U.S. patent application number 13/777149 was filed with the patent office on 2013-07-18 for amylin and amylin agonists for treating psychiatric diseases and disorders.
This patent application is currently assigned to Astrazeneca Pharmaceuticals LP. The applicant listed for this patent is Michael R. Hanley, Kevin D. LAUGERO, Christine M. Mack, David G. Parkes, Andrew A. Young. Invention is credited to Michael R. Hanley, Kevin D. LAUGERO, Christine M. Mack, David G. Parkes, Andrew A. Young.
Application Number | 20130184213 13/777149 |
Document ID | / |
Family ID | 37054153 |
Filed Date | 2013-07-18 |
United States Patent
Application |
20130184213 |
Kind Code |
A1 |
LAUGERO; Kevin D. ; et
al. |
July 18, 2013 |
Amylin and Amylin Agonists for Treating Psychiatric Diseases and
Disorders
Abstract
Methods and compositions for treating psychiatric diseases and
disorders are disclosed. The methods provided generally involve the
administration of an amylin or an amylin agonist to a subject in
order to treat psychiatric diseases and disorders, and conditions
associated with psychiatric diseases and disorders.
Inventors: |
LAUGERO; Kevin D.; (Davis,
CA) ; Hanley; Michael R.; (Corte Madera, CA) ;
Mack; Christine M.; (Ventura, CA) ; Parkes; David
G.; (Del Mar, CA) ; Young; Andrew A.;
(Research Triangle Park, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LAUGERO; Kevin D.
Hanley; Michael R.
Mack; Christine M.
Parkes; David G.
Young; Andrew A. |
Davis
Corte Madera
Ventura
Del Mar
Research Triangle Park |
CA
CA
CA
CA
NC |
US
US
US
US
US |
|
|
Assignee: |
Astrazeneca Pharmaceuticals
LP
Wilmington
DE
Amylin Pharmaceuticals, LLC
San Diego
CA
|
Family ID: |
37054153 |
Appl. No.: |
13/777149 |
Filed: |
February 26, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13112290 |
May 20, 2011 |
8389477 |
|
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13777149 |
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12641733 |
Dec 18, 2009 |
|
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13112290 |
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|
11910206 |
Feb 28, 2008 |
7671023 |
|
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PCT/US2006/012601 |
Mar 31, 2006 |
|
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12641733 |
|
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60667335 |
Mar 31, 2005 |
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60666681 |
Mar 31, 2005 |
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60675441 |
Apr 28, 2005 |
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60760583 |
Jan 20, 2006 |
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Current U.S.
Class: |
514/9.7 |
Current CPC
Class: |
A61P 25/30 20180101;
A61P 25/32 20180101; A61P 3/00 20180101; A61K 38/22 20130101; A61P
25/18 20180101; A61P 25/00 20180101; A61P 3/06 20180101; A61P 25/22
20180101; A61P 43/00 20180101; A61K 45/06 20130101; A61P 3/04
20180101; A61P 25/20 20180101; A61P 5/48 20180101; A61P 25/34
20180101; A61P 3/10 20180101; A61P 25/36 20180101; A61P 25/24
20180101 |
Class at
Publication: |
514/9.7 |
International
Class: |
A61K 38/22 20060101
A61K038/22; A61K 45/06 20060101 A61K045/06 |
Claims
1. A method for treating a substance addiction in a human in need
thereof comprising administering to the human a therapeutically
effective amount of a peptide comprising the amino acid sequence of
SEQ ID NO:6 to treat the substance addiction.
2. The method of claim 1, wherein the substance addiction is
alcohol addiction.
3. A method for treating a substance addiction in a human in need
thereof comprising administering to the human a therapeutically
effective amount of a peptide having at least 90% sequence identity
to SEQ ID NO:6 to treat the substance addiction.
4. The method of claim 3, wherein the peptide has at least 95%
sequence identity to SEQ ID NO:6.
5. The method of claim 3, wherein the substance addiction is
alcohol addiction.
6. A method for treating a mood disorder in a human in need thereof
comprising administering to the human a therapeutically effective
amount of a peptide having at least 90% sequence identity to SEQ ID
NO:6 to treat the mood disorder.
7. The method of claim 6, wherein the peptide has at least 95%
sequence identity to SEQ ID NO:6.
8. The method of claim 6, wherein the peptide comprises the amino
acid sequence of SEQ ID NO:6.
9. The method of claim 6, wherein the mood disorder is bipolar
disorder.
10. The method of claim 6, wherein the mood disorder is major
depressive disorder, dysthymic disorder, bipolar I disorder,
bipolar II disorder, or cyclothymic disorder.
11. The method of claim 6, further comprising administering to the
subject at least one other psychiatric medication.
12. The method of claim 6, further comprising administering to the
subject (i) a tricyclic antidepressant; (ii) a monoamine oxidase
inhibitor; (iii) a selective serotonin reuptake inhibitor; (iv) a
serotonin and noradrenaline reuptake inhibitor; (v) a second
generation antipsychotic medication; or (vi) a combination of two
or more thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/112,290 filed May 20, 2011, issued as U.S. Pat. No.
8,389,477, which is a continuation of U.S. application Ser. No.
12/641,733 filed Dec. 18, 2009, abandoned, which is a divisional of
U.S. application Ser. No. 11/910,206 filed Sep. 28, 2008, issued as
U.S. Pat. No. 7,671,023, which is a .sctn.371 of PCT Application
No. PCT/US06/12601 filed Mar. 31, 2006, which claims priority to
U.S. Application No. 60/760,583, filed Jan. 20, 2006; U.S.
Application No. 60/675,441, filed Apr. 28, 2005; U.S. Application
No. 60/667,335, filed Mar. 31, 2005; and U.S. Application No.
60/666,681, filed Mar. 31, 2005, the entire contents of each are
incorporated herein for all purposes.
TECHNICAL FIELD
[0002] This disclosure is in the medical field and in particular to
the fields of psychology and psychiatry, as well as health, diet
and nutrition.
BACKGROUND
[0003] Psychiatric diseases and disorders (also referred to as
mental illnesses or disorders) are described in resources such as
the American Psychiatric Association's Diagnostic and Statistical
Manual of Mental Disorders, or DSM-IV. Broad categories of mental
disorders include, but are not limited to, mood disorders, anxiety
disorders, schizophrenia and other psychotic disorders,
substance-related disorders, sleep disorders, somatoform disorders,
and eating disorders. Examples of mood disorders include bipolar
and depression. Other conditions falling within the broader
category of disorders described above can be found in the DSM-IV,
which is incorporated by reference in its entirety. These are
debilitating illnesses that affect millions of people and involve
astronomical costs, in terms of treatment, lost productivity, and
emotional toll.
[0004] In 2001, the National Institute of Mental Health published a
summary of statistics describing the prevalence of mental disorders
in America. In the report, it estimated that 22.1% of Americans
ages 18 and older suffer from a diagnosable mental disorder in a
given year (Reiger et al. (1993) Archives of General Psychiatry
50:85-94). When applied to the 1998 U.S. Census, the number of
people affected was 44.3 million.
[0005] Depressive disorders can encompass, among others illnesses,
major depressive disorder, dysthymic disorder and bipolar disorder.
About 9 to 9.5 percent of the U.S. population ages 18 and older
have a depressive condition. It has been reported that the direct
cost of depressive disorders is about $80 billion, with two-thirds
of it being borne by businesses. The indirect costs associated with
depressive disorders, such as lost productivity, are harder to
calculate because of events such as "presenteeism," described as
people at work but limited in their ability to produce or
participate (Durso, Employee Benefit News, December 2004).
[0006] Another psychiatric condition is anxiety disorders. These
disorders can include panic disorder, obsessive-compulsive
disorder, post-traumatic stress disorder generalized anxiety
disorder, and phobias. Approximately 19.1 million American adults
ages 18 to 54 (about 13.3% of people in this age group in a given
year) have an anxiety disorder.
[0007] Another common psychiatric condition is eating disorders.
There are three main types, anorexia nervosa, bulimia nervosa, and
binge-eating disorders. These are psychiatric conditions are often
linked to perceived notions about body image and are usually
independent of actual body weight or body mass index. The mortality
of people with anorexia has been estimated at 0.56 percent per
year, or approximately 5.6 percent per decade, which is about 12
times higher than the annual death rate due to all causes of death
among females ages 15-24 in the general population (Sullivan (1995)
American Journal of Psychiatry 152:1073-1074). It should be noted
that psychiatric illnesses usually present with elements of other
psychiatric disorders.
[0008] Another psychiatric condition is schizophrenia. In a given
year, over 2 million people are clinically diagnosed with
schizophrenia, and there is a lifetime prevalence of this disease
in approximately 1% of the U.S. population. Schizophrenia is a
chronic, debilitating disease that leaves an estimated 75% of
treated patients without ever achieving complete recovery.
Treatment of schizophrenia with the newer (atypical) antipsychotic
medications frequently comes with the side effect of weight gain
and possibly diabetes.
[0009] Exemplary types of schizophrenia include paranoid
schizophrenia. These persons are very suspicious of others and
often have grand schemes of persecution at the root of their
behavior. Hallucinations, and more frequently delusions, are a
prominent and common part of the illness. Persons with disorganized
schizophrenia (Hebephrenic Schizophrenia) are verbally incoherent
and may have moods and emotions that are not appropriate to the
situation. Hallucinations are not usually present with disorganized
schizophrenia. Catatonic schizophrenia is where a person is
extremely withdrawn, negative and isolated, and has marked
psychomotor disturbances. Residual schizophrenia is where a person
is not currently suffering from delusions, hallucinations, or
disorganized speech and behavior, but lacks motivation and interest
in day-to-day living. Schizoaffective disorder is where a person
has symptoms of schizophrenia as well as mood disorder such as
major depression, bipolar mania, or mixed mania. Undifferentiated
schizophrenia is where conditions meet the general diagnostic
criteria for schizophrenia but do not conform to any of the above
subtypes, or there are features of more than one of the subtypes
without a clear predominance of a particular set of diagnostic
characteristics.
[0010] Psychiatric diseases and disorders can be found in any age
group. Accordingly, these disorders can be found in young adults
and adults (defined herein as those aged 65 or under) as well as
infants, children, adolescents, and the elderly (defined herein as
over the age of 65). In fact, certain segments of the population
may be particularly prone to having a condition, such as eating
disorders in adolescents and young adults. The elderly may be
particularly susceptible to conditions such as depression.
[0011] Current treatments include psychosocial and behavioral
therapy, electroconvulsive therapy, and/or medication. A common
form of treatment for psychiatric illnesses, or at least a
component of the treatment, is the administration of medication.
Needed in the art are molecules that (1) effectively treat those
patients resistant to the current antidepressants (e.g.,
tricyclics, monoamine oxidase inhibitors, selective serotonin
reuptake inhibitors) (2) effectively treat depression, anxiety,
schizophrenia, or other psychiatric diseases or disorders without
the unwanted side effects of the current pharmaceuticals, (3) have
a faster onset of therapeutic action, and/or (4) improve physical
co-morbidities (e.g., diabetes, pain, weight gain) that often
present with and make more difficult the treatment of psychiatric
illnesses, such as depression, anxiety, and schizophrenia to name
just a few.
[0012] All references cited herein are incorporated by reference in
their entirety.
SUMMARY
[0013] In one general aspect, methods provided include the use of
amylin and its agonists, analogs, or derivatives in therapeutically
effective amounts for the treatment of a psychiatric disorder. In
certain embodiments, the psychiatric disorder is a mood disorder,
an anxiety disorder, schizophrenia or other psychotic disorder, a
substance-related disorder, a sleep disorder, a somatoform
disorder, and/or an eating disorder. In certain embodiments, the
psychiatric disorder is depression or bipolar disorder. In certain
embodiments, the psychiatric disorder is an obsessive-compulsive
disorder. In certain embodiments, methods provided may not include
treating an eating disorder. In other embodiments, methods provided
may not include treating anorexia. In certain embodiments, methods
provided may not include a somatoform disorder. In certain
embodiments, amylin and its agonists, analogs, or derivatives are
used to treat the underlying psychiatric condition of an eating
disorder. In certain embodiments, amylin and its agonists, analogs,
or derivatives are used to treat the underlying psychiatric
condition of a somatoform disorder. In certain embodiments, amylin
agonists may not include a calcitonin and/or a
calcitonin-gene-related peptide (CGRP). In still other embodiments,
amylin agonists may not include an analog of calcitonin and/or
CGRP.
[0014] In still another aspect, methods provided include treating a
psychiatric disorder in a subject desirous of, or in need of,
treatment comprising administering a therapeutically effective
amount of an amylin, its agonists, analogs, or derivatives to the
subject. In certain embodiments, the subject is overweight. In
other embodiments, the subject is obese. In still other
embodiments, the subject is lean, not overweight or obese. In still
other embodiments, the subject has a metabolic condition. In yet
other embodiments, the subject has diabetes, metabolic syndrome,
impaired glucose tolerance, or insulin resistance. In other
embodiments, amylin and its agonists, analogs, or derivatives are
beneficial in aiding the subject with their ability to modify food
preferences or food cravings.
[0015] In another general aspect, methods provided herein include
administration of a therapeutically effective amount of amylin, or
its agonist, analogs, and derivatives, in combination with a
conventional treatment for psychiatric disorders. In certain
embodiments, the combination includes the administration of
electroconvulsive therapy (ECT). In other embodiments, the
combination includes the administration of another psychiatric
medication. In still other embodiments, the psychiatric medication
is one or more of tricyclic antidepressants, monamine oxidase
inhibitors (MAOIs), selective serotonin reuptake inhibitors
(SSRIs), serotonin and noradrenaline reuptake inhibitors (SNRIs),
herbal antidepressants (e.g., St John's Wort or Hypericum), or
second generation antipsychotic medications (SGAs).
[0016] In another general aspect, methods provided herein include
treating an unwanted side effect of another psychiatric medication
comprising administering a therapeutically effective amount of
amylin, or its agonist, analogs, and derivatives, to a subject in
need thereof. In certain embodiments, the other psychiatric
medication is a second generation antipsychotic medication. In
certain embodiments, the unwanted side effect of the other
psychiatric medication is weight gain. In other embodiments, the
unwanted side effect of the other psychiatric medication is
diabetes.
[0017] In another general aspect, methods provided include treating
a psychiatric disorder comprising administering a therapeutically
effective amount of a compound that modulates behavioral pathways
through its modulatory actions on metabolic pathways or function,
including but not limited to glucose metabolism, lipid metabolism,
protein metabolism, and total energy metabolism. In certain
embodiments, the behavioral pathway is a corticotropin-releasing
factor (CRF) pathway that modulates at least one component of the
hypothalamic-pituitary-adrenal axis. In one embodiment, the
behavioral pathway is a dopamine pathway. In other embodiments, the
behavioral pathway is the 5HT.sub.1A pathway or any pathway
comprising the serotonergic system. In certain embodiments, the
metabolic or behavioral pathway is any one of glucoregulatory,
glucocorticoid responsive, or stress responsive. In certain
embodiments, the compound is an amylin or its agonist, analog or
derivative.
[0018] In still another aspect, methods provided include treating a
psychiatric disorder by administering a therapeutically effective
amount of a compound that modulates behavioral pathways through its
modulatory actions on food intake. In certain embodiments the
compound is amylin or its agonist, analog or derivative.
[0019] In another aspect, the disclosure provides for the use of an
amylin, its agonists, analogs, or derivatives for manufacture of a
medicament useful for treating psychiatric diseases and disorders
described herein. In another aspect, the disclosure provides for
the use of an amylin, its agonists, analogs, or derivatives for
manufacture of a medicament useful for treating unwanted side
effects of another psychiatric medication, for example, a second
generation antipsychotic medication.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1A and 1B are graphs depicting effects of amylin on
stress-induced feeding. In these graphs, R indicates animals
subjected to restraint stress and C indicates control animals not
subjected to restraint stress.
[0021] FIG. 2 is a graph depicting the effect of amylin and control
agents on stress-induced hyperthermia in mice.
[0022] FIG. 3 is a graph depicting the effect of amylin and control
agents on marble burying.
[0023] FIG. 4 is a graph depicting the effect of amylin and vehicle
control on animals undergoing the forced swim test.
[0024] FIG. 5 is a graph depicting the effect of amylin and control
agents on prepulse inhibition.
[0025] FIG. 6 is a graph depicting the effect of amylin and control
agents on phencyclidine (PCP)-induced locomotion.
[0026] FIGS. 7A and 7B are graphs depicting effects of clozapine on
weight. The graph in FIG. 7A depicts the effect of clozapine alone
on weight and the graph in FIG. 7B depicts the effect of the
combination of clozapine and amylin on weight.
DETAILED DESCRIPTION
[0027] It has now been discovered that amylin, amylin agonists,
amylin analogs, amylin agonist analogs, amylin derivatives, or a
combination thereof, may be able to modulate the stress systems
and/or the actions of CRF and/or glucocorticoids (GC), thereby
presenting novel pharmacotherapeutic options. As demonstrated
herein, amylin administration appears to reduce or protect against
stress and it effects (e.g., anxiety, obsessive-compulsive
behavior, depression, psychosis, changes in eating behavior). We
also demonstrate herein that, in specific animal behavioral tests,
amylin administration results in behavioral effects that include
anti-stress, anxiolytic, antidepressant, and antipsychotic
actions.
[0028] In exploring new medical treatments, it was noted that a
number of metabolic pathologies (e.g., diabetes, obesity) are
associated with behavioral dysfunction (e.g., major depression,
schizophrenia). Although these diseases are generally believed to
be co-morbid, there is recent evidence suggesting that the
behavioral and metabolic alterations are physiologically linked in
many cases (Laugero et al. (2001) Endocrinology 142:2796-2804;
Laugero et al. (2002) Endocrinology 143:4552-4562; Dallman et al.
(2003) Proc. Natl. Acad. Sci. USA 100:11696-11701; Laugero (2004)
Vitamins and Hormones, Volume 68, Litwack (ed.)). A common link
between these seemingly disparate disease states may be chronic
stress and the associated changes in brain CRF and the
adrenocortical steroid hormones, GC.
[0029] The CRF and GC molecules play a critical role in modulating
behavioral, neuroendocrine, autonomic, and metabolic function under
normal and stressful conditions. Chronic stress and the induction
of expression and activity of these molecules are highly associated
with behavioral diseases like anxiety and depression, and also with
some obesities and diabetes. There is evidence that links CRF and
adrenocortical abnormalities to the metabolic syndrome, autoimmune
inflammatory disorders, acute and chronic neurodegeneration, sleep
disorders, chronic pain, eating disorders, chronic anxiety
disorder, and major depression (Wong et al. (2000) Proc. Natl.
Acad. Sci. USA 97:325-330; Sarnyai et al. (2001) Pharmacol. Rev.
53:209-243; Heinrichs et al. (1999) Baillieres Best Pract. Res.
Clin. Endocrinol. Metab. 13:541-554; Chrousos (2000) Int. J. Obes.
Relat. Metab. Disord. 24:S50-S55; Peek et al. (1995) Ann. N.Y.
Acad. Sci. 771:665-676; Grammatopoulos et al. (1999) Lancet
354:1546-1549; Dallman et al. (2003) Proc. Natl. Acad. Sci. USA
100:11696-11701).
[0030] As demonstrated herein, amylin is shown to share properties
of anxiolytic, antidepressant, and antipsychotic agents in
behavioral testing. Thus, it has now been discovered that amylin
and amylin agonists may have the surprising ability to treat
psychiatric disorders. Psychiatric disorders that can be treated
include mood disorders, anxiety disorders, schizophrenia and other
psychotic disorders, substance-related disorders, sleep disorders,
somatoform disorders, and eating disorders. These compounds may be
particularly effective in treating psychiatric disorders that have
elements of metabolic disturbances, e.g., eating disorders, or in
treating patients with a psychiatric disorder or those with a
psychiatric disorder and who also suffer from a metabolic
disturbance.
