minute epoch for a total of 45 minutes. The cumulative length of
licking for each 2 minute time period was measured. An animal
receiving the requisite volume of vehicle was alternated with each
mouse given GAL-BBB2, Gal 1-16, or gabapentin. Animals were
euthanized following the conclusion of the experiment.
[0343] In a further experiment (in table below), two additional
galanin analogs with unique structural motifs (i.e., NAX 306-3 and
306-4) were found to be potent, as well as NAX 5055, in the mouse
formalin assay of inflammatory pain. These findings show that the
active pharmacophore is amenable to structural modifications.
TABLE-US-00023 TABLE 15 NAX Structure Active at: 5055
(Sar)WTLNSAGYLLGPKK(Lys-P)K 5 mg/kg ("flat phase II") (SEQ ID NO:
56) 306-3 (Sar)WTLNSAGYKK(Lys-P)K (SEQ ID 2.7 mg/kg ("flat phase
II") NO: 66) 306-4 (Sar)WTLNSAGY(Ahx)KK(Lys-P)K 2.9 mg/kg ("flat
phase II") (SEQ ID NO: 67)
[0344] Area under the curve (AUC) determination was made using the
GraphPad Prism Version 3.03. Total AUC is calculated for both the
test and control groups for both the acute and inflammatory phases.
The AUC for individual animals for each phase is also calculated
and converted to percentage of total AUC of control. The average
percentages and SEM for both the drug treated and control were
calculated and tested for significant difference.
[0345] b) Ligation of the Sciatic Nerve
[0346] Just prior to surgery, rats are treated subcutaneously with
0.1 to 0.5 mg/kg of the long acting opiate buprenorphine. Rats are
then anesthetized with pentobarbital and the depth of anesthesia
monitored by their response to a tail pinch and observation of the
depth of respiration. Sterile technique was used throughout the
surgery.
[0347] The upper thigh of each rat was shaved and wiped off with
ethanol and betadine. A small incision was made in the skin and
through the underlying muscle of the upper thigh until the sciatic
nerve was exposed. The nerve was then separated from the
surrounding connective tissue and slightly elevated by a pair of
fine, curved forceps. Approximately 1/3 to 1/2 of the nerve was
then tied off by passing a needle and nylon suture (7.0) through
the nerve. The muscle and skin incision was then closed sutured
separately with 5.0 suture and the animals were kept warm by
placing on a thermostatically controlled heat blanket until they
have recovered from the anesthesia. This procedure was conducted on
the right side (ipsilateral) while a sham surgery was performed on
the left hind leg (contralateral). The latter involves a similar
procedure with the exception that the sciatic nerve on this side
was only exposed and not ligated. Twelve hours post surgery, a
second dose of buprenorphine was administered to minimize any
discomfort from the surgical procedure. Rats were closely monitored
daily for the development of infection or untoward effects of the
surgery.
[0348] After an appropriate time for recovery (e.g., 7-14 days) the
animals were tested for the development of mechanical allodynia
(pain response to a non-noxious stimulus). For this study, the
animals placed in a bottomless plexiglass box placed on a wire mesh
(1/4'') platform. After a 30-60 minute acclimation period, a
baseline mechanical sensitivity was determined. This procedure was
done by applying a series of calibrated Von Frey fibers
perpendicularly to the plantar surface of each hind paw and holding
it there for about 6 secs with enough force to slightly bend the
fiber. After a positive response (withdrawal of the foot) is noted
a smaller diameter fiber was applied. This procedure was repeated
until a 50% threshold for withdrawal could be determined.
[0349] Following i.p. injection of 2 mg/kg GAL-BBB2 (n=8 rats per
drug) the mechanical threshold was assessed 30 min post-injection
and at various times thereafter (e.g., 1, 2, 4, and 6 h) to
determine the duration of action of the test compound. Results
obtained with GAL-BBB2 were compared to those obtained with 2 mg/kg
morphine, and 40 mg/kg gabapentin.
[0350] c) Results
[0351] A number of anticonvulsants have demonstrated efficacy in
the treatment of pain. Therefore, GAL-BBB2 was examined in the
mouse formalin model to assess whether it possessed analgesic
properties. In this test, GAL-BBB2 was found to significantly
reduce the pain associated with s.c. plantar formalin as estimated
by quantification of the time that an animal spends licking the
ipsilateral paw. As shown in FIG. 16, GAL-BBB2 (0.52-5 mg/kg)
produced a dose-dependent reduction in paw licking during both the
initial acute phase as well as the prolonged inflammatory phase. In
contrast, the un-modified native fragment Gal 1-16 was found to be
inactive following i.p. administration of a dose 4 times higher
than the highest dose of GAL-BBB2 tested (i.e., 20 mg/kg). In
addition, 5 mg/kg GAL-BBB2 (FIG. 16) was found to be equivalent to
a 10 mg/kg dose of gabapentin (FIG. 17).
[0352] As shown in FIG. 18, GAL-BBB2 displayed a time-dependent
increase in the threshold for mechanical allodynia in the sciatic
ligation model of chronic pain. Furthermore, GAL-BBB2 was
equi-potent to morphine and several fold more potent that
gabapentin in this test (inset to FIG. 18).
[0353] Collectively, the results obtained in these two established
models of pain show that GAL-BBB2 possesses potent pain relief in
rodent models of chronic pain.
4. Example 4: Galanin Analogs that Penetrate the
Blood-Brain-Barrier
[0354] Table 16 shows galanin analogs that can be used with the
compositions and methods disclosed herein:
TABLE-US-00024 (Sar)WTLNSAGY(D-Lys)(D-Lys)(Lys-P)(D-Lys) (SEQ ID
NO: Gal-BBB25 119) (Sar)WTLNSAGY(Ahx)(D-Lys)(D-Lys)(Lys-P)(D-Lys)
(SEQ Gal-BBB26 ID NO: 120)
(Sar)WTLNSAGY(7-Ahp)(D-Lys)(D-Lys)(Lys-P)(D-Lys) (SEQ Gal-BBB27 ID
NO: 121) (Sar)WTLNSAGY(3,5-dibromo-Tyr)LLGPKK(Lys-P)K (SEQ
Gal-BBB28 ID NO: 122) (Sar)WTLNSAGYLLGPHH(Lys-P)K (SEQ ID NO: 123)
Gal-BBB29 (Sar)WTLNSAGYLLGPKK(Cys-Mmt)K (SEQ ID NO: 124) Gal-BBB30
(Sar)WTLNSAGYLLGPKK(Lys-Biotin-aminocaproyl)K (SEQ Gal-BBB31 ID NO:
125) (Sar)WTLNSAGYLLGPKK(Lys-sterol)K (SEQ ID NO: 126) Gal-BBB32
(Sar)WTLNSAGYLLGPKK(Lys-decanoyl)K (SEQ ID NO: 127) Gal-BBB33
(Sar)WTLNSAGYLLGPKK(Lys-octanoyl)K (SEQ ID NO: 128) Gal-BBB34
(Sar)WTLNSAGYLLGPKK(Lys-linoyl)K (SEQ ID NO: 129) Gal-BBB35
(Sar)WTLNSAGYLLGPKK(Ser-melbiose)K (SEQ ID NO: 130) Gal-BBB36
(Sar)WTLNSAGYLLGPKK(Lys-adamentoyl)K (SEQ ID NO: Gal-BBB37 131)
(Sar)WTLNSAGYLLGPKK(Glu(.beta.-Lac-PEG.sub.3-amine))K (SEQ
Gal-BBB38 ID NO: 132) (Sar)WTLTSAGYLLGPKK(Lys-palmitoyl)K (SEQ ID
NO: 133) Gal-BBB39 (Sar)WTLLSAGYLLGPKK(Lys-palmitoyl)K (SEQ ID NO:
134) Gal-BBB40 (Sar)WTLDSAGYLLGPKK(Lys-palmitoyl)K (SEQ ID NO:
Gal-BBB41 135)
[0355] Lipophilicity and basicity contribute to increased
permeability of peptides through the BBB without the need for
specific transporters or carriers. The lipophilic character of a
peptide (measured by a log P value) may be altered by either
conjugation of a hydrophobic moiety (e.g., lipoamino acids), or
halogenation of aromatic residues. Regarding the basicity, the
Poduslo group showed that polyamine-modified proteins and peptides
cross the BBB more efficiently (Poduslo and Curran 1996; Poduslo
and Curran 1996; Poduslo, Curran et al. 1998; Poduslo, Curran et
al. 1999). Tamai and coworkers (Tamai, Sai et al. 1997) provided
evidence that the increased basicity of small peptides was an
important determinant of transport through the BBB via
absorptive-mediated endocytosis (AME).
[0356] Glycosylation appeared as a very efficient approach to
produce systemically-active opioid peptides (Elmagbari, Egleton et
al. 2004; Polt, Dhanasekaran et al. 2005). SAR studies showed that
the structure of saccharides was an important determinant of the
activity, but monosaccharides were generally less effective than
disaccharides. O-glycosylated serine with beta-melibiose or
beta-lactose were among the most efficient modifications that
yielded potent analgesic compounds.
[0357] Role of galanin and its receptors in epilepsy and
epileptogenesis. Neuropeptides are potent modulators of classic
neurotransmitters and neuronal excitability (Hokfelt, Broberger et
al. 2000). Coexistence of neuropeptides with classic
neurotransmitters in select neuronal populations implies that
neuronal excitability can be regulated through modification of
peptidergic transmission (Baraban and Tallent 2004). Under ambient
conditions, peptides are "silent" and exert little effect on normal
neurotransmission. In contrast, under conditions of excessively
high neuronal firing (as occurs in a seizure focus), neuropeptides
are released and exert a modulatory effect on
neurotransmission.
[0358] Galanin produces multiple effects in the brain (Hokfelt, Xu
et al. 1998; Lundstrom, Elmquist et al. 2005). Three galanin
receptor subtypes identified to date belong to the superfamily of G
protein coupled receptors (GPCR) (Branchek, Smith et al. 2000;
Lundstrom, Elmquist et al. 2005). Galanin receptor type 1 (GalR1)
is present in many brain areas, but displays the highest expression
in the hippocampus (Burgevin, Loquet et al. 1995). The galanin
receptor type 2 (GalR2) is as widely distributed as GalR1. In the
brain it is expressed in the hypothalamus, the hippocampus (dentate
gyrus>CA3>CA1), the amygdala, piriform cortex, basal
forebrain (medial septum/diagonal band), the cerebellum, and the
brainstem. Galanin receptor type 3 (GalR3) exhibits very restricted
expression in the brain. It is most abundant in the hypothalamus,
medial reticular formation and diagonal band, and is absent from
the hippocampus.
[0359] Since the pioneer work of Mazarati and coworkers (Mazarati,
Halaszi et al. 1992), there has been increasing evidence that
galanin is a potent anticonvulsant peptide. The acute
administration of galanin receptor agonists or virus-mediated
overexpression of galanin in the hippocampus has been found to
inhibit limbic status epilepticus, pentylenetetrazol and picrotoxin
seizures in rats and mice (Mazarati, Halaszi et al. 1992; Mazarati,
Liu et al. 1998; Saar, Mazarati et al. 2002; Haberman, Samulski et
al. 2003; Lin, Richichi et al. 2003; Bartfai, Lu et al. 2004).
Furthermore, the seizure threshold of galanin overexpressing
transgenic animals is increased in status epilepticus and kindling
models (Mazarati, Hohmann et al. 2000; Kokaia, Holmberg et al.
2001; Schlifke, Kuteeva et al. 2006).
[0360] In vitro, galanin inhibits glutamate release from the
hippocampus (Zini, Roisin et al. 1993; Mazarati, Hohmann et al.
2000). Results obtained from studies with GalR1 knockout mice and
rats treated with GalR2 peptide nucleic acid antisense suggests
that galanin exerts its anticonvulsant effect through an action at
both GalR1 and GalR2 (Mazarati, Lu et al. 2004; Mazarati, Lu et al.
2004). Furthermore, GalR2 is thought to play an important role in
the neuroprotective effects of galanin in hippocampal neurons
(Haberman et al., 2003; Mazarati et al., 2004a; Pirondi et al.,
2005; Elliot-Hunt et al., 2004; Lee et al., 2005; Hwang et al.,
2004).
[0361] It should be emphasized that galanin is effective in
preventing the expression of acute seizures and modifying the
development of epilepsy following various insults. For example,
several reports have shown that galanin can modify the damage
associated with limbic seizures and delay or prevent the
development of epilepsy (i.e. antiepileptogenic). Kokaia et al.
(Kokaia, Holmberg et al. 2001) reported delayed kindling in galanin
peptide overexpressing mice. Results from a recent study in a model
of rapid kindling show that hippocampal GalR2 coupled to Go,
protein exerts an antiepileptogenic effect independent of GIRK,
while GalR1 delays the acquisition of kindling by GIRK activation
(Mazarati, Lundstrom et al. 2006).
[0362] Structure-activity-relationships (SAR) in galanin and
galanin receptor ligands. Galanin was first discovered in 1983
(Tatemoto, Rokaeus et al. 1983). It is a 29-30 amino acid long
peptide with the following sequence:
TABLE-US-00025 Human (SEQ ID NO: 93)
GWTLNSAGYLLGPHAVGNHRSFSDKNGLTS-.sub.COOH Rat/mouse (SEQ ID NO: 94)
GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-.sub.NH2 Porcine (SEQ ID NO: 95)
GWTLNSAGYLLGPHAIDNHRSFHDKYGLA-.sub.NH2
[0363] The first N-terminal 14 residues (shaded) are highly
conserved among galanin sequences from different animal species
(Langel and Bartfai 1998). Since the
structure-activity-relationship of galanin has been extensively
studied we will only review those SAR results that are relevant to
this grant application. The N-terminal fragments of GAL, consisting
of the first 15 residues (analog GAL(1-15)) or 16 residues (analog
GAL(1-16)) have been shown to maintain high affinity toward galanin
receptors (Fisone, Berthold et al. 1989; Land, Langel et al. 1991).
