U.S. patent application number 13/651130 was filed with the patent office on 2013-02-07 for neuromedin and fn-38 peptides for psychotic diseases.
This patent application is currently assigned to Amylin Pharmaceuticals, LLC. The applicant listed for this patent is Michael R. Hanley, Kevin D. Laugero, Christine M. Mack, Paul McGonigle, David G. Parkes. Invention is credited to Michael R. Hanley, Kevin D. Laugero, Christine M. Mack, Paul McGonigle, David G. Parkes.
Application Number | 20130035294 13/651130 |
Document ID | / |
Family ID | 39682305 |
Filed Date | 2013-02-07 |
United States Patent
Application |
20130035294 |
Kind Code |
A1 |
Laugero; Kevin D. ; et
al. |
February 7, 2013 |
Neuromedin and FN-38 Peptides for Psychotic Diseases
Abstract
Methods and compositions for treating psychiatric diseases and
disorders are disclosed. The methods provided generally involve the
administration of an NMX peptide, an FNX peptide, or an NMX
receptor agonist, or analogs or derivatives thereof, 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 Madero, CA) ;
Mack; Christine M.; (Camarillo, CA) ; Parkes; David
G.; (Del Mar, CA) ; McGonigle; Paul;
(Villanova, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Laugero; Kevin D.
Hanley; Michael R.
Mack; Christine M.
Parkes; David G.
McGonigle; Paul |
Davis
Corte Madero
Camarillo
Del Mar
Villanova |
CA
CA
CA
CA
PA |
US
US
US
US
US |
|
|
Assignee: |
Amylin Pharmaceuticals, LLC
San Diego
CA
|
Family ID: |
39682305 |
Appl. No.: |
13/651130 |
Filed: |
October 12, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12524903 |
Mar 11, 2010 |
8309522 |
|
|
PCT/US2008/001500 |
Feb 4, 2009 |
|
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13651130 |
|
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Current U.S.
Class: |
514/17.7 |
Current CPC
Class: |
A61P 25/18 20180101;
A61K 38/22 20130101; A61P 25/22 20180101 |
Class at
Publication: |
514/17.7 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61P 25/22 20060101 A61P025/22; A61P 25/18 20060101
A61P025/18 |
Claims
1-15. (canceled)
16. A method for treating a psychotic disorder in a subject in need
thereof comprising administering to the subject a therapeutically
effective amount of a neuromedin peptide or an FN38 peptide to
treat the psychotic disorder.
17. The method of claim 16, wherein the psychotic disorder is
schizophrenia.
18. The method of claim 16, wherein the psychotic disorder is
schizophreniform disorder.
19. The method of claim 16, wherein the psychotic disorder is
schizoaffective disorder.
20. The method of claim 16, wherein the neuromedin peptide or the
FN38 peptide has at least 80% sequence identity to the peptide
comprising the amino acid sequence of
FLFHYSKTQKLGKSNVVEELQSPFASQSRGYFLFRPRN--NH.sub.2 (SEQ ID NO:5).
21. The method of claim 16, wherein the neuromedin peptide or the
FN38 peptide has at least 90% sequence identity to the peptide
comprising the amino acid sequence of
FLFHYSKTQKLGKSNVVEELQSPFASQSRGYFLFRPRN--NH.sub.2 (SEQ ID NO:5).
22. The method of claim 16, wherein the neuromedin peptide or the
FN38 peptide comprises the amino acid sequence of
FLFHYSKTQKLGKSNVVEELQSPFASQSRGYFLFRPRN--NH.sub.2 (SEQ ID NO:5).
23. The method of claim 16, wherein the neuromedin peptide or the
FN38 peptide comprises the amino acid sequence of: TABLE-US-00009
(SEQ ID NO: 11) YKVNEYQGPVAPSGGFFLFRPRN-NH.sub.2; (SEQ ID NO: 13)
SDEEVQVPGGVISNGYFLFRPRN-NH.sub.2; (SEQ ID NO: 14)
FLFHYSKTQKLGKSNSDEEVQVPGGVISNGYFLFRPRN-NH.sub.2; (SEQ ID NO: 31)
FLFHYSKTQKLGKSNVVEEFQSPFASQSRGYFLFRPRN-NH.sub.2; (SEQ ID NO: 51)
FLFHYSKTQKLGKSNEELQSPFASQSRGYFLFRPRN-NH.sub.2; (SEQ ID NO: 52)
FLFHYSKTQKLGKSNVVEELQSPSRGYFLFRPRN-NH.sub.2; (SEQ ID NO: 53)
FLFHYSKTQKLGKSNEELQSPSRGYFLFRPRN-NH.sub.2; (SEQ ID NO: 54)
YSKTQKLGKSNVVEELQSPFASQSRGYFLFRPRN-NH.sub.2; or (SEQ ID NO: 64)
YSKTQKLGKSNEELQSPSRGYFLERPRN-NH.sub.2.
24. The method of claim 16, wherein the neuromedin peptide or the
FN38 peptide comprises the amino acid sequence of SEQ ID NO:1, 2,
3, 4, 6, 7, 8, 9, 10, 12, 15, 16, or 17.
25. The method of claim 16, wherein the neuromedin peptide or the
FN38 peptide comprises the amino acid sequence of any one of SEQ ID
NOs:32-50, 55-63, and 65-76.
26. The method of claim 1.6, further comprising administering to
the subject a therapeutically effective amount of a tricyclic
antidepressant, a monoamine oxidase inhibitor, a selective
serotonin reuptake inhibitor, a serotonin and noradrenaline
reuptake inhibitor, or a second generation antipsychotic.
27. The method of claim 16, further comprising administering to the
subject a therapeutically effective amount of a second generation
antipsychotic.
28. A method for treating an anxiety disorder in a subject in need
thereof comprising administering to the subject a therapeutically
effective amount of a neuromedin peptide or an FN38 peptide to
treat the anxiety disorder.
29. The method of claim 28, wherein the anxiety disorder is a panic
disorder, a specific phobia, a social phobia, an
obsessive-compulsive disorder, a posttraumatic stress disorder, an
acute stress disorder, or a generalized anxiety disorder.
30. The method of claim 28, wherein the anxiety disorder is an
anxiety disorder due to a medical condition, a substance-induced
anxiety disorder, or an anxiety disorder not otherwise
specified.
31. The method of claim 28, wherein the neuromedin peptide or the
FN38 peptide has at least 80% sequence identity to the peptide
comprising the amino acid sequence of
FLFHYSKTQKLGKSNVVEELQSPFASQSRGYFLFRPRN--NH.sub.2 (SEQ ID NO:5).
32. The method of claim 28, wherein the neuromedin peptide or the
FN38 peptide comprises the amino acid sequence of
FLFHYSKTQKLGKSNVVEELQSPFASQSRGYFLFRPRN--NH.sub.2 (SEQ ID NO:5).
33. The method of claim 28, wherein the neuromedin peptide or the
FN38 peptide comprises the amino acid sequence of: TABLE-US-00010
(SEQ ID NO: 11) YKVNEYQGPVAPSGGFFLFRPRN-NH.sub.2; (SEQ ID NO: 13)
SDEEVQVPGGVISNGYFLFRPRN-NH.sub.2; (SEQ ID NO: 14)
FLFHYSKTQKLGKSNSDEEVQVPGGVISNGYFLFRPRN-NH.sub.2; (SEQ ID NO: 31)
FLFHYSKTQKLGKSNVVEEFQSPFASQSRGYFLFRPRN-NH.sub.2; (SEQ ID NO: 51)
FLFHYSKTQKLGKSNEELQSPFASQSRGYFLFRPRN-NH.sub.2; (SEQ ID NO: 52)
FLFHYSKTQKLGKSNVVEELQSPSRGYFLFRPRN-NH.sub.2; (SEQ ID NO: 53)
FLFHYSKTQKLGKSNEELQSPSRGYFLFRPRN-NH.sub.2; (SEQ ID NO: 54)
YSKTQKLGKSNVVEELQSPFASQSRGYFLFRPRN-NH.sub.2; or (SEQ ID NO: 64)
YSKTQKLGKSNEELQSPSRGYFLFRPRN-NH.sub.2.
34. The method of claim 28, wherein the neuron neuromedin peptide
or the FN38 peptide comprises the amino acid sequence of any one of
SEQ ID NOs:1-4, 6-10, 12, 15-17, 32-50, 55-63, and 65-76.
Description
TECHNICAL FIELD
[0001] 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
[0002] 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 (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 and for all
purposes. These are debilitating illnesses that affect millions of
people and involve astronomical costs, in terms of treatment, lost
productivity, and emotional toll.
[0003] 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.
[0004] 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).
[0005] 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.
[0006] Another common psychiatric condition is eating disorders.
There are three main types, anorexia nervosa, bulimia nervosa, and
binge-eating disorders. These psychiatric conditions are often
linked to perceived notions relating to 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.
[0007] 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.
[0008] 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 describes where a person is
extremely withdrawn, negative and isolated, and has marked
psychomotor disturbances. Residual schizophrenia describes 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 describes
where a person has symptoms of schizophrenia as well as mood
disorder such as major depression, bipolar mania, or mixed mania.
Undifferentiated schizophrenia describes 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.
[0009] 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.
[0010] 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. A
need remains for new and/or improved molecules and medications
effective to treat psychiatric diseases and disorders. In addition,
needed are molecules that effectively treat those patients
resistant to the current medications, effectively treat psychiatric
diseases or disorders without the unwanted side effects of the
current medications, have a faster onset of therapeutic action,
and/or improve physical co-morbidities (e.g., diabetes, pain,
weight gain) that often present with and make more difficult the
treatment of psychiatric illnesses.