[0031] More particular types of the above named disorders can be
found in the DSM-IV. The following are only examples of disorders
that may be treated by the methods disclosed herein. Examples
include mood disorders that may include depressive disorders and
bipolar disorders. They can further be characterized as major
depressive disorders, dysthymic disorder, bipolar I disorder,
bipolar II disorder, cyclothymic disorder, bipolar disorder not
otherwise specified, mood disorders due to a medical condition,
substance-induced mood disorder, or mood disorder not otherwise
specified. Anxiety disorders can include panic disorder, specific
phobia, social phobia, obsessive-compulsive disorder, posttraumatic
stress disorder, acute stress disorder, generalized anxiety
disorder, anxiety disorder due to a medical condition, substance
induced anxiety disorder and anxiety disorder not otherwise
specified.
[0032] Substance-related disorders include substance dependence,
substance addiction, substance-induced anxiety disorder, and
substance-induced mood disorder. Substance dependence and addiction
can occur with a variety of substances, including but not limited
to, alcohol, nicotine, cocaine, opioids, narcotics, hallucinogens,
amphetamines, phencyclidines, phencyclidine-like substances,
inhalants, and sedatives. Substance-induced anxiety disorder can
occur in response to substances which include, but not limited to,
caffeine, cannabis, cocaine, hallucinogens, amphetamines,
phencyclidines, phencyclidine-like substances, and inhalants.
Substance-induced mood disorder can occur in response to substances
which include, but not limited to cocaine, hallucinogens, opioids,
amphetamines, phencyclidines, phencyclidine-like substances, and
inhalants. Substance-related disorders can occur in response to one
substance or to a combination of substances, such as in
polysubstance-related disorder.
[0033] In some embodiments, methods provided include the treatment
of medication-induced psychiatric disorders or psychiatric
disorders that result from treatment of a disease. For example,
hedonistic homeostatic dysregulation is a neuropsychological
behavioral disorder recognized in patients with Parkinson's disease
undergoing dopamine replacement therapy. Dopamine replacement
therapy in these patients appears to stimulate central dopaminergic
pathways and lead to a behavioral disorder with some similarities
to that associated with stimulant addiction. Giovannoni et al.
(2000) J. Neurol. Neurosurg. Psychiatry 68:423-428.
[0034] Eating disorders can include anorexia nervosa, bulimia
nervosa, and eating disorders not otherwise specified. These eating
disorders may include binge eating. In certain embodiments, methods
provided are drawn to the treatment of the psychiatric illness
associated with the eating disorder. In other embodiments, methods
provided do not include the treatment of eating disorders. In
certain embodiments, methods provided do not include the treatment
of anorexia. In other embodiments, methods provided may be used for
treating the psychiatric illness associated with anorexic patients.
In certain embodiments, methods provided do not include the
treatment of binge eating.
[0035] In some embodiments, methods provided can be used to treat
subjects experiencing intermittent excessive behaviors (IEB). IEB
characterize a variety of disorders including, binge eating,
substance abuse, alcoholism, aberrant sexual conduct, and
compulsive gambling. IEB occur when occasional normal behavioral
excess is transformed into repetitive, intermittent, maladaptive
behavioral excess. Corwin (2006) Appetite 46:11-15.
[0036] In certain embodiments, methods provided may not include the
treatment of somatoform disorders. In certain embodiments, methods
provided may include somatoform disorders but do not include the
treatment of physical pain. In still other embodiments, methods
provided may include the treatment of the psychiatric illness
associated with pain.
[0037] In one general aspect, it is contemplated that compounds
that reduce or moderate stress, or regulate the stress pathway, may
be useful as pharmacotherapeutic agents. In another general aspect,
it is contemplated that compounds that can affect or regulate
metabolic disturbances as well as psychiatric or behavioral
processes would be useful as pharmacotherapeutic agents. In another
general aspect, it is contemplated that compounds that can
attenuate or reverse metabolic disturbances would be useful as
pharmacotherapeutic treatments of psychiatric diseases or
disorders. It is contemplated that compounds useful in the methods
provided may be amylin, amylin agonists, amylin analogs, and amylin
derivatives. In certain embodiments, amylin agonists may not
include calcitonin and/or CGRP.
[0038] It is theorized that medicines that not only treat the
psychiatric illness, but also alleviate the physical co-morbidities
of the illness, would be expected to elicit an increased rate of
treatment response and outcome success in patients with a
psychiatric illness. Physical co-morbidities, like diabetes,
exacerbate the morbidity that comes with psychiatric illness and
lead to a reduction in treatment response. Amylin and its agonists
may be particularly useful in the methods described herein because
of its anti-diabetic, anti-obesigenic, and appetite suppressant
effects. Amylin and its agonists may further be beneficial in
aiding the subject with their ability to modify food preferences
described in commonly-owned U.S. Pat. Application No. 60/666,681,
filed on Mar. 31, 2005, and PCT application attorney docket no.
0113-PCT-0, filed 31 Mar. 2006, the contents of each are
incorporated by reference in its entirety. These effects may
increase the rate of treatment response and outcome success in
certain patient populations who suffer a psychiatric illness and
who exhibit obesity, obesity-related disease, or eating disorders
(e.g., diabetes, metabolic syndrome, obesity, Cushing's syndrome,
Cushing's disease, atypical major depression, schizophrenia,
seasonal affective disorder, polycystic ovary syndrome,
post-traumatic stress disorder, night eating syndrome, bulimia
nervosa, binge eating disorder, and chronic fatigue syndrome). In
certain embodiments, the methods do not include treatment of
anorexia. In other embodiments, the methods include treating the
psychiatric illness associated with anorexia.
[0039] Still another general aspect includes the use of the
naturally occurring and peripherally secreted amylin peptide or an
amylin agonist, analog, or derivative, for the treatment of
psychiatric disorders. In some embodiments, methods for psychiatric
disorders in a subject are provided, wherein the method comprises
administering to a subject in need thereof, an amylin or an amylin
agonist, analog, or derivative in an amount effective to treat the
psychiatric disorder. In some instances, the psychiatric disorders
are of natural or unidentified etiology. In some instances, the
psychiatric disorder may result from medication for or treatment of
a different disease. Accordingly, in some embodiments, methods for
treating medication-induced psychiatric disorders or psychiatric
disorders that result from treatment of a disease in a subject are
provided, wherein the method comprises administering to a subject
in need thereof an amylin or an amylin agonist, analog, or
derivative in an amount effective to treat the psychiatric
disorder. Because the amylin peptide is a naturally secreted
hormone, it may reduce the side effect profile commonly present in
patients receiving the currently prescribed treatments and
pharmaceuticals for psychiatric disorders. Another general aspect
includes the use of compounds that can treat both the psychiatric
disease and metabolic disturbances present in a subject.
[0040] Amylin, amylin agonists, amylin analogs, or amylin
derivatives may also be superior to some other antidepressant,
anxiolytic, and/or antipsychotic agents, as it does not promote
weight gain and, in fact, may induce weight loss. This attribute of
amylin may lead to greater compliance among patients being treated
for psychiatric disease.
[0041] It is further contemplated that amylin or its agonists,
analogs or derivatives, may be used in conjunction with other
psychiatric medications or therapies, such as those conventionally
used to treat psychiatric disease, such as tricyclic
antidepressants and the monamine oxidase inhibitors (MAOIs),
selective serotonin reuptake inhibitors (SSRIs), serotonin and
noradrenaline reuptake inhibitors (SNRIs), herbal antidepressants
(e.g., St John's Wort or Hypericum), second generation
antipsychotic medications (SGAs), psychoanalysis,
cognitive-behavioral therapy, and interpersonal therapy. When used
in conjunction with other psychiatric medications or therapies,
administration of the amylin or amylin agonist may occur
concurrently or sequentially with the other psychiatric medications
or therapies. For example, the amylin or amylin agonist may be
administered during the same time period as the other psychiatric
medication, during an overlapping time period as the other
psychiatric medication, or in a time period that does not overlap
with administration of the other psychiatric medication. As a
combination or add-on therapy, the beneficial qualities of an
amylin or amylin agonist may counteract or moderate one or more of
the unwanted side effects of currently available medications, e.g.,
weight gain, diabetes.
[0042] For example, SGAs are effective therapeutics for the
treatment of symptoms associated with schizophrenia and related
psychotic conditions. Despite these advances in treating the
psychiatric condition, accumulating clinical data have revealed an
association between the use of SGAs and weight gain, diabetes, and
dyslipidemia (American Diabetes Association et al. (2004) Diabetes
Care 27: 596-601). Weight gain may be one contributing factor to a
patient's non-compliance with his medication. So, as good as any
medication may be, it does not provide any benefit to a patient
that is not taking it, or not taking it properly. Exemplary SGAs
such as clozapine and olanzapine have been identified as being
likely to produce weight gain; in addition, these two SGAs have
also been associated with increased risk for both diabetes and
dyslipidemia. The ability of amylin to effectively reduce body
weight gain induced by clozapine treatment is demonstrated herein
in Example 3. In addition, amylin and amylin agonists are also able
to treat or aid in the treatment of diabetes and dyslipidemia. The
amylin agonist, pramlintide, has been approved by the FDA as an
adjunct therapy to insulin in the treatment of diabetes (type 1 and
type 2). Accordingly, when used with other psychiatric medications,
amylin and amylin agonists may not only provide an additional
treatment to the psychiatric condition, but also be able to
counteract at least a negative side effect of those other
psychiatric medications.
[0043] As used herein, a "subject" may include any mammal,
including humans. A "subject" may also include pets (e.g., dogs,
cats, horses), as well as other animals. Subjects may have at least
one of the psychiatric disorders described herein. Subjects who may
benefit from the methods disclosed herein may be overweight or
obese; however, they may also be lean. They may have a metabolic
disorder or condition in addition to a psychiatric disorder.
Exemplary metabolic disorders include diabetes, metabolic syndrome,
insulin-resistance, and dyslipidemia. Subjects can be of any age.
Accordingly, these disorders can be found in young adults and
adults (defined herein as those aged 65 or under) as well as
infants, children, adolescents, and the elderly (defined herein as
over the age of 65). In fact, certain segments of the population
may be particularly prone to having a particular condition, such as
eating disorders in adolescents and young adults. The elderly may
be particularly susceptible to conditions such as depression.
[0044] As used herein, and as well-understood in the art,
"treatment" is an approach for obtaining beneficial or desired
results, including clinical results. "Treating" or "palliating" a
disease, disorder, or condition means that the extent, undesirable
clinical manifestations of a condition, or both, of a disorder or a
disease state are lessened and/or time course of the progression is
slowed or lengthened, as compared to not treating the disorder. For
purposes of the methods disclosed herein, beneficial or desired
clinical results include, but are not limited to, alleviation or
amelioration of one or more symptoms, diminishment of extent of
disorder, stabilized (i.e., not worsening) state of disorder, delay
or slowing of disorder progression, amelioration or palliation of
the disorder, and remission (whether partial or total), whether
detectable or undetectable. "Treatment" can also mean prolonging
survival as compared to expected survival if not receiving
treatment. Further, treating does not necessarily occur by
administration of one dose, but often occurs upon administration of
a series of doses. Thus, a therapeutically effective amount, an
amount sufficient to palliate, or an amount sufficient to treat a
disease, disorder, or condition may be administered in one or more
administrations.
[0045] As used herein, the singular form "a", "an", and "the"
includes plural references unless otherwise indicated or clear from
context. For example, as will be apparent from context, "an" amylin
agonist can include one or more amylin agonists.
[0046] Without wishing to be bound by theory, amylin may exert its
psychiatric/behavioral effects by modulating GC-responsive sites
(such as the brain) and impact functions that are normally
modulated by GCs (e.g., CFR) expression and activity, behavior,
autonomic nervous system activity, neuroendocrine function, and
metabolism). Without wishing to be bound by theory, amylin, its
agonists, analogs, or derivatives, may block or reduce the
behavioral (e.g., anxiety, depression) and metabolic (e.g.,
feeding, obesigenic) effects of GCs, brain CRF, or chronic stress.
In addition, amylin may have some antidepressant, anxiolytic,
and/or antipsychotic activities that are not directed related to
it's anti-diabetic or anti-obesity activities. Again, without
wishing to be bound by theory, amylin may, in part, be working by
modulating, or otherwise, affecting stress pathways and/or the CRF
and/or GC regulatory actions on stress-mediated behavioral,
autonomic, neuroendocrine, and metabolic responses.
[0047] Stress, GC, and CRF seem to have an intricate and
complicated relationship in psychological and metabolic function.
Stress has profound effects on neuroendocrine (e.g.,
hypothalamic-pituitary-adrenal (HPA) axis), autonomic, behavioral
(e.g., anxiety, depression, substance abuse, feeding), and
metabolic (e.g., fat deposition, energy utilization) function in
man and animals (Dallman et al. (2002) Hormones, Brain, and
Behavior pp 571-631, San Diego, Calif. USA: Academic Press.). All
of these effects are modulated by GCs. The neuropeptide CRF
mediates many of the stress-induced responses, including acute
inhibition of food intake and anxiety (Krahn et al. (1986) Brain
Research Bulletin 17:285-289; Dunn et al. (1990) Brain Res. Brain
Res. Rev. 15:71-100; Smagin et al. (1999) Am. J. Physiol.
276:R1461-1468; Koob (1999) Biol. Psychiatry 46:1167-1180). GC and
CRF activity are tightly interdependent and, together, make up a
functional and well-described physiological system that controls
behavioral, autonomic, neuroendocrine, and metabolic function
(Dallman et al. (2003) Proc. Natl. Acad. Sci. USA
100:11696-11701).
[0048] Disruptions in the GC and CRF relationship, such as those
caused by stress, can have profound effects on health. Through the
central actions of chronically elevated glucocorticoid hormones
(cortisol in man, corticosterone in rodents), chronic stress
promotes palatable feeding (e.g., simple sugar, fat). When
palatable food is available, as it abundantly is in modern
societies, chronic stress increases the proportion of palatable
calories consumed (Pecoraro et al. (2004) Endocrinology
145:3754-3762; Laugero (2004) Vitamins and Hormones, Volume 68. Ed.
Gerald Litwack; Laugero et al. (2002) Endocrinology 143:4552-4562).
The significance of this response, under the background of elevated
GCs, is increased fat deposition in the abdominal or truncal region
(Dallman et al. (2003) Proc. Natl. Acad. Sci. USA 100:11696-11701),
which is a significant and independent risk factor for the
development of cardiovascular disease (Bjorntorp (1993) Obesity
Research 1:206; Carr et al. (2004) Diabetes 53:2087-2094). In fact,
endogenous GC elevation, as in Cushing's patients, or through
exogenous administration, as in Lupus patients, cause metabolic
syndrome (truncal obesity, insulin resistance, hypertension,
hypertriglyceridemia) (Stewart (2003) The Adrenal Cortex, Williams
Textbook of Endocrinology, 10.sup.th edition, Saunders Publishing,
U.S.). Furthermore, truncal obesity is characterized by increased
glucocorticoid activity, and it has been hypothesized that, through
the activation of adrenocortical synthesis and secretion of GCs,
chronic stress plays a significant role in the development of
truncal obesity and the metabolic syndrome (Bjorntorp (2001) Obes.
Rev. 2:73-86; Rosmond et al. (2000) Obes. Res. 8:445-450; Bjorntorp
(1997) Nutrition 13:795-803; Dallman et al. (2002) Hormones, Brain,
and Behavior pp 571-631, San Diego, Calif. USA: Academic Press.;
Dallman et al. (2003) Proc. Natl. Acad. Sci. USA
100:11696-11701).
[0049] Hyper- and hypo-cortisolemia are also well-documented
features of psychiatric disease, and patients suffering from the
metabolic syndrome show signs of abnormal cortisol secretion and
are more likely to present with a psychiatric disease such as major
depression. Chronic stress and the associated increase in central
CRF activity are believed to play a critical role in the
development of clinical depression, anxiety disorders, substance
abuse, eating disorders, and metabolic syndrome (Chrousos et al.
(2000) Int. J. Obes. Relat. Metab. Disord. 24:S50-S55; Koob (1999)
Biol. Psychiatry 46:1167-1180). In fact, small molecule CRF
antagonists are currently being investigated for their therapeutic
actions in patients with major depression, general anxiety
disorder, eating disorders, and other stress related
pathologies.
[0050] Amylin is a hormone that is co-secreted with insulin from
the pancreatic .beta.-cell and that has numerous metabolic effects,
including glucoregulatory actions. The glucoregulatory actions of
amylin may be related to its effects on gastric emptying,
suppression of inappropriately elevated postprandial glucagon
secretion, and inhibition of food intake (Young (1997) Curr. Opin.
in Endocrinology and Diabetes 4:282-290). An amylin agonist analog,
pramlintide, is being developed for the treatment for type 1 and 2
diabetes (Baron et al. (2002) Curr. Drug Targets Immune Endocr.
Metabol. Disord. 2:63-82) and has recently been approved by the FDA
for such use. Because of its anti-diabetic effects, amylin is also
a good candidate for treating the metabolic syndrome. Moreover,
amylin is a candidate for an anti-obesity drug for humans due to
its effect on weight in animals (Mack et al. (2003) Diabetes 52
(Suppl. 1) A389).
[0051] Amylin agonists include amylin agonist analogs, examples of
which are described in U.S. Pat. No. 5,686,411; U.S. Pat. No.
6,610,824; U.S. Pat. No. 5,998,367; U.S. Pat. No. 6,087,334; U.S.
Provisional Application No. 60/617,468, filed Oct. 8, 2004; and PCT
Application No. PCT/US2005/004631, all of which are incorporated
herein by reference. In certain embodiments, methods provided may
not include the use of calcitonins. In certain embodiments, the
calcitonin is salmon calcitonin. In other embodiments, methods
provided may not include the use of CGRP. In still other
embodiments, methods provided may not include the use of analogs of
CGRP or calcitonin. Accordingly, it is contemplated that methods
provided may include a proviso that excludes CGRP, calcitonin, or
their analogs.
[0052] By "amylin" is meant the human peptide hormone referred to
as amylin and secreted from the beta cells of the pancreas, and
species variations thereof, examples of which are described in U.S.
Pat. No. 5,234,906, the contents of which are hereby incorporated
by reference. More particularly, amylin is a 37-amino acid
polypeptide hormone normally co-secreted with insulin by pancreatic
beta cells in response to nutrient intake (see, e.g., Koda et al.
(1992) Lancet 339:1179-1180). In this sense, "amylin," "wild-type
amylin," and "native amylin," i e , unmodified amylin, are used
interchangeably. Amylin is also sometimes referred to as
"IAPP."
[0053] By "agonist" is meant a compound which elicits a biological
activity of amylin, for example, having a potency better than
amylin, or within five orders of magnitude (plus or minus) of
potency compared to amylin, for example 4, 3, 2, or 1 order of
magnitude, when evaluated by art-known measures such as, for
example, receptor binding/competition studies as described
herein.
[0054] In one embodiment, the term agonist refers to a compound
which elicits a biological effect similar to that of native amylin,
for example a compound (1) having activity in a food intake,
gastric emptying, pancreatic secretion, or weight loss assay (PCT
Application No. PCT/US2005/004631, filed on Feb. 11, 2005, and
incorporated by reference) similar to the native human reference
peptide, and/or (2) which binds specifically in a reference
receptor assay or in a competitive binding assay with amylin. In
one embodiment, the agonists will bind in such assays with an
affinity of better than 1 .mu.M, and, in another embodiment, with
an affinity of better than 1-5 nM. Such agonists may comprise a
polypeptide comprising an active fragment of amylin or a small
chemical molecule. In some embodiments, an agonist is a peptide,
not a small chemical molecule. It is, however, contemplated that in
certain embodiments, salmon calcitonin, calcitonin, CGRP, and/or
their respective analogs may be excluded from the scope of amylin
agonist with proviso language. In certain embodiments, an amylin
agonist is not a small chemical molecule and small chemical
molecules may be excluded from the scope of amylin agonist with
proviso language.
[0055] Agonists include amylin analogs and amylin derivatives. By
"analog" is meant a peptide whose sequence is derived from that of
amylin including insertions, substitutions, extensions, and/or
deletions, having at least some amino acid identity to amylin or
region of an amylin peptide. Analogs may have at least 50 or 55%
amino acid sequence identity with a native amylin, or at least 70%,
80%, 90%, or 95% amino acid sequence identity with a native amylin.