As shown in the following table, systematic truncation of GAL(1-16)
results in a gradual decrease of the affinity toward its receptors
(Land, Langel et al. 1991).
TABLE-US-00026 TABLE 17 Effects of truncation of GAL(1-16) fragment
on its affinity towards galanin receptors. K.sub.D Fragment
Sequence [.mu.M] 1-16 GWTLNSAGYLLGPHAI 0.007 (SEQ ID NO: 96) 1-14
GWTLNSAGYLLGPH 0.15 (SEQ ID NO: 97) 1-12 GWTLNSAGYLLG (SEQ 3 ID NO:
98) 1-10 GWTLNSAGYL (SEQ ID 25 NO: 99) 1-9 GWTLNSAGY (SEQ ID 100
NO: 100)
[0364] In the same study, the authors demonstrated that the Gly1,
Trp2, Asn5, Tyr9 and Gly12 residues are important for high affinity
binding of the GAL(1-16) analog to the galanin receptors.
Alanine-walk analogs of GAL(1-16) indicated that replacement of the
these residues affects their affinity toward GalR2, rather than
GalR1 (Carpenter, Schmidt et al. 1999). An alanine-shaving approach
revealed that a sequence spanning from Tyr9 to His14 was critical
for recognition of galanin receptors (Jureus, Langel et al.
1997).
[0365] A very extensive SAR study of the GAL(1-16) analog was
described by (Pooga, Jureus et al. 1998). Modification of Gly1 or
Trp2 resulted in a significant loss of affinity toward galanin
receptors. All analogs of GAL(1-13) with Lys14 epsilon-NH group
coupled to different groups varying in size retained high affinity.
Based on these results, the C-terminal part of GAL(1-13) can
accommodate relatively bulky groups without compromising binding
properties. In summary, the SAR of galanin and its truncated
analogs indicate that Trp2, Asn5 and Tyr9 are key residues for
protecting an interaction between galanin and GalR1 and GarR2
receptors. Mutagenesis and modeling studies show that Trp2
interacts with Phe282 of hGalR1, whereas Tyr9 with His264 (Kask et
al, 1996; Berthold et al 1997; Church et al, 2002).
[0366] As a result of extensive structure-activity relationship
studies, a number of peptide-based galanin analogs, both agonists
and antagonists, have been synthesized and functionally
characterized. The binding properties of selected galanin receptor
ligands is summarize in Table 18.
TABLE-US-00027 TABLE 18 Selected galanin receptor ligands and their
binding properties to different galanin receptor subtypes.
Affinity.sup.a, K.sub.D [nM] Ligand hGalR1 hGalR2 hGalR3 Agonists
hGAL(1-29) 9.4 8.6 7.1 GAL(1-16) 8.5 8.3 6.5 GAL(2-11) 879 1.8 n.d.
Agonists that cross blood-brain-barrier Galnon 12,000 24,000 n.d.
Galmic 34,000 >100,000 n.d. Analog 5055.sup.b ~9 ~6 n.d.
Antagonists M35 0.1 2 15 M15 (galantide) 0.3 1 40 .sup.aK.sub.D
values were compiled from (Branchek, Smith et al. 2000; Lundstrom,
Elmquist et al. 2005; Lundstrom, Sollenberg et al. 2005;
Sollenberg, Lundstrom et al. 2006). Galnon and galmic are two
non-peptide galanin receptor agonists that have become very useful
pharmacological tools to study the effects of galanin receptors in
the CNS (Saar, Mazarati et al. 2002; Bartfai, Lu et al. 2004;
Badie-Mahdavi, Behrens et al. 2005; Lu, Barr et al. 2005; Schlifke,
Kuteeva et al. 2006). However, as recently stated: "The drawback of
galnon and galmic are that they are low affinity (micromolar
affinities), non-receptor subtype selective, and interacting with
other pharmacologically important targets . . ." (Lu, Lundstrom et
al. 2005).
[0367] Rational design and chemical synthesis of NAX 5055, a
galanin analog that penetrates the BBB. As disclosed herein,
galanin is a 30-amino-acid neuropeptide, and it has been
demonstrated that the N-terminal fragment GAL(1-16) is still a
highly potent agonist at the hippocampal galanin receptor (Fisone,
Berthold et al. 1989). Results obtained to date were obtained with
a truncated GAL(1-16) analog (FIG. 31) that has been modified in a
way to increase metabolic stability, and improve permeability
through the BBB.
[0368] Based on available structure-activity relationship data, the
following modifications were introduced to the GAL(1-16) analog to
improve its metabolic stability and permeability through the BBB:
(1) Gly1 residue was replaced by Sarcosine. N-methylation of Gly1
does not affect galanin receptor affinity (Rivera Baeza, Kask et
al. 1994). Furthermore, aminopeptidases N are known to degrade
neuropeptides, and thus capping of the N-terminal amino group is
likely to decrease the rate of metabolic degradation of
Sarcosine-containing analogs; (2) His14 and Ala15 were replaced by
Lys residues. Amidated Lys residue was added to the C-terminus.
These additional positive charges increase BBB permeability
mediated through adsorptive-mediated endocytosis (Tamai, Sai et al.
1997); (3) Val16 was replaced by Lysine-palmitoyl (Lys-palm)
residue. This long, hydrophobic can increase passive diffusion, and
provide additional resistance to metabolic degradation (Yuan, Wang
et al. 2005). NAX 5055 was chemically synthesized on a solid
support using the standard Fmoc protocols and automated peptide
synthesizer.
[0369] Pharmacological Properties of NAX 5055.
[0370] To date, NAX 5055 has been evaluated in a radioligand
binding assay and a battery of in vivo acute seizure tests. The
results obtained from these investigations show that the approach
disclosed herein can yield potent, high affinity galanin receptor
modulators that penetrate the BBB.
[0371] NAX 5055 Retains High Affinity Towards GalR1 and GalR2.
[0372] Affinity of NAX 5055 for human hGalR1 and hGalR2 was
confirmed in a preliminary radioligand binding study that was
conducted by MDS-PS contract screening company (report#1077561,
reference: MDSPS 231510 and 231600). In this study, hGalR1 was
expressed in HEK-293 cells, whereas hGalR2 was expressed in CHO-K1
cells and human [.sup.125I]galanin was used as the radioligand. NAX
5055 retained high affinity toward both subtypes with an estimated
Ki of 9 nM for hGalR1 and 6 nM for hGalR2.
[0373] NAX 5055 Displays Potent Anticonvulsant Activity Following
Systemic Administration.
[0374] NAX 5055 was initially tested in the Frings audiogenic
seizure (AGS)-susceptible mouse model of reflex epilepsy following
i.p. administration of 4 mg/kg. At various times after
administration (i.e., 15, 30, 60, 120, and 240 min) each mouse was
placed into a cylindrical test chamber fitted with an audio
transducer and challenged with a high-intensity sound stimulus (110
dB, 11 KHz for 20 sec).
[0375] Animals not Displaying Tonic Hind-Limb Extension were
Considered Protected.
[0376] As shown in FIG. 24, the results obtained from this
experiment demonstrate that NAX5055 displays a time-dependent
anticonvulsant effect that is rapid in onset (within 30 min) and
moderate in duration (between two and four hours). In a subsequent
dose-response study, anticonvulsant efficacy was quantitated at the
time to peak effect (i.e., 1 h). The calculated median effective
dose (i.e., ED50) and 95% confidence intervals obtained from a
Probit analysis of the dose-response data was 3.2 (2.3-6.1) mg/kg.
When tested one-hour after i.p. administration, NAX 5055 (4 mg/kg),
but not the native GAL(1-16) fragment (20 mg/kg) was effective in
blocking sound-induced seizures in the Frings mouse.
[0377] NAX 5055 was also tested in two well-established seizure
models; i.e., the maximal electroshock seizure (model of
generalized tonic-clonic epilepsy) and the s.c. Metrazol-seizure
test (model of generalized myoclonic epilepsy). For this study, 4
mg/kg NAX 5055 was administered i.p. and mice were tested one-hour
later for protection against tonic-extension (maximal electroshock)
and clonic (s.c. Metrazol) seizures. NAX 5055 was minimally active
(25% protection) in the s.c. Metrazol seizure test and was
completely inactive in the maximal electroshock seizure test
(results not shown). Although these results might be interpreted as
negative from a clinical efficacy point of view, the profile of NAX
5055 described thus far is virtually identical to that of the novel
antiepileptic drug levetiracetam which was introduced in 2000 for
the treatment of human partial seizures.
[0378] Thus, in an effort to expand the anticonvulsant profile of
NAX 5055, we have also evaluated it in another
levetiracetam-sensitive acute seizure model; i.e., the 6 Hz
psychomotor test. The 6 Hz seizure test is evolving as a unique
model for differentiating potential anticonvulsant compounds that
might be useful for the treatment of refractory partial epilepsy
(Barton, Klein et al. 2001; White 2003). NAX 5055 is very potent
following in this model of pharmaco-resistant epilepsy following
i.p. administration (Table 19). Unlike levetiracetam and even
valproic acid, the potency of NAX 5055 is retained as the stimulus
intensity is increased from 22 to 44 mA. Thus, NAX 5055 is
relatively unique among the anticonvulsant drugs tested in the 6 Hz
test in that it remains very potent at all three current
intensities evaluated. In contrast, the potency of the other drugs
decreases as the stimulation intensity is increased from 22 to 44
mA. NAX 5055 was subsequently tested in the mouse 6 Hz limbic
seizure model following subcutaneous (s.c.) administration.
Interestingly, activity is preserved following s.c. administration
(FIG. 26). The finding that there was only a slight rightward shift
in the ED50 of NAX following s.c. administration shows that NAX
5055 possesses good bioavailability.
[0379] In an effort to confirm that the native peptide fragment is
active when it has access to the brain, a subsequent study was
conducted wherein NAX 5055 and GAL (1-16) were both administered
directly into the ventricular space. Results obtained from this
intracerebroventricular (i.c.v.) study are summarized in FIG. 22.
As shown in FIG. 22, both analogs were very potent (i.e.,
ED.sub.50's:0.07 and 1.7 nmoles for NAX 5055 and GAL(1-16)
respectively) following i.c.v. administration. It is interesting to
note that NAX 5055 may actually be more potent and efficacious than
the native peptide fragment GAL(1-16).
TABLE-US-00028 TABLE 19 PHARMACOLOGY OF 6 Hz MODEL AED 22 mA 32 mA
44 mA Phenytoin 9.4 (4.7-14.9) >60 >60 Lamotrigine 4.4
(2.2-6.6) >60 >60 Ethosuximide 86.9 (37.8-156) 167 (114-223)
600 Levetiracetam 4.6 (1.1-8.7) 19.4 (9.9-36.0) 1089 (787-2650)
Valporic acid 41.5 (16.1-68.8) 126 (94.5-152) 310 (258-335) NAX
5055 <4 2.9 (2.2-4.0) 5.5 (4.6-8.5)
[0380] Structure-Activity Relationships of the NAX 5055 analog. SAR
studies have been carried out in an effort to better understand
structural determinants of its activity following systemic
administration. First, it was tested whether individual chemical
modifications at the C-terminus could produce comparable effects as
compared to the combination of both modifications. As shown below,
none of individual modifications had a long-lasting and potent
anticonvulsant activity, as compared to NAX 5055. The analog 1105-2
exhibited lower potency (ED.sub.50=3.8 mg/kg, as compared to 0.8
mg/kg for 5055), shorter duration of action and toxicity (motor
impairment) not observed in 5055. Presence of the Lys-palm residue
was insufficient to produce any observable anticonvulsant activity.
These results strongly suggested that the combination of both
modifications is very important for pharmacological properties of
the 5055 analog.
TABLE-US-00029 TABLE 20 % Protection at 1, 2 and 4 hours Analog
Structure afforded by a dose of 4 mg/kg, i.p. Gal(1-16)
GWTLNSAGYLLGPHAV (SEQ ID NO: Not active 1) 5055
(Sar)WTLNSAGYLLGPKK(Lys-P)K 100%, 100%, 0% (SEQ ID NO: 56) 1105-2
(Sar)WTLNSAGYLLGPKKKK (SEQ ID 30%, 0%, 0%, toxic NO: 60) 306-5
(Sar)WTLNSAGYLLGPHA(Lys-P) Not active (SEQ ID NO: 68) Note: all
analogs, including NAX 5055 are amidated at the C-terminus.
[0381] Secondly, a role of Lys residues at the C-terminus was
investigated by replacing Lys with .sub.DLys, or Arg, or by
changing a number of Lys residues. Replacing Lys with Arg only
slightly changed the activity of the analog. Replacing Lys with its
isomer .sub.DLys resulted in a longer-lasting analog (1205-2) with
comparable potency (ED.sub.50=1.2 mg/kg). Replacing Gly12-Pro13
with two additional Lys residues maintained the anticonvulsant
activity, but also generated toxicity. Results from these
experiments are summarized below in Table 21:
TABLE-US-00030 % Protection at 1, 2 and 4 hours NAX Structure
afforded by a dose of 4 mg/kg, i.p. 5055
(Sar)WTLNSAGYLLGPKK(Lys-P)K (SEQ 100%, 100%, 0% ID NO: 56) 1205-2
(Sar)WTLNSAGYLLGP.sub.DK.sub.DK(Lys-P).sub.DK 100%, 50%, 75% (SEQ
ID NO: 69) 1205-3 (Sar)WTLNSAGYLLGPRR(Lys-P)R (SEQ 100%, 75%, 0% ID
NO: 70) 1205-4 (Sar)WTLNSAGYLLKKKK(Lys-P)K (SEQ 75%, 100%, 66%,
toxic ID NO: 71)
[0382] Next, functional consequences of central truncation of NAX
5055 were determined. As shown in Table 22, a truncation of
Gal(1-16) by "G12,P13" or "L10,L11,G12,P13" reduced the affinity
toward galanin receptors by at least two orders of magnitude.