[0011] All references cited herein are incorporated by reference in
their entirety and for all purposes.
SUMMARY
[0012] In a first aspect, methods provided include the use of an
NMX peptide, an FNX peptide, or an NMX receptor agonist, or an
analog or a derivative thereof, in a therapeutically effective
amount for the treatment of a psychiatric disorder. In certain
embodiments, the psychiatric disorder is an anxiety disorder,
schizophrenia or other psychotic disorder. In certain embodiments,
the psychiatric disorder is an obsessive-compulsive disorder. In
certain embodiments, NMX peptides, FNX peptides, or NMX receptor
agonists, or analogs or derivatives thereof, are used to treat the
underlying psychiatric condition of an eating disorder.
[0013] In another aspect, methods provided herein include
administration of a therapeutically effective amount of an NMX
peptide, an FNX peptide, or an NMX receptor agonist, or an analog
or a derivative thereof, in combination with a conventional
treatment for psychiatric disorders, to a subject in need thereof.
In certain embodiments, the combination includes the administration
of electroconvulsive therapy (ECT). In other embodiments, the
combination includes the administration of at least one other
medication for treating a psychiatric disease or disorder. In still
other embodiments, the at least one other medication for treating a
psychiatric disease or disorder is one or more of tricyclic
antidepressants, monoamine 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). In some embodiments, the combination includes
the administration of an amylin or its agonists, analogs, or
derivatives, as the at least one other medication for treating a
psychiatric disease or disorder. In some embodiments, the at least
one other medication for treating a psychiatric disease or disorder
is not an amylin or its agonists, analogs, or derivatives.
[0014] In another aspect, methods provided herein include treating
an unwanted side effect of another psychiatric medication
comprising administering a therapeutically effective amount of an
NMX peptide, an FNX peptide, or an NMX receptor agonist, or an
analog or a derivative thereof, to a subject in need thereof. In
certain embodiments, the other psychiatric medication is an SGA
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.
[0015] In 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 NMX peptide, an FNX peptide, or an NMX receptor
agonist, or an analog or a derivative thereof, 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.
[0016] In another 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.
In certain embodiments, the behavioral pathway is the 5HT.sub.1A
pathway or any pathway comprising the serotonergic system. In
certain embodiments, the behavioral pathway is stress responsive.
In certain embodiments, the compound is an NMX peptide, an FNX
peptide, or an NMX receptor agonist, or an analog or a derivative
thereof.
[0017] In another aspect, the disclosure provides for the use of an
NMX peptide, an FNX peptide, or an NMX receptor agonist, or an
analog or a derivative thereof, for manufacture of a medicament
useful for treating psychiatric diseases and disorders described
herein. In yet another aspect, the disclosure provides for the use
of an NMX peptide, an FNX peptide, or an NMX receptor agonist, or
an analog or a derivative thereof, for manufacture of a medicament
useful for treating unwanted side effects of another psychiatric
medication, for example without limitation, an SGA. Further to any
aspect or embodiment described herein, additionally contemplated is
the use of a compound described herein for manufacture of a
medicament useful for treating psychiatric diseases and disorders,
wherein in certain embodiments, the psychiatric disorder is an
anxiety disorder, schizophrenia or other psychotic disorder.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0018] FIG. 1 depicts the effect of FN-38 (SEQ ID NO:5) and control
agents on stress-induced hyperthermia in mice, as described in
Example 2. Legend: Water (open); buspirone, 15 mg/kg (striped);
FN-38 (filled).
[0019] FIG. 2 depicts the effect of FN-38 (SEQ ID NO:5) and control
agents on marble burying, as described in Example 2. Legend: Water
(open); chlordiazepoxide (CDP) (striped); FN-38 (filled).
[0020] FIG. 3 depicts the effect of FN-38 (SEQ ID NO:5) and control
agents on phencyclidine (PCP)-induced locomotion, as described in
Example 2. Legend (initial-subsequent injection contents):
water-water (diamonds); water-PCP (boxes); FN-38-PCP
(triangles).
[0021] FIG. 4 depicts the effect of 10 mg/kg FN-38 (SEQ ID NO:5)
and control agents on prepulse inhibition at the prepulse stimulus
intensities of 74, 78, and 82 dB, as described in Example 2.
DETAILED DESCRIPTION
[0022] It has now been discovered that an NMX peptide, an FNX
peptide, or an NMX receptor agonist, or an analog or a derivative
thereof, or a combination thereof, present novel
pharmacotherapeutic options. For example as demonstrated herein,
compound FN-38 (FLFHYSKTQKLGKSNVVEELQSPFASQSRGYFLFRPRN, SEQ ID
NO:5) amide is shown to share properties of anxiolytic and
antipsychotic agents in behavioral testing. "FN-38 peptide" and
like terms refer to FN-38 and analogs based thereon. Administration
of FN-38 to animals results in behavioral effects that include
anti-stress, anxiolytic, and antipsychotic actions. Thus, an NMX
peptide, an FNX peptide, or an NMX receptor agonist, or an analog
or a derivative thereof, may have the surprising ability to treat
psychiatric disorders. Psychiatric disorders that can be treated
include anxiety disorders and schizophrenia and other psychotic
disorders. These compounds may be particularly effective in
treating psychiatric disorders that have elements of metabolic
disturbances, e.g., in treating subjects with a psychiatric
disorder or those with a psychiatric disorder and who also suffer
from a metabolic disturbance. More particular types of the above
named disorders can be found in the DSM-IV. The following are
examples without limitation of disorders that may be treated by the
methods disclosed herein.
[0023] In some embodiments, methods provided can be used to treat
subjects with anxiety disorder. Examples include 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.
[0024] In some embodiments, methods provided can be used to treat
subjects with schizophrenia and other psychotic disorders.
Schizophrenia and other psychotic disorders feature a mixture of
generally characteristic signs and symptoms, both positive and
negative. Positive symptoms of schizophrenia and other psychotic
disorders appear to reflect an excess or distortion of normal
functions, whereas the negative symptoms appear to reflect a
diminution or loss of normal functions. Positive symptoms include,
but are not limited to, delusions, hallucinations, disorganized
thinking or thought disorder, grossly disorganized behavior, and
catatonic motor behavior. Positive symptoms may comprise two
distinct dimensions: the "psychotic dimension" includes delusions
and hallucinations and the "disorganization dimension" includes
disorganized speech and behavior. Negative symptoms include, but
are not limited to, affective flattening, alogia, and avolition.
Affective flattening is generally displayed as restrictions in the
range and intensity of emotional expression. Alogia is generally
displayed as restrictions in the fluency and productivity of
thought and speech. Avolition is generally displayed as
restrictions in the initiation of goal-directed behavior.
[0025] Schizophrenia and other psychotic disorders include
schizophrenia, schizophreniform disorder, schizoaffective disorder,
delusional disorder, brief psychotic disorder, shared psychotic
disorder, psychotic disorder due to a general medical condition,
substance-induced psychotic disorder and psychotic disorder not
otherwise specified. Schizoaffective disorder involves
characteristic symptoms of schizophrenia and a major depressive,
manic, or mixed depressive and manic episode.
[0026] In some instances, a psychiatric disorder may result from
use of a particular substance or drug. In some embodiments, methods
provided include the treatment of subjects with substance-induced
psychiatric disorders. Substance-induced anxiety disorder can occur
in response to substances which include, but are not limited to,
caffeine, cannabis, cocaine, hallucinogens, amphetamines,
phencyclidines, phencyclidine-like substances, and inhalants.
Substance-induced psychotic disorder can occur in response to
substances which include, but are not limited to, cocaine,
hallucinogens, narcotics, 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.
[0027] In some instances, the psychiatric disorder may result from
medication for or treatment of a different disease (other than the
psychiatric 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. In some embodiments, methods provided include the
treatment of medication-induced anxiety disorders or
medication-induced psychotic disorders that result from treatment
of a disease. In some embodiments, methods provided include the
treatment of anxiety or anxiousness associated with taking a
medication, such as, a prescription medication, an over-the-counter
medication, or an herbal remedy or medication. For example,
psychiatric side-effects such as anxiety, depression, and psychosis
are commonly associated with interferon therapy in patients with
chronic hepatitis C disease (Kraus et al., 2005, World J.
Gastroenterol. 11:1769-1774; Neri et al., 2006, Clin. Drug
Investig. 26:655-662).
[0028] In some embodiments, methods provided can be used to treat
subjects with personality disorders, including, but not limited to,
schizoid personality disorder and schizotypal personality disorder.
Individuals with schizoid personality disorder may also experience
symptoms of depression and/or transient psychotic episodes,
particularly in response to stress. Individuals with schizotypal
personality disorder may also experience symptoms of anxiety,
depression, and/or transient psychotic episodes.
[0029] In certain embodiments, methods provided are drawn to the
treatment of the psychiatric illness associated with an 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 subjects. In certain
embodiments, methods provided do not include the treatment of binge
eating.
[0030] In certain embodiments, methods for treating psychiatric
disorders in a subject are provided, wherein the method comprises
administering to a subject in need thereof an NMX peptide, an FNX
peptide, or an NMX receptor agonist, or an analog or a derivative
thereof, in an amount effective to treat the psychiatric disorder.