In one embodiment, such analogs may comprise conservative or
non-conservative amino acid substitutions (including non-natural
amino acids and L and D forms). Amylin agonist analogs are analogs
as herein described and function as an amylin agonist.
[0056] A "derivative" is defined as a molecule having the amino
acid sequence of a native amylin or analog, but additionally having
a chemical modification of one or more of its amino acid side
groups, .alpha.-carbon atoms, terminal amino group, or terminal
carboxylic acid group. A chemical modification includes, but is not
limited to, adding chemical moieties, creating new bonds, and
removing chemical moieties. Modifications at amino acid side groups
include, without limitation, acylation of lysine .epsilon.-amino
groups, N-alkylation of arginine, histidine, or lysine, alkylation
of glutamic or aspartic carboxylic acid groups, and deamidation of
glutamine or asparagine. Modifications of the terminal amino
include, without limitation, the desamino, N-lower alkyl,
N-di-lower alkyl, constrained alkyls (e.g. branched, cyclic, fused,
adamantyl) and N-acyl modifications. Modifications of the terminal
carboxy group include, without limitation, the amide, lower alkyl
amide, constrained alkyls (e.g. branched, cyclic, fused, adamantyl)
alkyl, dialkyl amide, and lower alkyl ester modifications. Lower
alkyl is C1-C4 alkyl. Furthermore, one or more side groups, or
terminal groups, may be protected by protective groups known to the
ordinarily-skilled synthetic chemist. The .alpha.-carbon of an
amino acid may be mono- or dimethylated.
[0057] Human amylin (hAmylin or h-amylin) has the following amino
acid sequence: Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln
Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-S-
er-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr (SEQ ID NO:1). Rat amylin
(rAmylin) has the following sequence:
KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY (SEQ ID NO:2). The use of
amylins from any species is contemplated.
[0058] Amylin agonists contemplated in the use in the methods
disclosed herein include those described in U.S. Pat. Nos.
5,686,411, 6,114,304, and 6,410,511, and PCT Application
Publication No. WO 93/10146, the contents of which are herein
incorporated by reference in their entirety. Such compounds include
those having formula I:
.sup.1A.sub.1-X-Asn-Thr-.sup.5Ala-Thr-Y-Ala-Thr-.sup.10Gln-Arg-Leu-B.sub.-
1-Asn-.sup.15Phe-Leu-C.sub.1-D.sub.1-E.sub.1-.sup.20F.sub.1-G.sub.1-Asn-H.-
sub.1-Gly-.sup.25I.sub.1-J.sub.1-Leu-K.sub.1-L.sub.1-.sup.30Thr-M.sub.1-Va-
l-Gly-Ser-.sup.35Asn-Thr-Tyr (SEQ ID NO:3); wherein A.sub.1 is Lys,
Ala, Ser or hydrogen; B.sub.1 is Ala, Ser or Thr; C.sub.1 is Val,
Leu or Ile; D.sub.1 is His or Arg; E.sub.1 is Ser or Thr; F.sub.1
is Ser, Thr, Gln or Asn; G.sub.1 is Asn, Gln or His; H.sub.1 is
Phe, Leu or Tyr; I.sub.1 is Ala or Pro; J.sub.1 is Ile, Val, Ala or
Leu; K.sub.1 is Ser, Pro, Leu, Ile or Thr; L.sub.1 is Ser, Pro or
Thr; M.sub.1 is Asn, Asp, or Gln; X and Y are independently
selected amino acid residues having side chains which are
chemically bonded to each other to form an intramolecular
linkage.
[0059] The C-terminal portion can be amino, alkylamino,
dialkylamino, cycloalkylamino, arylamino, aralkylamino, alkyloxy,
aryloxy, aralkyloxy or carboxyl. Suitable side chains for X and Y
include groups derived from alkyl sulfhydryls which may form
disulfide bonds; alkyl acids and alkyl amines which may form cyclic
lactams; alkyl aldehydes or alkyl halides and alkylamines which may
condense and be reduced to form an alkyl amine bridge; or side
chains which may be connected to form an alkyl, alkenyl, alkynyl,
ether or thioether bond. Preferred alkyl chains include lower alkyl
groups having from about 1 to about 6 carbon atoms.
[0060] An additional aspect, compositions and methods of use
provided herein are directed to agonist analogues of SEQ ID NO:3
which are not bridged, and wherein X and Y are independently
selected from Ala, Ser, Cys, Val, Leu and Ile or alkyl, aryl, or
aralkyl esters and ethers of Ser or Cys.
[0061] Exemplary compounds include, but are not limited to
des-.sup.1Lys-h-amylin (SEQ ID NO:4), .sup.28Pro-h-amylin (SEQ ID
NO:5), .sup.25,28,29pro-h-amylin (SEQ ID NO:6),
.sup.18Arg.sup.25,28Pro-h-amylin (SEQ ID NO:7), and
des-.sup.1Lys.sup.18Arg.sup.25,28Pro-h-amylin (SEQ ID NO:8), which
all show amylin activity in vivo in treated test animals, (e.g.,
provoking marked hyperlactemia followed by hyperglycemia). In
addition to having activities characteristic of amylin, certain of
the compounds provided herein have also been found to possess more
desirable solubility and stability characteristics when compared to
human amylin. Examples of these compounds include
.sup.25Pro.sup.26Val.sup.28,29Pro-h-amylin (SEQ ID NO:9),
25,28,29Pro-h-amylin, and .sup.18Arg.sup.25,28Pro-h-amylin.
[0062] Other compounds include .sup.18Arg.sup.25,28,29Pro-h-amylin
(SEQ ID NO:10), des-.sup.1Lys.sup.18Arg.sup.25,28,29Pro-h-amylin
(SEQ ID NO:11), des-.sup.1Lys.sup.25,28,29Pro-h-amylin (SEQ ID
NO:12), .sup.25Pro.sup.26Val.sup.28,29Pro-h-amylin (SEQ ID NO:13),
.sup.23Leu.sup.25Pro.sup.26Val.sup.28,29Pro-h-amylin (SEQ ID
NO:14), .sup.23Leu.sup.25Pro.sup.26Val.sup.28Pro-h-amylin (SEQ ID
NO:15),
des-.sup.1Lys.sup.23Leu.sup.25Pro.sup.26Val.sup.28Pro-h-amylin (SEQ
ID NO:16),
.sup.18Arg.sup.23Leu.sup.25Pro.sup.26Val.sup.28Pro-h-amylin (SEQ ID
NO:17), .sup.18Arg.sup.23Leu.sup.25,28,29Pro-h-amylin (SEQ ID
NO:18), .sup.18Arg.sup.23Leu.sup.25,28Pro-h-amylin (SEQ ID NO:19),
.sup.17Ile.sup.23Leu.sup.25,28,29Pro-h-amylin (SEQ ID NO:20),
.sup.17Ile.sup.25,28,29Pro-h-amylin (SEQ ID NO:21),
des-.sup.1Lys.sup.17Ile.sup.23Leu.sup.25,28,29Pro-h-amylin (SEQ ID
NO:22), .sup.17Ile.sup.18Arg.sup.23Leu-h-amylin (SEQ ID NO:23),
.sup.17Ile.sup.18Arg.sup.23Leu.sup.26Val.sup.29Pro-h-amylin (SEQ ID
NO:24),
.sup.17Ile.sup.18Arg.sup.23Leu.sup.25Pro.sup.26Val.sup.28,29Pro-h-
-amylin (SEQ ID NO:25),
.sup.13Thr.sup.21His23Leu.sup.26Ala.sup.28Leu.sup.29Pro.sup.31Asp-h-amyli-
n (SEQ ID NO:26),
.sup.13Thr.sup.21His.sup.23Leu.sup.26Ala.sup.29Pro.sup.31Asp-h-amylin
(SEQ ID NO:27),
des-.sup.1Lys.sup.13Thr.sup.21His.sup.23Leu.sup.26Ala.sup.28Pro.sup.31Asp-
-h-amylin (SEQ ID NO:28),
.sup.13Thr.sup.18Arg.sup.21His.sup.23Leu.sup.26Ala.sup.29Pro.sup.31Asp-h--
amylin (SEQ ID NO:29),
.sup.13Thr.sup.18Arg.sup.21His.sup.23Leu.sup.28,29Pro.sup.31Asp-h-amylin
(SEQ ID NO:30), and
.sup.13Thr.sup.18Arg.sup.21His.sup.23Leu.sup.25Pro.sup.26Ala.sup.28,29Pro-
.sup.31Asp-h-amylin (SEQ ID NO:31).
[0063] Amylin agonists contemplated in the use in the methods
disclosed herein include Amylin Family Polypeptide-6 (AFP-6)
analogs as described in U.S. Provisional Application No. 60/617,468
and PCT Application No. PCT/US05/036456, which are herein
incorporated by reference in their entirety. A mature AFP-6
peptide, also known as intermedin, has the following amino acid
sequence TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQDSAP VDPSSPHSY (SEQ ID
NO:32). The AFP-6 or AFP-6 analogs may or may not be amidated at
the C-terminal end. Such AFP-6 analogs include those having formula
II:
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-QVQNLSHRL-
WQL-X.sub.21-X.sub.22-X.sub.23-X.sub.24-X.sub.25-X.sub.26-X.sub.27-X.sub.2-
8-SAPV-X.sub.33-PSSPHSY (SEQ ID NO:33) wherein X.sub.1 is absent,
TQAQLLRVG (SEQ ID NO:34), any of one or more consecutive amino
acids of SEQ ID NO:34, N-aryl, or N-acyl with a substituent
selected from a C1-C18 alkyl, a substituted alkyl or a heteroaryl
moiety; X.sub.2 is M, S, C, substituted L, K, D, or E, where the
side chain can be linked via an amide bond, or any amino acid that
can form a bond with X.sub.8, for example a disulfide or an amide
bond; X.sub.3 is V, D, L, G, N, A, or S; X.sub.4 is V, D, L, G, N,
A, S or T; X.sub.5 is V, D, L, G, N, A, or S; X.sub.6 is V, D, L,
G, N, A, S, or absent; X.sub.7 is T, S, Hse (homoSER), Ahb
((S)-2-Amino-3-hydroxy-3-methybutanoic acid) or (Ahp)
(2R,3R)-2-Amino-3-hydroxy-4-methylpentanoic acid; X.sub.8 is M, S,
C, substituted L, K, D, or E, or any amino acid that can form a
bond with X.sub.2, for example a disulfide or an amide bond;
X.sub.21 is M, G, P, A, or absent; X.sub.22 is M, G, P, A, or
absent; X.sub.23 is M, G, P, A, or absent; X.sub.24 is M, G, P, A,
or absent; X.sub.25 is M, G, P, A, or absent; X.sub.26 is R or
absent, wherein when X.sub.26 is absent, X.sub.27 is absent;
X.sub.27 is Q or absent, wherein when X.sub.27 is absent, X.sub.26
is absent; X.sub.28 is D or E; X.sub.33 is D or E; and biologically
active fragments thereof.
[0064] In other embodiments, AFP-6 analogs comprise, or the active
region consists of, compounds having an amino acid sequence of
formula (III):
X.sub.1-X.sub.2-QNLSHRLWQL-X.sub.13-X.sub.14-X.sub.15-X.sub.16-X.sub.17-X-
.sub.18-X.sub.19-X.sub.20-SAPV-X.sub.25-PSSPHSY (SEQ ID NO:35)
wherein X.sub.1 is Q or absent; X.sub.2 is V or absent; X.sub.13 is
M, G, P, A, or absent; X.sub.14 is M, G, P, A, or absent; X.sub.15
is M, G, P, A, or absent; X.sub.16 is M, G, P, A, or absent;
X.sub.17 is M, G, P, A, or absent; X.sub.18 is R or absent, wherein
when X.sub.18 is absent, X.sub.19 is absent; X.sub.19 is Q or
absent, wherein when X.sub.19 is absent, X.sub.18 is absent;
X.sub.20 is D or E; X.sub.25 is D or E; and biologically active
fragments thereof.
[0065] Amino acid sequences of exemplary AFP-6 analogs for use in
the disclosed methods include:
TABLE-US-00001 (SEQ ID NO: 36)
RVGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO: 37)
GCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO: 38)
CVLGTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO: 39)
QVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO: 40)
VQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO: 41)
VQNLSHRL-QLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO: 42)
TQAQLLRVGCVLGTCQVQNLSHRLWQLRQDSAPVDPSSPHSY (SEQ ID NO: 43)
TQAQLLRVGCVLGTCQVQNLSHRLWQLDSAPVDPSSPHSY (SEQ ID NO: 44)
VGCVLGTCQVQNLSHRLWQLRQDSAPVDPSSPHSY (SEQ ID NO: 45)
CVLGTCQVQNLSHRLWQLRQESAPVEPSSPHSY (SEQ ID NO: 46)
TQAQLLRVGCSNLSTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO: 47)
TQAQLLRVGCNTATCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO: 48)
RVGCGNLSTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO: 49)
TQAQLLRVGCDTATCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO: 50)
TQAQLLRVGCGNLSTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO: 51)
TQAQLLRVGMVLGTMQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO: 52)
GMVLGTMQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO: 53)
VGMVLGTMQVQNLSHRLWQLRQDSAPVDPSSPHSY (SEQ ID NO: 54)
RVGCGNLSTCQVQNLSHRLWQLMGPAGRQDSAPVDPSSPHSY (SEQ ID NO: 55)
VGCGNLSTCQVQNLSHRLWQLRQDSAPVDPSSPHSY (SEQ ID NO: 56)
V-CNTA-TCQVQNLSHRLWQLRQDSAPVDPSSPHSY (SEQ ID NO: 57)
GCNTATCQVQNLSHRLWQLRQDSAPVDPSSPHSY (SEQ ID NO: 58)
TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQESAPVEPSSPHSY (SEQ ID NO: 59)
TQAQLLRVGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVEPSSPHSY (SEQ ID NO: 60)
GTMQVQNLSHRLWQLRQDSAPVEPSSPHSY (SEQ ID NO: 61)
VGCVLGTCQVQNLSHRLWQLMGPAGRQDSAPVEPSSPHSY (SEQ ID NO: 62)
VGCVLGTCQVQNLSHRLWQLRQDSAPVEPSSPHSY (SEQ ID NO: 63)
GCNTATCQVQNLSHRLWQLRQDSAPVEPSSPHSY (SEQ ID NO: 64)
GCSNLSTCQVQNLSHRLWQLRQDSAPVEPSSPHSY (SEQ ID NO: 65)
GCGNLSTCQVQNLSHRLWQLRQDSAPVEPSSPHSY (SEQ ID NO: 66)
GCVLGTCQVQNLSHRLWQLRQESAPVEPSSPHSY.
[0066] Amylin agonists contemplated in the use in the methods
disclosed herein include analogs identified in U.S. Pat. No.
6,087,334, the contents of which is hereby incorporated by
reference. Such useful amylin agonists include analogs of formula
IV:
X.sub.1-Xaa.sub.1-X.sub.2-Xaa.sub.2-X.sub.3-Xaa.sub.3-X.sub.4-Xaa.sub.4-X-
.sub.5-Xaa.sub.5-X.sub.6 (SEQ ID NO:67) wherein X.sub.1 is Lys, Arg
or absent; X.sub.2 is Xaa.sub.6Xaa.sub.7Xaa.sub.8Xaa.sub.9 (SEQ.
ID. NO.68) or Z-Xaa.sub.10SerThr, provided that if X.sub.2 is
Z-Xaa.sub.10SerThr, then X.sub.1 and Xaa.sub.1 are both absent;
X.sub.3 is AlaThr, AlaSer, SerMet, GluThr or ValThr; X.sub.4 is
ArgLeuAla, HisLeuAla, ArgIleAla, LysIleAla, ArgMetAla, HisMetAla,
LysMetAla or ArgLeuThr; X.sub.5 is PheLeu, PheIle, PheMet, TyrLeu,
TyrIle, TyrMet, TrpIle or TrpMet; X.sub.6 is ArgSerSerGlyTyr (SEQ
ID NO:69), LysSerSerGlyTyr (SEQ ID NO:70), HisSerSerGlyTyr (SEQ ID
NO:71), ProSerSerGlyTyr (SEQ ID NO:72), ArgSerArgGlyTyr (SEQ ID
NO:73), ArgThrSerGlyTyr (SEQ ID NO:74), ArgAlaSerGlyTyr (SEQ ID
NO:75), AlaSerSerGlyTyr (SEQ ID NO:76), ArgSerAlaGlyTyr (SEQ ID
NO:77), HisSerAlaGlyTyr (SEQ ID NO:78), ArgSerGlyTyr (SEQ ID
NO:79), ArgSer, LysSer, HisSer, ArgThr, ProSer or Arg; Xaa.sub.1 is
Cys or absent; Xaa.sub.2 is Cys or Ala; Xaa.sub.3 is Gln, Ala or
Asn; Xaa.sub.4 is Asn, Ala or Gln; Xaa.sub.5 is Val, Ala, Ile, Met,
Leu, PentylGly, or t-butylGly; Xaa.sub.6 is Asn, Gln or Asp;
Xaa.sub.7 is Thr, Ser, Met, Val, Leu or Ile; Xaa.sub.8 is Ala or
Val; Xaa.sub.9 is Thr or Ser; Xaa.sub.10 is Leu, Val, Met or Ile; Z
is an alkanoyl group of about 1 to about 8 carbon atoms or absent;
and pharmaceutically acceptable salts thereof.
[0067] It should be noted that throughout the application that
alternatives are written in Markush groups, for example, each amino
acid position that contains more than one possible amino acid. It
is specifically contemplated that each member of the Markush group
should be considered separately, thereby comprising another
embodiment, and the Markush group is not to be read as a single
unit.
[0068] Amylin agonists contemplated in the use in the methods
disclosed herein include the amylin family peptides and analogs
described in PCT Application No. PCT/US2005/00463 1, which is
herein incorporated by reference in its entirety. Such useful
amylin agonists may include analogs comprising an amino acid
sequence of formula V: Xaa.sub.1 X Xaa.sub.3 Xaa.sub.4 Xaa.sub.5
Xaa.sub.6 Y Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Xaa.sub.11 Xaa.sub.12
Xaa.sub.13 Xaa.sub.14 Xaa.sub.15 Xaa.sub.16 Xaa.sub.17 Xaa.sub.18
Xaa.sub.19 Xaa.sub.20 Xaa.sub.21 Xaa.sub.22 Xaa.sub.23 Xaa.sub.24
Xaa.sub.25 Xaa.sub.26 Xaa.sub.27 Xaa.sub.28 Xaa.sub.29 Xaa.sub.30
Xaa.sub.31 Xaa.sub.32 (SEQ ID NO:80) wherein Xaa.sub.1 is A, C, hC
(homoCys), D, E, F, I, L, K, hK (homoLys), R, hR (homoArg), S,
Hse(homoSer), T, G, Q, N, M, Y, W, P, Hyp (hydroxyPro), H, V or
absent; Xaa.sub.3 is A, D, E, N, Q, G, V, R, K, hK, hR, H, I, L, M,
or absent; Xaa.sub.4 is A, I, L, S, Hse, T, V, M, or absent;
Xaa.sub.5 is A, S, T, Hse, Y, V, I, L, or M; Xaa.sub.6 is T, A, S,
Hse, Y, V, I, L, or M; Xaa.sub.8 is A, V, I, L, F, or M; Xaa.sub.9
is L, T, S, Hse, V, I, or M; Xaa.sub.10 is G, H, Q, K, R, N, hK, or
hR; Xaa.sub.11 is K, R, Q, N, hK, hR, or H; Xaa.sub.12 is L, I, V,
F, M, W, or Y; Xaa.sub.13 is A, F, Y, N, Q, S, Hse, orT; Xaa.sub.14
is A, D, E, G, N, K, Q, R, H, hR, or hK; Xaa.sub.15 is A, D, E, F,
L, S, Y, I, V, or M; Xaa.sub.16 is L, F, M, V, Y, or I; Xaa.sub.17
is H, Q, N, S, Hse, T, or V; Xaa.sub.18 is K, hK, R, hR, H, u
(Cit), or n (Orn); Xaa.sub.19 is F, L, S, Hse, V, I, T, or absent;
Xaa.sub.20 is H, R, K, hR, hK, N, Q, or absent; Xaa.sub.21 is T, S,
Hse, V, I, L, Q, N, or absent; Xaa.sub.22 is F, L, M, V, Y, or I;
Xaa.sub.23 is P or Hyp; Xaa.sub.24 is P, Hyp, R, K, hR, hK, or H;
Xaa.sub.25 is T, S, Hse, V, I, L, F, or Y; Xaa.sub.26 is N, Q, D,
or E; Xaa.sub.27 is T, V, S, F, I, or L; Xaa.sub.28 is G or A;
Xaa.sub.29 is S, Hse, T, V, I, L, or Y; Xaa.sub.30 is E, G, K, N,
D, R, hR, hK, H, or Q; Xaa.sub.31 is A, T, S, Hse, V, I, L, F, or
Y; and Xaa.sub.32 is F, P, Y, Hse, S, T, or Hyp; wherein X and Y
are capable of creating a bond and are independently selected
residues having side chains which are chemically bonded to each
other to form an intramolecular linkage such as disulfide bonds;
amide bond; alkyl acids and alkyl amines which may form cyclic
lactams; alkyl aldehydes or alkyl halides and alkylamines which may
condensed and be reduced to form an alkyl amine or imine bridge; or
side chains which may be connected to form an alkyl, alkenyl,
alkynyl, ether or thioether bond.