Systematic central truncations of the 5055 analog (analogs 1205-5
and 306-3) produced only slightly lower potency in the 6 Hz model
(ED50=2.7 mg/kg both analogs, as compared to 0.8 mg/kg for the 5055
analog) and a shorter duration of action.
TABLE-US-00031 TABLE 22 % Protection at 1, 2 and 4 hours NAX
Structure afforded by a dose of 4 mg/kg, i.p. 5055
(Sar)WTLNSAGYLLGPKK(Lys-P)K (SEQ 100%, 100%, 0% ID NO: 56) 1205-5
(Sar)WTLNSAGYLLKK(Lys-P)K (SEQ ID 100%, 25%, 0% NO: 72) 306-3
(Sar)WTLNSAGYKK(Lys-P)K (SEQ ID 75%, 25%, 0% NO: 66)
[0383] Introduction of backbone spacers, such as 6-aminohexanoic
acid, were also explored between the galanin fragment and the KKKpK
motif might affect the anticonvulsant activity of the analogs. The
analogs 306-2 and 306-4 maintained its anticonvulsant activity
(ED.sub.50=2.95 mg/kg for 306-4). Interestingly, the 306-4 analog
appeared very active in the second response phase of the
inflammatory pain assay in mice at the 2.95 mg/kg dose (ED50 in the
6 Hz model), suggesting that the potency of its anticonvulsant and
antinociceptive activity may not be directly correlated.
TABLE-US-00032 TABLE 23 % Protection at 1, 2 and 4 hours NAX
Structure afforded by a dose of 4 mg/kg, i.p. 5055
(Sar)WTLNSAGYLLGPKK(Lys-P)K (SEQ 100%, 100%, 0% ID NO: 56) 306-2
(Sar)WTLNSAGYLLGP(Ahx)KK(Lys-P)K 100%, 75%, 0% (SEQ ID NO: 65)
306-4 (Sar)WTLNSAGY(Ahx)KK(Lys-P)K (SEQ 50%, 0%, 0% ID NO: 67)
[0384] Lastly, the activity of the 5055 analog missing the
N-terminal sarcosine residue was examined (analog 1205-1:
WTLNSAGYLLGPKK(Lys-P)K). This analog was designed based on the
galanin agonist analog Gal(2-11), also known as AR-M1896: the
assumption was that the removal of the first residue would reduce
the affinity of the analog toward GalR1 subtype, while maintaining
the high affinity toward GalR2 (see binding data for the Gal(2-11)
analog in Table 21). The 1205-1 analog had reduced potency in the 6
Hz limbic seizure model (ED.sub.50=5.7 mg/kg).
[0385] In summary, the SAR results show that a combination of
cationization and lipidization is superior over individual chemical
modifications.
[0386] a) Design and Chemical Synthesis of Galanin Analogs that
Penetrate the BBB.
[0387] Based on the current SAR results, the synthesis and
characterization of the analogs containing a combination of
chemical modifications is carried out. A new strategy for
cationization is employed: polyamine-based compounds, such as
spermine, or lipo-polyamine conjugates are used. Since
glycosylation of peptides is a well-established strategy to improve
their BBB penetration, glycosylated galanin analogs are
employed.
[0388] Cationization.
[0389] Positively charged Lys residues and their combinations with
Lys-palm can improve delivery of the analogs into the CNS. Examples
of these relatively conservative analogs are shown in Table 24.
TABLE-US-00033 TABLE 24 Analogs with modifications of the KKKpK
motif. All analogs are amidated at the C-terminus. Analogs
Rationale (Sar)WTLNSAGYLLGPKK(Lys-P)K NAX 5055 (shown as a
reference) (SEQ ID NO: 56) (Sar)WTLNSAGYLLGP(Orn)(Orn)(Lys-
Replacing Lys with Ornithine (Orn) results in shorter P)(Orn) (SEQ
ID NO: 73) side chains by one methylene group. This addresses a
relative role of hydrophobicity in the BBB-penetration of NAX 5055.
(Sar)WTLNSAGYLLGP(Dab)(Dab)(Lys- Replacing Lys with
2,4-diaminobutyric acid (Dab) P)(Dab) (SEQ ID NO: 74) results in
shorter side chains by two methylene groups. See the rationale
above. (Sar)WTLNSAGYLLGP.sub.bK.sub.bK(Lys-P).sub.bK beta-homo-Lys
is (1) metabolically stable and (2) more (SEQ ID NO: 75)
hydrophobic. (Sar)WTLNSAGYLLGPHH(Lys-P)H Lys-to-His replacement
decreases basicity of the (SEQ ID NO: 76) analog
(Sar)WTLNSAGYLLGP(Lys-P)KKK Changing relative positions of the Lys
and Lys-palm (SEQ ID NO: 77) residues (Sar)WTLNSAGYLLGPK(Lys-P)KK
As above (SEQ ID NO: 78) (Sar)WTLNSAGYLLGPKKK(Lys-P) As above (SEQ
ID NO: 79)
[0390] Since cationization is critical for the penetration of the
galanin analogs through the BBB, galanin analogs containing
polyamines are explored. The rationale behind exploring analogs
containing spermine is to replace several Lys residues (present in
the NAX 5055) with a single molecule carrying several amine groups,
while lacking peptide bonds (reduced susceptibility for proteolysis
and a lack of hydrogen-bonding donors/acceptors. Several galanin
analogs are synthesized in which spermine is either a part of the
backbone or a side chain. Table 25 summarizes structures and a
rationale for the galanin-spermine analogs.
TABLE-US-00034 TABLE 25 NAX 5055 analogs containing spermine as a
backbone spacer or as a side chain. SpermineS is
1,5,10,14-tetra-azaquatrodecan- N4-succinamic acid. All analogs are
amidated at the C-terminus. Analog Rationale
(Sar)WTLNSAGY(SpermineS)(Lys-P) (SEQ ID Spermine-N4succinamic acid
has 18 backbone NO: 80) atoms, replacing in principle 6 AA.
SpermineS plays a role as a backbone spacer replacing "LLGPKK" in
NAX 5055. (Sar)WTLNSAGYLLGPKK(Lys-P)- Replacing of the Lys17 with
spermine will (SpermineS) (SEQ ID NO: 81) increase basicity of the
analog. (Sar)WTLNSAGYLLGPKK(Glu-Spermine)K Replacing of the
Lys-palm with spermine (SEQ ID NO: 82) coupled to side chain of
glutamic acid. (Sar)WTLNSAGYLLGPKK(Lys-Spermine- Replacing Lys-palm
moiety with spermine- Palmitoyl)K (SEQ ID NO: 83) palmitoyl will
increase basicity of the analog.
[0391] Lipidization.
[0392] In the next set of analogs (Table 26), the effects of
lipidization in the position 16 on the penetration of the analogs
through the BBB are explored.
TABLE-US-00035 TABLE 26 Analogs with modifications of in position
16. All analogs are amidated at the C-terminus. Analogs Rationale
(Sar)WTLNSAGYLLGPKK Testing effects of replacing (TDA)K the
Lys-palmityoyl moiety (SEQ ID NO: 84) with a shorter lipoamino
acid: tetradecanoic moiety. (Sar)WTLNSAGYLLGPKK Replacing the
Lys-palmityoyl (NorL)K moiety with norleucine is a (SEQ ID NO: 85)
"truncated" analog missing palmitoyl residue (and .quadrature.amino
group).
[0393] Glycosylation.
[0394] Two glycosylated galanin analogs containing alpha-mannosyl
or beta-melibiose serine residues in the position 16 are
synthesized first. Thus, in these analogs, the saccharide moiety
replaces the Lys-palm residue.
TABLE-US-00036 TABLE 27 Glycosylated galanin analogs. All analogs
are amidated at the C-terminus. Analogs Rationale
(Sar)WTLNSAGYLLGPKK NAX 5055 (shown as a reference) (Lys-P)K (SEQ
ID NO: 56) (Sar)WTLNSAGYLLGPKK Testing effects of replacing (Man)K
Lys-palmityoyl moiety with a (SEQ ID NO: 86) monosaccharide
derivative: L-Ser-alpha-Mannose. (Sar)WTLNSAGYLLGPKK Testing
effects of replacing (Mel)K Lys-palmityoyl moiety with a (SEQ ID
NO: 87) disaccharide derivative: L-Ser-beta-Melibiose. Rationale as
above.
[0395] Backbone Spacers.
[0396] Effects of backbone spacers on the anticonvulsant activity
of the NAX-5055 based analogs are explored. Main advantages of
replacing parts of the peptidic backbone with nonpeptidic-based
spacers are: (1) reducing molecular size that should improve the
BBB-permeability, (2) reducing susceptibility of proteolytic
degradation, and (3) a lack of hydrogen bond donors/acceptors.
Examples of these analogs are shown in Table 28.
TABLE-US-00037 TABLE 28 NAX 5055 analogs containing extended
glycine backbone spacers. All analogs are amidated at the
C-terminus. Analogs Spacer (Sar)WTLNSAGYLL(1PEG)KK(Lys-P)K
5-amino-3- (SEQ ID NO: 88) oxapemtanoic acid
(Sar)WTLNSAGYLL(5AVA)KK(Lys-P)K 5-aminovaleric acid (SEQ ID NO: 89)
(Sar)WTLNSAGYL(2PEG)KK(Lys-P)K 8-amino-3,6- (SEQ ID NO: 90)
dioxaoctanoic acid (Sar)WTLNSAGYL(8AOA)KK(Lys-P)K 8-aminooctanoic
(SEQ ID NO: 91) acid (Sar)WTLNSAGY(1PEG)(5AVA)KK 5-amino-3-
(Lys-P)K (SEQ ID NO: 92) oxapemtanoic acid, 5-aminovaleric acid
[0397] Chemical Synthesis of the Analogs.
[0398] All analogs are synthesized using Fmoc-based solid-phase
peptide synthesis (SPPS) protocols and an automated peptide
synthesizer. All building blocks for SPPS are commercially
available, including Fmoc protected backbone spacers, glycoamino
acids, spermine, spermine-succinamic acid or spermine-palimitoyl
(Sussex Research Laboratories, Iris Biotech, NeoMPS, Chem-Impex).
Coupling methods will be performed as described previously (Fields
and Noble 1990; Albericio 2000). The For the analogs in which to
spermine or its derivatives are conjugated in the position 16, the
analogs have incorporated glutamic acid protected with
gamma-2-phenylisopropyl ester (0-2-PhiPr). The side chain carboxyl
is deprotected on-resin using 1% TFA in dichloromethane. Coupling
of spermine or its palmitoyl derivative (Fmoc/Boc protected on
other amino groups) is carried out using
1,3-diisopropylcarbodiimide (DIC). The peptides are removed from
solid support, washed and precipitated with MTBE. The analogs are
purified using a diphenyl preparative reversed-phase HPLC. Linear
gradient of acetonitrile (in 0.1% TFA) from 20% to 90% of 90%
acetonitrile/10% water/0.1% TFA in 15 minutes is used for elution.
Analogs are quantified using molar absorbance coefficient 7,000 at
280 nm (1 Tpr 5,600 and 1 Tyr 1,400). These procedures have been
efficient for preparing NAX 5055 and similar analogs with
individual batches ranging from 10 to 50 milligrams.
[0399] b) In Vitro Characterization of Galanin Analogs that Cross
BBB.
[0400] In order to characterize biological activity of the
synthesized galanin analogs, binding constants for GalR1 and GalR2
receptors are determined. The three main objectives were: (1) to
assess what effect chemical modifications introduced to GAL(1-16)
change affinity toward galanin receptors, (2) to determine the
selectivity profile for galanin analogs, and (3) to develop a
reliable screening assay of high-affinity galanin analogs that
penetrate the BBB. It has been shown that NAX 5055 retains low
nanomolar affinity toward both GalR1 and GalR2.
[0401] A fluorescence-based binding assay with europium-labeled
galanin (Delfia assay from Perkin Elmer) can be used. This assay,
previously described by (Valenzano, Miller et al. 2000), is
validated and used to characterize binding properties of all
previously synthesized analogs. The main advantage of the
fluorescence-based binding assay over more traditionally used
radioligand binding assay is to avoid drawbacks of radioactivity
(health, disposal, short shelf-life, long duration of acquiring
signals), making such assays more friendly for medium- and
high-throughput screening. Labeling receptor ligands with
lanthanides offers an advantage of high sensitivity due to their
long fluorescence lifetimes. Using time-resolved fluorescence
detection with a delay of 400 microseconds, less that 1 femtomole
of europium can be detected in a single well.
[0402] Two galanin receptors are acquired from Perkin Elmer or
Multispan, Inc. in the form of membrane preparations.
Europium-labeled galanin (Eu-Galanin) is purchased from Perkin
Elmer. Binding reactions are carried out with 10 micrograms of
membrane protein (concentrations) in a volume of 60 microliters of
the binding buffer (EDTA, BSA, PEG in a hypotonic buffer).