Certain embodiments contemplate the use of the naturally occurring
and peripherally secreted NMX peptide, FNX peptide, or an NMX
receptor agonist for the treatment of the psychiatric disorder. In
some instances, the psychiatric disorders are of natural or
unidentified etiology.
[0031] In certain embodiments, it is contemplated that compounds
that reduce or moderate stress, or regulate the stress pathway, may
be useful as pharmacotherapeutic agents. In other embodiments, 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 yet
other embodiments, it is contemplated that compounds that can
attenuate or reverse metabolic disturbances would be useful as
pharmacotherapeutic treatments of psychiatric diseases or
disorders. Certain embodiments contemplate the use of compounds
that can treat both the psychiatric disease and metabolic
disturbances present in a subject. It is contemplated that
compounds useful in the methods provided may be NMX peptides, FNX
peptides, or NMX receptor agonists, or analogs or derivatives
thereof.
[0032] Without wishing to be bound by theory, it is believed 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 subjects with a psychiatric disease or disorder.
Physical co-morbidities, for example without limitation obesity,
exacerbate the morbidity that attends psychiatric disease or
disorder and lead to a reduction in treatment response. NMX
peptides, FNX peptides, or NMX receptor agonists, or analogs or
derivatives thereof, may be particularly useful in the methods
described herein because of anti-obesigenic and appetite
suppressant effects. These effects may increase the rate of
treatment response and outcome success in certain subject
populations who suffer a psychiatric disease or disorder and who
exhibit obesity, obesity-related disease, or eating disorders, for
example without limitation 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.
[0033] NMX peptides, FNX peptides, or NMX receptor agonists, or
analogs or derivatives thereof, may be superior to some other
anxiolytic and/or antipsychotic agents, for example, as certain
compounds contemplated herein do not promote weight gain and, in
fact, may induce weight loss. This attribute of NMX peptides, FNX
peptides, or NMX receptor agonists or analogs or derivatives
thereof, may lead to greater compliance among subjects being
treated for psychiatric disease or disorder. Central administration
of NMU to rats and peripheral (intraperitoneal) administration of
NMX and FNX peptides to mice inhibited food intake by the animals.
See, for example, Wren et al., 2002, Endocrinology 143:4227-4234
and commonly-owned PCT Patent Application No. PCT/US2006/047953 (WO
2007/075439), incorporated herein by reference in its entirety and
for all purposes.
[0034] It is further contemplated that an NMX peptide, an FNX
peptide, or an NMX receptor agonist, or an analog or a derivative
thereof, may be used in conjunction with at least one other
medication or therapy for treating a psychiatric disease or
disorder, including, but not limited to, those conventionally used
to treat psychiatric disease, such as tricyclic antidepressants and
the monoamine oxidase inhibitors (MAOIs), selective serotonin
reuptake inhibitors (SSRIs), serotonin and noradrenaline reuptake
inhibitors (SNRIs), herbal antidepressants (e.g., St John's Wort or
Hypericum), SGAs, psychoanalysis, cognitive-behavioral therapy, and
interpersonal therapy.
[0035] Second generation antipsychotics (SGAs) (also known as
"atypical antipsychotics") are a class of medication with a broad
spectrum of neurotransmitter activity, having affinity for not only
the dopamine D.sub.2, but also D.sub.1, D.sub.3, and D.sub.4
receptors, as well as for serotonin, adrenergic, histamine, and/or
opiate receptors. SGAs can be well tolerated, having fewer and less
severe side effects compared with other antipsychotics, and with
few to no extrapyramidal side effects at clinical doses. Many of
these newer medications are also more effective at treating the
negative, cognitive, and affective symptoms. Thus, SGAs are now
considered the first-line interventions for psychotic disorders.
One of the atypical agents, clozapine, is clearly the most
effective antipsychotic, but clozapine is reserved as a second-line
agent, indicated only after other medications have failed or in
patients at high risk for suicidal behavior, because it can cause
agranulocytosis (American Diabetes Association, American
Psychiatric Association, American Association of Clinical
Endocrinologists, North American Association for the Study of
Obesity, J. Clin. Psychiatry., 2004, 65(2):267-72; Leo, et al.,
2000, Prim. Care Companion J. Clin. Psychiatry 2(6):194-204). SGAs
are widely prescribed, to approximately 3 percent of the U.S.
population, for treatment of schizophrenia, bipolar disorder,
depression and dementia. However, concern has arisen regarding
weight gain, obesity and an increased risk of diabetes associated
with the use of SGAs (Ader, et al., 2005, Diabetes 54:862-871).
[0036] In some embodiments, the NMX, FNX, or NMX receptor agonist,
or analog or derivative thereof, may be used in conjunction with an
amylin or its agonists, analogs, or derivatives, as the at least
one other medication for treating a psychiatric disease or
disorder. Examples of amylin, amylin agonists, amylin analogs, and
derivatives thereof, for such a use include those described in U.S.
Pat. Nos. 5,686,411, 6,610,824, 5,998,367, 6,087,334, 6,114,304,
6,410,511; and PCT Application Publication Nos. WO 93/10146, WO
2006/042242, WO 2006/083254, and WO 2006/105527, all of which are
incorporated herein by reference in their entireties and for all
purposes. In certain embodiments, use of amylin agonists may not
include the use of calcitonins. In certain embodiments, the
calcitonin is salmon calcitonin. In other embodiments, use of
amylin agonists may not include the use of CGRP. In still other
embodiments, use of amylin agonists may not include the use of
analogs of CGRP or calcitonin. Accordingly, it is contemplated that
use of amylin agonists provided may include a proviso that excludes
CGRP, calcitonin, or analogs thereof. In some embodiments, the at
least one other psychiatric medication is not an amylin, an amylin
agonist, an amylin analog, or an amylin derivative.
[0037] When used in conjunction with other medications or therapies
for treating a psychiatric disease or disorder, administration of
the NMX peptide, FNX peptide, or NMX receptor agonist, or analog or
derivative thereof, may occur concurrently or sequentially with the
other medication(s), therapy or therapies. For example, the NMX
peptide, FNX peptide, or NMX receptor agonist, or analog or
derivative thereof, 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 NMX peptide, an FNX peptide, or an NMX receptor
agonist, or an analog or a derivative thereof, may counteract or
moderate one or more unwanted side effects of currently available
medications, for example without limitation anxiety, weight gain,
diabetes, and the like.
[0038] For example, SGAs are effective therapeutics for the
treatment of symptoms associated with schizophrenia and related
psychotic conditions. Despite advances in treating the psychiatric
condition afforded by SGAs, 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 non-compliance of a subject with a
medication regimen. So, as good as any medication may be, it does
not provide any benefit to a subject 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.
Additionally, these two SGAs have also been associated with
increased risk for both diabetes and dyslipidemia. The ability of
an NMX peptide, an FNX peptide, or an NMX receptor agonist, or an
analog or a derivative thereof, to effectively reduce body weight
gain induced by clozapine treatment is of use to the recipient.
Accordingly, an NMX peptide, an FNX peptide, or an NMX receptor
agonist, or an analog or a derivatives thereof, is also able to
treat or aid in the treatment of diabetes and dyslipidemia.
Accordingly, when used with other psychiatric medications, an NMX
peptide, an FNX peptide, or an NMX receptor agonist, or an analog
or a derivative thereof, 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.
[0039] An NMX peptide, an FNX peptide, or an NMX receptor agonist,
or an analog or a derivative thereof may have anxiolytic and/or
antipsychotic activities that are not directed related an
anti-obesity activity of the compound.
[0040] As used herein, a "subject" may include any mammal,
including humans. A "subject" may also include pets and commercial
animals (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).
[0041] 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.
[0042] As used herein, the term "therapeutically effective amount"
means an amount of active compound in the composition that will
elicit a biological response that is sought in a cell, tissue,
system, and/or subject (including a human being), which includes
without limitation, alleviation and/or prevention of the symptom(s)
of a disorder or condition being treated and/or prevented. As used
herein, the term "symptom(s)" refers to any marker(s) of the
condition, disease or disorder (collectively referred to herein as
a "condition" unless context dictates otherwise) which can be
observed directly or indirectly and can include, but is not limited
to, physiological response(s) and/or the expression of particular
biomarker(s) (e.g., protein(s), peptide(s), nucleic acid(s),
metabolites, molecule(s), etc.) associated with a disorder or
condition, and/or the progression of a disorder or condition.
[0043] As used herein, the terms "protein", "polypeptide" or
"peptide" include any molecule that comprises five or more amino
acids. It is well known in the art that proteins may undergo
modification, including post-translational modifications such as,
but not limited to, disulfide bond formation, glycosylation,
phosphorylation, or oligomerization. Thus, as used herein, the term
"protein" or "peptide" includes any protein or peptide that is
modified by any biological or non-biological process. In certain
contexts, as used herein, a "peptide" refers to a polymer
comprising less than about 200 amino acid residues, less than about
100 amino acid residues, less than about 50 amino acid residues, or
less than about 40 amino acids. Generally, "peptides" as used
herein do not include polyamino acids unless explicitly referred to
as such. Also, generally, unless context dictates otherwise, as
used herein the term "peptide", "polypeptide" and "protein" are
used herein interchangeably.
[0044] 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" FNX
peptide can include one or more FNX peptides. The term "about" in
the context of a numeric value can refer to the numerical value
+/-10% thereof.