[0069] Alkyl chains may include lower alkyl groups having from
about 1 to about 6 carbon atoms. In certain embodiments, the
intramolecular linkage may be a disulfide, amide, imine, amine,
alkyl or alkene bond. In certain embodiments, X and Y are
independently selected from Ser, Asp, Glu, Lys, Orn, or Cys. In
certain embodiments, X and Y are Cys and Cys. In other embodiments,
X and Y are Ser and Ser. In still other embodiments, X and Y are
Asp and Lys or Lys and Asp.
[0070] Useful amylin agonists may also include analogs comprising
the amino acid sequence of formula VI: Xaa.sub.1 Xaa.sub.2
Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6 Xaa.sub.7 Xaa.sub.8
Xaa.sub.9 Xaa.sub.10 Xaa.sub.11 Xaa.sub.12 Xaa.sub.13 Xaa.sub.14
Xaa.sub.15 Xaa.sub.16 Xaa.sub.17 Xaa.sub.18 Xaa.sub.19 Xaa.sub.20
Xaa.sub.21 Xaa.sub.22 P Xaa.sub.24 T N Xaa.sub.27 G S Xaa.sub.30
Xaa.sub.31 Xaa.sub.32 (SEQ ID NO:81) wherein Xaa.sub.1 is A, C, D,
F, I, K, S, T, or absent; Xaa.sub.2 is C, D, S, or absent;
Xaa.sub.3 is A, D, N, or absent; Xaa.sub.4 is A, L, T, or absent;
Xaa.sub.5 is A or S; Xaa.sub.6 is T, A, S, or V; Xaa.sub.7 is C, K,
or A; Xaa.sub.8 is A, V, L, orM; Xaa.sub.9 is L or T; Xaa.sub.10 is
G, H, or Q; Xaa.sub.11 is K, R, Q, or hArg; Xaa.sub.12 is L, W, or
Y; Xaa.sub.13 is A, F, N, Q, S, or T; Xaa.sub.14 is A, D, E, G, N,
K, Q, or R; Xaa.sub.15 is A, D, E, F, L, S, or Y; Xaa.sub.16 is L,
or F; Xaa.sub.17 is H, Q, S, or V; Xaa.sub.18 is K, R, hArg, u
(Cit), or n (Orn); Xaa.sub.19 is F, L, S, or absent; Xaa.sub.20 is
H, Q, or absent; Xaa.sub.21 is T, N, or absent; Xaa.sub.22 is F, L,
M, V, or Y; Xaa.sub.24 is P or R; Xaa.sub.27 is T or V; Xaa.sub.30
is E, G, K, or N; Xaa.sub.31 is A or T; and Xaa.sub.32 is F, P, or
Y.
[0071] Useful amylin agonists may also include analogs comprising
the amino acid sequence of formula VII: Xaa.sub.1 Xaa.sub.2
Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 T Xaa.sub.7 Xaa.sub.8 Xaa.sub.9
Xaa.sub.10 Xaa.sub.11 L Xaa.sub.13 Xaa.sub.14 Xaa.sub.15 L
Xaa.sub.17 Xaa.sub.18 Xaa.sub.19 Xaa.sub.20 Xaa.sub.21 Xaa.sub.22 P
Xaa.sub.24 T N Xaa.sub.27 G S Xaa.sub.30 Xaa.sub.31 Xaa.sub.32,
(SEQ ID NO:82) wherein Xaa.sub.1 is A, C, F, I, K, S, or absent;
Xaa.sub.2 is C, D, or S; Xaa.sub.3 is A, D or N; Xaa.sub.4 is A, L
or T; Xaa.sub.5 is A or S; Xaa.sub.7 is C or K; Xaa.sub.8 is A or
V; Xaa.sub.9 is L or T; Xaa.sub.10 is G, H, or Q; Xaa.sub.11 is K,
R, or hArg; Xaa.sub.13 is A, F, N, S, or T; Xaa.sub.14 is A, D, E,
G, N, Q, or R; Xaa.sub.15 is A, E, F, L, S, or Y; Xaa.sub.17 is H,
S, or V; Xaa.sub.18 is K, R, hArg, u (Cit), or n (Orn); Xaa.sub.19
is F, L, or S; Xaa.sub.20 is H or Q; Xaa.sub.21 is T or N;
Xaa.sub.22 is F, L, M, V, or Y; Xaa.sub.24 is P or R; Xaa.sub.27 is
T, or V; Xaa.sub.30 is E, G, K, or N; Xaa.sub.31 is A, or T; and
Xaa.sub.32 is F, P, or Y.
[0072] Useful amylin agonists may also include analogs comprising
the amino acid sequence of formula VIII: Xaa.sub.1 Xaa.sub.2
Xaa.sub.3 Xaa.sub.4 Xaa.sub.5 Xaa.sub.6 Xaa.sub.7 Xaa.sub.8
Xaa.sub.9 Xaa.sub.10 Xaa.sub.11 Xaa.sub.12 Xaa.sub.13 Xaa.sub.14
Xaa.sub.15 Xaa.sub.16 Xaa.sub.17 Xaa.sub.18 Xaa.sub.19 Xaa.sub.20
Xaa.sub.21 Xaa.sub.22 P Xaa.sub.24 T N Xaa.sub.27 G S Xaa.sub.30
Xaa.sub.31 Xaa.sub.32 (SEQ ID NO:83) wherein Xaa.sub.1 is A, C, D,
F, K, T, or absent; Xaa.sub.2 is A, C, D, S, or absent; Xaa.sub.3
is A, D, N, or absent; Xaa.sub.4 is A, L, T, or absent; Xaa.sub.5
is A or S; Xaa.sub.6 is A, S, T, or V; Xaa.sub.7 is A, C, or K;
Xaa.sub.8 is A, L, M, or V; Xaa.sub.9 is L or T; Xaa.sub.10 is G,
H, or Q; Xaa.sub.11 is K, Q, or R; Xaa.sub.12 is L, W, or Y;
Xaa.sub.13 is A, N, Q, S, or T; Xaa.sub.14 is A, D, E, G, K, N, Q,
or R; Xaa.sub.15 is A, D, E, F, L, S, or Y; Xaa.sub.16 is F or L;
Xaa.sub.17 is H, Q, S or V; Xaa.sub.18 is K, or R; Xaa.sub.19 is F,
L, S, or absent; Xaa.sub.20 is H, K, Q, or absent; Xaa.sub.21 is Q,
T, or absent; Xaa.sub.22 is F, L, or Y; Xaa.sub.24 is P or R;
Xaa.sub.27 is T or V; Xaa.sub.30 is E, K or N; Xaa.sub.31 is A or
T; and Xaa.sub.32 is F, Y, or absent.
[0073] In a general aspect, the sequence of formula V, VI, VII, or
VIII further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or
more modifications of substitutions, insertions, deletions,
elongations and/or derivatizations. In certain embodiments, the
sequence of formula V, VI, VII, or VIII comprises a deletion at
position 24. In certain embodiments, the sequence of formula V, VI,
or VII comprises a Val inserted between amino acids at positions 22
and 23. In other embodiments, the sequence of formula V, VI, or VII
comprises a Gln inserted between positions 22 and 23. In still
other embodiments, the sequence of formula V, VI, or VII comprises
a sequence of Gln-Thr-Tyr between positions 22 and 23. In yet other
embodiments, the sequence of formula V, VI, or VII comprises a
sequence of Leu-Gln-Thr-Tyr (SEQ ID NO:84) between positions 22 and
23. In another general aspect, the modifications of formula V, VI,
or VII may be at the N-terminal end. In certain embodiments, the
N-terminal portion of formula V, VI, or VII has an added
octylglycine. In other embodiments, the N-terminal portion of
formula V, VI, or VII has an added isocap. Other embodiments are
described in PCT Application No. PCT/US2005/004631 and incorporated
by reference.
[0074] Exemplary compounds described with reference to human amylin
(SEQ ID NO:1; hAmylin), rat amylin (SEQ ID NO:2; rAmylin), and
salmon calcitonin (sCT) CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP (SEQ ID
NO:85) with modifications at the position(s) indicated include,
(1-7 hAmylin)(.sup.18Arg 8-27 sCT)(33-37 hAmylin) (SEQ ID NO:86);
(1-7 hAmylin)(.sup.11,18Arg.sup.22Leu 8-27sCT)(33-37 hAmylin) (SEQ
ID NO:87); (1-7 hAmylin)(.sup.11,18Arg.sup.24Pro 8-27 sCT)(33-37
hAmylin) (SEQ ID NO:88); (1-7 hAmylin)(.sup.11,18Arg 8-24
sCT)(30-37 hAmylin) (SEQ ID NO:89); (1-7 hAmylin)(.sup.11,18Arg
8-21 sCT)(27-37 rAmylin) (SEQ ID NO:90);
(.sup.8Val.sup.9Leu.sup.10Gly 1-15 hAmylin)(.sup.18Arg 16-27
sCT)(31-37 hAmylin) (SEQ ID NO:91); (.sup.1Ala 1-7
hAmylin)(.sup.11,18Arg 8-27 sCT)(33-37 hAmylin) (SEQ ID NO:92);
(.sup.3Ala 1-7 hAmylin)(.sup.11,18Arg 8-27 sCT)(33-37 hAmylin) (SEQ
ID NO:93); (.sup.4Ala 1-7 hAmylin)(.sup.11,18Arg 8-27 sCT)(33-37
hAmylin) (SEQ ID NO:94); (.sup.6Ala 1-7 hAmylin)(.sup.11,18Arg 8-27
sCT)(33-37 hAmylin) (SEQ ID NO:95); (.sup.2Ala.sup.11,18Arg 1-27
sCT)(33-37 hAmylin) (SEQ ID NO:96); (Isocap-.sup.7Ala.sup.11,18Arg
5-27 sCT)(33-37 hAmylin) (SEQ ID NO:97); (.sup.4Ala.sup.11,18Arg
1-27 sCT)(33-37 hAmylin) (SEQ ID NO:98); (.sup.5Ala.sup.11,18Arg
1-27 sCT)(33-37 hAmylin) (SEQ ID NO:99); (.sup.6Ala.sup.11,18Arg
1-27 sCT)(33-37 hAmylin) (SEQ ID NO:100); (1-7 hAmylin)(.sup.11Arg
8-27 sCT)(33-37 hAmylin) (SEQ ID NO:101);
(.sup.13Ser.sup.14Gln.sup.15Glu 1-16
hAmylin)(.sup.17Arg.sup.30Asn.sup.32Tyr 17-32 sCT) (SEQ ID NO:102);
(.sup.3Ala.sup.11,18Arg 1-27 sCT)(33-37 hAmylin) (SEQ ID NO:103);
(Acetyl-2,7Agy.sup.11,18Arg 1-27 sCT)(33-37 hAmylin) (SEQ ID
NO:104); (Acetyl-.sup.2,7Agy 1-7 hAmylin)(.sup.11,18Arg 8-27
sCT)(33-37 hAmylin) (SEQ ID NO:105);
(Isocap-.sup.7Ala.sup.10Aib.sup.11Lys(For).sup.17Aib.sup.18Lys(For)
5-27 sCT)(33-37 hAmylin) (SEQ ID NO:106);
(Isocap-.sup.7Ala.sup.10Aib.sup.11Lys(For).sup.17Aib.sup.18Lys(For)
5-24sCT)(30-37 hAmylin) (SEQ ID NO:107);
(Isocap-.sup.7Ala.sup.10Aib.sup.11Lys(For).sup.17Aib.sup.18Lys(For)
5-22 sCT)(.sup.28,29Pro 28-37 hAmylin) (SEQ ID NO:108);
(Isocap-.sup.7Ala.sup.10Aib.sup.11Lys(For).sup.17Aib.sup.18Lys(For)
5-21 sCT)(.sup.28,29Pro 27-37 hAmylin) (SEQ ID NO:109); (1-7
hAmylin)(LLQQWQKLLQKLKQ (SEQ ID
NO:110))(.sup.28Pro.sup.29Arg.sup.32Thr 27-37 hAmylin) (SEQ ID
NO:111); (1-7 hAmylin)(LLQQLQKLLQKLKQY (SEQ ID
NO:112))(.sup.28Pro.sup.29Arg.sup.32Thr 28-37 hAmylin) (SEQ ID
NO:113); (.sup.6Ser 1-7 hAmylin)(.sup.11,18Arg 8-27 sCT)(33-37
hAmylin) (SEQ ID NO:114); (.sup.6Val 1-7 hAmylin)(.sup.11,18Arg
8-27 sCT)(33-37 hAmylin) (SEQ ID NO:115); (1-7
hAmylin)(.sup.11,18Arg 8-18 sCT)(.sup.28Pro.sup.29Arg.sup.32Thr
27-37 hAmylin) (SEQ ID NO:116); (1-7 hAmylin)(.sup.11Arg 8-17
sCT)(.sup.28Pro.sup.29Arg.sup.32Thr 27-37 hAmylin) (SEQ ID NO:117);
(1-7 hAmylin)(.sub.11Arg 8-16
sCT)(.sup.27Tyr.sup.28Pro.sup.29Arg.sup.32Thr Thr 27-37 hAmylin)
(SEQ ID NO:118); (1-7 hAmylin)(.sup.11Arg
8-15sCT)(.sup.27Tyr.sup.28Pro.sup.29Arg.sup.32Thr 27-37 hAmylin)
(SEQ ID NO:119); (1-7 hAmylin)(.sub.11Arg 8-14
sCT)(.sup.27Tyr28Pro.sup.29 Arg.sup.32 Thr 27-37 hAmylin) (SEQ ID
NO:120); (1-7 hAmylin)(.sup.11,18Lys(For) 8-27 sCT)(33-37 hAmylin)
(SEQ ID NO:121); (.sup.6D-Thr 1-7 hAmylin)(.sup.11,18Arg 8-27
sCT)(33-37 hAmylin) (SEQ ID NO:122); (Acetyl-1-7
hAmylin)(.sup.11,18Lys(PEG5000) 8-27 sCT)(33-37 hAmylin) (SEQ ID
NO:123); (Acetyl-.sup.1Ala 1-7
hAmylin)(.sup.11Lys(PEG5000).sup.18Arg 8-27 sCT)(33-37 hAmylin)
(SEQ ID NO:124); (Acetyl-.sup.1Ala 1-7
hAmylin)(.sup.11Arg.sup.18Lys(PEG5000) 8-27 sCT)(33-37 hAmylin)
(SEQ ID NO:125); (1-7 hAmylin)(.sup.11,18Arg 8-21 sCT)(19-27
sCT)(33-37 hAmylin) (SEQ ID NO:126); (1-7 hAmylin)(.sup.11,18Arg
8-21 sCT)(.sup.18Leu 18-27 sCT)(33-37 hAmylin) (SEQ ID NO:127);
(1-7 hAmylin)(8-27 sCT)(33-37 hAmylin) (SEQ ID NO:128); (.sup.5Ser
1-7 hAmylin)(.sup.11,18Arg 8-27 sCT)(33-37 hAmylin) (SEQ ID
NO:129); (1-12 hAmylin)(.sup.18Arg 13-27 sCT)(33-37 hAmylin) (SEQ
ID NO:130); (1-12 hAmylin)(.sup.18Arg 13-24 sCT)(30-37 hAmylin)
(SEQ ID NO:131); (.sup.5Ser.sup.15Glu.sup.18Arg 1-18hAmylin)(19-24
sCT)(30-37 hAmylin) (SEQ ID NO:132; (.sup.6Hse 1-7
hAmylin)(.sup.11,18Arg 8-27 sCT)(33-37 hAmylin) (SEQ ID NO:133);
(.sup.6Ahb 1-7 hAmylin)(.sup.11,18Arg 8-27 sCT)(33-37 hAmylin) (SEQ
ID NO:134); (.sup.6Ahp 1-7hAmylin)(.sup.11,18Arg 8-27 sCT)(33-37
hAmylin) (SEQ ID NO:135); .sup.6Thr(OPO.sub.3H.sub.2) 1-7
hAmylin)(.sup.11,18Arg 8-27 sCT)(33-37 hAmylin) (SEQ ID NO:136);
(.sup.7Ala.sup.11,18Arg 5-27 sCT)(33-37 hAmylin) (SEQ ID NO:137);
(1-7 hAmylin)(.sup.11,18Orn 8-27 sCT)(33-37 hAmylin) (SEQ ID
NO:138); (1-7 hAmylin)(.sup.11,18Cit 8-27 sCT)(33-37 hAmylin) (SEQ
ID NO:139); (1-7 hAmylin)(.sup.11,18homoLys 8-27 sCT)(33-37
hAmylin) (SEQ ID NO:140); (L-Octylglycine-1-7hAmylin)(.sup.11,18Arg
8-27 sCT)(33-37 hAmylin) (SEQ ID NO:141);
(N-3,6-dioxaoctanoyl-1-7-hAmylin)(.sup.11,18Arg 8-27 sCT)(33-37
hAmylin) (SEQ ID NO:142);
(cyclo(1-7)-.sup.1Asp.sup.7Lys.sup.11,18Arg 1-27 sCT)(33-37
hAmylin) (SEQ ID NO:143); (cyclo(2-7)-.sup.2Asp.sup.7Lys 1-7
hAmylin)(.sup.11,18Arg 8-27 sCT)(33-37 hAmylin) (SEQ ID NO:144);
(cyclo (2-7) hAmylin)(.sup.11,18Arg 8-27 sCT)(33-37 hAmylin) (SEQ
ID NO:145); (1-7 hAmylin)(.sup.11,18Arg 8-27 sCT)(33-37
hAmylin-9Anc) (SEQ ID NO:146); (1-7 hAmylin)(.sup.11,18Arg 8-27
sCT)(33-37 hAmylin-L-octylglycine) (SEQ ID NO:147);
(N-isocaproyl-1-7-hAmylin)(.sup.11,18Arg 8-27sCT)(33-37 hAmylin)
(SEQ ID NO:148); (1-7 hAmylin)(.sup.11,18homoArg 8-27 sCT)(33-37
hAmylin) (SEQ ID NO:149); (.sup.1Phe 1-7 hAmylin)(.sup.11,18Arg
8-27 sCT)(33-37 hAmylin) (SEQ ID NO:150); (1-7
hAmylin)(.sup.11,18Arg 8-24 sCT)(.sup.32Thr 30-37 hAmylin) (SEQ ID
NO:151); (1-7 hAmylin)(.sup.11,18Arg 8-27 sCt)(33-37 hAmylinlin)
(SEQ ID NO:152); (.sup.15Glu.sup.18Arg 1-18 hAmylin)(19-24
sCT)(30-37 hAmylin) (SEQ ID NO:153);
(.sup.13Ala.sup.14Asp.sup.15Phe 1-18 hAmylin)(19-23 sCT)(30-37
hAmylin) (SEQ ID NO:154); and (2-18 hAmylin)(19-23 sCT)(30-36
hAmylin) (SEQ ID NO:155). Peptides useful in the compositions and
methods provided herein, like those above, can be in the acid or
amide form.