Saturation binding curves are generated with a range of Eu-galanin
from 0.01 to 10 nM ligand concentration. For determining binding
constants, 0.2 nM Eu-Galanin is incubated with membranes for 2
hours. The reactions is terminated by a rapid filtration through
AcroWell filter plates using a vacuum box, and washed three times
with 300 mL of hypotonic buffer. Enhancement solution is added to
each well and the TRF signal is recorded on Victor3
spectrofluorimeter with TFR.
[0403] c) In Vivo Characterization of Galanin Analogs that Cross
the BBB
[0404] The anticonvulsant activity of galanin-based neuropeptides
in a pharmaco-resistant model of epilepsy and their
antiepileptogenic properties.
[0405] Selected analogs in the 6 Hz limbic seizure model of
pharmaco-resistant partial epilepsy are characterized. The potency
of the analogs in this model of pharmaco-resistant epilepsy is
determined by generating dose-response curves following i.p.
administration. All compounds are administered i.p. or in 0.9% NaCl
in a volume of 0.01 ml/g body weight. In addition to the acute
efficacy studies in the 6 Hz seizure test, the ability of NAX 5055
and other galanin analogs to prevent kindling acquisition in the
mouse corneal kindling model of partial epilepsy is analyzed
(Matagne and Klitgaard 1998).
[0406] Anticonvulsant Testing.
[0407] The ability of each analog to prevent seizures induced by 6
Hz corneal stimulation (3 sec duration) is assessed at three
different stimulus intensities (i.e., 22, 32, and 44 mA). The 6 Hz
seizure is characterized by a minimal clonic phase that is followed
by stereotyped, automatistic behaviors described originally as
being similar to the aura of human patients with partial seizures
(Toman, Everett et al. 1952; Barton, Klein et al. 2001). Animals
not displaying this behavior are considered protected. As mentioned
above, the 6 Hz seizure becomes more resistant to block by
antiepileptic drugs as the current is increased from 22 mA to 44
mA. Activity for each of the analogs in the 6 Hz seizure test is
quantitated at the time to peak effect following i.p. according to
the methods described by (Barton, Klein et al. 2001). For this
test, a drop of anesthetic/electrolyte solution (0.5% tetracaine
hydrochloride in 0.9% saline) is applied to the eyes of each animal
prior to placement of the corneal electrodes. For the time to peak
effect studies, a total of 20 CF-1 mice is employed. Groups of 4
mice are tested at various times (i.e., 0.25, 0.5, 1, 2, and 4 h).
For the dose-response studies, groups of at least eight mice are
tested with various doses of the candidate peptide until at least
two points have been established between the limits of 100%
efficacy and 0% efficacy. The dose of drug required to produce the
median effective dose (i.e., ED.sub.50) in 50% of the animals
exposed, the 95% confidence interval, the slope of the regression
line, and the S.E.M. of the slope is calculated by a computer
program based on the method described by Finney (Finney 1971;
Finney 1971).
[0408] Acquisition of Corneal Kindling.
[0409] NAX 5055 and a select number of other galanin analogs are
evaluated for their ability to prevent the acquisition of kindling
in the mouse corneal kindling model as described by (Matagne and
Klitgaard 1998). Daily electrical stimulation via the cornea
results in a stable kindled state within 15-20 days. This is an
ideal model to initially assess the ability of selected galanin
analogs to prevent kindling acquisition because the amount of
peptide required is much lower than what is required for a rat
kindling study. Those compounds found to be effective in preventing
the acquisition of kindling are subsequently evaluated in a more
traditional rat kindling model; i.e., amygdala kindled rat. For the
proposed studies two groups of mice (n=8 mice per group) are
randomized to receive either vehicle or peptide prior to each
kindling stimulation. Each peptide is administered i.p. at a dose
that approximates the ED.sub.50 for prevention of 6 Hz (44 mA)
seizures. At the time to peak effect (obtained from the 6 Hz
study), they are stimulated via corneal electrodes with a
subconvulsive current (50 Hz, 3 mA, for 3.0 sec) and observed for
the presence or absence of seizure activity. Seizure activity is
scored according to the criteria established by Racine et al.,
(1972). Animals receive two stimulations per day until they display
their first Stage 5 behavioral seizure and then once daily until
they display stable secondarily generalized seizures (5 consecutive
Stage 4-5 seizures). Peptide treatment continues in the
experimental group until the point that the control mice become
fully kindled. At this point, mice in both groups are permitted a
one-week stimulus and treatment-free week. On the 8.sup.th day,
mice in both groups are stimulated in the absence of peptide or
vehicle and their seizure score recorded.
[0410] Mice in the vehicle-treated group can reach a fully kindled
state by the end of the second full week of stimulation (FIGS. 32
and 33). Furthermore, given that i.c.v. galanin has been previously
found to prevent the development of kindling (Mazarati, Lundstrom
et al. 2006), mice in the experimental group do not kindle or
kindle at a rate slower than vehicle-treated mice. Therefore, the
seizure score on the last day of the active kindling study can be
at 1 or less and the seizure score would remain low following the
wash-out period; e.g., treated (antiepileptogenic). If the
post-washout period seizure score of the experimental group is not
significantly different from the vehicle-treated group, it can be
concluded that the effect of the peptide during the active
treatment period was due to its anticonvulsant effect; e.g.,
treated (anticonvulsant). When an antiepileptogenic effect is
observed, all mice in both the vehicle- and treated-groups are
kindled in the absence of drug to see whether the previously
treated mice kindle in the absence of peptide.
5. Example 5: Design and Chemical Synthesis of Analogs
[0411] a) Neuropeptide Analogs that Penetrate the BBB.
[0412] Table 29 summarizes structures of several neuropeptide
analogs. For each of the analogs, the "KKK.sub.pK" motif is
attached to either N- or C-terminus during the solid-phase peptide
synthesis. Below, provided is a brief rationale for designing
analogs of each neuropeptide.
TABLE-US-00038 TABLE 29 Structures of selected neuropeptides.
Neuropeptide Structure SOM Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-
Thr-Phe-Thr-Ser-Cys (SEQ ID NO: 30) Octreotide-NH2
.sub.DPhe-Cys-Phe-.sub.DTrp-Lys-Thr-Cys- Thr-NH.sub.2 (SEQ ID NO:
101) DSIP Trp-Ala-Gly-Gly-Asp-Phe-Ser-Gly-Glu (SEQ ID NO: 102)
Dynorphin Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg- A(1-16)
Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln (SEQ ID NO: 103) NPY(13-36)
PAEDLARYYSALRAYINLITRQRY- NH.sub.2 (SEQ ID NO: 104)
[0413] Somatostatin and its subtype-selective analog, octreotide,
can "accommodate" the N-terminal extensions without compromising
their bioactivity (Dasgupta and Mukherjee 2000; Dasgupta, Singh et
al. 2002; Na, Murry et al. 2003). N-terminal acetylation of the
RC-160 analog resulted in an increase of the CNS concentrations of
this somatostatin analog (Banks, Schally et al. 1990). Octreotide
can also penetrate the BBB to some extent without additional
modifications (Fricker, Nobmann et al. 2002).
[0414] DSIP.
[0415] It has been shown that the N-terminal extensions did not
affect antiepileptic activity of DSIP, whereas the C-terminal
extensions resulted in inactive analogs. Thus, DSIP analogs can be
created with vectors attached to the N-terminus.
[0416] The "KKK.sub.pK" motif is introduced to the N-terminus of
SOM, octreotide or DSIP. Attaching the "KKK.sub.pK" motif using
three distinct spacers (Gly, 6-aminohexanoix acid (Ahx), Ahx-Gly)
minimizes the possibility that the bulky Lys-palm residue can
affect interactions with a target receptor. The following analogs
are synthesized:
TABLE-US-00039 (SEQ ID NO: 105) KK(K.sub.p)K-(neuropeptide) (SEQ ID
NO: 106) KK(K.sub.p)KG-(neuropeptide) (SEQ ID NO: 107)
KK(K.sub.p)K(Ahx)-(neuropeptide) (SEQ ID NO: 108)
KK(K.sub.p)K(Ahx)G-(neuropeptide)
[0417] Dynorphin A(1-16) can be truncated or modified at the
C-terminus, without significant reduction of the affinity toward
opioid receptors (Lapalu, Moisand et al. 1997; Naqvi, Haq et al.
1998; Schlechtingen, DeHaven et al. 2003). Thus, similar to galanin
analogs that penetrate the BBB, dynorphin A (1-16) analogs are
synthesized in which the last several residues are replaced by the
"KKKpK" motif. The following analogs are shown:
TABLE-US-00040 (SEQ ID NO: 109)
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-
Lys-Lys-(Lys-palm)-Lys-NH.sub.2 (SEQ ID NO: 110)
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-
Lys-Lys-Lys-(Lys-palm)-Lys-NH.sub.2 (SEQ ID NO: 111)
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Lys-
Lys-(Lys-palm)-Lys-NH.sub.2 (SEQ ID NO: 112)
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-
Lys-(Ahx)-Lys-Lys-(Lys-palm)-Lys-NH.sub.2
[0418] Neuropeptide Y analogs are designed based on data from
numerous SAR studies carried out in the laboratory of Prof Annette
G. Beck-Sickinger (comprehensive reviews (Beck-Sickinger and Jung
1995; Cabrele and Beck-Sickinger 2000)). Specifically, the design
is based on .sup.99mTc-labeled NPY analogs that were synthesized
and tested as tumor imaging agents (Langer, La Bella et al. 2001).
The truncated NPY analog (Ac-[Ahx5-24,K4(99mTc(CO)3-PADA),A26]-NPY,
containing bulky 2-picolylamine-N,N-diacetic acid with chelated
.sup.99mTc was very potent against Y2 receptor subtype (IC.sub.50=1
nM). Thus, 2-picolylamine-N,N-diacetic acid can be replaced with
Lys-palmitoyl residue. The following two NPY analogs are shown:
TABLE-US-00041 (SEQ ID NO: 113)
KKK(K.sub.p)(Ahx)RAYINLITRQRY-NH.sub.2 (SEQ ID NO: 114)
KK(K.sub.p)K(Ahx)RAYINLITRQRY-NH.sub.2
[0419] Four analogs of NPY(13-36) are also produced with the
N-terminal extensions:
TABLE-US-00042 (SEQ ID NO: 115) KK(K.sub.p)K-[NPY(13-36)] (SEQ ID
NO: 116) KK(K.sub.p)KG-[NPY(13-36)] (SEQ ID NO: 117)
KK(K.sub.p)K(Ahx)-[NPY(13-36)] (SEQ ID NO: 118)
KK(K.sub.p)K(Ahx)G-[NPY(13-36)]
[0420] All analogs are synthesized using standard Fmoc-based
solid-phase peptide synthesis (SPPS) protocols and an automated
peptide synthesizer. All building blocks for SPPS are commercially
available, including Fmoc protected aminohexanoic acid,
Lys-palmitoyl, and glycoamino acids (Sussex Research Laboratories,
Iris Biotech, NeoMPS, Chem-Impex). Coupling methods are performed
as described previously (Fields and Noble 1990; Albericio 2000).
The peptides are removed from solid support with reagent K, washed
and precipitated with MTBE. The analogs are purified using a
diphenyl preparative reversed-phase HPLC. Linear gradient are used
for elution. Initial and final concentrations of 90%
acetonitrile/10% water/0.1% TFA is determined for each analog based
on their retention times from analytical HPLC analysis. Analogs are
quantified using molar absorbance coefficient calculated for each
analog at 280 nm (Trp =5,600 and Tyr =1,400). These procedures have
been efficient for preparing galanin analogs in quantities ranging
from 10 to 50 milligrams. For oxidation of the disulfide bridge in
somatostatin or octreotide analogs, Clear-Ox resin is used (Darlak,
Wiegandt Long et al. 2004; Green and Bulaj 2006). Octreotide
analogs described herein were oxidized with yields exceeding 95%.
Final oxidation products are purified by preparative HPLC.
[0421] Anticonculsant Activity of Neuropeptide Analogs.
[0422] Analogs in the 6 Hz limbic seizure model of
pharmaco-resistant partial epilepsy are characterized. The activity
of the analogs following i. c. v. and i.p. administration can be
determined. The strategy is to test the analogs i. c. v. (2 nmoles)
first: active analogs are further tested using i.p. bolus
injections (4 mg/kg). Anticonvulsant activity of dynorphin A(1-16),
NPY and NPY(13-36) is also tested at 2 nmoles, following i.c.v.
administration. These results serve as a reference for screening
their modified analogs.
[0423] Anticonvulsant Testing.
[0424] The ability of each analog to prevent seizures induced by 6
Hz corneal stimulation (3 sec duration) is assessed at 32 mA
stimulus intensity. The 6 Hz seizure is characterized by a minimal
clonic phase that is followed by stereotyped, automatistic
behaviors described originally as being similar to the aura of
human patients with partial seizures (Toman, Everett et al. 1952;
Barton, Klein et al. 2001). Animals not displaying this behavior
are considered protected. Activity for each of the analogs in the 6
Hz seizure test are quantitated at the time to peak effect
following i.c.v or i.p. according to the methods described by
(Barton, Klein et al. 2001). For this test, a drop of
anesthetic/electrolyte solution (0.5% tetracaine hydrochloride in
0.9% saline) is applied to the eyes of each animal prior to
placement of the corneal electrodes. For the time to peak effect
studies, a total of 20 CF-1 mice are employed. Groups of 4 mice are
tested at various times (i.e., 0.25, 0.5, 1, 2, and 4 h). All
compounds are administered in 0.9% NaCl in a volume of 0.01 ml/g
body weight.
[0425] Design and Chemical Synthesis of Glycosylated Neuropeptide
Analogs.
[0426] Glycosylation appeared very effective in improving
penetration of opioid peptides through the BBB (Elmagbari, Egleton
et al. 2004; Polt, Dhanasekaran et al. 2005). FIG. 34 shows
structures of sugar residues that were used for enkephalin analogs.