[0045] "NMX peptide" refers to a neuromedin U (NMU), a neuromedin S
(NMS), or an FN-38 peptide, including FNX peptides as described
herein. The polypeptide may be obtained or derived from any
species. Thus, the term includes the human full-length amino acid
peptides, and species variations thereof, including for example
without limitation murine, hamster, chicken, bovine, rat, and dog
polypeptides. In this sense the descriptors "wild-type," "native"
and "unmodified" are used interchangeably.
[0046] By "NMX Receptor agonist" is meant any compound, including
peptide, peptide-like compounds and small molecules, that elicits
similar biological activities as FN-38 and acts on a known NMU or
NMS receptor, e.g., NMUR1 or NMUR2.
[0047] FNX peptides for use in the methods provided herein include
those described in commonly-owned PCT Patent Application No.
PCT/US2006/047953 (WO 2007/075439), which is incorporated herein by
reference in its entirety and for all purposes. In particular, the
NMX and FNX peptides described in WO 2007/075439 are incorporated
herein by reference and for all purposes.
[0048] Exemplary NMX peptides include, but are not limited to, the
peptides provided in Table 1.
TABLE-US-00001 TABLE 1 Exemplary NMX peptides SEQ ID NO:
Description Sequence 1 human NMU FLFHYSKTQKLGKSNVVEEFQSPFASQSRG
YFLFRPRNGRRSAGF 2 rat NMU FLFHYSKTQKLGNSNVVEYQGPVAPSGGFF variant
LFRPRN CAD52851 3 tree frog NMU FLFHYSKSHDSGNSDITEEVQVPGGVISNG
variant YFLFRPRN CAD52850 4 chicken NMU
FLFHYSKTHDSGNSDVREDLQGTGGIQSRG variant YFFFRPRN 5 human FN-38
FLFHYSKTQKLGKSNVVEELQSPFASQSRG YFLFRPRN 6 FN-38(1-28)
FLFHYSKTQKLGKSNVVEELQSPFASQS 7 FN-38(1-15) FLFHYSKTQKLGKSN 8 human
NMS ILQRGSGTAAVDFTKKDHTATWGRPFFLFR PRN 9 rat NMS
LPRLLHTDSRMATIDFPKKDPTTSLGRPFF LFRPRN
[0049] Additional examples of NMX peptides include, but are not
limited to, the peptides provided in Table 2.
TABLE-US-00002 TABLE 2 Additional exemplary NMX peptides SEQ ID NO:
Description Sequence 10 porcine NMU U8 YFLFRPRN (octapeptide) 11
rat NMU-23 YKVNEYQGPVAPSGGFFLFRPRN 12 human NMU U9 GYFLFRPRN 13
tree frog SN-23 SDEEVQVPGGVISNGYFLFRPRN
[0050] Other exemplary peptides include the peptides provided in
Table 3.
TABLE-US-00003 TABLE 3 Other exemplary peptides SEQ ID NO:
Description Sequence 14 FN-38 FLFHYSKTQKLGKSNSDEEVQVPGGVISNG
(1-15)- YFLFRPRN SN-23 15 FN-38 FLFHYSKTQKLGKSNSDEEVQVPGGVISNG
(1-15)- SN-23(des- octa- peptide) 16 FN-38(des-
FLFHYSKTQKLGKSNVVEELQSPFASQSRG octa- peptide) 17 human
FRVDEEFQSPFASQSRGYFLFRPRN NMU25 FN-38(1-15)-SN-23 (SEQ ID NO: 14)
is a hybrid of tree frog SN-23 NMU (treefrog SN-23 (SEQ ID NO: 13)
and human FN-38(1-15) (SEQ ID NO: 7).
[0051] NMX peptides, FNX peptides, and NMX receptor agonists, and
analogs and derivatives thereof which are peptides may or may not
be amidated at the C-terminal end. The term "des-octapeptide" in
the context of a peptide described herein refers to deletion of the
residues forming the C-terminal octapeptide.
[0052] As used herein, the names of some compounds indicate both
the peptide upon which the compound is based (e.g., the base
peptide) and the modification(s) made to the base peptide sequence.
"Base peptide," "base reference peptide," "reference peptide" and
like terms refer to a peptide which serves as a basis for analogous
peptides having, for example, insertions, substitutions,
extensions, and/or deletions of the amino acid sequence of the base
peptide, as known in the art. For example, "FN-38(1-15),"
"FN-38.sub.1-15" and the like refer to a peptide based on the
sequence of amino acids 1-15 of FN-38. An amino acid preceded by a
superscript number indicates that the amino acid so indicated
replaces the amino acid normally present at the amino acid position
of the superscript in the base peptide sequence. For example,
"FN-38-.sup.31F," "FN-38-(.sup.31F)" and "[.sup.31F]-FN-38" and the
like refer to a peptide based on the sequence of FN-38 having Phe
at residue 31. The term "des-" preceding one or more amino acids
indicates that the named amino acids normally present at the
positions of the superscripts in the base peptide sequence are
deleted. For example, "FN-38 des-(.sup.24F-.sup.27 Q)" and
"des-(.sup.24F-.sup.27Q)-FN-38" refer to a peptide based on the
sequence of FN-38 having amino acids Phe through Gln at positions
24 through 27 deleted.
[0053] In some embodiments, FNX peptides comprise an amino acid
sequence of Formula I (F1-P) or Formula II (F2-P), as described in
PCT Patent Application No. PCT/US2006/047953 (WO 2007/075439),
incorporated herein by reference in its entirety and for all
purposes.
[0054] In Formula I peptides, the F1 portion is a des-octapeptide
portion of FN-38 or analog, derivative or chimera thereof. An
exemplary F1 portion is FLFHYSKTQKLGKSNVVEELQSPFASQSRG (SEQ ID
NO:16).
[0055] In Formula II peptides, the F2 portion is a des-octapeptide
portion of FN-38 or SN-23, or hybrid, analog, derivative or chimera
thereof. An exemplary F2 portion is FLFHYSKTQKLGKSNSDEEVQVPGGVISNG
(SEQ ID NO:15).
[0056] Exemplary octapeptide sequences ("P") for use in peptides
comprising Formula I or Formula II include, but are not limited to,
those provided in Table 4.
TABLE-US-00004 TABLE 4 Exemplary octapeptide sequences ("P") SEQ ID
NO: Sequence 18 YFLFRPRN 19 YFLYRPRN 20 YFVFRPRN 21 FFLFRPRN 22
YFLVRPRN 23 YFFFRPRN 24 YFLFHPRN 25 YFLFRPHN 26 YFLFR(beta turn
mimic B)RN
[0057] Additional examples of octapeptide sequences ("P") with
multiple substitutions or modification to increase its
hydrophobicity and/or its positive charge include, but are not
limited to, the peptides provided in Table 5.
TABLE-US-00005 TABLE 5 Additional exemplary octapeptide sequences
("P") SEQ ID NO: Sequence 27 FFFYHPHN 28 FFFFRPRN 29 FFFFKHHN 30
FFFFK(beta turn mimic B)HN
[0058] In certain embodiments, FNX peptides have one of the
octapeptide sequences ("P"). In some embodiments, FNX peptides have
two, three, four, five, or six of the octapeptide substitutions or
modifications shown herein. In some embodiments, a P region
octapeptide does not have a histidine substituting for either or
both arginine residues. In some embodiments, a P region octapeptide
does not have a turn mimic substituting for proline. Exemplary
analogs of FNX peptide FN-38 (SEQ ID NO:5) having a P region
sequence as described herein and an F1 region of FN-38 include
without limitation the peptides provided in Table 6.
TABLE-US-00006 TABLE 6 Exemplary analogs of FNX peptide FN-38 SEQ
ID NO: Description Sequence 31 FN-38-(.sup.31F)
FLFHYSKTQKLGKSNVVEELQSPFASQSRG FFLFRPRN 32 FN-38-(.sup.34V)
FLFHYSKTQKLGKSNVVEELQSPFASQSRG YFLVRPRN 33 FN-38-(.sup.33F)
FLFHYSKTQKLGKSNVVEELQSPFASQSRG YFFFRPRN 34 FN-38-(.sup.35H)
FLFHYSKTQKLGKSNVVEELQSPFASQSRG YFLFHPRN 35 FN-38-(.sup.37H)
FLFHYSKTQKLGKSNVVEELQSPFASQSRG YFLFRPHN 36 FN-38-.sup.36
FLFHYSKTQKLGKSNVVEELQSPFASQSRG (beta turn YFLFR (beta turn mimic B)
RN mimic B) 37 FN-38- FLFHYSKTQKLGKSNVVEELQSPFASQSRG (.sup.31F
.sup.33F FFFYHPHN .sup.34Y .sup.35H .sup.37H) 38
FN-38-(.sup.31,33F) FLFHYSKTQKLGKSNVVEELQSPFASQSRG FFFFRPRN 39
FN-38-(.sup.31F .sup.33F FLFHYSKTQKLGKSNVVEELQSPFASQSRG .sup.35K
.sup.36H .sup.37H) FFFFKHHN 40 FN-38 (.sup.31F
FLFHYSKTQKLGKSNVVEELQSPFASQSRG .sup.33F .sup.35K FFFFK (beta turn
mimic B) HN .sup.36(beta turn mimic B) .sup.37H)
[0059] Exemplary analogs of Formula II having a P sequence and the
F2 region of FN-38(1-15)-SN-23 (SEQ ID NO:14) include, but are not
limited to the peptides provided in Table 7.