[0075] Exemplary peptides also for use in the compositions and
methods provided herein include:
TABLE-US-00002 (SEQ ID NO: 156) KCNTATCVLGKLSQELHRLQTYPRTNTGSNTY
(SEQ ID NO: 157) KCNTATCVLGRLSQELHRLQTLPRTNTGSNTY (SEQ ID NO: 158)
KCNTATCVLGRLSQELHRLQTYPPTNTGSNTY (SEQ ID NO: 159)
KCNTATCVLGRLSQELHRLQTYPRTNVGSNTY (SEQ ID NO: 160)
KCNTATCVLGRLSQELHRLQTLPPTNVGSNTY (SEQ ID NO: 161)
KCNTATCVLGRLANFLHRLQTYPRTNTGSNTY (SEQ ID NO: 162)
ACNTATCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 163)
KCNAATCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 164)
KCNTAACVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 165)
CANLSTCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 166)
isocaproyl-STAVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 167)
CSNASTCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 168)
CSNLATCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 169)
CSNLSACVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 170)
KCNTATCVLGRLSQELHKLQTYPRTNTGSNTY (SEQ ID NO: 171)
KCNTATCVLGRLSQELHRLQTYPRTNTGSGTP (SEQ ID NO: 172)
CSALSTCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 173)
Ac-(Agy)SNLST(Agy)VLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 174)
Ac-K(Agy)NTAT(Agy)VLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 175)
Isocaproyl-STAVL(Aib)RLSQELRLQTYPRTNTGSGTP (SEQ ID NO: 176)
Isocaproyl-STAVLG[K(For)]LSQELH[K(For)]LQTYPRTNTGSGTP (SEQ ID NO:
177) Isocaproyl-STAVL(Aib)[K(For)]LSQEL(Aib)[K(For)]LQTYPRTNTGSNTY
(SEQ ID NO: 178)
Isocaproyl-STAVL(Aib)[K(For)]LSQEL(Aib)[K(For)]LQTYPRTNVGSNTY (SEQ
ID NO: 179) KCNTATCLLQQLQKLLQKLKQYPRTNTGSNTY (SEQ ID NO: 180)
KCNTASCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 181)
KCNTAVCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 182)
KCNTATCVLGRLSQELHRYPRTNTGSNTY (SEQ ID NO: 183)
KCNTATCVLG[K(For)]LSQELH[K(For)L]QTYPRTNTGSNTY (SEQ ID NO: 184)
KCNTA(d-Thr)CVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 185)
KCNTA(dAh)CVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 186)
Ac-ACNTATCVLGRLSQELHK(PEG5000)LQTYPRTNTGSNTY (SEQ ID NO: 187)
KCNTATCVLGRLSQELHRLQTLQTYPRTNTGSNTY (SEQ ID NO: 188)
KCNTATCVLGRLSQELHRLQTLLQTYPRTNTGSNTY (SEQ ID NO: 189)
KCNTATCVLGKLSQELHKLQTYPRTNTGSNTY (SEQ ID NO: 190)
KCNTSTCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 191)
KCNTATCATQRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 192)
KCNTATCATQRLSQELHRLQTYPRTNVGSNTY (SEQ ID NO: 193)
KCNTSTCATQRLANELVRLQTYPRTNVGSNTY (SEQ ID NO: 194)
KCNTA(Hse)CVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 195)
KCNTA(Ahb)CVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 196)
KCNTA(Ahp)CVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 197)
KCNTAT(OPO.sub.3H.sub.2)CVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 198)
KCNTATCVLG(Orn)LSQELH(Orn)LQTYPRTNTGSNTY (SEQ ID NO: 199)
KCNTATCVLG(Cit)LSQELH(Cit)LQTYPRTNTGSNTY (SEQ ID NO: 200)
KCNTATCVLG(hK)LSQELH(hK)LQTYPRTNTGSNTY (SEQ ID NO: 201)
L-OctylglycineKCNTATCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 202)
N-3,6-dioxaoctanoyl-CNTATCVLGRLSQELHRLQTVPRTNTGSNTY (SEQ ID NO:
203) KCNTATCMLGRYTQDFHRLQTYPRTNTGSNTY (SEQ ID NO: 204)
DSNLSTKVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 205)
KDNTATKVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 206)
CNTATCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 207)
KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY(9Anc) (SEQ ID NO: 208)
KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY(L-octylglycine) (SEQ ID NO: 209)
N-isocaproyl-KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 210)
KCNTATCVLG(hR)LSQELH(hR)LQTYPRTNTGSNTY (SEQ ID NO: 211)
FCNTATCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 212)
KCNTATCVLGRLSQELH(Cit)LQTYPRTNTGSNTY (SEQ ID NO: 213)
KCNTATCVLGRLSQELH(Orn)LQTYPRTNTGSNTY (SEQ ID NO: 214)
ICNTATCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 215)
1-Octylglycine-CNTATCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 216)
Isocaproyl-CNTATCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 217)
KCNTATCVLG(Cit)LSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 218)
KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY(4ABU) (SEQ ID NO: 219)
Isocaproyl-KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY(4ABU) (SEQ ID NO: 220)
KCNTSTCATQRLANELVRLQTYPRTNVGSEAF (SEQ ID NO: 221)
KCNTATCVLGRLSQELHRLQTYPTNVGSEAF (SEQ ID NO: 222)
KCNTATCVLGRLSRSLHRLQTYPRTNTGSNTY (SEQ ID NO: 223)
KCNTATCVTHRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 224)
KCNTATCVLGRLADFLHRLQTYPRTNTGSNTY (SEQ ID NO: 225)
CNTATCVLGRLSQELHRLQTYPRTNTGSNT (SEQ ID NO: 226)
KCNTATCVLGRLSQELHRLQNFVPRTNTGSNTY (SEQ ID NO: 227)
KCNTATCVLGRLSQELHRLQTYPRTNTGSETF (SEQ ID NO: 228)
ACDTATCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 229)
KCNTATCVLGRLSQELHRLQTYPRTNTGSKAF (SEQ ID NO: 230)
KCDTATCVTHRLAGLLSRSQTYPRTNTGSNTY (SEQ ID NO: 231)
KCNTATCVLGRLADALHRLQTYPRTNTGSNTY (SEQ ID NO: 232)
KCNTATCVLGRLAAFLHRLQTYPRTNTGSNTY (SEQ ID NO: 233)
SCNTATCVLGRLADFLHRLQTYPRTNTGSNTY (SEQ ID NO: 234)
KCNTATCVLGRLSQELHRLQTMPRTNTGSNTY (SEQ ID NO: 235)
KCNTATCVLGRLSQELHRLQTVPRTNTGSNTY (SEQ ID NO: 236)
KCNTATCVLGRLNEYLHRLQTYPRTNTGSNTY (SEQ ID NO: 237)
SCNTATCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 238)
KCNTATCVLGRLTEFLHRLQTYPRTNTGSNTY (SEQ ID NO: 239)
KCNTATCVLGRLAEFLHRLQTYPRTNTGSNTY (SEQ ID NO: 240)
KCNTATCVLGRLTDYLHRLQTYPRTNTGSNTY (SEQ ID NO: 241)
KCNTATCVLGRLAQFLHRLQTYPRTNTGSNTY (SEQ ID NO: 242)
KCNTATCVLGRLADFLHRFQTFPRTNTGSNTY (SEQ ID NO: 243)
KCNTATCVLGRLADFLHRFHTFPRTNTGSNTY (SEQ ID NO: 244)
KCNTATCVLGRLADFLHRFQTFPRTNTGSGTP (SEQ ID NO: 245)
CNTATCVLGRLADFLHRLQTYPRTNTGSNTY (SEQ ID NO: 246)
KCDTATCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 247)
KCNTATCVLGRLFDFLHRLQTYPRTNTGSNTY (SEQ ID NO: 248)
KCNTATCVLGRLAAALHRLQTYPRTNTGSNTY (SEQ ID NO: 249)
TCDTATCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 250)
CSNLSTCATQRLANELVRLQTYPRTNVGSNTY (SEQ ID NO: 251)
KCNTATCATQRLANELVRLQTYPRTNVGSNTY (SEQ ID NO: 252)
CSNLSTCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO: 253)
KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY.
[0076] In some embodiments, compounds comprising the amino acid
sequence KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY (SEQ ID NO:253) are of
particular use in the disclosed methods.
[0077] Derivatives of the agonists and analogs are also included
within the methods provided in which the stereochemistry of
individual amino acids may be inverted from (L)/S to (D)/R at one
or more specific sites. Also included within the methods provided
are the agonists and analogs modified by glycosylation of Asn, Ser
and/or Thr residues. Compounds useful in the methods provided may
also be biologically active fragments of the peptides (native,
agonist, analog, and derivative) herein described.
[0078] Agonist and analogs of amylin that contain less peptide
character are included within the methods provided. Such peptide
mimetics may include, for example, one or more of the following
substitutions for --CO--NH-- amide bonds: depsipeptides
(--CO--O--), iminomethylenes (--CH.sub.2--NH--), trans-alkenes
(--CH.dbd.CH--), beta-enaminonitriles (--C(.dbd.CH--CN)--NH--),
thioamides (--CS--NH), thiomethylenes (--S--CH.sub.2-- or
--CH.sub.2--S--), methylenes (--CH.sub.2--C2-) and retro-amides
(--NH--CO--).
[0079] Compounds for use in the methods provided form salts with
various inorganic and organic acids and bases. Such salts include
salts prepared with organic and inorganic acids, for example, HCl,
HBr, H.sub.2SO.sub.4, H.sub.3PO.sub.4, trifluoroacetic acid, acetic
acid, formic acid, methanesulfonic acid, toluenesulfonic acid,
maleic acid, fumaric acid and camphorsulfonic acid. Salts prepared
with bases include, for example, ammonium salts, alkali metal salts
(such as sodium and potassium salts) and alkali earth salts (such
as calcium and magnesium salts). In certain embodiments, the
compounds form acetate, hydrochloride, and trifluoroacetate
salts.
[0080] Amylin agonists useful in the compositions and methods
provided herein may also include fragments of amylin and its
analogs as described above as well as those described in EP 289287,
the contents of which are herein incorporated by reference. Amylin
agonists analogs may also be compounds having at least 60, 65, 70,
75, 80, 85, 90, 95, or 99% amino acid sequence identity to SEQ ID
NO:1, or any of the amylin analogs specifically described herein
having amylin activity. Amylin agonists also include small chemical
molecules and non-peptide molecules, for example those based on
small molecule chemistry. In some embodiments, amylin agonists are
not small chemical molecules.
[0081] "Amylin activity" as used herein may include at least one of
the activities known in the art as described below. Amylin activity
may also include the ability of amylin to modulate the stress
response, affect GC and/or affect CFR activity in a body. Desirable
amylin agonists or amylin analogs may have at least one property
shared by the antipsychotic, antidepressant, and anxiolytic agents
used herein in Examples 2 and 3. Amylin agonist analogs also
include insertions, deletions, extensions, truncations, and/or
substitutions in at least one or more amino acid positions of SEQ
ID NO:1 or any of the amylin analogs specifically described herein.
The number of amino acid insertions, deletions, or substitutions
may be at least 5, 10, 15, 20, or 25 amino acid insertions,
deletions, or substitutions. The number of amino acid insertions,
deletions, or substitutions may be not more than 5, 10, 15, 20, 25,
or 30 amino acid insertions, deletions, or substitutions.
Insertions, extensions, or substitutions may be with other natural
amino acids, synthetic amino acids, peptidomimetics, or other
chemical compounds. It is contemplated that in certain embodiments,
amylin agonists, useful in certain embodiments, may not include
calcitonins and/or CGRPs. For example, in certain embodiments,
calcitonins and/or CGRPs may be excluded from the scope of amylin
agonist in the treatment of mood disorders, anxiety disorders or
substance-related disorders. However, calcitonins and/or CGRPs may
be included in the scope of amylin agonists for other conditions
like sleeping or eating disorders. Similarly, in certain
embodiments, amylin agonists, useful in methods provided, may not
include calcitonin and/or CGRP analogs.
[0082] In general, amylin agonists or amylin agonist analogs are
recognized as referring to compounds which, by directly or
indirectly interacting or binding with one or more receptors,
mimics an action of amylin. They may also be referred to as
amylinomimetics.
[0083] Activity as amylin agonists and/or analogs can be confirmed
and quantified by performing various screening assays, including
the nucleus accumbens receptor binding assay, the soleus muscle
assay, a gastric emptying assay, or by the ability to induce
hypocalcemia or reduce postprandial hyperglycemia in mammals.
Methods of testing compounds for amylin activity are known in the
art. Exemplary screening methods and assays for testing amylin
agonists are described in U.S. Pat. Nos. 5,264,372 and 5,686,411,
which are incorporated herein by reference.
[0084] The receptor binding assay, a competition assay that
measures the ability of compounds to bind specifically to
membrane-bound amylin receptors. A preferred source of the membrane
preparations used in the assay is the basal forebrain which
comprises membranes from the nucleus accumbens and surrounding
regions. Compounds being assayed compete for binding to these
receptor preparations with .sup.125I Bolton Hunter rat amylin.
Competition curves, wherein the amount bound (B) is plotted as a
function of the log of the concentration of ligand, are analyzed by
computer using analyses by nonlinear regression to a 4-parameter
logistic equation (INPLOT program, GraphPAD Software, San Diego,
Calif.) or the ALLFIT program of DeLean et al. (ALLFIT, Version 2.7
(NIH, Bethesda, Md. 20892)). Munson et al. (1980) Anal. Biochem.
107:220-239.
[0085] Assays of biological activity of amylin agonists/analogs in
the soleus muscle may be performed using previously described
methods (Leighton et al. (1988) Nature 335:632-635; Cooper et al.
(1988) Proc. Natl. Acad. Sci. USA 85:7763-7766), in which amylin
agonist activity may be assessed by measuring the inhibition of
insulin-stimulated glycogen synthesis. In brief, an exemplary
method includes soleus muscle strips prepared from 12-h fasted male
Wistar rats. The tendons of the muscles are ligated before
attachment to stainless steel clips. Muscle strips are
pre-incubated in Erlenmeyer flasks containing 3.5 ml Krebs-Ringer
bicarbonate buffer, 7 mM
N-2-hydroxyethyl-peperazine-N'-2-ethane-sulphonic acid, pH 7.4, and
5.5 mM pyruvate. Flasks are sealed and gassed continuously with
O.sub.2 and CO.sub.2 in the ratio 19:1 (v/v). After pre-incubation
of muscles in this medium for 30 min at 37.degree. C. in an
oscillating water bath, the muscles strips are transferred to
similar vials containing identical medium (except pyruvate) with
added [U-.sup.14C] glucose (0.5 .mu.Ci/ml) and insulin (100
.mu.U/ml). The flasks are sealed and re-gassed for an initial 15
min in a 1-h incubation. At the end of the incubation period,
muscles are blotted and rapidly frozen in liquid N.sub.2. The
concentration of lactate in the incubation medium can be determined
spectrophotometrically and [U-.sup.14C] glucose incorporation in
glycogen measured.
[0086] Methods of measuring the rate of gastric emptying are
disclosed in, for example, Young et al. (1995) Diabetologia
38:642-648. In a phenol red method, conscious rats receive by
gavage an acoloric gel containing methyl cellulose and a phenol red
indicator. Twenty minutes after gavage, animals are anesthetized
using halothane, the stomach exposed and clamped at the pyloric and
lower esophageal sphincters, removed and opened into an alkaline
solution. Stomach content may be derived from the intensity of the
phenol red in the alkaline solution, measured by absorbance at a
wavelength of 560 nm. In a tritiated glucose method, conscious rats
are gavaged with tritiated glucose in water. The rats are gently
restrained by the tail, the tip of which is anesthetized using
lidocaine. Tritium in the plasma separated from tail blood is
collected at various timepoints and detected in a beta counter.
Test compounds are normally administered about one minute before
gavage.
[0087] Amylin agonist compounds may exhibit activity in the
receptor binding assay on the order of less than about 1 to 5 nM,
in some embodiments less than about 1 nM and in some embodiments
less than about 50 .mu.M. In the soleus muscle assay, amylin
agonist compounds may show EC.sub.50 values on the order of less
than about 1 to 10 micromolar. In the gastric emptying assays,
amylin agonist compounds show ED.sub.50 values on the order of less
than 100 .mu.g/rat.
[0088] In one exemplary method of making the compounds, compounds
provided herein may be prepared using standard solid-phase peptide
synthesis techniques, for example using an automated or
semiautomated peptide synthesizer. Typically, using such
techniques, an .alpha.-N-carbamoyl protected amino acid and an
amino acid attached to the growing peptide chain on a resin are
coupled at room temperature in an inert solvent such as
dimethylformamide, N-methylpyrrolidinone or methylene chloride in
the presence of coupling agents such as dicyclohexylcarbodiimide
and 1-hydroxybenzotriazole in the presence of a base such as
diisopropylethylamine The .alpha.-N-carbamoyl protecting group is
removed from the resulting peptide-resin using a reagent such as
trifluoroacetic acid or piperidine, and the coupling reaction
repeated with the next desired N-protected amino acid to be added
to the peptide chain. Suitable N-protecting groups are well known
in the art, with t-butyloxycarbonyl (tBoc) and
fluorenylmethoxycarbonyl (Fmoc) being preferred herein. Other
methods of synthesizing or expressing amylin and amylin agonists
and purifying them are known to the skilled artisan.
[0089] The amylin, amylin agonists, amylin analogs, and amylin
derivatives (herein referred to in this section as the "compounds")
may be administered alone or in combination with pharmaceutically
acceptable carriers or excipients, in either single or multiple
doses. These pharmaceutical compounds may be formulated with
pharmaceutically acceptable carriers or diluents as well as any
other known adjuvants and excipients in accordance with
conventional techniques such as those disclosed in Remington's
Pharmaceutical Sciences by E. W. Martin. See also Wang et al.
(1988) Journal of Parenteral Science and Technology Technical
Report No. 10, Supp. 42:2S.
[0090] In general, the compounds may be formulated into a stable,
safe pharmaceutical composition for administration to a patient.
Pharmaceutical formulations contemplated for use in the methods
described herein may comprise approximately 0.01 to 6.0% (w/v), or
0.05 to 1.0%, of the compound; approximately 0.02 to 0.5% (w/v) of
an acetate, phosphate, citrate or glutamate buffer allowing a pH of
the final composition of from about 3.0 to about 7.0; approximately
1.0 to 10% (w/v) of a carbohydrate or polyhydric alcohol tonicifier
and, optionally, approximately 0.005 to 1.0% (w/v) of a
preservative selected from the group consisting of m-cresol, benzyl
alcohol, methyl-, ethyl-, propyl- and butyl-parabens and phenol.
Such a preservative is generally included if the formulated peptide
is to be included in a multiple use product.
[0091] In a particular embodiment, a pharmaceutical formulation may
contain a range of concentrations of the compound, e.g., between
about 0.01% to about 98% (w/v), or between about 1 to about 98%
(w/v), or between 80% and 90% (w/v), or between about 0.01% to
about 50% (w/v), or between about 10% to about 25% (w/v) in this
embodiment. A sufficient amount of water for injection may be used
to obtain the desired concentration of solution.
[0092] Additional tonicifying agents such as sodium chloride, as
well as other known excipients, may also be present, if desired. In
some cases, such excipients are useful in maintenance of the
overall tonicity of the formulation. An excipient may be included
in the presently described formulations at various concentrations.
For example, an excipient may be included in the concentration
range from about 0.02% to about 20% (w/w), about 0.02% and 0.5%
(w/w), about 0.02% to about 10% (w/w), or about 1% to about 20%
(w/w). In addition, similar to the present formulations themselves,
an excipient may be included in solid (including powdered), liquid,
semi-solid or gel form.
[0093] As described herein, a variety of liquid vehicles are
suitable for use in the present peptide formulations, for example,
water or an aqueous/organic solvent mixture or suspension. The
pharmaceutical formulations may be composed in various forms, e.g.,
solid, semisolid or liquid. The term "solid", as used herein, is
meant to encompass all normal uses of this term including, for
example, powders and lyophilized formulations. The presently
described formulations may be lyophilized.