Introduction of .beta.-melibiose into the peptide produced analog
with the best analgesic potency following i. v. administration.
[0427] .beta.-melibiose-Ser residue can be introduced into selected
neuropeptide analogs in place of either the full-length "KKKpK"
motif or Lys-palmitoyl residue (as described above). For chemical
synthesis of the glycosylated analogs, an Fmoc-protected
peracetyl-.beta.-melibiose-Ser derivative available from Sussex
Research is used. The standard solid-phase synthesis protocol is
applied. Deacetylation of melibiose residue is accomplished by
pH-controlled (pH 10) incubation of analogs in 10 mM sodium
methoxide in methanol for 4-5 hours. The analogs are purified by
preparative HPLC and dried in a speedvac prior to testing their
anticonvulsant activity.
6. Example 6: Effects of NAX5055 on Mouse Corneal Kindling
Acquisition
[0428] The ability of the blood-brain-barrier penetrant
galanin-based neuropeptide NAX-5055 to prevent the development of
corneal kindling was studied in CF#1 mice. The mouse corneal
kindling model is a non-intrusive animal model of partial epilepsy
wherein mice receive an initially subconvulsive current (3 mA) for
3 seconds via corneal electrodes twice daily for several days.
Vehicle treated mice usually require between 12-16 days to reach a
stable Stage 5 seizure; i.e., a secondary generalized focal
seizure. In the present study, 16 mice were randomized to one of
two experimental groups; saline or NAX-5055. Mice in the NAX-5055
group received an intraperitoneal (i.p.) injection of NAX-5055 (4
mg/kg, n=8) 12 hours and 1 hour prior to their first corneal
stimulation and 1 hour prior to each subsequent stimulation. In
contrast, mice in the vehicle group received 0.9% NaCl (n=8) one
hour prior each corneal stimulation. Prior to each stimulation a
drop of 0.5% tetracaine in 0.9% saline was applied to the cornea.
Following stimulation seizure activity was scored on a scale of 0-5
as established by Racine (1972). Treatment was continued until
control animals consistently displayed stage 5 seizures.
[0429] As shown in FIGS. 32 and 33, mice in the NAX-5055 group
segregated into two separate populations; i.e., those that were
sensitive (n=3) and those that were insensitive to NAX-5055 (n=5)
treatment. Sensitivity was defined as those mice that failed to
display a Stage 5 seizure during the period of stimulation.
NAX-5055 sensitive animals required a significantly greater number
of stimulations to reach stage 1, 2 and 3 compared to both controls
and NAX-5055 insensitive animals. Furthermore, upon re-challenge
after a one-week stimulation and peptide free period, NAX-5055
sensitive animals required significantly more stimulations to reach
stage 4/5 seizures compared to controls and NAX-5055 insensitive
animals. These results show that NAX-5055 can delay kindling
acquisition and thus may be useful for the early treatment of
patients at risk for the development of epilepsy following a given
brain insult. (Racine R J. Modification of seizure activity by
electrical stimulation. II. Motor seizure. Electroencephalogr Clin
Neurophysiol. 1972 March; 32(3):281-94.)
[0430] Table 30 shows anticonvulsant activity of octreotide or DSIP
analogs following systemic delivery.
TABLE-US-00043 % Protection at 30 min (4 Analogs mg/kg, i.p.)
Octreotide analogs
.sub.DPhe-Cys-Phe-.sub.DTrp-Lys-Thr-Cys-Thr-N.sub.2 50%
Lys-Lys-Lys(palm)-Lys-Ahx-.sub.DPhe- 100%
Cys-Phe-.sub.DTrp-Lys-Thr-Cys-Thr-NH.sub.2 DSIP analogs
Trp-Ala-Gly-Gly-Asp-Phe-Ser-Gly-Glu 0%
Ahx-Gly-Gly-Trp-Ala-Gly-Gly-Asp-Phe- 50% Ser-Gly-Glu (100% after 2
hours)
[0431] Table 31 shows additional Delta Sleep Inducing Peptides:
TABLE-US-00044 Analog Structure DSIP-BBB8 (Ahx)GGWAGGDASGE (SEQ ID
NO: 136) DSIP-BBB99 (Palm)GGWAGGDASGE (SEQ ID NO: 137) DSIP-BBB100
(K.sub.p)GGWAGGDASGE (SEQ ID NO: 138) DSIP-BBB101
KKK(K.sub.p)GGWAGGDASGE (SEQ ID NO: 139) DSIP-BBB102
KK(K.sub.p)KGGWAGGDASGE (SEQ ID NO: 140) DSIP-BBB103 KKKGGWAGGDASGE
(SEQ ID NO: 141) DSIP-BBB104 (DK)(DK)(DK)(Kp)GGWAGGDASGE (SEQ ID
NO: 58)
G. SEQUENCES
TABLE-US-00045 [0432] SEQ ID NO: 1 (Wild type Galanin) Gly Trp Thr
Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Ala Val (GAL-BBB1) SEQ
ID NO: 2 Sar-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-
Gly-Pro-His-(Lys-palm)-Tle-NH2 (where Sar is sarcosine, Tle is
tert-Leucine and Lys-palm is lysine residue coupled with palmityoyl
moiety via epsilon amino group and --NH2 denotes amidation at the
C-terminus) (GAL-BBB2) SEQ ID NO: 3
Sar-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-
Gly-Pro-Lys-Lys-(Lys-palm)-Lys-NH2 (where Sar is sarcosine, Tle is
tert-Leucine and Lys-palm is lysine residue coupled with palmityoyl
moiety via epsilon amino group and --NH2 denotes amidation at the
C-terminus) (From Table 11) SEQ ID NO: 4 Sar Trp Thr Leu Asn Ser
Ala Gly Tyr Leu Leu Gly Pro Lys Lys Lys-P Lys (From Table 11) SEQ
ID NO: 5 Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Lys
Lys-P Lys (From Table 11) SEQ ID NO: 6 Sar Trp Thr Leu Asn Ser Ala
Gly Tyr Leu Leu Lys Lys Lys-P Lys (From Table 11) SEQ ID NO: 7 Sar
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Lys Lys Lys-P Lys (From Table
11) SEQ ID NO: 8 Sar Trp Thr Leu Asn Ser Ala Gly Tyr Lys Lys Lys-P
Lys (From Table 12) SEQ ID NO: 9 Sar Trp Thr Leu Asn Ser Ala Gly
Tyr Leu Leu Gly Pro Lys Lys Lys-P Lys (From Table 12) SEQ ID NO: 10
Sar Trp Xaa Xaa Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Lys-P
Lys (From Table 12) SEQ ID NO: 11 Sar Trp Thr Leu Asn Xaa Xaa Gly
Tyr Leu Leu Gly Pro Lys Lys Lys-P Lys (From Table 12) SEQ ID NO: 12
Sar Trp Thr Leu Asn Ser Xaa Xaa Tyr Leu Leu Gly Pro Lys Lys Lys-P
Lys (From Table 12) SEQ ID NO: 13 Sar Trp Thr Leu Asn Ser Ala Gly
Tyr Xaa Xaa Gly Pro Lys Lys Lys-P Lys (From Table 12) SEQ ID NO: 14
Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Xaa Xaa Pro Lys Lys Lys-P
Lys (From Table 12) SEQ ID NO: 15 Sar Trp Thr Leu Asn Ser Ala Gly
Tyr Leu Leu Xaa Xaa Lys Lys Lys-P Lys (From Table 12) SEQ ID NO: 16
Sar Trp Thr Leu Asn Xaa Xaa Xaa Tyr Leu Leu Gly Pro Lys Lys Lys-P
Lys (From Table 12) SEQ ID NO: 17 Sar Trp Xaa Xaa Asn Xaa Xaa Xaa
Tyr Leu Leu Gly Pro Lys Lys Lys-P Lys (In the sequences form Table
12 (SEQ ID NOS: 9-17) "Xaa" represents a spacer and can be any
length). (From Table 13) SEQ ID NO: 18 Sar Trp Thr Leu Asn Ser Ala
Gly Tyr Leu Leu Gly Pro Lys Lys Lys-P Lys (From Table 13) SEQ ID
NO: 19 Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Lys Lys
Lys-P Lys (From Table 13) SEQ ID NO: 20 Sar Trp Thr Leu Asn Ser Ala
Gly Tyr Leu Leu Lys Lys Lys Lys Lys-P Lys (From Table 13) SEQ ID
NO: 21 Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys
Lys Lys (From Table 13) SEQ ID NO: 22 Sar Trp Thr Leu Asn Ser Ala
Gly Tyr Leu Leu Gly Pro Lys Lys-P Lys Lys (From Table 13) SEQ ID
NO: 23 Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys-P
Lys Lys Lys (From Table 13) SEQ ID NO: 24 Sar Trp Thr Leu Asn Ser
Ala Gly Tyr Leu Leu Gly Pro D-Lys D-Lys Lys-P D-Lys (From Table 13)
SEQ ID NO: 25 Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro
h-Lys h-Lys Lys-P h-Lys (From Table 13) SEQ ID NO: 26 Sar Trp Thr
Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro DAB DAB Lys-P DAB (From
Table 13) SEQ ID NO: 27 Sar Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu
Gly Pro Lys Lys TDA Lys (From Table 13) SEQ ID NO: 28 Sar Trp Thr
Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys DPA Lys
[0433] In the above sequences from Table 3 (SEQ ID NOS: 18-28), TDA
is 2-amino-tetradecanoic acid, DAB is diaminoobyturicc acid, D-Lys
is D-isomer of Lys and h-Lys is homo-Lys, DPA is
3,3-diphenylalanine
TABLE-US-00046 (From Table 13) SEQ ID NO: 29 Sar Trp Thr Leu Asn
Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Lys-P Lys X where X
denotes: 12-amino-dodecanoic acid or 2-amino-tetradecanoic acid.
(Somatostatin Wild Type) SEQ ID NO: 30 Ala Gly Cys Lys Asn Phe Phe
Trp Lys Thr Phe Thr Ser Cys (From Table 5) SEQ ID NO: 31 Ala Ala
Cys Lys DAB Phe Phe D-Trp Lys DAP Phe DAP DAP Cys (From Table 5)
SEQ ID NO: 32 Ala Gly Cys Lys-P Lys-P Phe Phe D-Trp Lys Thr Phe Thr
Ser Cys (From Table 5) SEQ ID NO: 33 Ala Gly Cys Lys Asn Cl-Phe
Cl-Phe D-Trp Lys Thr Cl-Phe Thr Ser Cys (From Table 5) SEQ ID NO:
34 Ala Ala Cys Lys DAB Phe Phe L-Trp Lys DAP Phe DAP DAP Cys (From
Table 5) SEQ ID NO: 35 Ala Gly Cys Lys-P Lys-P Phe Phe L-Trp Lys
Thr Phe Thr Ser Cys (From Table 5) SEQ ID NO: 36 Ala Gly Cys Lys
Asn Cl-Phe Cl-Phe L-Trp Lys Thr Cl-Phe Thr Ser Cys
[0434] In the above sequences from Table 5, DAB is diaminobutyric
acid; DAP is diaminopropionic acid; Lys-palm is Lys-palmitoyl; and
Cl-Phe is chloro-Phe.
TABLE-US-00047 (From Table 9) SEQ ID NO: 37 Sar Trp Thr Leu Asn Ser
Ala Gly Tyr Leu Leu Gly Lys-p Lys-p Lys-p Val (From Table 9) SEQ ID
NO: 38 Sar Trp DAP Leu Asn DAP DAP Gly Tyr Leu Leu Gly DAB His DAP
DAB (From Table 9) SEQ ID NO: 39 Sar Trp Thr Leu Asn Ser Ala Gly
Cl-Tyr Leu Leu Gly Pro His Ala Val (Octreotide with modifications)
SEQ ID NO: 40 D-Phe Cys Phe D-Trp Lys Thr Cys Thr(ol) (SOM-BBB1)
SEQ ID NO: 41 (NN3APG)(AHX)AGCKNFFWKTFTSC (SOM-BBB2) SEQ ID NO: 42
(NN3APG)(AHX)AGCKNFF(DW)KT(Cl-Phe)T(Dap)C (SOM-BBB3) SEQ ID NO: 43
W(AHX)KKCKNFF(DW)KT(Cl-Phe)(Dab)(Dab)C (SOM-BBB21) SEQ ID NO: 44
KK(Lys-P)K(AHX)(DF)CF(DW)KTC-Thr(ol) (SOM-BBB22) SEQ ID NO: 45
KKK(Lys-P)K(AHX)(AHX)(DF)CF(DW)KTC-Thr(ol) (SOM-BBB23) SEQ ID NO:
46 (Lys-P)KK(Lys-P)K(AHX)(DF)CF(DW)KTC-Thr(ol) (SOM-BBB24) SEQ ID
NO: 47 KK(Lys-P)K(AHX)KK(Lys-P)K(AHX)(DF)CF(DW)KTC- Thr(ol)
(SOM-BBB25) SEQ ID NO: 48
(PFHA)K(DK)K(ACPA)KK(Lys-P)K(AHX)(DF)CF(DW)KTC- Thr(ol)
[0435] For SEQ ID NOS: 41-48, (AHX) is aminohexanoic acid;
(Dab)=diaminobutyric acid; (Dap)=diaminopropionic acid;
(Tle)=tert-Leucine; (Cl-Phe)=4-chlorophenylalanine;
(NN3APG)=N,N-bis(3-aminopropyl)glycine; (AHX)=aminohexanoic acid;
(Lys-P)=Lys-palmitoyl; Thr(ol)=Threoninol; DK, DF, DW denotes
D-isomer; PFHA is 2H, 2H, 3H, 3H-perfluoroheptanoic acid; and ACPA
is 8-aminocaprylic acid.