TABLE-US-00007 TABLE 7 Exemplary analogs of Formula II SEQ ID NO:
Description Sequence 41 FN-38(1-15)- FLFHYSKTQKLGKSNSDEEVQVPGGVISNG
SN-23-.sup.31F FFLFRPRN 42 FN-38(1-15)-
FLFHYSKTQKLGKSNSDEEVQVPGGVISNG SN-23-.sup.34V YFLVRPRN 43
FN-38(1-15)- FLFHYSKTQKLGKSNSDEEVQVPGGVISNG SN-23-.sup.33F YFFFRPRN
44 FN-38(1-15)- FLFHYSKTQKLGKSNSDEEVQVPGGVISNG SN-23-.sup.35H
YFLFHPRN 45 FN-38(1-15)- FLFHYSKTQKLGKSNSDEEVQVPGGVISNG
SN-23-.sup.37H YFLFRPHN 46 FN-38(1-15)-
FLFHYSKTQKLGKSNSDEEVQVPGGVISNG SN-23-.sup.36(beta YFLFR (beta turn
mimic B) RN turn mimic B) 47 FN-38(1-15)-
FLFHYSKTQKLGKSNSDEEVQVPGGVISNG SN-23- FFFYHPHN
.sup.31F.sup.33F.sup.34Y.sup.35H.sup.37H 48 FN-38(1-15)-
FLFHYSKTQKLGKSNSDEEVQVPGGVISNG SN-23-.sup.31,33F FFFFRPRN 49
(FN-38(1-15)- FLFHYSKTQKLGKSNSDEEVQVPGGVISNG SN-23- FFFFKHHN
.sup.31,33F.sup.35K.sup.36,37H 50 (FN-38(1-15)-
FLFHYSKTQKLGKSNSDEEVQVPGGVISNG SN-23- FFFFK (beta turn mimic B) HN
.sup.31,33F.sup.35K.sup.36 (beta turn mimic B).sup.37H
[0060] In certain embodiments, FNX peptides for use in the methods
provided herein have one or more amino acid deletions or deleted
regions, for example without limitation, the deletions and deleted
regions shown herein. In other embodiments, an FNX peptide has two
such deleted regions. In other embodiments, an FNX peptide has at
least one amino acid deletion, the amino acid being any one of the
amino acids contained within any of the deleted regions shown
below. In other embodiments, one, two, three, four, or five amino
acids are deleted. Accordingly, depending on the length of the
parent peptide, the FNX peptide may be at least or equal to 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43
residues in length or any combination thereof, e.g., at least 10
but no more than 15 residues. In certain embodiments, the deleted
amino acids are of the amino acids contained in any of the deleted
regions shown herein.
[0061] Exemplary FNX peptides with amino acids deletions include,
but are not limited to, the deletion and/or substitution analogs of
FN-38 (SEQ ID NO:5) and FN-38(1-15)-SN-23 (SEQ ID NO:14) provided
in Table 8, wherein dashes indicate locations of deleted amino acid
residues.
TABLE-US-00008 TABLE 8 Exemplary FNX peptides with amino acids
deletions SEQ ID NO: Description Sequence 51 FN-38 des-
FLFHYSKTQKLGKSN--EELQSPFASQSRG (.sup.16V-.sup.17V) YFLFRPRN 52
FN-38 des- FLFHYSKTQKLGKSNVVEELQSP----SRG (.sup.24F-.sup.27Q)
YFLFRPRN 53 FN-38 des- FLFHYSKTQKLGKSN--EELQSP----SRG
(.sup.16V-.sup.17V, YFLFRPRN .sup.24F-.sup.27Q) 54 FN-38 des-
----YSKTQKLGKSNVVEELQSPFASQSRG (.sup.1F-.sup.4H) YFLFRPRN 55 FN-38
des- FLFHY----KLGKSNVVEELQSPFASQSRG (.sup.6S-.sup.9Q) YFLFRPRN 56
FN-38 des- FLFHYSKTQKLG-------LQSPFASQSRG (.sup.13K-.sup.19E)
YFLFRPRN 57 FN 38 des- F-------QKLGKSNVVEELQSPFASQSRG
(.sup.2L-.sup.8T) YFLFRPRN 58 FN-38 des-
FLFHYS-----------------FASQSRG (.sup.7K-.sup.23P) YFLFRPRN 59 FN-38
des- FLFHYSKTQKLGKSN--------------G (.sup.16V-.sup.29R) YFLFRPRN 60
FN-38 des- FLFHYSKTQKLGKSN------------SRG (.sup.16V-.sup.27Q)
YFLFRPRN 61 FN-38 des- FLFHYSKTQKLGKSN--EELQSP----SRG
(.sup.16V-.sup.17V, YFLFKPRN .sup.24F-.sup.27Q) .sup.35K 62 FN-38
des- FLFHYS-----------------------G (.sup.7K-.sup.29R) YFLFRPRN 63
FN-38 des- ---------KLGKSNVVEELQSPFASQSRG (.sup.1F-.sup.9Q)
YFLFRPRN 64 FN-38 des- ----YSKTQKLGKSN--EELQSP----SRG
(.sup.1F-.sup.4H, YFLFRPRN .sup.16V-.sup.17V, .sup.24F-.sup.27Q) 65
FN-38 des- FLFHYS-----------------------G (.sup.7K-.sup.29R) YFLFR
(beta turn mimic B) RN .sup.36(beta turn mimic B) 66 FN-38 des-
FLFHYS-----------------------G (.sup.7K-.sup.29R), .sup.31F, FFLFR
(beta turn mimic B) HN .sup.36(beta turn mimic B) .sup.37H 67 FN-38
des- FLFHYS-----------------------G (.sup.7K-.sup.29R), .sup.35K
YFLFKPRN 68 FN-38 des- FLFHYS-----------------------G
(.sup.7K-.sup.29R), .sup.31F FFLFRPRN 69 FN-38 des-(.sup.7K-
FLFHYS-----------------------G .sup.29R), .sup.31F, .sup.35K
FFLFKPRN 70 FN-38(1-15)- FLFHYSKTQKLGKSN--EEVQVPGGVISNG SN-23 des-
YFLFRPRN (.sup.16S-.sup.17D)) 71 FN-38(1-15)-
FLFHYSKTQKLGKSNSDEEVQVP----SNG SN-23 des- YFLFRPRN
(.sup.24G-.sup.27I)) 72 FN-38(1-15)- FLFHYSKTQKLGKSN--EEVQVP----SNG
SN-23 des- YFLFRPRN (.sup.16S-.sup.17D, .sup.24G-.sup.27I) 73
FN-38(1-15)- ----YSKTQKLGKSNVVEELQSPFASQSRG SN-23 des- YFLFRPRN
(.sup.1F-.sup.4H) 74 FN-38(1-15)- FLFHY----KLGKSNVVEELQSPFASQSRG
SN-23 des- YFLFRPRN (.sup.6S-.sup.9Q) 75 FN-38(1-15)-
FLFHYSKTQKLG-------VQVPGGVISNG SN-23 des- YFLFRPRN
(.sup.13K-.sup.19E) 76 FN-38(1-15)- F-------QKLGKSNSDEEVQVPGGVISNG
SN-23 des- YFLFRPRN (.sup.2L-.sup.8T)
[0062] NMX peptides, FNX peptides, and NMX receptor agonists, and
analogs or derivatives thereof, that contain modified 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 (--C(S)--NH--), thiomethylenes (e.g., --S--CH.sub.2--,
--CH.sub.2--S--), methylenes (--CH.sub.2--), alkylenes (e.g.,
--(CH.sub.2).sub.n--, n>1) and retro-amides (--NH--CO--).
[0063] By "agonist" is meant a compound which elicits a biological
activity of a reference peptide. In certain aspects, an agonist has
a greater potency than the reference peptide, or within five orders
of magnitude (plus or minus) of potency compared to the reference
peptide, for example 4, 3, 2, or 1 order of magnitude, when
evaluated by art-known measures such as, for example, receptor
activation studies, ligand binding/competition studies, receptor
binding/competition studies. In one aspect, an agonist will bind in
such assays with an affinity of greater than about 1 .mu.M, and in
certain aspects, with an affinity of greater than about 1-5 nM. An
agonist can be a fragment of a reference peptide which retains
potency or displays enhanced potency compared to the reference
peptide and/or can be an analog of the reference peptide. In one
aspect, an agonist can modulate the therapeutic efficacy, scope,
duration of action, physicochemical properties, and/or other
pharmacokinetic properties of a bioactive peptide or receptor
molecule.
[0064] As used herein, "analog" refers to a peptide which sequence
is derived from that of the base reference peptide, e.g., NMU,
FN-38, etc., and includes insertions, substitutions, extensions,
and/or deletions of the reference amino acid sequence, for example
having at least 50% or 55% amino acid sequence identity with the
base reference peptide. In certain embodiments, an analog may have
at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%,
90%, 92%, 94%, 95%, 96%, 97%, 98% or even 99% amino acid sequence
identity with the base reference peptide. In one embodiment, such
analogs may comprise conservative or non-conservative amino acid
substitutions (including non-natural amino acids and L and D
forms). A "conversative" amino acid substitution maintains charge,
hydrophobicity and/or other amino acid property. Exemplary
conservative substitutions include without limitation Ile for Leu,
Arg for Lys, Tyr for Phe, and the like as well known in the art.
Analogs include compounds having agonist and compounds having
antagonist activity. As used herein "analog" further refers to
bioactive peptides or proteins that are structurally related to a
parent peptide by amino acid sequence but which may differ from the
parent in a characteristic of interest such as for example without
limitation bioactivity, solubility, resistance to proteolysis, and
the like. In certain embodiments, analogs have activities between
about 1% to about 10,000%, about 10% to about 1000%, and about 50%
to about 500% of the bioactivity of the parental peptide.