[0094] The terms buffer, buffer solution and buffered solution,
when used with reference to hydrogen-ion concentration or pH, refer
to the ability of a system, particularly an aqueous solution, to
resist a change of pH on adding acid or alkali, or on dilution with
a solvent. Characteristic of buffered solutions, which undergo
small changes of pH on addition of acid or base, is the presence
either of a weak acid and a salt of the weak acid, or a weak base
and a salt of the weak base. An example of the former system is
acetic acid and sodium acetate. The change of pH is slight as long
as the amount of hydronium or hydroxyl ion added does not exceed
the capacity of the buffer system to neutralize it.
[0095] The stability of a peptide formulation is enhanced by
maintaining the pH of the formulation in the range of about 3.0 to
about 7.0 when in liquid form. In one embodiment, the pH of the
formulation is maintained in the range of about 3.5 to 5.0, or
about 3.5 to 6.5, or about 3.7 to 4.3, or about 3.8 to 4.2. A
particular pH may be about 4.0. While not seeking to be bound by
this theory, it is presently understood that, in some embodiments,
where the pH of the pharmaceutical formulation exceeds 5.5,
chemical degradation of the peptide may be accelerated such that
the shelf life is less than about two years.
[0096] In some embodiments, the buffer used in the practice of the
present methods is an acetate buffer (typically at a final
formulation concentration of from about 1-5 mM, e.g., 1.5 mM, to
about 60 mM), phosphate buffer (typically at a final formulation
concentration of from about 1-5 mM, e.g., 1.5 mM, to about 30 mM)
or glutamate buffer (typically at a final formulation concentration
of from about 1-5 mM, e.g., 1.5 mM, to about 60 mM). In one
embodiment, the buffer is acetate at a final formulation
concentration of from about 5 mM to about 30 mM.
[0097] A stabilizer may be included in the present formulation but,
and importantly, is not necessarily needed. If included, however, a
stabilizer useful in the practice of the present formulation is a
carbohydrate or a polyhydric alcohol. A suitable stabilizer useful
in the practice of the present methods is approximately 1.0 to 10%
(w/v) of a carbohydrate or polyhydric alcohol. The polyhydric
alcohols and carbohydrates share the same feature in their
backbones, i.e., --CHOH--CHOH--, which is responsible for
stabilizing the proteins. The polyhydric alcohols include such
compounds as sorbitol, mannitol, glycerol, and polyethylene glycols
(PEGs). These compounds are straight-chain molecules. The
carbohydrates, such as mannose, ribose, sucrose, fructose,
trehalose, maltose, inositol, and lactose, on the other hand, are
cyclic molecules that may contain a keto or aldehyde group. These
two classes of compounds have been demonstrated to be effective in
stabilizing protein against denaturation caused by elevated
temperature and by freeze-thaw or freeze-drying processes. In
embodiments in which the subjects have diabetes, suitable
carbohydrates include: galactose, arabinose, lactose or any other
carbohydrate which does not have an adverse affect on a diabetic
patient, i.e., the carbohydrate is not metabolized to form
unacceptably large concentrations of glucose in the blood. Such
carbohydrates are well known in the art as suitable for diabetics.
Sucrose and fructose are suitable for use with the compound in
non-diabetic subjects.
[0098] In certain embodiments, if a stabilizer is included, the
compound is stabilized with a polyhydric alcohol such as sorbitol,
mannitol, inositol, glycerol, xylitol, and polypropylene/ethylene
glycol copolymer, as well as various PEGs of molecular weight 200,
400, 1450, 3350, 4000, 6000, and 8000). Mannitol is an example of a
particular polyhydric alcohol. Another useful feature of the
lyophilized formulations described herein is the maintenance of the
tonicity of the lyophilized formulations with the same formulation
component that serves to maintain their stability. Mannitol is a
particular polyhydric alcohol used for this purpose.
[0099] The United States Pharmacopeia (USP) states that
anti-microbial agents in bacteriostatic or fungistatic
concentrations must be added to preparations contained in multiple
dose containers. They must be present in adequate concentration at
the time of use to prevent the multiplication of microorganisms
inadvertently introduced into the preparation while withdrawing a
portion of the contents with a hypodermic needle and syringe, or
using other invasive means for delivery, such as pen injectors.
Anti-microbial agents should be evaluated to ensure compatibility
with all other components of the formula, and their activity should
be evaluated in the total formula to ensure that a particular agent
that is effective in one formulation is not ineffective in another.
It is not uncommon to find that a particular anti-microbial agent
will be effective in one formulation but not effective in another
formulation.
[0100] A preservative is, in the common pharmaceutical sense, a
substance that prevents or inhibits microbial growth and may be
added to pharmaceutical formulations for this purpose to avoid
consequent spoilage of the formulation by microorganisms. While the
amount of the preservative is not great, it may nevertheless affect
the overall stability of the peptide.
[0101] While the preservative for use in the pharmaceutical
compositions can range from 0.005 to 1.0% (w/v), the typical range
for each preservative, alone or in combination with others, is:
benzyl alcohol (0.1-1.0%), or m-cresol (0.1-0.6%), or phenol
(0.1-0.8%) or combination of methyl (0.05-0.25%) and ethyl- or
propyl- or butyl-(0.005%-0.03%) parabens. The parabens are lower
alkyl esters of para-hydroxybenzoic acid. A detailed description of
each preservative is set forth in "Remington's Pharmaceutical
Sciences" as well as Pharmaceutical Dosage Forms: Parenteral
Medications, Vol. 1, 2nd ed., Avis et al. Ed., Mercel Dekker, New
York, N.Y. (1992).
[0102] Pramlintide, .sup.25,28,29Pro-h-amylin, does not have a
tendency to adsorb onto the glass in a glass container when in a
liquid form, therefore, a surfactant is not required to further
stabilize the pharmaceutical formulation. However, with regard to
compounds which do have such a tendency when in liquid form, a
surfactant should be used in their formulation. These formulations
may then be lyophilized. Surfactants frequently cause denaturation
of protein, both of hydrophobic disruption and by salt bridge
separation. Relatively low concentrations of surfactant may exert a
potent denaturing activity, because of the strong interactions
between surfactant moieties and the reactive sites on proteins.
However, judicious use of this interaction can stabilize proteins
against interfacial or surface denaturation. Surfactants which
could further stabilize the peptide may optionally be present in
the range of about 0.001 to 0.3% (w/v) of the total formulation and
include polysorbate 80 (i.e., polyoxyethylene(20) sorbitan
monooleate), CHAPS.RTM. (i.e.,
3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonate),
BRIJ.RTM. (e.g., Brij 35, which is (polyoxyethylene (23) lauryl
ether), poloxamer, or another non-ionic surfactant.
[0103] It may also be desirable to add sodium chloride or other
salt to adjust the tonicity of the pharmaceutical formulation,
depending on the tonicifier selected. However, this is optional and
depends on the particular formulation selected. Parenteral
formulations typically may be isotonic or substantially
isotonic.
[0104] A suitable vehicle for parenteral products is water. Water
of suitable quality for parenteral administration can be prepared
either by distillation or by reverse osmosis. Water for injection
is the preferred aqueous vehicle for use in the pharmaceutical
injectable formulations.
[0105] It is possible that other ingredients may be present in the
pharmaceutical formulations. Such additional ingredients may
include, e.g., wetting agents, emulsifiers, oils, antioxidants,
bulking agents, tonicity modifiers, chelating agents, metal ions,
oleaginous vehicles, proteins (e.g., human serum albumin, gelatin
or proteins) and a zwitterion (e.g., an amino acid such as betaine,
taurine, arginine, glycine, lysine and histidine). Additionally,
polymer solutions, or mixtures with polymers provide the
opportunity for controlled release of the peptide. Such additional
ingredients, of course, should not adversely affect the overall
stability of the pharmaceutical formulation provided herein.
[0106] Containers are also an integral part of the formulation of
an injection and may be considered a component, for there is no
container that is totally inert, or does not in some way affect the
liquid it contains, particularly if the liquid is aqueous.
Therefore, the selection of a container for a particular injection
must be based on a consideration of the composition of the
container, as well as of the solution, and the treatment to which
it will be subjected. Adsorption of the peptide to the glass
surface of the vial can also be minimized, if necessary, by use of
borosilicate glass, for example, Wheaton Type I borosilicate glass
#33 (Wheaton Type 1-33) or its equivalent (Wheaton Glass Co.).
Other vendors of similar borosilicate glass vials and cartridges
acceptable for manufacture include Kimbel Glass Co., West Co.,
Bander Glas GMBH and Forma Vitrum. The biological and chemical
properties of the compound may be stabilized by formulation and
lyophilization in a Wheaton Type 1-33 borosilicate serum vial to a
final concentration of 0.1 mg/ml and 10 mg/ml of the compound in
the presence of 5% mannitol, and 0.02% Tween 80.
[0107] In order to permit introduction of a needle from a
hypodermic syringe into a multiple-dose vial and provide for
resealing as soon as the needle is withdrawn, the open end of each
vial is typically sealed with a rubber stopper closure held in
place by an aluminum band. Stoppers for glass vials, such as, West
4416/50, 4416/50 (Teflon faced) and 4406/40, Abbott 5139 or any
equivalent stopper can be used as the closure for pharmaceutical
for injection. These stoppers are compatible with the peptide as
well as the other components of the formulation. These stoppers
pass the stopper integrity test when tested using patient use
patterns, e.g., the stopper can withstand at least about 100
injections. Alternatively, the peptide can be lyophilized in vials,
syringes or cartridges for subsequent reconstitution. Liquid
formulations provided herein can be filled into one or two
chambered cartridges, or one or two chamber syringes.
[0108] Each of the components of the pharmaceutical formulation
described above is known in the art and is described in
Pharmaceutical Dosage Forms: Parenteral Medications, Vol. 1, 2nd
ed., Avis et al. Ed., Mercel Dekker, New York, N.Y. 1992, which is
incorporated by reference in its entirety herein.
[0109] The manufacturing process for the above liquid formulations
generally involves compounding, sterile filtration and filling
steps. The compounding procedure involves dissolution of
ingredients in a specific order (for example, preservative followed
by stabilizer/tonicity agents, buffers and peptide) or dissolving
at the same time.
[0110] Alternative formulations, e.g., non-parenteral, may not
require sterilization. However, if sterilization is desired or
necessary, any suitable sterilization process can be used in
developing the peptide pharmaceutical formulation provided herein.
Typical sterilization processes include filtration, steam (moist
heat), dry heat, gases (e.g., ethylene oxide, formaldehyde,
chlorine dioxide, propylene oxide, beta-propiolacctone, ozone,
chloropicrin, peracetic acid methyl bromide and the like), exposure
to a radiation source, and aseptic handling. Filtration is an
examplary method of sterilization for liquid formulations described
herein. The sterile filtration involves filtration through 0.45
.mu.m and 0.22 .mu.m (1 or 2) which may be connected in series.
After filtration, the solution is filled into appropriate vials or
containers.
[0111] In one embodiment, the liquid pharmaceutical formulations
are intended for parenteral administration. Suitable routes of
administration include intramuscular, intravenous, subcutaneous,
intradermal, intraarticular, intrathecal and the like. The
subcutaneous route of administration is one particular route.
Mucosal delivery is also particularly suitable. These mucosal
routes include, but are not limited to, oral, nasal, sublingual,
pulmonary and buccal routes which may include administration of the
peptide in liquid, semi-solid or solid form. Administration via
these routes requires substantially more peptide to obtain the
desired biological effects due to decreased bioavailability
compared to parenteral delivery. In addition, parenteral controlled
release delivery can be achieved by forming polymeric
microcapsules, matrices, solutions, implants and devices and
administering them parenterally or by surgical means. Examples of
controlled release formulations are described in U.S. Pat. Nos.
6,368,630, 6,379,704, and 5,766,627, which are incorporated herein
by reference. These dosage forms may have a lower bioavailability
due to entrapment of some of the peptide in the polymer matrix or
device. See e.g., U.S. Pat. Nos. 6,379,704, 6,379,703, and
6,296,842.
[0112] The compounds may be provided in dosage unit form containing
an amount of the compound that will be effective in one or multiple
doses to treat or help in treating the psychiatric disease and/or
unwanted side effects of the psychiatric treatment/medication. As
will be recognized by those in the field, an effective amount of
therapeutic agent will vary with many factors including the age and
weight of the patient, the patient's physical condition, the
condition to be treated, and other factors.
[0113] However, typical doses may contain from a lower limit of
about 1 .mu.g, 5 .mu.g, 10 .mu.g, 50 .mu.g to 100 .mu.g to an upper
limit of about 100 .mu.g, 500 .mu.g, 1 mg, 5 mg, 10 mg, 50 mg or
100 mg of the pharmaceutical compound per day. Also contemplated
are other dose ranges such as 0.1 .mu.g to 1 mg of the compound per
dose. Thus, exemplary doses may be 30, 60, 120, 240, or 360 .mu.g
of the compound per dose. The doses per day may be delivered in
discrete unit doses or provided continuously in a 24 hour period,
or any portion of that 24 hour period. The number of doses per day
may be from 1 to about 4 doses per day, although it could be more.
Continuous delivery can be in the form of continuous infusions.
Exemplary doses and infusion rates include from 0.005 nmol/kg to
about 20 nmol/kg per discrete dose or from about 0.01/pmol/kg/min
to about 10 pmol/kg/min in a continuous infusion. These doses and
infusions can be delivered by intravenous administration (i.v.) or
subcutaneous administration (s.c.). Exemplary total dose/delivery
of the pharmaceutical composition given i.v. may be about 2 .mu.g
to about 8 mg per day, whereas total dose/delivery of the
pharmaceutical composition given s.c may be about 6 .mu.g to about
16 or 24 mg per day.
[0114] The following Examples are provided to illustrate, but not
limit, the invention.
EXAMPLES
Example 1
[0115] Male, Sprague-Dawley.RTM. rats were submitted to a sugar
withdrawal paradigm and observed for effects of stress on sugar
intake. Briefly, rats were implanted with ALZET.RTM. osmotic pumps
containing vehicle or rat amylin (300 .mu.g/kg/d). All rats were
provided with ad libitum access to standard chow, water, and a 30%
sucrose drink. Subsequently, the sucrose drink was removed and half
of the rats were subjected, daily, to 3 h of mild restraint stress
for 3 successive days. After 3 days, sucrose was provided and its
daily average consumption was measured over 4 days. Chow intake was
also measured over the 3 days of withdrawal and stress and over the
following 4 days when sucrose was re-introduced. No restraint was
applied during the 4 days of sucrose re-introduction. Results of
this assay are shown in FIG. 1 where * is P<0.05 by ANOVA and
Fisher LSD post-hoc analyses.
[0116] In this model, chronic stress stimulates the proportion of
total calories taken in as sugar. As shown in FIG. 1A, stress
induced by restraint significantly increased the average sucrose to
chow consumption ratio (as % of baseline) (saline, R) over control
consumption without stress inducement (saline, C). FIG. 1A also
shows that amylin administration prevents the increase in the
average sucrose to chow consumption ratio expected as a result of
the stress induced by restraint. Compare the sucrose to chow ratio
(expressed as a percentage of baseline) between amylin-administered
stress-induced rats (amylin, R) and amylin-administered
non-stressed control rats (amylin, C). Also compare the sucrose to
chow ratio between amylin-administered stress-induced rats (amylin,
R) and control vehicle (saline)-administered stress-induced rats
(saline, R). As shown, amylin administration reduces this
stress-induced response. Accordingly, amylin administration appears
to reduce or protect against stress and its effects.
[0117] FIG. 1B depicts the same results from the restraint stress
assay from a different perspective, that of chow consumption. It is
believed that while stress increases palatable feeding (e.g.,
simple sugars), stress decreases consumption of less palatable
foods (e.g., standard chow), a typical response to stress. In line
with this belief, stress without amylin administration
significantly decreased chow consumption in animals undergoing the
stress of restraint. Compare, for example, the chow consumption
between saline-treated, restrained animals and saline-treated,
control animals in FIG. 1B. Amylin administration also protects
against this behavioral response to stress, as indicated by the
food intake data in FIG. 1B.
[0118] As previously described, the acute effect of stress on food
intake is mediated by a central CFR pathway (Smagin et al. (1999)
Am. J. Physiol. 276:R1461-1468). This result may have behavioral
implications since CRF administered into the brain of animals
causes anxiety (Dunn et al. (1990) Brain Res. Brain Res. Rev.
15:71-100), and chronic stress and the associated increase in
central CRF activity are believed to play a critical role in the
development of clinical depression and anxiety disorders (Chrousos
(2000) Int. J. Obes. Relat. Metab. Disord. 24:S50-S55; Koob (1999)
Biol. Psychiatry 46:1167-1180). Hence, amylin appears to impact a
central stress pathway known to mediate behavioral states like
depression and anxiety and may be of use as a therapeutic to treat
the same.
Example 2
[0119] To confirm the interpretation of the findings outlined under
Example 1, a variety of animal behavior assays were performed to
test for anxiolytic, antidepressant, and antipsychotic effects of
amylin administration. The behavorial assays performed use
art-accepted, animal models that demonstrate properties
characteristic of the respective clinical condition (e.g., anxiety,
depression, schizophrenia, obsessive-compulsive disorder) and
hence, show face validity. These specific behavioral tests are
known to be sensitive to anxiolytic, antidepressant, or
antipsychotic drugs. For these assays, rat amylin was administered
to mice at doses ranging from 0.1 to 10 mg/kg, intraperitoneally or
via a subcutaneously implanted osmotic pump (ALZET.RTM.) containing
vehicle or rat amylin, and their performance in the assay was
assessed.
Stress-Induced Hyperthermia
[0120] Body temperature and emotional state are closely related in
humans, and stress-induced hyperthermia (SIH) in mice is considered
to have predictive validity for certain human anxiety/stress
disorders. The SIH assay assesses the effect of anxiolytics or test
agents on stress-induced hyperthermia and measures the intrinsic
effects of these drugs on core body temperature of the animal. See,
for example, Zethof et al. (1994) Physiol. Behav. 55:109-115.
Anxiolytics blunt the increase in body temperature, or hyperthermic
response, following stress exposure. The animals were treated with
rat amylin (0.1, 1.0 or 10 mg/kg) or control agents (vehicle or 10
mg/kg chlordiazepoxide) 60 minutes before the assay. Mice were
subjected to two sequential rectal temperature measurements ten
minutes apart. The stress from the first measurement induces
hyperthermia which was measured by the second temperature
measurement. The difference between the two temperatures (AT) was
the stress-induced hyperthermia. Results of this assay are shown in
FIG. 2 where * is P<0.05. As shown in FIG. 2, administration
amylin, like that of the anxiolytic positive control,
chlordiazepoxide (CDP), blunted the SIH response. The SIH test
results demonstrate the anxiolytic activity of amylin
administration. Amylin was also administered chronically at 300
.mu.g/kg/d via subcutaneously implanted pumps and the animals were
subjected to the SIH test. At both 1 week and 4 weeks of
administration, chronic amylin infusion significantly blunted the
SIH response, as did chronically infused CDP at 10 mg/kg/d.
Marble Burying
[0121] Marble burying is used as a model for both anxiety and
obsessive-compulsive disorder. See, for example, Chaki et al.
(2003) J. Pharmacol. Exp. Ther. 304:818-826. Anxiolytics suppress
marble burying activity. Mice were injected with the test agent
(rat amylin at 0.1, 1.0 or 10 mg/kg, 20/kg buspirone, or vehicle)
15-30 minutes prior to the test. Mice were then placed individually
in clean cages containing 5-cm of hard wood bedding and 20 marbles
spaced evenly in rows of five. The number of marbles buried in 30
minutes was recorded. Results of this assay are shown in FIG. 3
where * is P<0.05. As shown in FIG. 3, administration of amylin,
like that of the anxiolytic positive control, buspirone, reduced
the number of marbles buried. The reduction in marble burying with
amylin at 10 mg/kg and buspirone at 20 mg/kg were statistically
significant. Buspirone is a partial 5-HT1A agonist and an known
anxiolytic agent. The marble burying assay results demonstrate the
anxiolytic activity and the anti-obsessive compulsive activity of
amylin administration.