TABLE-US-00048 GAL-BBB3 SEQ ID NO: 49 WTLNSAGYLLGPKKXK-NH2 GAL-BBB4
SEQ ID NO: 50 Sar-WTLNSAGYLLGP(D-Lys)(D-Lys)X(D-Lys)-NH.sub.2
GAL-BBB5 SEQ ID NO: 51 Sar-WTLNSAGYLLGPRRXR-NH2 GAL-BBB6 SEQ ID NO:
52 Sar-WTLNSAGYLLGPHHXH-NH2 GAL-BBB7 SEQ ID NO: 53
Sar-WTLNSAGYLLKKKKXK-NH2 GAL-BBB8 SEQ ID NO: 54
Sar-WTLNSAGYLLKKXK-NH2 In SEQ ID NOS: 49-54, Sar is sarcosine and X
is Lys-palmitoyl residue. DSIP-BBB8 SEQ ID NO: 55 (AHX)GGWAGGDASGE
SEQ ID NO: 56 (Sar)WTLNSAGYLLGPKK(Lys-P)K SEQ ID NO: 57
WTLNSAGYLLGPKK(Lys-P)K SEQ ID NO: 58 (DK)(DK)(DK)(Kp)GGWAGGDASGE
SEQ ID NO: 59 (Sar)WTLNSAGYLLGPRR(Lys-P)R SEQ ID NO: 60
(Sar)WTLNSAGYLLKKKK(Lys-P)K SEQ ID NO: 61 (Sar)WTLNSAGYLLGPKKKK SEQ
ID NO: 62 (Sar)WTLNSAGYLLKK(Lys-P)K SEQ ID NO: 63
(Sar)WTLNSAGYKK(Lys-P)K SEQ ID NO: 64
(Sar)WTLNSAGYLLGP(Ahx)KK(Lys-P)K SEQ ID NO: 65
(Sar)WTLNSAGY(Ahx)KK(Lys-P)K SEQ ID NO: 66 (Sar)WTLNSAGYKK(Lys-P)K
SEQ ID NO: 67 (Sar)WTLNSAGY(Ahx)KK(Lys-P)K SEQ ID NO: 68
(Sar)WTLNSAGYLLGPHA(Lys-P) SEQ ID NO: 69
(Sar)WTLNSAGYLLGP.sub.DK.sub.DK(Lys-P).sub.DK SEQ ID NO: 70
(Sar)WTLNSAGYLLGPRR(Lys-P)R SEQ ID NO: 71
(Sar)WTLNSAGYLLKKKK(Lys-P)K SEQ ID NO: 72 (Sar)WTLNSAGYLLKK(Lys-P)K
SEQ ID NO: 73 (Sar)WTLNSAGYLLGP(Orn)(Orn)(Lys-P)(Orn) SEQ ID NO: 74
(Sar)WTLNSAGYLLGP(Dab)(Dab)(Lys-P)(Dab) SEQ ID NO: 75
(Sar)WTLNSAGYLLGPbKbK(Lys-P)bK SEQ ID NO: 76
(Sar)WTLNSAGYLLGPHH(Lys-P)H SEQ ID NO: 77
(Sar)WTLNSAGYLLGP(Lys-P)KKK SEQ ID NO: 78
(Sar)WTLNSAGYLLGPK(Lys-P)KK SEQ ID NO: 79
(Sar)WTLNSAGYLLGPKKK(Lys-P) SEQ ID NO: 80
(Sar)WTLNSAGY(SpermineS)(Lys-P) SEQ ID NO: 81
(Sar)WTLNSAGYLLGPKK(Lys-P)-(SpermineS) SEQ ID NO: 82
(Sar)WTLNSAGYLLGPKK(Glu-Spermine)K SEQ ID NO: 83
(Sar)WTLNSAGYLLGPKK(Lys-Spermine-Palmitoyl)K SEQ ID NO: 84
(Sar)WTLNSAGYLLGPKK(TDA)K SEQ ID NO: 85 (Sar)WTLNSAGYLLGPKK(NorL)K
SEQ ID NO: 86 (Sar)WTLNSAGYLLGPKK(Man)K SEQ ID NO: 87
(Sar)WTLNSAGYLLGPKK(Mel)K SEQ ID NO: 88
(Sar)WTLNSAGYLL(1PEG)KK(Lys-P)K SEQ ID NO: 89
(Sar)WTLNSAGYLL(5AVA)KK(Lys-P)K SEQ ID NO: 90
(Sar)WTLNSAGYL(2PEG)KK(Lys-P)K SEQ ID NO: 91
(Sar)WTLNSAGYL(8AOA)KK(Lys-P)K SEQ ID NO: 92
(Sar)WTLNSAGY(1PEG)(5AVA)KK(Lys-P)K SEQ ID NO: 93
GWTLNSAGYLLGPHAVGNHRSFSDKNGLTS-.sub.COOH SEQ ID NO: 94
GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-.sub.NH2 SEQ ID NO: 95
GWTLNSAGYLLGPHAIDNHRSFHDKYGLA-.sub.NH2 SEQ ID NO: 96
GWTLNSAGYLLGPHAI SEQ ID NO: 97 GWTLNSAGYLLGPH SEQ ID NO: 98
GWTLNSAGYLLG SEQ ID NO: 99 GWTLNSAGYL SEQ ID NO: 100 GWTLNSAGY
[0436] In the above sequences:
[0437] Ahx=aminohexanoic acid
[0438] .sub.DK=D-isomer of lysine
[0439] om=ornithine
[0440] Dab=2,4 diaminobutyric acid
[0441] .sub.bK=beta-homo-lysine
[0442] spermineS=spermine-N.sub.4 succinic acid
[0443] TDA=tetradecanoic acid
[0444] NorL=norleucine
[0445] man=L-Ser-beta-melibiose
[0446] 1-PEG=5-amino-3-oxapemtanoic acid
[0447] SAVA=5-aminovaleric acid
[0448] 2-PEG=8-amino-3,6-dioxaoctanoic acid
[0449] 8AOA=8-amino octanoic acid.
TABLE-US-00049 SEQ ID NO: 101
.sub.DPhe-Cys-Phe-.sub.DTrp-Lys-Thr-Cys-Thr-NH.sub.2 SEQ ID NO: 102
Trp-Ala-Gly-Gly-Asp-Phe-Ser-Gly-Glu SEQ ID NO: 103
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-
Lys-Trp-Asp-Asn-Gln SEQ ID NO: 104
PAEDLARYYSALRAYINLITRQRY-NH.sub.2 SEQ ID NO: 105 KK(Kp)KX (where X
can be any length and any amino acid) SEQ ID NO: 106 KK(Kp)KGX
(where X can be any length or any amino acid) SEQ ID NO: 107
KK(Kp)K(Ahx)-(neuropeptide) where X can be any length or any amino
acid) SEQ ID NO: 108 KK(Kp)K(Ahx)G-(neuropeptide) (where X can be
any length or any amino acid) SEQ ID NO: 109
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-
Lys-Lys-(Lys-palm)-Lys-NH.sub.2 SEQ ID NO: 110
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-
Lys-Lys-Lys-(Lys-palm)-Lys-NH.sub.2 SEQ ID NO: 111
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Lys-
Lys-(Lys-palm)-Lys-NH.sub.2 SEQ ID NO: 112
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-
Lys-(Ahx)-Lys-Lys-(Lys-palm)-Lys-NH.sub.2 SEQ ID NO: 113
KKK(Kp)(Ahx)RAYINLITRQRY-NH.sub.2 SEQ ID NO: 114
KK(Kp)K(Ahx)RAYINLITRQRY-NH.sub.2 SEQ ID NO: 115 KK(Kp)K-[X(13-36)]
(wherein X(13-36) represents a neuropeptide Y analog) SEQ ID NO:
116 KK(Kp)KG-[X(13-36)] (wherein X(13-36) represents a neuropeptide
Y analog) SEQ ID NO: 117 KK(Kp)K(Ahx)-[X(13-36)] (wherein X(13-36)
represents a neuropeptide Y analog) SEQ ID NO: 118
KK(Kp)K(Ahx)G-[X(13-36)] (wherein X(13-36) represents a
neuropeptide Y analog) SEQ ID NO: 119
(Sar)WTLNSAGY(D-Lys)(D-Lys)(Lys-P)(D-Lys) SEQ ID NO: 120
(Sar)WTLNSAGY(Ahx)(D-Lys)(D-Lys)(Lys-P)(D-Lys) SEQ ID NO: 121
(Sar)WTLNSAGY(7-Ahp)(D-Lys)(D-Lys)(Lys-P)(D-Lys) SEQ ID NO: 122
(Sar)WTLNSAGY(3,5-dibromo-Tyr)LLGPKK(Lys-P)K SEQ ID NO: 123
(Sar)WTLNSAGYLLGPHH(Lys-P)K SEQ ID NO: 124
(Sar)WTLNSAGYLLGPKK(Cys-Mmt)K SEQ ID NO: 125
(Sar)WTLNSAGYLLGPKK(Lys-Biotin-aminocaproyl)K SEQ ID NO: 126
(Sar)WTLNSAGYLLGPKK(Lys-sterol)K SEQ ID NO: 127
(Sar)WTLNSAGYLLGPKK(Lys-decanoyl)K SEQ ID NO: 128
(Sar)WTLNSAGYLLGPKK(Lys-octanoyl)K SEQ ID NO: 129
(Sar)WTLNSAGYLLGPKK(Lys-linoyl)K SEQ ID NO: 130
(Sar)WTLNSAGYLLGPKK(Ser-melbiose)K SEQ ID NO: 131
(Sar)WTLNSAGYLLGPKK(Lys-adamentoyl)K SEQ ID NO: 132
(Sar)WTLNSAGYLLGPKK(Glu(.beta.-Lac-PEG3-amine))K SEQ ID NO: 133
(Sar)WTLTSAGYLLGPKK(Lys-palmitoyl)K SEQ ID NO: 134
(Sar)WTLLSAGYLLGPKK(Lys-palmitoyl)K SEQ ID NO: 135
(Sar)WTLDSAGYLLGPKK(Lys-palmitoyl)K SEQ ID NO: 136 (Ahx)GGWAGGDASGE
SEQ ID NO: 137 (Palm)GGWAGGDASGE SEQ ID NO: 138 (Kp)GGWAGGDASGE SEQ
ID NO: 139 KKK(Kp)GGWAGGDASGE SEQ ID NO: 140 KK(Kp)KGGWAGGDASGE SEQ
ID NO: 141 KKKGGWAGGDASGE
[0450] For SEQ ID NOS: 58 and 135-141, (.sub.DK) is D-Lys,
(K.sub.p) is Lys-palmitoyl, Axh is aminohexanoic acid, Palm is
palmitic acid.