[0065] As contemplated herein, NMX analogs may be compounds having
at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%,
90%, 92%, 94%, 95%, 96%, 97%, 98% or even 99% amino acid sequence
identity to any NMX peptide amino acid sequence described herein.
In some embodiments, NMX analogs may also be compounds having at
least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%,
92%, 94%, 95%, 96%, 97%, 98% or even 99% amino acid sequence
identity to any NMX peptide amino acid sequence described herein
and having NMX activity. In some embodiments, an NMX analog may be
a compound having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%,
84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or even 99% amino
acid sequence identity to human NMU and having NMX activity.
[0066] In certain embodiments, FNX analogs may be compounds having
at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%,
90%, 92%, 94%, 95%, 96%, 97%, 98% or even 99% amino acid sequence
identity to any of the FNX peptide amino acid sequence described
herein. In some embodiments, FNX analogs contemplated herein may
also be compounds having at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or even
99% amino acid sequence identity to any of the FNX peptide amino
acid sequence described herein and having FNX activity. In some
embodiments, an FNX analog may be a compounds having at least 50%,
55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%,
95%, 96%, 97%, 98% or even 99% amino acid sequence identity to the
FN-38 (SEQ ID NO:5) amino acid sequence and having FNX activity. In
other embodiments, an FNX analog may be a compound having at least
50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%,
94%, 95%, 96%, 97%, 98% or even 99% amino acid sequence identity to
FN-38(1-15)-SN-23 amino acid sequence (SEQ ID NO:14) and having FNX
activity.
[0067] In certain embodiments, NMX analogs include those with
insertions, deletions, extensions, truncations, and/or
substitutions in at least one or more amino acid positions of any
of the NMX peptides or analogs described herein. FNX analogs also
include those with insertions, deletions, extensions, truncations,
and/or substitutions in at least one or more amino acid positions
of any of the FNX peptides or analogs described herein. The number
of amino acid insertions, deletions, or substitutions may be at
least 1, 2, 3, 4, 5, 10, 15, 20 or even 25 amino acid insertions,
deletions, or substitutions. In certain embodiments, the number of
amino acid insertions, deletions, or substitutions may be not more
than 1, 2, 3, 4, 5, 10, 15, 20, 25 or even 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.
[0068] "Derivative" refers to a molecule having the amino acid
sequence of a native or parent NMX, FNX, NMX receptor agonist, or
analog thereof, and additionally having a chemical modification of
one or more amino acid side groups, .alpha.-carbon atoms, terminal
amino group, or terminal carboxylic acid group. Contemplated
chemical modification includes, but is not limited to, adding
chemical moieties, creating new bonds, and removing chemical
moieties. The peptides may be derivatized by chemical alterations
such as amidation, glycosylation, acylation, sulfation,
phosphorylation, acetylation, and cyclization. Such chemical
alterations may be obtained through chemical or biochemical
methodologies, as well as through in vivo processes, or any
combination thereof. 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 group
include without limitation, the desamino, N-lower alkyl, N-di-lower
alkyl, constrained alkyls (e.g. branched, cyclic, fused, adamantyl)
and N-acyl modifications as known in the art. 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 C.sub.1-C.sub.4 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. NMX and FNX peptides, and analogs and derivatives
thereof include acid as well as amide forms of the peptides.
[0069] Derivatives of the peptides 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 peptides 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.
[0070] Derivatives of the peptides and analogs described herein may
also include conjugation to one or more polymers or small molecule
substituents. One type of polymer conjugation is linkage or
attachment of polyethylene glycol (PEG) polymers, polyamino acids
(e.g., poly-his, poly-arg, poly-lys, etc.) and/or fatty acid chains
of various lengths to the N- or C-terminus or amino acid residue
side chains of an NMX or FNX peptide. Small molecule substituents
include lower alkyls, alkyls and constrained alkyls (e.g.,
branched, cyclic, fused, adamantyl), and aromatic groups. In
addition, basic residues such as R and K may be replaced with homoR
and homoK, citrulline, or ornithine to improve metabolic stability
of the peptide.
[0071] Compounds useful in the methods provided may further include
additional amino acids, chemicals, or moieties that do not affect
the biological activity or function of the peptide but may perform
other functions, such as aiding purification (e.g., histidine tag),
detection (e.g., biotin), increasing solubility or half-life (e.g.,
pegylation) or expression (e.g., secretion signal peptide).
[0072] By "amino acid," "amino acid residue" and like terms are
meant natural amino acids, unnatural amino acids, and modified
amino acid, all in their D and L stereoisomers if their structure
allows such stereoisomeric forms. Natural amino acids include
alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid
(Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu),
glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu),
Lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro),
serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and
valine (Val). Unnatural amino acids include, but are not limited to
azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid,
beta-alanine, aminopropionic acid, 2-aminobutyric acid,
4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid,
2-aminoisobutyric acid, 3-aminoisbutyric acid, 2-aminopimelic acid,
tert-butylglycine, 2,4-diaminoisobutyric acid, desmosine,
2,2'-diaminopimelic acid, 2,3-diaminopropionic acid,
N-ethylglycine, N-ethylasparagine, homolysine, homoproline,
homoserine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline,
4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine,
N-methylglycine, N-methylisoleucine, N-methylpentylglycine,
N-methylvaline, naphthalanine, norvaline, norleucine, ornithine,
pentylglycine, pipecolic acid and thioproline, homolysine,
homoarginine, homoserine, citrulline, ornithine,
N.sub..epsilon.-formyllysine. Modified amino acids include the
natural and unnatural amino acids which are chemically blocked,
reversibly or irreversibly, or modified on the N-terminal amino
group or their side chain groups, for example without limitation,
methionine sulfoxide, methionine sulfone, S (carbo) amino group or
side chain functional group which has been chemically codified to
another functional group. For example, aspartic acid-(beta-methyl
ester) is a modified amino acid of aspartic acid; N-ethylglycine is
a modified amino acid of glycine; or alanine carboxamide is a
modified amino acid of alanine. Additional residues that can be
incorporated are described, for example without limitation, in
Sandberg et al., 1998, J. Med. Chem. 41:2481-2491.
[0073] In certain embodiments, the NMX peptide, FNX peptide, NMX
receptor agonist, or analog or derivative thereof contemplated
herein may include substitutions of one or more unnatural and/or
non-amino acids, e.g., amino acid mimetics. In certain embodiments,
the non-amino acids are turn mimetics or linker moieties. Exemplary
linker moieties include without limitation --NH--X--CO--, wherein
X.dbd.(CH.sub.2).sub.n (wherein n can be 2-20),
--NH--CH.sub.2CH.sub.2(--O--CH.sub.2CH.sub.2--O--).sub.m--CH.su-
b.2--CO-- (wherein m=1-5,) and other linker moieties known in the
art. Preferred linker molecules include aminocaproyl ("Aca"),
.beta.-alanyl, and 8-amino-3,6-dioxaoctanoyl. In certain
embodiments, turn mimetics contemplated herein are .beta.-turn
mimetics as known in the art. Certain .beta.-turn mimetics are
available commercially (e.g., BioQuadrant Inc, Quebec, Canada) and
have been described in the literature. See Gu et al., 2003,
Tetrahedron Letters 44: 5863-6; Bourguet et al., 2003, Bioorganic
& Medicinal Chemistry Letters 13: 1561-4; Grieco et al., 2002,
Tetrahedron Letters 43: 6297-9; Souers et al., 2001, Tetrahedron
57: 7431-48; Tsai et al., 1999, Bioorganic & Medicinal
Chemistry 7: 29-38; Virgilio et al., 1997, Tetrahedron 53: 6635-44.
Preferred .beta.-turn mimetics include beta turn mimic A
(N-(3S,6S,9S)-2-oxo-3-amino-1-azabicyclo[4.3.0]-nonane-9-carboxylic
acid) and beta turn mimic B
(N-(3S,6S,9R)-2-oxo-3-amino-7-thia-1-azabicyclo[4.3.0]-nonane-9-carboxyli-
c acid) illustrated herein.
##STR00001##
[0074] "Sequence identity," as is well understood in the art, is a
relationship between two or more polypeptide sequences or two or
more polynucleotide sequences, as determined by comparing the
sequences. In the art, "identity" can also refer to the degree of
sequence relatedness between polypeptide or polynucleotide
sequences, as determined by the match between strings of such
sequences. Identity can be readily calculated by known methods
including, but not limited to, those described in Computational
Molecular Biology, Lesk, ed., Oxford University Press, New York
(1988); Biocomputing: Informatics and Genome Projects, Smith, ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part I, Griffin et al., eds., Humana Press, New Jersey (1994);
Sequence Analysis in Molecular Biology, von Heinje, Academic Press
(1987); Sequence Analysis Primer, Gribskov et al., eds., Stockton
Press, New York (1991); and Carillo et al., 1988, SIAM J Applied
Math 48:1073. Methods to determine identity are designed to give
the largest match between the sequences tested. Moreover, methods
to determine identity are codified in publicly available programs.