Forced Swim Test
[0122] The forced swim test (FST) is a commonly used paradigm to
evaluate antidepressant activity of drugs. This test is based on
measurement of the animal's floating time in a tank filled with
water. When rats or mice are forced to swim in a deep cylinder with
tepid water they become nearly immobile and cease trying to escape.
This characteristic immobile posture is thought to reflect a
depressive-like state and is readily influenced by a wide variety
of antidepressants. See, for example, Hedou et al. (2001)
Pharmacol., Biochem. Behav. 70:65-76, Chaki et al. (2003) J.
Pharmacol. Exp. Ther. 304:818-826, and Porsolt et al. (1977) Nature
266:730-732. Antidepressants decrease the immobility time in the
FST. Rat amylin or vehicle was delivered continuously for two weeks
to mice by subcutaneously implanted osmotic pumps prior to the FST.
On day one, the mice were placed in the water tank for a 15 minute
pre-swim session. On day two, the mice were placed back into the
water tank for assessment of climbing, swimming, and immobility
over a 5 minute trial session. Results of the FST are shown in FIG.
4 where * is P<0.05. As shown in FIG. 4, administration of rat
amylin significantly reduced the time spent in the immobile state.
Accordingly, the FST results demonstrate the antidepressant
activity of amylin administration.
Prepulse Inhibition
[0123] The prepulse inhibition (PPI) test measures the reflex
response to externally applied auditory stimulation (acoustic
startle response) and is related to the deficiency in sensory-motor
gating capacity seen in schizophrenia. The acoustic startle reflex
is a very basic response to strong exteroceptive stimuli and is
widely used to assess sensorimotor reactivity in animals and
humans. A weak auditory stimulus (prepulse, 74-82 dB) given prior
to the strong acoustic stimulus (120 dB) blunts the startle
response. This blunting of the startle response is referred to as
prepulse inhibition. See, for example, Conti et al. (2005)
Behavioral Neuroscience 119:1052-1060. Antipsychotics increase the
ability of the prepulse stimulus to blunt the startle response to
the strong stimulus. Some psychotomimetic agents, such as
phencyclidine (PCP) and ketamine, can actually reduce the percent
prepulse inhibition and stimulate a psychotic-like state in
animals, which can be antagonized by antipsychotic agents.
[0124] Mice were injected with the test agent (rat amylin at 0.1,
1.0 or 10 mg/kg, or vehicle) 15 prior to the test or with
haloperidol at 1 mg/kg 30 minutes prior to the test. The mice were
placed into an animal holder and the holder placed onto a
transducer platform in an acoustic chamber. A weak auditory
stimulus (prepulse) of 74, 78 and 82 dB was given prior to the
strong acoustic stimulus (120 dB). The amount of the animal's
"reaction" to the strong stimulus was recorded. Results of the PPI
assay are shown in FIG. 5 where * is P<0.05. As shown in FIG. 5,
administration of amylin at 10 mg/kg, like that of the
antipsychotic positive control halperidol, significantly increased
the percent of prepulse inhibition at all prepulse levels tested
(74, 78, and 82 dB). Halperidol is a dopamine receptor antagonist
and a first generation antipsychotic agent. The PPI test results
demonstrate the antipsychotic effects of amylin administration.
Phencyclidine (PCP)-Induced Locomotion
[0125] The PCP-induced locomotion test is used with the open field
activity chambers and measures locomotion, rearing, and stereotypic
activity under amphetamine/PCP-induced conditions. The test has
predictive validity for some antipsychotic drugs that normalize the
hyperactivity and stereotypic behavior seen with amphetamine and
PCP. See, for example, Williams et al. (2006) Prog.
Neuropsychopharmacol. Biol. Psychiatry 30:239-243. Mice were
injected with the test agent (rat amylin at 0.1, 1.0 or 10 mg/kg, 3
mg/kg clozapine (CZP), or vehicle) 15-30 minutes prior injection
with 5 mg/kg PCP. The animals were then placed in the center of an
open field and activity was recorded for 60 minutes. Results of
this assay are shown in FIG. 6 where * is P<0.05. As shown in
FIG. 6, administration of amylin, like the antipsychotic positive
control CZP, significantly reduced the total distance traveled
across all types assessed (total, central, and peripheral) in the
PCP-induced locomotion test. Amylin, like CZP, reduced rearing
activity and PCP-induced rearing activity. Administration of amylin
alone did not affect baseline activity whereas CPZ alone did affect
baseline activity. CZP is an atypical, second generation
antipsychotic drug has a mixed receptor profile, including the
dopamine receptor. CPZ has superior antipsychotic activity, with
less motor side effects. The PCP-induced locomotion test results
demonstrate the antipsychotic activity of amylin
administration.
Example 3
[0126] The effect of amylin administration on weight gain induced
by the second generation antipsychotic clozapine was examined
Adult, male Sprague-Dawley.RTM. rats (diet=58% kcal from fat) were
implanted subcutaneously in the interscapular region with osmotic
pumps that continuously delivered vehicle or drug for 4 weeks. In
one experiment, rats were treated with vehicle or clozapine (0.025
and 0.25 mg/kg/day). In a second experiment, rats were treated with
clozapine (0.25 mg/kg/day) or clozapine (0.25 mg/kg/day) in
conjunction with rat amylin (10 .mu.g/kg/day). FIG. 7A shows the
increased weight gain of clozapine treated-rats compared to
vehicle-treated rats. FIG. 7B shows that when amylin was
co-administered with clozapine, amylin prevented the weight gain
observed in rats treated with clozapine alone.
[0127] While the foregoing description discloses the present
invention, with examples provided for the purpose of illustration,
it will be understood that the practice of the present invention
encompasses all of the usual variations, adaptations, or
modifications as being within the scope of the claimed invention.
Therefore, descriptions and examples should not be construed as
limiting the scope of the invention, which is delineated by the
appended claims.
Sequence CWU 1
1
253137PRTHomo sapiens 1Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala
Ile Leu Ser Ser Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
237PRTRattus sp. 2Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu
Ala Asn Phe Leu 1 5 10 15 Val Arg Ser Ser Asn Asn Leu Gly Pro Val
Leu Pro Pro Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
337PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 3Xaa Xaa Asn Thr Ala Thr Xaa Ala Thr Gln Arg
Leu Xaa Asn Phe Leu 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Asn Xaa Gly Xaa
Xaa Leu Xaa Xaa Thr Xaa Val 20 25 30 Gly Ser Asn Thr Tyr 35
436PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 4Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu
Ala Asn Phe Leu Val 1 5 10 15 His Ser Ser Asn Asn Phe Gly Ala Ile
Leu Ser Ser Thr Asn Val Gly 20 25 30 Ser Asn Thr Tyr 35
537PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 5Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala
Ile Leu Pro Ser Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
637PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 6Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Pro
Ile Leu Pro Pro Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
737PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 7Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Val Arg Ser Ser Asn Asn Phe Gly Pro
Ile Leu Pro Ser Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
836PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 8Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu
Ala Asn Phe Leu Val 1 5 10 15 Arg Ser Ser Asn Asn Phe Gly Pro Ile
Leu Pro Ser Thr Asn Val Gly 20 25 30 Ser Asn Thr Tyr 35
937PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 9Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Pro
Val Leu Pro Pro Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
1037PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 10Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Val Arg Ser Ser Asn Asn Phe Gly Pro
Ile Leu Pro Pro Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
1136PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 11Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu
Ala Asn Phe Leu Val 1 5 10 15 Arg Ser Ser Asn Asn Phe Gly Pro Ile
Leu Pro Pro Thr Asn Val Gly 20 25 30 Ser Asn Thr Tyr 35
1236PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 12Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu
Ala Asn Phe Leu Val 1 5 10 15 His Ser Ser Asn Asn Phe Gly Pro Ile
Leu Pro Pro Thr Asn Val Gly 20 25 30 Ser Asn Thr Tyr 35
1337PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 13Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Pro
Val Leu Pro Pro Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
1437PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 14Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Val His Ser Ser Asn Asn Leu Gly Pro
Val Leu Pro Pro Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
1537PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 15Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Val His Ser Ser Asn Asn Leu Gly Pro
Val Leu Pro Ser Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
1636PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 16Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu
Ala Asn Phe Leu Val 1 5 10 15 His Ser Ser Asn Asn Leu Gly Pro Val
Leu Pro Ser Thr Asn Val Gly 20 25 30 Ser Asn Thr Tyr 35
1737PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 17Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Val Arg Ser Ser Asn Asn Leu Gly Pro
Val Leu Pro Ser Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
1837PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 18Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Val Arg Ser Ser Asn Asn Leu Gly Pro
Ile Leu Pro Pro Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
1937PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 19Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Val Arg Ser Ser Asn Asn Leu Gly Pro
Ile Leu Pro Ser Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
2037PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 20Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Ile His Ser Ser Asn Asn Leu Gly Pro
Ile Leu Pro Pro Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
2137PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 21Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Ile His Ser Ser Asn Asn Phe Gly Pro
Ile Leu Pro Pro Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
2236PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 22Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu
Ala Asn Phe Leu Ile 1 5 10 15 His Ser Ser Asn Asn Leu Gly Pro Ile
Leu Pro Pro Thr Asn Val Gly 20 25 30 Ser Asn Thr Tyr 35
2337PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 23Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Ile Arg Ser Ser Asn Asn Leu Gly Ala
Ile Leu Ser Ser Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
2437PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 24Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Ile Arg Ser Ser Asn Asn Leu Gly Ala
Val Leu Ser Pro Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
2537PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 25Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Ile Arg Ser Ser Asn Asn Leu Gly Pro
Val Leu Pro Pro Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
2637PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 26Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Thr Asn Phe Leu 1 5 10 15 Val His Ser Ser His Asn Leu Gly Ala
Ala Leu Leu Pro Thr Asp Val 20 25 30 Gly Ser Asn Thr Tyr 35
2737PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 27Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Thr Asn Phe Leu 1 5 10 15 Val His Ser Ser His Asn Leu Gly Ala
Ala Leu Ser Pro Thr Asp Val 20 25 30 Gly Ser Asn Thr Tyr 35
2836PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 28Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu
Thr Asn Phe Leu Val 1 5 10 15 His Ser Ser His Asn Leu Gly Ala Ala
Leu Pro Ser Thr Asp Val Gly 20 25 30 Ser Asn Thr Tyr 35
2937PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 29Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Thr Asn Phe Leu 1 5 10 15 Val Arg Ser Ser His Asn Leu Gly Ala
Ala Leu Ser Pro Thr Asp Val 20 25 30 Gly Ser Asn Thr Tyr 35
3037PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 30Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Thr Asn Phe Leu 1 5 10 15 Val Arg Ser Ser His Asn Leu Gly Ala
Ile Leu Pro Pro Thr Asp Val 20 25 30 Gly Ser Asn Thr Tyr 35
3137PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 31Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Thr Asn Phe Leu 1 5 10 15 Val Arg Ser Ser His Asn Leu Gly Pro
Ala Leu Pro Pro Thr Asp Val 20 25 30 Gly Ser Asn Thr Tyr 35
3247PRTHomo sapiens 32Thr Gln Ala Gln Leu Leu Arg Val Gly Cys Val
Leu Gly Thr Cys Gln 1 5 10 15 Val Gln Asn Leu Ser His Arg Leu Trp
Gln Leu Met Gly Pro Ala Gly 20 25 30 Arg Gln Asp Ser Ala Pro Val
Asp Pro Ser Ser Pro His Ser Tyr 35 40 45 3348PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
33Thr Gln Ala Gln Leu Leu Arg Val Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1
5 10 15 Gln Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Xaa Xaa Xaa
Xaa 20 25 30 Xaa Xaa Xaa Xaa Ser Ala Pro Val Xaa Pro Ser Ser Pro
His Ser Tyr 35 40 45 349PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 34Thr Gln Ala Gln Leu Leu
Arg Val Gly 1 5 3532PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 35Xaa Xaa Gln Asn Leu Ser His Arg Leu
Trp Gln Leu Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Ser Ala Pro
Val Xaa Pro Ser Ser Pro His Ser Tyr 20 25 30 3641PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
36Arg Val Gly Cys Val Leu Gly Thr Cys Gln Val Gln Asn Leu Ser His 1
5 10 15 Arg Leu Trp Gln Leu Met Gly Pro Ala Gly Arg Gln Asp Ser Ala
Pro 20 25 30 Val Asp Pro Ser Ser Pro His Ser Tyr 35 40
3739PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 37Gly Cys Val Leu Gly Thr Cys Gln Val Gln Asn
Leu Ser His Arg Leu 1 5 10 15 Trp Gln Leu Met Gly Pro Ala Gly Arg
Gln Asp Ser Ala Pro Val Asp 20 25 30 Pro Ser Ser Pro His Ser Tyr 35
3838PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 38Cys Val Leu Gly Thr Cys Gln Val Gln Asn Leu
Ser His Arg Leu Trp 1 5 10 15 Gln Leu Met Gly Pro Ala Gly Arg Gln
Asp Ser Ala Pro Val Asp Pro 20 25 30 Ser Ser Pro His Ser Tyr 35
3932PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 39Gln Val Gln Asn Leu Ser His Arg Leu Trp Gln
Leu Met Gly Pro Ala 1 5 10 15 Gly Arg Gln Asp Ser Ala Pro Val Asp
Pro Ser Ser Pro His Ser Tyr 20 25 30 4031PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
40Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Met Gly Pro Ala Gly 1
5 10 15 Arg Gln Asp Ser Ala Pro Val Asp Pro Ser Ser Pro His Ser Tyr
20 25 30 4130PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 41Val Gln Asn Leu Ser His Arg Leu Gln
Leu Met Gly Pro Ala Gly Arg 1 5 10 15 Gln Asp Ser Ala Pro Val Asp
Pro Ser Ser Pro His Ser Tyr 20 25 30 4242PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
42Thr Gln Ala Gln Leu Leu Arg Val Gly Cys Val Leu Gly Thr Cys Gln 1
5 10 15 Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Arg Gln Asp Ser
Ala 20 25 30 Pro Val Asp Pro Ser Ser Pro His Ser Tyr 35 40
4340PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 43Thr Gln Ala Gln Leu Leu Arg Val Gly Cys Val
Leu Gly Thr Cys Gln 1 5 10 15 Val Gln Asn Leu Ser His Arg Leu Trp
Gln Leu Asp Ser Ala Pro Val 20 25 30 Asp Pro Ser Ser Pro His Ser
Tyr 35 40 4435PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 44Val Gly Cys Val Leu Gly Thr Cys Gln
Val Gln Asn Leu Ser His Arg 1 5 10 15 Leu Trp Gln Leu Arg Gln Asp
Ser Ala Pro Val Asp Pro Ser Ser Pro 20 25 30 His Ser Tyr 35
4533PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 45Cys Val Leu Gly Thr Cys Gln Val Gln Asn Leu
Ser His Arg Leu Trp 1 5 10 15 Gln Leu Arg Gln Glu Ser Ala Pro Val
Glu Pro Ser Ser Pro His Ser 20 25 30 Tyr 4648PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
46Thr Gln Ala Gln Leu Leu Arg Val Gly Cys Ser Asn Leu Ser Thr Cys 1
5 10 15 Gln Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Met Gly Pro
Ala 20 25 30 Gly Arg Gln Asp Ser Ala Pro Val Asp Pro Ser Ser Pro
His Ser Tyr 35 40 45 4747PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 47Thr Gln Ala Gln Leu Leu
Arg Val Gly Cys Asn Thr Ala Thr Cys Gln 1 5 10 15 Val Gln Asn Leu
Ser His Arg Leu Trp Gln Leu Met Gly Pro Ala Gly 20 25 30 Arg Gln
Asp Ser Ala Pro Val Asp Pro Ser Ser Pro His Ser Tyr 35 40 45
4842PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 48Arg Val Gly Cys Gly Asn Leu Ser Thr Cys Gln
Val Gln Asn Leu Ser 1 5 10 15 His Arg Leu Trp Gln Leu Met Gly Pro
Ala Gly Arg Gln Asp Ser Ala 20 25 30 Pro Val
Asp Pro Ser Ser Pro His Ser Tyr 35 40 4947PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
49Thr Gln Ala Gln Leu Leu Arg Val Gly Cys Asp Thr Ala Thr Cys Gln 1
5 10 15 Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Met Gly Pro Ala
Gly 20 25 30 Arg Gln Asp Ser Ala Pro Val Asp Pro Ser Ser Pro His
Ser Tyr 35 40 45 5048PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 50Thr Gln Ala Gln Leu Leu
Arg Val Gly Cys Gly Asn Leu Ser Thr Cys 1 5 10 15 Gln Val Gln Asn
Leu Ser His Arg Leu Trp Gln Leu Met Gly Pro Ala 20 25 30 Gly Arg
Gln Asp Ser Ala Pro Val Asp Pro Ser Ser Pro His Ser Tyr 35 40 45
5147PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 51Thr Gln Ala Gln Leu Leu Arg Val Gly Met Val
Leu Gly Thr Met Gln 1 5 10 15 Val Gln Asn Leu Ser His Arg Leu Trp
Gln Leu Met Gly Pro Ala Gly 20 25 30 Arg Gln Asp Ser Ala Pro Val
Asp Pro Ser Ser Pro His Ser Tyr 35 40 45 5239PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
52Gly Met Val Leu Gly Thr Met Gln Val Gln Asn Leu Ser His Arg Leu 1
5 10 15 Trp Gln Leu Met Gly Pro Ala Gly Arg Gln Asp Ser Ala Pro Val
Asp 20 25 30 Pro Ser Ser Pro His Ser Tyr 35 5335PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
53Val Gly Met Val Leu Gly Thr Met Gln Val Gln Asn Leu Ser His Arg 1
5 10 15 Leu Trp Gln Leu Arg Gln Asp Ser Ala Pro Val Asp Pro Ser Ser
Pro 20 25 30 His Ser Tyr 35 5442PRTArtificial SequenceDescription
of Artificial Sequence Synthetic construct 54Arg Val Gly Cys Gly
Asn Leu Ser Thr Cys Gln Val Gln Asn Leu Ser 1 5 10 15 His Arg Leu
Trp Gln Leu Met Gly Pro Ala Gly Arg Gln Asp Ser Ala 20 25 30 Pro
Val Asp Pro Ser Ser Pro His Ser Tyr 35 40 5536PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
55Val Gly Cys Gly Asn Leu Ser Thr Cys Gln Val Gln Asn Leu Ser His 1
5 10 15 Arg Leu Trp Gln Leu Arg Gln Asp Ser Ala Pro Val Asp Pro Ser
Ser 20 25 30 Pro His Ser Tyr 35 5634PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
56Val Cys Asn Thr Ala Thr Cys Gln Val Gln Asn Leu Ser His Arg Leu 1
5 10 15 Trp Gln Leu Arg Gln Asp Ser Ala Pro Val Asp Pro Ser Ser Pro
His 20 25 30 Ser Tyr 5734PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 57Gly Cys Asn Thr Ala Thr
Cys Gln Val Gln Asn Leu Ser His Arg Leu 1 5 10 15 Trp Gln Leu Arg
Gln Asp Ser Ala Pro Val Asp Pro Ser Ser Pro His 20 25 30 Ser Tyr
5847PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 58Thr Gln Ala Gln Leu Leu Arg Val Gly Cys Val
Leu Gly Thr Cys Gln 1 5 10 15 Val Gln Asn Leu Ser His Arg Leu Trp
Gln Leu Met Gly Pro Ala Gly 20 25 30 Arg Gln Glu Ser Ala Pro Val
Glu Pro Ser Ser Pro His Ser Tyr 35 40 45 5947PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
59Thr Gln Ala Gln Leu Leu Arg Val Gly Cys Val Leu Gly Thr Cys Gln 1
5 10 15 Val Gln Asn Leu Ser His Arg Leu Trp Gln Leu Met Gly Pro Ala
Gly 20 25 30 Arg Gln Asp Ser Ala Pro Val Glu Pro Ser Ser Pro His
Ser Tyr 35 40 45 6030PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 60Gly Thr Met Gln Val Gln
Asn Leu Ser His Arg Leu Trp Gln Leu Arg 1 5 10 15 Gln Asp Ser Ala
Pro Val Glu Pro Ser Ser Pro His Ser Tyr 20 25 30 6140PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
61Val Gly Cys Val Leu Gly Thr Cys Gln Val Gln Asn Leu Ser His Arg 1
5 10 15 Leu Trp Gln Leu Met Gly Pro Ala Gly Arg Gln Asp Ser Ala Pro
Val 20 25 30 Glu Pro Ser Ser Pro His Ser Tyr 35 40
6235PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 62Val Gly Cys Val Leu Gly Thr Cys Gln Val Gln
Asn Leu Ser His Arg 1 5 10 15 Leu Trp Gln Leu Arg Gln Asp Ser Ala
Pro Val Glu Pro Ser Ser Pro 20 25 30 His Ser Tyr 35
6334PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 63Gly Cys Asn Thr Ala Thr Cys Gln Val Gln Asn
Leu Ser His Arg Leu 1 5 10 15 Trp Gln Leu Arg Gln Asp Ser Ala Pro
Val Glu Pro Ser Ser Pro His 20 25 30 Ser Tyr 6435PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
64Gly Cys Ser Asn Leu Ser Thr Cys Gln Val Gln Asn Leu Ser His Arg 1
5 10 15 Leu Trp Gln Leu Arg Gln Asp Ser Ala Pro Val Glu Pro Ser Ser
Pro 20 25 30 His Ser Tyr 35 6535PRTArtificial SequenceDescription
of Artificial Sequence Synthetic construct 65Gly Cys Gly Asn Leu
Ser Thr Cys Gln Val Gln Asn Leu Ser His Arg 1 5 10 15 Leu Trp Gln
Leu Arg Gln Asp Ser Ala Pro Val Glu Pro Ser Ser Pro 20 25 30 His
Ser Tyr 35 6634PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 66Gly Cys Val Leu Gly Thr Cys Gln Val
Gln Asn Leu Ser His Arg Leu 1 5 10 15 Trp Gln Leu Arg Gln Glu Ser
Ala Pro Val Glu Pro Ser Ser Pro His 20 25 30 Ser Tyr
6722PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 67Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa 20
684PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 68Xaa Xaa Xaa Xaa 1 695PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
69Arg Ser Ser Gly Tyr 1 5 705PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 70Lys Ser Ser Gly Tyr 1 5
715PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 71His Ser Ser Gly Tyr 1 5 725PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
72Pro Ser Ser Gly Tyr 1 5 735PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 73Arg Ser Arg Gly Tyr 1 5
745PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 74Arg Thr Ser Gly Tyr 1 5 755PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
75Arg Ala Ser Gly Tyr 1 5 765PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 76Ala Ser Ser Gly Tyr 1 5
775PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 77Arg Ser Ala Gly Tyr 1 5 785PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
78His Ser Ala Gly Tyr 1 5 794PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 79Arg Ser Gly Tyr 1
8032PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 80Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 8132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
81Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1
5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Thr Asn Xaa Gly Ser Xaa Xaa
Xaa 20 25 30 8232PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 82Xaa Xaa Xaa Xaa Xaa Thr Xaa Xaa Xaa
Xaa Xaa Leu Xaa Xaa Xaa Leu 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Pro
Xaa Thr Asn Xaa Gly Ser Xaa Xaa Xaa 20 25 30 8332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
83Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1
5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Thr Asn Xaa Gly Ser Xaa Xaa
Xaa 20 25 30 844PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 84Leu Gln Thr Tyr 1
8532PRTOncorhynchus sp. 85Cys Ser Asn Leu Ser Thr Cys Val Leu Gly
Lys Leu Ser Gln Glu Leu 1 5 10 15 His Lys Leu Gln Thr Tyr Pro Arg
Thr Asn Thr Gly Ser Gly Thr Pro 20 25 30 8632PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
86Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Lys Leu Ser Gln Glu Leu 1
5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr
Tyr 20 25 30 8732PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 87Lys Cys Asn Thr Ala Thr Cys Val Leu
Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Leu Pro
Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30 8832PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
88Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1
5 10 15 His Arg Leu Gln Thr Tyr Pro Pro Thr Asn Thr Gly Ser Asn Thr
Tyr 20 25 30 8932PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 89Lys Cys Asn Thr Ala Thr Cys Val Leu
Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro
Arg Thr Asn Val Gly Ser Asn Thr Tyr 20 25 30 9032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
90Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1
5 10 15 His Arg Leu Gln Thr Leu Pro Pro Thr Asn Val Gly Ser Asn Thr
Tyr 20 25 30 9134PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 91Lys Cys Asn Thr Ala Thr Cys Val Leu
Gly Arg Leu Ala Asn Phe Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro
Arg Thr Asn Thr Asn Val Gly Ser Asn 20 25 30 Thr Tyr
9232PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 92Ala Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 9332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
93Lys Cys Ala Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1
5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr
Tyr 20 25 30 9432PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 94Lys Cys Asn Ala Ala Thr Cys Val Leu
Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro
Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30 9532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
95Lys Cys Asn Thr Ala Ala Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1
5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr
Tyr 20 25 30 9632PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 96Cys Ala Asn Leu Ser Thr Cys Val Leu
Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro
Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30 9728PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
97Ser Thr Ala Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg Leu Gln 1
5 10 15 Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25
9832PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 98Cys Ser Asn Ala Ser Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 9932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
99Cys Ser Asn Leu Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1
5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr
Tyr 20 25 30 10032PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 100Cys Ser Asn Leu Ser Ala Cys Val Leu
Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro
Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30 10132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
101Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Lys Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 10232PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 102Lys Cys Asn Thr Ala Thr
Cys Ala Thr Gln Arg Leu Ser Gln Glu Leu 1 5 10 15 Arg Lys Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
10332PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 103Cys Ser Ala Leu Ser Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 10432PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
104Cys Xaa Asn Leu Ser Thr Xaa Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 10532PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 105Lys Xaa Asn Thr Ala Thr
Xaa Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
10628PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 106Ser Thr Ala Val Leu Xaa Lys Leu Ser Gln Glu
Leu Xaa Lys Leu Gln 1 5 10 15 Thr Tyr Pro Arg Thr Asn Thr Gly Ser
Asn Thr Tyr 20 25 10728PRTArtificial SequenceDescription of
Artificial Sequence
Synthetic construct 107Ser Thr Ala Val Leu Xaa Lys Leu Ser Gln Glu
Leu Xaa Lys Leu Gln 1 5 10 15 Thr Tyr Pro Arg Thr Asn Val Gly Ser
Asn Thr Tyr 20 25 10828PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 108Ser Thr Ala Val Leu Xaa
Lys Leu Ser Gln Glu Leu Xaa Lys Leu Gln 1 5 10 15 Thr Tyr Pro Pro
Thr Asn Val Gly Ser Asn Thr Tyr 20 25 10928PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
109Ser Thr Ala Val Leu Xaa Lys Leu Ser Gln Glu Leu Xaa Lys Leu Gln
1 5 10 15 Thr Leu Pro Pro Thr Asn Val Gly Ser Asn Thr Tyr 20 25
11021PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 110Lys Cys Asn Thr Ala Thr Cys Leu Leu Gln Gln
Trp Gln Lys Leu Leu 1 5 10 15 Gln Lys Leu Lys Gln 20
11111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 111Leu Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr
1 5 10 11222PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 112Lys Cys Asn Thr Ala Thr Cys Leu Leu
Gln Gln Leu Gln Lys Leu Leu 1 5 10 15 Gln Lys Leu Lys Gln Tyr 20
11310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 113Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 1 5
10 11432PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 114Lys Cys Asn Thr Ala Ser Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 11532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
115Lys Cys Asn Thr Ala Val Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 11629PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 116Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Pro
Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 11728PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
117Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Leu Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25
11827PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 118Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 11926PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 119Lys Cys Asn Thr Ala Thr Cys Val Leu
Gly Arg Leu Ser Gln Glu Tyr 1 5 10 15 Pro Arg Thr Asn Thr Gly Ser
Asn Thr Tyr 20 25 12025PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 120Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Tyr Pro 1 5 10 15 Arg Thr Asn Thr
Gly Ser Asn Thr Tyr 20 25 12132PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 121Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Lys Leu Ser Gln Glu Leu 1 5 10 15 His Lys Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
12232PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 122Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 12332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
123Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Lys Leu Ser Gln Glu Leu
1 5 10 15 His Lys Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 12432PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 124Ala Cys Asn Thr Ala Thr
Cys Val Leu Gly Lys Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
12532PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 125Ala Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Lys Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 12635PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
126Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Leu Gln Thr Tyr Pro Arg Thr Asn Thr
Gly Ser 20 25 30 Asn Thr Tyr 35 12736PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
127Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Leu Leu Gln Thr Tyr Pro Arg Thr Asn
Thr Gly 20 25 30 Ser Asn Thr Tyr 35 12832PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
128Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Lys Leu Ser Gln Glu Leu
1 5 10 15 His Lys Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 12932PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 129Lys Cys Asn Thr Ser Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
13032PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 130Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 13132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
131Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Val Gly Ser Asn
Thr Tyr 20 25 30 13232PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 132Lys Cys Asn Thr Ser Thr
Cys Ala Thr Gln Arg Leu Ala Asn Glu Leu 1 5 10 15 Val Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Val Gly Ser Asn Thr Tyr 20 25 30
13332PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 133Lys Cys Asn Thr Ala Xaa Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 13432PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
134Lys Cys Asn Thr Ala Xaa Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 13532PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 135Lys Cys Asn Thr Ala Xaa
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
13632PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 136Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 13728PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
137Ser Thr Ala Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg Leu Gln
1 5 10 15 Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25
13832PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 138Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Xaa
Leu Ser Gln Glu Leu 1 5 10 15 His Xaa Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 13932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
139Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Xaa Leu Ser Gln Glu Leu
1 5 10 15 His Xaa Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 14032PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 140Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Xaa Leu Ser Gln Glu Leu 1 5 10 15 His Xaa Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
14133PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 141Xaa Lys Cys Asn Thr Ala Thr Cys Val Leu Gly
Arg Leu Ser Gln Glu 1 5 10 15 Leu His Arg Leu Gln Thr Tyr Pro Arg
Thr Asn Thr Gly Ser Asn Thr 20 25 30 Tyr 14232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
142Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 14332PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 143Asp Ser Asn Leu Ser Thr
Lys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
14432PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 144Lys Asp Asn Thr Ala Thr Lys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 14531PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
145Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His
1 5 10 15 Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr
Tyr 20 25 30 14633PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 146Lys Cys Asn Thr Ala Thr Cys Val Leu
Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro
Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30 Xaa 14733PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
147Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 Xaa 14832PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 148Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
14932PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 149Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Xaa
Leu Ser Gln Glu Leu 1 5 10 15 His Xaa Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 15032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
150Phe Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 15132PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 151Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
15232PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 152Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 15332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
153Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Glu Leu
1 5 10 15 Val Arg Leu Gln Thr Tyr Pro Arg Thr Asn Val Gly Ser Asn
Thr Tyr 20 25 30 15431PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 154Lys Cys Asn Thr Ala Thr
Cys Ala Thr Gln Arg Leu Ala Asp Phe Leu 1 5 10 15 Val His Leu Gln
Thr Tyr Pro Thr Asn Val Gly Ser Asn Thr Tyr 20 25 30
15529PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 155Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu
Ala Asn Phe Leu Val 1 5 10 15 His Leu Gln Thr Tyr Pro Thr Asn Val
Gly Ser Asn Thr 20 25 15632PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 156Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Lys Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
15732PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 157Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Leu Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 15832PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
158Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Pro Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 15932PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 159Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Val Gly Ser Asn Thr Tyr 20 25 30
16032PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 160Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Leu Pro Pro Thr
Asn Val Gly Ser Asn Thr Tyr 20
25 30 16132PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 161Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ala Asn Phe Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 16232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
162Ala Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 16332PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 163Lys Cys Asn Ala Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
16432PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 164Lys Cys Asn Thr Ala Ala Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 16532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
165Cys Ala Asn Leu Ser Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 16628PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 166Ser Thr Ala Val Leu Gly
Arg Leu Ser Gln Glu Leu His Arg Leu Gln 1 5 10 15 Thr Tyr Pro Arg
Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 16732PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
167Cys Ser Asn Ala Ser Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 16832PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 168Cys Ser Asn Leu Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
16932PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 169Cys Ser Asn Leu Ser Ala Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 17032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
170Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Lys Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 17132PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 171Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro 20 25 30
17232PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 172Cys Ser Ala Leu Ser Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 17332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
173Xaa Ser Asn Leu Ser Thr Xaa Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 17432PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 174Lys Xaa Asn Thr Ala Thr
Xaa Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
17527PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 175Ser Thr Ala Val Leu Xaa Arg Leu Ser Gln Glu
Leu Arg Leu Gln Thr 1 5 10 15 Tyr Pro Arg Thr Asn Thr Gly Ser Gly
Thr Pro 20 25 17628PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 176Ser Thr Ala Val Leu Gly Lys Leu Ser
Gln Glu Leu His Lys Leu Gln 1 5 10 15 Thr Tyr Pro Arg Thr Asn Thr
Gly Ser Gly Thr Pro 20 25 17728PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 177Ser Thr Ala Val Leu Xaa
Lys Leu Ser Gln Glu Leu Xaa Lys Leu Gln 1 5 10 15 Thr Tyr Pro Arg
Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 17828PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
178Ser Thr Ala Val Leu Xaa Lys Leu Ser Gln Glu Leu Xaa Lys Leu Gln
1 5 10 15 Thr Tyr Pro Arg Thr Asn Val Gly Ser Asn Thr Tyr 20 25
17932PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 179Lys Cys Asn Thr Ala Thr Cys Leu Leu Gln Gln
Leu Gln Lys Leu Leu 1 5 10 15 Gln Lys Leu Lys Gln Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 18032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
180Lys Cys Asn Thr Ala Ser Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 18132PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 181Lys Cys Asn Thr Ala Val
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
18229PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 182Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Tyr Pro Arg Thr Asn Thr Gly
Ser Asn Thr Tyr 20 25 18332PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 183Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Lys Leu Ser Gln Glu Leu 1 5 10 15 His Lys Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
18432PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 184Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 18532PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
185Lys Cys Asn Thr Ala Xaa Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 18632PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 186Ala Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Lys Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
18735PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 187Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Leu Gln Thr Tyr
Pro Arg Thr Asn Thr Gly Ser 20 25 30 Asn Thr Tyr 35
18836PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 188Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Leu Leu Gln Thr
Tyr Pro Arg Thr Asn Thr Gly 20 25 30 Ser Asn Thr Tyr 35
18932PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 189Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Lys
Leu Ser Gln Glu Leu 1 5 10 15 His Lys Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 19032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
190Lys Cys Asn Thr Ser Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 19132PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 191Lys Cys Asn Thr Ala Thr
Cys Ala Thr Gln Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
19232PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 192Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Val Gly Ser Asn Thr Tyr 20 25 30 19332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
193Lys Cys Asn Thr Ser Thr Cys Ala Thr Gln Arg Leu Ala Asn Glu Leu
1 5 10 15 Val Arg Leu Gln Thr Tyr Pro Arg Thr Asn Val Gly Ser Asn
Thr Tyr 20 25 30 19432PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 194Lys Cys Asn Thr Ala Xaa
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
19532PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 195Lys Cys Asn Thr Ala Xaa Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 19632PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
196Lys Cys Asn Thr Ala Xaa Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 19732PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 197Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
19832PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 198Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Xaa
Leu Ser Gln Glu Leu 1 5 10 15 His Xaa Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 19932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
199Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Xaa Leu Ser Gln Glu Leu
1 5 10 15 His Xaa Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 20032PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 200Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Xaa Leu Ser Gln Glu Leu 1 5 10 15 His Xaa Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
20133PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 201Xaa Lys Cys Asn Thr Ala Thr Cys Val Leu Gly
Arg Leu Ser Gln Glu 1 5 10 15 Leu His Arg Leu Gln Thr Tyr Pro Arg
Thr Asn Thr Gly Ser Asn Thr 20 25 30 Tyr 20231PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
202Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu His
1 5 10 15 Arg Leu Gln Thr Val Pro Arg Thr Asn Thr Gly Ser Asn Thr
Tyr 20 25 30 20332PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 203Lys Cys Asn Thr Ala Thr Cys Met Leu
Gly Arg Tyr Thr Gln Asp Phe 1 5 10 15 His Arg Leu Gln Thr Tyr Pro
Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30 20432PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
204Asp Ser Asn Leu Ser Thr Lys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 20532PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 205Lys Asp Asn Thr Ala Thr
Lys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
20631PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 206Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu
Ser Gln Glu Leu His 1 5 10 15 Arg Leu Gln Thr Tyr Pro Arg Thr Asn
Thr Gly Ser Asn Thr Tyr 20 25 30 20733PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
207Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 Xaa 20833PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 208Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30 Xaa
20932PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 209Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 21032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
210Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Xaa Leu Ser Gln Glu Leu
1 5 10 15 His Xaa Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 21132PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 211Phe Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
21232PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 212Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1
5 10 15 His Xaa Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr
Tyr 20 25 30 21332PRTArtificial SequenceDescription of Artificial
Sequence Synthetic construct 213Lys Cys Asn Thr Ala Thr Cys Val Leu
Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Xaa Leu Gln Thr Tyr Pro
Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30 21432PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
214Ile Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 21532PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 215Xaa Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
21631PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 216Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu
Ser Gln Glu Leu His 1 5 10 15 Arg Leu Gln Thr Tyr Pro Arg Thr Asn
Thr Gly Ser Asn Thr Tyr 20 25 30 21732PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
217Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Xaa Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 21833PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 218Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30 Xaa
21933PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 219Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 Xaa 22032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
220Lys Cys Asn Thr Ser Thr Cys Ala Thr Gln Arg Leu Ala Asn Glu Leu
1 5 10 15 Val Arg Leu Gln Thr Tyr Pro Arg Thr Asn Val Gly Ser Glu
Ala Phe 20 25 30 22131PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 221Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Thr Asn Val Gly Ser Glu Ala Phe 20 25 30
22232PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 222Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Arg Ser Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 22332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
223Lys Cys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 22432PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 224Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ala Asp Phe Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
22530PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 225Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu
Ser Gln Glu Leu His 1 5 10 15 Arg Leu Gln Thr Tyr Pro Arg Thr Asn
Thr Gly Ser Asn Thr 20 25 30 22633PRTArtificial SequenceDescription
of Artificial Sequence Synthetic construct 226Lys Cys Asn Thr Ala
Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu
Gln Asn Phe Val Pro Arg Thr Asn Thr Gly Ser Asn Thr 20 25 30 Tyr
22732PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 227Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Glu Thr Phe 20 25 30 22832PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
228Ala Cys Asp Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 22932PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 229Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Lys Ala Phe 20 25 30
23032PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 230Lys Cys Asp Thr Ala Thr Cys Val Thr His Arg
Leu Ala Gly Leu Leu 1 5 10 15 Ser Arg Ser Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 23132PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
231Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ala Asp Ala Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 23232PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 232Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ala Ala Phe Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
23332PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 233Ser Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ala Asp Phe Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 23432PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
234Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Met Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 23532PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 235Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Val Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
23632PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 236Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Asn Glu Tyr Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 23732PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
237Ser Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 23832PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 238Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Thr Glu Phe Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
23932PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 239Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ala Glu Phe Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 24032PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
240Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Thr Asp Tyr Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 24132PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 241Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ala Gln Phe Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
24232PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 242Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ala Asp Phe Leu 1 5 10 15 His Arg Phe Gln Thr Phe Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 24332PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
243Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ala Asp Phe Leu
1 5 10 15 His Arg Phe His Thr Phe Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 24432PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 244Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ala Asp Phe Leu 1 5 10 15 His Arg Phe Gln
Thr Phe Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro 20 25 30
24531PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 245Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu
Ala Asp Phe Leu His 1 5 10 15 Arg Leu Gln Thr Tyr Pro Arg Thr Asn
Thr Gly Ser Asn Thr Tyr 20 25 30 24632PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
246Lys Cys Asp Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 24732PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 247Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Phe Asp Phe Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
24832PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 248Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ala Ala Ala Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 24932PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
249Thr Cys Asp Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 25032PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 250Cys Ser Asn Leu Ser Thr
Cys Ala Thr Gln Arg Leu Ala Asn Glu Leu 1 5 10 15 Val Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Val Gly Ser Asn Thr Tyr 20 25 30
25132PRTArtificial SequenceDescription of Artificial Sequence
Synthetic construct 251Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Glu Leu 1 5 10 15 Val Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Val Gly Ser Asn Thr Tyr 20 25 30 25232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic construct
252Cys Ser Asn Leu Ser Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu
1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn
Thr Tyr 20 25 30 25332PRTArtificial SequenceDescription of
Artificial Sequence Synthetic construct 253Lys Cys Asn Thr Ala Thr
Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
* * * * *