Sequence CWU 1
1
149116PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic Construct 1Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu
Gly Pro His Ala Val1 5 10 15 216PRTArtificial SequenceDescription
of Artificial Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES15Xaa = Lys-palmMOD_RES16Xaa =
tert-LeucineMOD_RES16Amidation at the C-terminus 2Xaa Trp Thr Leu
Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Xaa Xaa1 5 10 15
317PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa =
Lys-palmMOD_RES17Amidation at the C-terminus 3Xaa Trp Thr Leu Asn
Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Xaa1 5 10 15
Lys417PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa = Lys-palm 4Xaa
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Xaa1 5 10
15 Lys516PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES15Xaa = Lys-palm 5Xaa
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Lys Xaa Lys1 5 10
15 615PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES14Xaa = Lys-palm 6Xaa
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Lys Lys Xaa Lys1 5 10 15
714PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES13Xaa = Lys-palm 7Xaa
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Lys Lys Xaa Lys1 5 10
813PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES12Xaa = Lys-palm 8Xaa
Trp Thr Leu Asn Ser Ala Gly Tyr Lys Lys Xaa Lys1 5 10
916PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa = Lys-palm 9Xaa
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Xaa1 5 10
15 1017PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES(3)...(4)Xaa =
represents spacer between residue 2 and residue 5MOD_RES16Xaa =
Lys-palm 10Xaa Trp Xaa Xaa Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys
Lys Xaa1 5 10 15 Lys1117PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES(6)...(7)Xaa = represents spacer between residue 5 and
residue 8MOD_RES16Xaa = Lys-palm 11Xaa Trp Thr Leu Asn Xaa Xaa Gly
Tyr Leu Leu Gly Pro Lys Lys Xaa1 5 10 15 Lys1217PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES(7)...(8)Xaa = represents spacer
between residue 6 and residue 9MOD_RES16Xaa = Lys-palm 12Xaa Trp
Thr Leu Asn Ser Xaa Xaa Tyr Leu Leu Gly Pro Lys Lys Xaa1 5 10 15
Lys1317PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES(10)...(11)Xaa =
represents spacer between residue 9 and residue 12MOD_RES16Xaa =
Lys-palm 13Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Xaa Xaa Gly Pro Lys
Lys Xaa1 5 10 15 Lys1417PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES(11)...(12)Xaa = represents spacer between residue 10
and residue 13MOD_RES16Xaa = Lys-palm 14Xaa Trp Thr Leu Asn Ser Ala
Gly Tyr Leu Xaa Xaa Pro Lys Lys Xaa1 5 10 15 Lys1517PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES(12)...(13)Xaa = represents
spacer between residue 11 and residue 14MOD_RES16Xaa = Lys-palm
15Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Xaa Xaa Lys Lys Xaa1
5 10 15 Lys1617PRTArtificial SequenceDescription of Artificial
Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES(6)...(8)Xaa = represents spacer between residue 5 and
residue 9MOD_RES16Xaa = Lys-palm 16Xaa Trp Thr Leu Asn Xaa Xaa Xaa
Tyr Leu Leu Gly Pro Lys Lys Xaa1 5 10 15 Lys1717PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES(3)...(4)Xaa = represents spacer
between residue 2 and residue 5MOD_RES(6)...(8)Xaa = represents
spacer between residue 5 and residue 9MOD_RES16Xaa = Lys-palm 17Xaa
Trp Xaa Xaa Asn Xaa Xaa Xaa Tyr Leu Leu Gly Pro Lys Lys Xaa1 5 10
15 Lys1817PRTArtificial SequenceDescription of Artificial Sequence
= Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa = Lys-palm
18Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Xaa1
5 10 15 Lys1917PRTArtificial SequenceDescription of Artificial
Sequence = Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa =
Lys-palm 19Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Lys
Lys Xaa1 5 10 15 Lys2017PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES16Xaa = Lys-palm 20Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Lys Lys Lys Lys Xaa1 5 10 15 Lys2117PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGly 21Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro Lys Lys Lys1 5 10 15 Lys2217PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES15Xaa = Lys-palm 22Xaa Trp Thr
Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Xaa Lys1 5 10 15
Lys2317PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES14Xaa = Lys-palm 23Xaa
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Xaa Lys Lys1 5 10
15 Lys2417PRTArtificial SequenceDescription of Artificial Sequence
= Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES14, 15, 17Xaa =
D-LysMOD_RES16Xaa = Lys-palm 24Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro Xaa Xaa Xaa1 5 10 15 Xaa2517PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES14, 15, 17Xaa =
homo-LysMOD_RES16Xaa = Lys-palm 25Xaa Trp Thr Leu Asn Ser Ala Gly
Tyr Leu Leu Gly Pro Xaa Xaa Xaa1 5 10 15 Xaa2617PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES14, 15, 17Xaa = DbuMOD_RES16Xaa
= Lys-palm 26Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro
Xaa Xaa Xaa1 5 10 15 Xaa2717PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES16Xaa = 2-amino-tetradecanoic acid 27Xaa Trp Thr Leu
Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Xaa1 5 10 15
Lys2817PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa =
3,3-diphenylalanine 28Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu
Gly Pro Lys Lys Xaa1 5 10 15 Lys2918PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa = Lys-palmMOD_RES18Xaa =
12-amino-dodecanoic acid or 2-amino-tetradecanoic acid 29Xaa Trp
Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Xaa1 5 10 15
Lys Xaa3014PRTArtificial SequenceDescription of Artificial Sequence
= Synthetic Construct 30Ala Gly Cys Lys Asn Phe Phe Trp Lys Thr Phe
Thr Ser Cys1 5 10 3114PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES5Xaa =
DbuVARIANT8Xaa = D-TrpMOD_RES10, 12, 13Xaa = Dpr 31Ala Ala Cys Lys
Xaa Phe Phe Xaa Lys Xaa Phe Xaa Xaa Cys1 5 10 3214PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES4, 5Xaa = Lys-palmVARIANT8Xaa = D-Trp 32Ala Gly Cys
Xaa Xaa Phe Phe Xaa Lys Thr Phe Thr Ser Cys1 5 10 3314PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES6, 7, 11Xaa = Chloro-PheVARIANT8Xaa = D-Trp 33Ala
Gly Cys Lys Asn Xaa Xaa Xaa Lys Thr Xaa Thr Ser Cys1 5 10
3414PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES5Xaa = DbuMOD_RES10, 12, 13Xaa = Dpr
34Ala Ala Cys Lys Xaa Phe Phe Trp Lys Xaa Phe Xaa Xaa Cys1 5 10
3514PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES4, 5Xaa = Lys-palm 35Ala Gly Cys Xaa Xaa
Phe Phe Trp Lys Thr Phe Thr Ser Cys1 5 10 3614PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES6, 7, 11Xaa = Chloro-Phe 36Ala Gly Cys Lys Asn Xaa
Xaa Trp Lys Thr Xaa Thr Ser Cys1 5 10 3716PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES13, 14, 15Xaa = Chloro-Phe 37Xaa
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Xaa Xaa Xaa Val1 5 10
15 3816PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES3, 6, 7, 15Xaa =
DprMOD_RES13, 16Xaa = Dbu 38Xaa Trp Xaa Leu Asn Xaa Xaa Gly Tyr Leu
Leu Gly Xaa His Xaa Xaa1 5 10 15 3916PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES9Xaa = Chloro-Phe 39Xaa Trp Thr
Leu Asn Ser Ala Gly Xaa Leu Leu Gly Pro His Ala Val1 5 10 15
408PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructVARIANT1Xaa = D-PheVARIANT4Xaa =
D-TrpVARIANT8Xaa = Threoninol 40Xaa Cys Phe Xaa Lys Thr Cys Xaa1 5
4116PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = N,N-bis(3aminopropyl)
glycineMOD_RES2Xaa = aminohexanoic acid 41Xaa Xaa Ala Gly Cys Lys
Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys1 5 10 15 4216PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = N,N-bis(3aminopropyl) glycineMOD_RES2Xaa =
aminohexanoic acidVARIANT10Xaa = D-TrpMOD_RES13Xaa =
Chloro-PheMOD_RES15Xaa = Dpr 42Xaa Xaa Ala Gly Cys Lys Asn Phe Phe
Xaa Lys Thr Xaa Thr Xaa Cys1 5 10 15 4316PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES2Xaa = aminohexanoic acidVARIANT10Xaa =
D-TrpMOD_RES13Xaa = Chloro-PheMOD_RES14, 15Xaa = Dpr 43Trp Xaa Lys
Lys Cys Lys Asn Phe Phe Xaa Lys Thr Xaa Xaa Xaa Cys1 5 10 15
4413PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES3Xaa = Lys-palmMOD_RES5Xaa =
aminohexanoic acidVARIANT6Xaa = D-PheVARIANT9Xaa =
D-TrpMOD_RES13Xaa = Threoninol 44Lys Lys Xaa Lys Xaa Xaa Cys Phe
Xaa Lys Thr Cys Xaa1 5 10 4515PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES4Xaa =
Lys-palmMOD_RES6, 7Xaa = aminohexanoic acidVARIANT8Xaa =
D-PheVARIANT11Xaa = D-TrpMOD_RES15Xaa = Threoninol 45Lys Lys Lys
Xaa Lys Xaa Xaa Xaa Cys Phe Xaa Lys Thr Cys Xaa1 5 10 15
4614PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1, 4Xaa = Lys-palmMOD_RES6Xaa =
aminohexanoic acidVARIANT7Xaa = D-PheVARIANT10Xaa =
D-TrpMOD_RES14Xaa = Threoninol 46Xaa Lys Lys Xaa Lys Xaa Xaa Cys
Phe Xaa Lys Thr Cys Xaa1 5 10 4718PRTArtificial SequenceDescription
of Artificial Sequence = Synthetic ConstructMOD_RES3, 8Xaa =
Lys-palmMOD_RES5, 10Xaa = aminohexanoic acidVARIANT11Xaa =
D-PheVARIANT14Xaa = D-TrpMOD_RES18Xaa = Threoninol 47Lys Lys Xaa
Lys Xaa Lys Lys Xaa Lys Xaa Xaa Cys Phe Xaa Lys Thr1 5 10 15 Cys
Xaa4818PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = 2H, 2H, 3H 3H-perfluoroheptanoic
acidVARIANT3Xaa = D-LysMOD_RES5Xaa = 8-aminocaprylic
acidMOD_RES8Xaa = Lys-palmMOD_RES10Xaa = aminohexanoic
acidVARIANT11Xaa = D-PheVARIANT14Xaa = D-TrpMOD_RES18Xaa =
Threoninol 48Xaa Lys Xaa Lys Xaa Lys Lys Xaa Lys Xaa Xaa Cys Phe
Xaa Lys Thr1 5 10 15 Cys Xaa4916PRTArtificial SequenceDescription
of Artificial Sequence = Synthetic ConstructMOD_RES15Xaa =
Lys-palmMOD_RES16Amidation at the C-terminus 49Trp Thr Leu Asn Ser
Ala Gly Tyr Leu Leu Gly Pro Lys Lys Xaa Lys1 5 10 15
5017PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyVARIANT14, 15, 17Xaa =
D-LysMOD_RES16Xaa = Lys-palm 50Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro Xaa Xaa Xaa1 5 10 15 Xaa5117PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa =
Lys-palmMOD_RES17Amidation at the C-terminus 51Xaa Trp Thr Leu Asn
Ser Ala Gly Tyr Leu Leu Gly Pro Arg Arg Xaa1 5 10 15
Arg5217PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa =
Lys-palmMOD_RES17Amidation at the C-terminus 52Xaa Trp Thr Leu Asn
Ser Ala Gly Tyr Leu Leu Gly Pro His His Xaa1 5 10 15
His5317PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa =
Lys-palmMOD_RES17Amidation at the C-terminus 53Xaa Trp Thr Leu Asn
Ser Ala Gly Tyr Leu Leu Lys Lys Lys Lys Xaa1 5 10 15
Lys5415PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES14Xaa =
Lys-palmMOD_RES15Amidation at the C-terminus 54Xaa Trp Thr Leu Asn
Ser Ala Gly Tyr Leu Leu Lys Lys Xaa Lys1 5 10 15 5512PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = aminohexanoic acid 55Xaa Gly Gly Trp Ala Gly
Gly Asp Ala Ser Gly Glu1 5 10 5617PRTArtificial SequenceDescription
of Artificial Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES16Xaa = Lys-palm 56Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro Lys Lys Xaa1 5 10 15 Lys5716PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES15Xaa = Lys-palm 57Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro Lys Lys Xaa Lys1 5 10 15 5815PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructVARIANT1, 2, 3Xaa = D-LysMOD_RES4Xaa = Lys-palm 58Xaa Xaa
Xaa Xaa Gly Gly Trp Ala Gly Gly Asp Ala Ser Gly Glu1 5 10 15
5917PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa = Lys-palm 59Xaa
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Arg Arg Xaa1 5 10
15 Arg6017PRTArtificial SequenceDescription of Artificial Sequence
= Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa = Lys-palm
60Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Lys Lys Lys Lys Xaa1
5 10 15 Lys6117PRTArtificial SequenceDescription of Artificial
Sequence = Synthetic ConstructMOD_RES1Xaa = MeGly 61Xaa Trp Thr Leu
Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Lys1 5 10 15
Lys6215PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES14Xaa = Lys-palm 62Xaa Trp Thr
Leu Asn Ser Ala Gly Tyr Leu Leu Lys Lys Xaa Lys1 5 10 15
6313PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES12Xaa = Lys-palm 63Xaa
Trp Thr Leu Asn Ser Ala Gly Tyr Lys Lys Xaa Lys1 5 10
6418PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES14Xaa = aminohexanoic
acidMOD_RES17Xaa = Lys-palm 64Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro Xaa Lys Lys1 5 10 15 Xaa Lys6518PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES14Xaa = aminohexanoic
acidMOD_RES17Xaa = Lys-palm 65Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro 1 5 10 Xaa Lys Lys Xaa Lys 156613PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES12Xaa = Lys-palm 66Xaa Trp Thr
Leu Asn Ser Ala Gly Tyr Lys Lys Xaa Lys1 5 10 6714PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES10Xaa = aminohexanoic
acidMOD_RES13Xaa = Lys-palm 67Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Xaa Lys Lys Xaa Lys1 5 10 6816PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES16Xaa = Lys-palm 68Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro His Ala Xaa1 5 10 15 6917PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyVARIANT14, 15, 17Xaa =
D-LysMOD_RES16Xaa = Lys-palm 69Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro Xaa Xaa Xaa1 5 10 15 Xaa7017PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa = Lys-palm 70Xaa Trp Thr
Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Arg Arg Xaa1 5 10 15
Arg7117PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa = Lys-palm 71Xaa
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Lys Lys Lys Lys Xaa1 5 10
15 Lys7215PRTArtificial SequenceDescription of Artificial Sequence
= Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES14Xaa = Lys-palm
72Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Lys Lys Xaa Lys1 5 10