Computer programs which can be used to determine identity between
two sequences include, but are not limited to, GCG (Devereux et
al., 1984, Nucleic Acids Research 12:387; suite of five BLAST
programs, three designed for nucleotide sequences queries (BLASTN,
BLASTX, and TBLASTX) and two designed for protein sequence queries
(BLASTP and TBLASTN) (Coulson, 1994, Trends in Biotechnology
12:76-80; Birren et al., 1997, Genome Analysis 1:543-559). The
BLAST X program is publicly available from NCBI and other sources
(BLAST Manual, Altschul et al., NCBI NLM NIH, Bethesda, Md. 20894;
Altschul et al., 1990, J. Mol. Biol. 215:403-410). The well known
Smith Waterman algorithm can also be used to determine identity.
For all percent identity calculations contemplated herein, percent
identity is determined by analysis methods and tools well known in
the art, for example without limitation the AlignX.RTM. module in
Vector NTI.RTM. (Invitrogen; Carlsbad, Calif.), and the like.
[0075] Parameters for polypeptide sequence comparison typically
include the following: Algorithm: Needleman & Wunsch, 1970, J.
Mol. Biol. 48:443-453; Comparison matrix: BLOSSUM62 from Hentikoff
& Hentikoff, 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919;
Gap Penalty: 12; Gap Length Penalty: 4. A program that can be used
with these parameters is publicly available as the "gap" program
from Genetics Computer Group ("GCG"), Madison, Wis. The above
parameters along with no penalty for end gap are the default
parameters for peptide comparisons. In one embodiment, the BLASTP
program of NCBI is used with the default parameters of no
compositional adjustment, expect value of 10, word size of 3,
BLOSUM62 matrix, gap extension cost of 11, end gap extension cost
of 1, dropoff (X) for blast extension (in bits) 7, X dropoff value
for gapped alignment (in bits) 15, and final X dropoff value for
gapped alignment (in bits) 25.
[0076] Parameters for nucleic acid molecule sequence comparison
include the following: Algorithm: Needleman & Wunsch, Id.;
Comparison matrix: matches--+10; mismatches=0; Gap Penalty: 50; Gap
Length Penalty: 3. As used herein, "% identity" is determined using
the above parameters as the default parameters for nucleic acid
molecule sequence comparisons and the "gap" program from GCG,
version 10.2.
[0077] As used herein, the term "bioactive" refers to an ability to
elicit a biological response that is sought in a cell, tissue,
system, and/or subject (including a human being), e.g., a bioactive
peptide is one which can be provided in a therapeutically effective
amount. For example, in one aspect, a bioactive peptide has
biological activity in at least one in vivo hormonal and/or
signaling pathway. Biological activity may be evaluated through
target receptor binding assays, or through studies that monitor a
physiological or behavioral indication, and/or through the
measurement of relevant biomarkers, as is known in the art.
[0078] In certain embodiments, the FNX peptides can have comparable
or higher potency in the treatment and/or prevention of the disease
and conditions described herein as compared to native FN-38
polypeptides. In other embodiments, the FNX peptide can have less
(e.g., may be 2, 3, 4, or even 5 times less), though still
effective, potency in the treatment and/or prevention of the above
described conditions, but may possess other desirable
characteristics over native FN-38, e.g., increased stability or
solubility, less side effects, combination of biological
activities, and/or ease in manufacturing, formulating, or use.
[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] "NMX peptide activity," "FNX peptide activity," and "NMX
receptor agonist activity" as used herein may include at least one
of the activities described herein or known in the art for these
compounds. Desirable NMX peptides, FNX peptides, NMX receptor
agonists, or analogs or derivatives thereof, may have at least one
property shared by the antipsychotic and anxiolytic agents
described herein.
[0081] Activity as NMX peptides, FNX peptides, NMX receptor
agonists, and/or analogs or derivatives thereof, can be confirmed
and quantified by performing various screening assays, including
receptor (e.g., NMUR1 or NMUR2) binding assays, food intake assays,
gastric emptying assays, gastric acid secretion assays, energy
expenditure assays, smooth muscle contractility assays, calcium
signaling assays in cells expressing NMU receptors, blood pressure
assays, heart rate assays, or nociceptive assays. Assays for
testing compounds for NMX peptide, an FNX peptide, or NMX receptor
agonist activity are known in the art (for example, Brighton et
al., 2004, Pharmacological Rev. 56:231-248; Westfall et al., 2002,
J. Pharmacol. Exp. Ther. 301:987-992; Wren et al., 2002,
Endocrinology 143:4227-4234; Mondal et al., 2003, Am. J. Physiol.
Gastrointest. Liver Physiol. 284:963-969; Yu et al., 2003,
Neuroscience 120:467-474; Ida et al., 2005, Endocrinology
146:4217-4223; Mori et al., 2005, EMBO J. 24:325-335. Exemplary
screening methods and assays for testing NMX peptides, FNX
peptides, or NMX receptor agonists are also described in PCT Patent
Application No. PCT/US2006/047953 (WO 2007/075439), which is
incorporated herein by reference in its entirety and for all
purposes.
[0082] The NMX peptides, FNX peptides, or NMX receptor agonists, or
analogs thereof, may be prepared using chemical peptide synthesis
techniques known in the art, e.g., using an automated or
semi-automated peptide synthesizer, standard recombinant
techniques, or both. Derivatives of the NMX peptides, FNX peptides,
or NMX receptor agonists, or analogs thereof, may be produced using
standard chemical, biochemical, and/or in vivo methodologies as
known in the art.
[0083] The NMX peptides, FNX peptides, or NMX receptor agonists, or
analogs or derivatives thereof, may be synthesized in solution or
on a solid support in accordance with conventional techniques.
Various automated synthesizers are commercially available and may
be used in accordance with known protocols. See, e.g., Stewart et
al., 1984, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical
Co.; Tam et al., 1983, J. Am. Chem. Soc. 105: 6442; Merrifield,
1986, Science 232: 341-347; and Barany et al., 1979, The Peptides,
Gross et al., eds., Academic Press, NY, 1-284. Solid phase peptide
synthesis may be carried out 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) as examples. For example, solid
phase peptide synthesis may be carried out with an automated
peptide synthesizer (e.g., Model 430A, Applied Biosystems Inc.,
Foster City, Calif.) using the NMP/HOBt (Option 1) system and tBoc
or Fmoc chemistry with capping (see, Applied Biosystems User's
Manual for the ABI 430A Peptide Synthesizer, Version 1.3, B Jul. 1,
1988, section 6:49-70). Peptides may also be assembled using an
Advanced ChemTech Synthesizer (Model MPS 350, Louisville, Ky.).
Peptides may be purified by RP-HPLC (preparative and analytical)
using, e.g., a Waters.RTM. DELTA-PREP.TM. 3000 system (Waters
Corp., Milford, Mass.) and a C.sub.4, C.sub.8, or C.sub.18
preparative column (10.mu., 2.2.times.25 cm; Grace Vydac, Hesperia,
Calif.). The peptide can be readily synthesized and then screened
in assays designed to identify peptides with particular activities.
Other methods of synthesizing and purifying peptides are known to
the skilled artisan.
[0084] The NMX peptides, FNX peptides, or NMX receptor agonists, or
analogs or derivatives thereof, disclosed herein may alternatively
be produced by recombinant techniques well known in the art. See,
e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory
Manual, 2d ed., Cold Spring Harbor, N.Y. The peptides produced by
recombinant technologies may be expressed from a polynucleotide.
One skilled in the art will appreciate that the polynucleotides,
including DNA and RNA, that encode the various fragments of the
peptides may be obtained from the wild-type cDNA, taking into
consideration the degeneracy of codon usage, or may be engineered
as desired, for example using techniques such as amplification by
PCR and site-directed mutagenesis. These polynucleotide sequences
may incorporate codons facilitating transcription and translation
of mRNA in microbial hosts. Such manufacturing sequences may
readily be constructed according to the methods well known in the
art. The polynucleotides above may also optionally encode an
N-terminal methionyl residue. The polynucleotides above may also
optionally encode a C-terminal glycyl residue for proper amide
formation. Non-peptide compounds useful in composition and methods
provided herein may be prepared by art-known methods. For example,
phosphate-containing amino acids and peptides containing such amino
acids may be prepared using methods known in the art. See, e.g.,
Bartlett et al., 1986, Bioorg. Chem. 14: 356-377.
[0085] A variety of cell types may be used to contain and express a
peptide coding sequence including, for example, bacteria, yeast,
algae, insect cells, plant cells, and animal cells such as
mammalian and avian cells. A variety of expression vector/host
systems may be used, including, but are not limited to
microorganisms such as bacteria transformed with recombinant
bacteriophage, plasmid or cosmid DNA expression vectors; yeast
transformed with yeast expression vectors; insect cell systems
infected with virus expression vectors (e.g., baculovirus); plant
cell systems transfected with virus expression vectors (e.g.,
cauliflower mosaic virus (CaMV); tobacco mosaic virus (TMV) or
transformed with bacterial expression vectors (e.g., Ti or pBR322
plasmid); or animal cell systems. Mammalian cells and cell lines
that are useful in recombinant protein productions include, but are
not limited to, VERO (African green monkey kidney) cells, HeLa
cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as
COS-7), WI38 (human lung fibroblasts), baby hamster kidney (BHK)
cells, HepG2, 3T3, RIN, Madin-Darby canine kidney epithelial (MDCK)
cells, A549, PC12, K562 and 293 cells. Exemplary protocols for the
recombinant expression of polypeptides are well known in the
art.
[0086] Host cell strains may be chosen for a particular ability to
process the expressed peptide or produce certain post-translation
modifications that will be useful in providing peptide activity.