15 7317PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES14, 15, 17Xaa =
OrnMOD_RES16Xaa = Lys-palm 73Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro Xaa Xaa Xaa1 5 10 15 Xaa7417PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES14, 15, 17Xaa = DbuMOD_RES16Xaa
= Lys-palm 74Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro
Xaa Xaa Xaa1 5 10 15 Xaa7517PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES14, 15, 17Xaa = beta-homo-lysineMOD_RES16Xaa = Lys-palm
75Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Xaa Xaa Xaa 1
5 10 15 Xaa7617PRTArtificial SequenceDescription of Artificial
Sequence = Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa =
Lys-palm 76Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His
His Xaa1 5 10 15 His7717PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES14Xaa = Lys-palm 77Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro Xaa Lys Lys1 5 10 15 Lys7817PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES15Xaa = Lys-palm 78Xaa Trp Thr
Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Xaa Lys1 5 10 15
Lys7917PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES17Xaa = Lys-palm 79Xaa
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Lys1 5 10
15 Xaa8011PRTArtificial SequenceDescription of Artificial Sequence
= Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES10Xaa = Spermine-N4
succinic acidMOD_RES11Xaa = Lys-palm 80Xaa Trp Thr Leu Asn Ser Ala
Gly Tyr Xaa Xaa1 5 10 8117PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES16Xaa = Lys-palmMOD_RES17Xaa = Spermine-N4 succinic
acid 81Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys
Xaa1 5 10 15 Xaa8217PRTArtificial SequenceDescription of Artificial
Sequence = Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa =
Glu-spermine 82Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro
Lys Lys Xaa1 5 10 15 Lys8317PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES16Xaa = Lys-spermine-palm 83Xaa Trp Thr Leu Asn Ser Ala
Gly Tyr Leu Leu Gly Pro Lys Lys Xaa1 5 10 15 Lys8417PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa = Tetradecanoic acid 84Xaa
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Xaa1 5 10
15 Lys8517PRTArtificial SequenceDescription of Artificial Sequence
= Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa = Nle 85Xaa
Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Xaa1 5 10
15 Lys8617PRTArtificial SequenceDescription of Artificial Sequence
= Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa =
L-Ser-alpha-mannose 86Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu
Gly Pro Lys Lys Xaa1 5 10 15 Lys8717PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa = L-Ser-beta-melibose
87Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Xaa1
5 10 15 Lys8816PRTArtificial SequenceDescription of Artificial
Sequence = Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES12Xaa =
5-amino-3-oxapemtanoic acidMOD_RES15Xaa = Lys-palm 88Xaa Trp Thr
Leu Asn Ser Ala Gly Tyr Leu Leu Xaa Lys Lys Xaa Lys1 5 10 15
8916PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES12Xaa = 5-aminovaleric
acidMOD_RES15Xaa = Lys-palm 89Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Xaa Lys Lys Xaa Lys1 5 10 15 9015PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES11Xaa = 8-amino-3,
6-dioxaoctanoic acidMOD_RES14Xaa = Lys-palm 90Xaa Trp Thr Leu Asn
Ser Ala Gly Tyr Leu Xaa Lys Lys Xaa Lys1 5 10 15 9115PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES11Xaa = 8-amino octanoic
acidMOD_RES14Xaa = Lys-palm 91Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Xaa Lys Lys Xaa Lys1 5 10 15 9215PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES10Xaa = 5-amino-3-oxapemtanoic
acidMOD_RES11Xaa = 5-aminovaleric acidMOD_RES14Xaa = Lys-palm 92Xaa
Trp Thr Leu Asn Ser Ala Gly Tyr Xaa Xaa Lys Lys Xaa Lys1 5 10 15
9330PRTHomo sapien 93Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu
Gly Pro His Ala Val1 5 10 15 Gly Asn His Arg Ser Phe Ser Asp Lys
Asn Gly Leu Thr Ser 20 25 30 9429PRTMurinaeAMIDATION29Amidation at
the C-terminus 94Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly
Pro His Ala Ile1 5 10 15 Asp Asn His Arg Ser Phe Ser Asp Lys His
Gly Leu Thr 20 25 9529PRTSus scrofa 95Gly Trp Thr Leu Asn Ser Ala
Gly Tyr Leu Leu Gly Pro His Ala Ile1 5 10 15 Asp Asn His Arg Ser
Phe His Asp Lys Tyr Gly Leu Ala 20 25 9616PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic Construct
96Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Ala Ile1
5 10 15 9714PRTArtificial SequenceDescription of Artificial
Sequence = Synthetic Construct 97Gly Trp Thr Leu Asn Ser Ala Gly
Tyr Leu Leu Gly Pro His1 5 10 9812PRTArtificial SequenceDescription
of Artificial Sequence = Synthetic Construct 98Gly Trp Thr Leu Asn
Ser Ala Gly Tyr Leu Leu Gly1 5 10 9910PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic Construct
99Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu1 5 10 1009PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic Construct
100Gly Trp Thr Leu Asn Ser Ala Gly Tyr1 5 1018PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructVARIANT1Xaa = D-PheVARIANT4Xaa = D-TrpAMIDATION8Amidation
at the C terminus 101Xaa Cys Phe Xaa Lys Thr Cys Thr1 5
1029PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic Construct 102Trp Ala Gly Gly Asp Phe Ser Gly Glu1 5
10317PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic Construct 103Tyr Gly Gly Phe Leu Arg Arg Ile Arg Pro Lys
Leu Lys Trp Asp Asn1 5 10 15 Gln10424PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructAMIDATION24Amidation at the C terminus 104Pro Ala Glu Asp
Leu Ala Arg Tyr Tyr Ser Ala Leu Arg Ala Tyr Ile1 5 10 15 Asn Leu
Ile Thr Arg Gln Arg Tyr 20 1055PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES3Xaa =
Lys-palmVARIANT5Xaa = any amino acid 105Lys Lys Xaa Lys Xaa1 5
1066PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES3Xaa = Lys-palmVARIANT6Xaa = any amino
acid 106Lys Lys Xaa Lys Gly Xaa1 5 1076PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES3Xaa = Lys-palmMOD_RES5Xaa = Aminohexanoic
acidVARIANT6Xaa = any amino acid 107Lys Lys Xaa Lys Xaa Xaa1 5
1087PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES3Xaa = Lys-palmMOD_RES5Xaa =
Aminohexanoic acidVARIANT7Xaa = any amino acid 108Lys Lys Xaa Lys
Xaa Gly Xaa1 5 10916PRTArtificial SequenceDescription of Artificial
Sequence = Synthetic ConstructMOD_RES15Xaa =
Lys-palmAMIDATION16Amidation at the C terminus 109Tyr Gly Gly Phe
Leu Arg Arg Ile Arg Pro Lys Leu Lys Lys Xaa Lys1 5 10 15
11017PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES16Xaa = Lys-palmAMIDATION17Amidation at
the C terminus 110Tyr Gly Gly Phe Leu Arg Arg Ile Arg Pro Lys Leu
Lys Lys Lys Xaa1 5 10 15 Lys11115PRTArtificial SequenceDescription
of Artificial Sequence = Synthetic ConstructMOD_RES14Xaa =
Lys-palmAMIDATION15Amidation at the C terminus 111Tyr Gly Gly Phe
Leu Arg Arg Ile Arg Pro Lys Lys Lys Xaa Lys1 5 10 15
11218PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES14Xaa = Aminohexanoic acidMOD_RES17Xaa =
Lys-palmAMIDATION18Amidation at the C terminus 112Tyr Gly Gly Phe
Leu Arg Arg Ile Arg Pro Lys Leu Lys Xaa Lys Lys1 5 10 15 Xaa
Lys11317PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES4Xaa = Lys-palmMOD_RES5Xaa =
Aminohexanoic acidAMIDATION17Amidation at the C terminus 113Lys Lys
Lys Xaa Xaa Arg Ala Tyr Ile Asn Leu Ile Thr Arg Gln Arg1 5 10 15
Tyr11417PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES3Xaa = Lys-palmMOD_RES5Xaa =
Aminohexanoic acidAMIDATION17Amidation at the C terminus 114Lys Lys
Xaa Lys Xaa Arg Ala Tyr Ile Asn Leu Ile Thr Arg Gln Arg1 5 10 15
Tyr1155PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES3Xaa = Lys-palmMOD_RES5Xaa = any amino
acid 115Lys Lys Xaa Lys Xaa1 5 1166PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES3Xaa = Lys-palmMOD_RES6Xaa = any amino acid 116Lys
Lys Xaa Lys Gly Xaa1 5 1176PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES3Xaa =
Lys-palmMOD_RES5Xaa = Aminohexanoic acidMOD_RES6Xaa = any amino
acid 117Lys Lys Xaa Lys Xaa Xaa1 5 1187PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES3Xaa = Lys-palmMOD_RES5Xaa = Aminohexanoic
acidMOD_RES7Xaa = any amino acid 118Lys Lys Xaa Lys Xaa Gly Xaa1 5
11913PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyVARIANT10, 11, 13Xaa =
D-LysMOD_RES12Xaa = Lys-palm 119Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Xaa Xaa Xaa Xaa1 5 10 12014PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES10Xaa = Aminohexanoic acidVARIANT11, 12, 14Xaa =
D-LysMOD_RES13Xaa = Lys-palm 120Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Xaa Xaa Xaa Xaa Xaa1 5 10 12114PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES10Xaa = 7-AhpVARIANT11, 12, 14Xaa = D-LysMOD_RES13Xaa =
Lys-palm 121Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Xaa Xaa Xaa Xaa
Xaa1 5 10 12218PRTArtificial SequenceDescription of Artificial
Sequence = Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES10Xaa =
3,5-dibromo-TyrMOD_RES17Xaa = Lys-palm 122Xaa Trp Thr Leu Asn Ser
Ala Gly Tyr Xaa Leu Leu Gly Pro Lys Lys1 5 10 15 Xaa
Lys12317PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa = Lys-palm
123Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His His Xaa1
5 10 15 Lys12417PRTArtificial SequenceDescription of Artificial
Sequence = Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa =
4-methyltrityl 124Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly
Pro Lys Lys Xaa1 5 10 15 Lys12517PRTArtificial SequenceDescription
of Artificial Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES16Xaa = Lys-Biotin-aminocaproyl 125Xaa Trp Thr Leu Asn
Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Xaa1 5 10 15
Lys12617PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa = Lys-sterol
126Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Xaa1
5 10 15 Lys12717PRTArtificial SequenceDescription of Artificial
Sequence = Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa =
Lys-decanoyl 127Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro
Lys Lys Xaa1 5 10 15 Lys12817PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES16Xaa = Lys-octanoyl 128Xaa Trp Thr Leu Asn Ser Ala Gly
Tyr Leu Leu Gly Pro Lys Lys Xaa1 5 10 15 Lys12917PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa = Lys-linoyl 129Xaa Trp
Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Xaa1 5 10 15
Lys13017PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa = Ser-melbiose
130Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Xaa1
5 10 15 Lys13117PRTArtificial SequenceDescription of Artificial
Sequence = Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa =
Lys-adamentoyl 131Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly
Pro Lys Lys Xaa1 5 10 15 Lys13217PRTArtificial SequenceDescription
of Artificial Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES16Xaa = Glu(Beta-Lac-PEG3-amine) 132Xaa Trp Thr Leu Asn
Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Xaa1 5 10 15
Lys13317PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa = Lys-palm
133Xaa Trp Thr Leu Thr Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Xaa1
5 10 15 Lys13417PRTArtificial SequenceDescription of Artificial
Sequence = Synthetic ConstructMOD_RES1Xaa = MeGlyMOD_RES16Xaa =
Lys-palm 134Xaa Trp Thr Leu Leu Ser Ala Gly Tyr Leu Leu Gly Pro Lys
Lys Xaa1 5 10 15 Lys13517PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES1Xaa =
MeGlyMOD_RES16Xaa = Lys-palm 135Xaa Trp Thr Leu Asp Ser Ala Gly Tyr
Leu Leu Gly Pro Lys Lys Xaa1 5 10 15 Lys13612PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = Aminohexanoic acid 136Xaa Gly Gly Trp Ala
Gly Gly Asp Ala Ser Gly Glu1 5 10 13712PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES1Xaa = palmitic acid 137Xaa Gly Gly Trp Ala Gly Gly
Asp Ala Ser Gly Glu1 5 10 13812PRTArtificial SequenceDescription of
Artificial Sequence = Synthetic ConstructMOD_RES1Xaa = Lys-palm
138Xaa Gly Gly Trp Ala Gly Gly Asp Ala Ser Gly Glu1 5 10
13915PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES4Xaa = Lys-palm 139Lys Lys Lys Xaa Gly
Gly Trp Ala Gly Gly Asp Ala Ser Gly Glu1 5 10 15 14015PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic
ConstructMOD_RES3Xaa = Lys-palm 140Lys Lys Xaa Lys Gly Gly Trp Ala
Gly Gly Asp Ala Ser Gly Glu1 5 10 15 14114PRTArtificial
SequenceDescription of Artificial Sequence = Synthetic Construct
141Lys Lys Lys Gly Gly Trp Ala Gly Gly Asp Ala Ser Gly Glu1 5 10
14212PRTArtificial SequenceDescription of Artificial Sequence =
Synthetic ConstructMOD_RES1Xaa = Aminohexanoic acid 142Xaa Gly Gly
Trp Ala Gly Gly Asp Phe Ser Gly Glu1 5 10 14316PRTArtificial
SequenceSynthetic constructMOD_RES1Xaa = MeGly 143Xaa Trp Thr Leu
Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Ala Val1 5 10
1514414PRTArtificial SequenceSynthetic constructMOD_RES1Xaa =
MeGlyMOD_RES10Xaa = Aminohexanoic acidVARIANT11, 12, 13, 14Xaa =
D-Lys 144Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Xaa Xaa Xaa Xaa Xaa1 5
1014517PRTArtificial SequenceSynthetic constructMOD_RES1Xaa = MeGly
145Xaa Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Lys1
5 10 15Lys14616PRTArtificial SequenceSynthetic
constructMOD_RES15Xaa = Lys-palm 146Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro Lys Lys Xaa Lys1 5 10 1514716PRTArtificial
SequenceSynthetic constructMOD_RES1Xaa = MeGlyMOD_RES13, 14, 16Xaa
= beta-homo-lysineMOD_RES15Xaa = Lys-palm 147Xaa Trp Thr Leu Asn
Ser Ala Gly Tyr Leu Leu Gly Xaa Xaa Xaa Xaa1 5 10
151488PRTArtificial SequenceSynthetic constructMOD_RES1Xaa =
D-PheMOD_RES4Xaa = D-Trp 148Xaa Cys Phe Xaa Lys Thr Cys Thr1
514913PRTArtificial SequenceSynthetic constructMOD_RES3Xaa =
Lys-palmMOD_RES5Xaa = aminohexanoic acidMOD_RES6Xaa =
D-PheMOD_RES9Xaa = D-Trp 149Lys Lys Xaa Lys Xaa Xaa Cys Phe Xaa Lys
Thr Cys Thr1 5 10
* * * * *
References