Such modifications of the polypeptide include, but are not limited
to, acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, acylation, and amidation, for example, carboxy-terminal
amidation. Post-translational processing, which cleaves a "prepro"
form of the polypeptide, may also be important for correct
insertion, folding and/or function. Different host cells, such as
CHO, HeLa, MDCK, 293, WI38, and the like, have specific cellular
machinery and characteristic mechanisms for such post-translational
activities, and may be chosen to ensure the correct modification
and processing of the introduced foreign protein.
[0087] The NMX peptides, FNX peptides, or NMX receptor agonists, or
analogs or derivatives thereof, described herein may also be
produced using chemical ligation schemes known in the art,
including those described, for example, in U.S. Application
Publication Nos. 2003-0191291, 2003-0208046, and 2004-0115774.
Chemical ligation refers to a chemoselective reaction involving the
covalent joining of two chemical moieties, each of which moieties
bears a mutually reactive functional group that is uniquely capable
of forming a non-reversible covalent bond with the other. Unique,
mutually reactive, functional groups present on the first and
second components can be used to render the ligation reaction
chemoselective. For example, the chemical ligation of peptides and
polypeptides involves the chemoselective reaction of peptide or
polypeptide segments bearing compatible unique, mutually reactive,
C-terminal and N-terminal amino acid residues. Chemical ligation
includes covalent ligation of (1) a first peptide or polypeptide
bearing a uniquely reactive C-terminal group with (2) a second
peptide or polypeptide bearing a uniquely reactive N-terminal
group, where the C-terminal and N-terminal reactive groups form a
non-reversible covalent bond therein between. It also includes
N-terminal to N-terminal and C-terminal to C-terminal ligation. In
particular, chemical ligation includes any chemoselective reaction
chemistry that can be applied to ligation of unprotected peptide
segments. Several different chemistries have been utilized for this
purpose, examples of which include native chemical ligation, oxime
forming chemical ligation, thioester forming ligation (Schnolzer et
al., 1992, Science 256:221-225; Gieselman et al., 2001, Org. Lett.
3:1331-1334), thioether forming ligation (Englebretsen et al.,
1995, Tot. Leffs. 36:8871-8874), hydrazone forming ligation
(Gaertner, et al., 1994, Bioconj. Chem. 5:333-338), and
thiazolidine forming ligation and oxazolidine forming ligation
(Zhang et al., 1998, Proc. Natl. Acad. Sci. USA 95:9184-9189; PCT
Publication No. WO 95/00846; U.S. Pat. No. 5,589,356); and
Staudinger amide forming chemical ligation (Saxon et al., 2000,
Org. Lett. 2:2141-2143).
[0088] Reaction conditions for a given ligation chemistry are
generally selected to maintain the desired interaction of the
peptide or polypeptide segments employed for ligation. For example,
pH and temperature, water-solubility of the ligation components,
ratio of the first segment to the second segment, water content and
composition of the reaction mixture can be varied to optimize
ligation. Addition or exclusion of reagents that solubilize the
ligation segments to different extents may further be used to
control the specificity and rate of the desired ligation reaction,
i.e., control exposure and presentation of reactive groups by
manipulating solubility of the peptide or polypeptide segments.
Reaction conditions are readily determined by assaying for the
desired chemoselective reaction product compared to one or more
internal and/or external controls. These methodologies have proven
a robust methodology for generating a native amide bond at the
ligation site.
[0089] Methods useful in the synthesis of peptides and polypeptides
backbones are described in, for example, U.S. Application
Publication Nos. 2004-0138412 (extended native chemical ligation),
2003-0208046 (pseudo-native chemical ligation), 2005-0261473
(carboxy protection strategies for acidic C-terminal amino acids in
chemical ligation to eliminate formation of unwanted side
products), 2005-0064538 and 2005-0113563 (native chemical ligation
with improved efficiency of ligation and chemical ligation with
three or more components); in PCT Application Publication Nos.
WO2004/105685 (aqueous-compatible solid phase chemical ligation
using a displaceable linker) and WO2004/060925 (multiplex polymer
ligation with water-soluble polymeric protecting groups and their
replacement with desired adducts); and in U.S. Pat. Nos. 6,307,018
and 6,184,344 (native chemical ligation), 6,326,468 (solid phase
native chemical ligation), 6,217,873 (polyoxime compounds),
6,174,530 (homogenous polyoxime compositions), 6,001,364
(hetero-polyoxime compounds), and 6,451,543 (lipid-matrix assisted
synthesis). In general, synthesis of a peptide or polypeptide
backbone by chemical ligation involves selection of suitable
ligation sites that are chosen based on the ligation chemistry
selected for assembling the various polypeptide backbone segments,
the reversible (or cleavable) polymer attachment chemistry chosen
for a given target peptide, and the particular polymer attachment
sites. When native chemical ligation is employed, cysteine ligation
sites are determined by scanning the target polypeptide backbone
amino acid sequence for suitable naturally occurring cysteine
residue. When "extended native chemical ligation" is employed,
ligation sites can be selected by scanning the target polypeptide
backbone amino acid sequence for suitable naturally occurring
ligation site junctions that permit robust ligations. Because
extended native chemical ligation is not limited to ligation at
cysteine residues, any number of residues may serve as the ligation
site junction. In some instances, a combination of native and
extended native chemical ligation may be part of the design.
[0090] In some embodiments, native chemical ligation is used to
generate part or all of the full-length polypeptide chain.
Cysteines present in the naturally occurring protein or peptide
backbone can be used as the chemical ligation sites. Alternatively,
where a desired ligation junction is devoid of a suitable cysteine,
the non-cysteine amino acid at that position can be replaced with a
cysteine or a cysteine can be inserted so as to permit native
chemical ligation at that site. If desired, the newly introduced
cysteine can be converted to a pseudo amino acid residue
corresponding to the original amino acid at that position.
Formation of a pseudo amino acid by conversion of a cysteine at
native chemical ligation sites is referred to "pseudo native
chemical ligation." Alternatively, when the cysteine is introduced
at a site for polymer protecting group modification, the side chain
thiol can be exploited for the attachment of a thiol reactive
water-soluble polymer construct, provided that all other cysteines
in the target polypeptide that one does not wish to modify are
protected. In another embodiment, extended native chemical ligation
can be utilized to generate part or all of the full-length
polypeptide. Peptides used for thioester-mediated ligation, such as
for native chemical ligation, can be made following standard
protocols as well, for example see U.S. Pat. Nos. 6,307,018 and
6,184,344.
[0091] It may be desirable to purify the NMX peptides, FNX
peptides, or NMX receptor agonists, or analogs or derivatives
thereof, generated by the methods described herein. Peptide
purification techniques are well known to those of skill in the
art. These techniques involve, at one level, the crude
fractionation of the cellular milieu to polypeptide and
non-polypeptide fractions. Having separated the polypeptide from
other proteins, the polypeptide of interest may be further purified
using chromatographic and electrophoretic techniques to achieve
partial or complete purification (or purification to homogeneity).
Purification techniques include, for example, precipitation with
ammonium sulfate, PEG, antibodies, and the like; heat denaturation,
followed by centrifugation; chromatography steps such as ion
exchange, gel filtration, reverse phase, hydroxylapatite and
affinity chromatography; isoelectric focusing; gel electrophoresis;
and combinations of such and other techniques. Analytical methods
particularly suited to the preparation of a pure peptide are
ion-exchange chromatography, exclusion chromatography,
polyacrylamide gel electrophoresis, and isoelectric focusing. A
particularly efficient method of purifying peptides is reverse
phase HPLC, followed by characterization of purified product by
liquid chromatography/mass spectrometry (LC/MS) and Matrix-Assisted
Laser Desorption Ionization (MALDI) mass spectrometry. Additional
confirmation of purity is obtained by determining amino acid
analysis. As is generally known in the art, it is believed that the
order of conducting the various purification steps may be changed,
or that certain steps may be omitted, and still result in a
suitable method for the preparation of a substantially purified
protein or peptide.
[0092] As used herein, the term "purified peptide" is intended to
refer to a composition, isolated from other components, wherein the
peptide is purified to any degree relative to its naturally
obtainable state. A purified peptide therefore also refers to a
peptide, free from the environment in which it may naturally occur.
Generally, "purified" will refer to an NMX peptide, an FNX peptide,
or an NMX receptor agonist, or an analogs or derivative thereof,
that has been subjected to fractionation to remove various other
components, and which composition substantially retains a
biological activity. Where the term "substantially purified" is
used, this designation will refer to a composition in which the
peptide forms the major component of the composition, such as
constituting about 50%, about 60%, about 70%, about 80%, about 90%,
about 95% or more of the peptide in the composition.
[0093] There is no general requirement that the peptides always be
provided in their most purified state. Indeed, it is contemplated
that less substantially purified products will have utility in
certain embodiments. Partial purification may be accomplished by
using fewer purification steps in combination, or by utilizing
different forms of the same general purification scheme. For
example, it is appreciated that a cation-exchange column
chromatography performed, utilizing an HPLC apparatus, will
generally result in a greater "-fold" purification than the same
technique utilizing a low pressure chromatography system. Methods
exhibiting a lower degree of relative purification may have
advantages in total recovery of protein product, or in maintaining
the activity of the peptide. In some embodiments, a combination of
anion exchange and immunoaffinity chromatography may be used to
produce purified peptide compositions described herein.
[0094] The NMX peptides, FNX peptides, or NMX receptor agonists, or
analogs or derivatives thereof (herein referred to as the
"compounds provided"), may be administered alone or in combination
with pharmaceutically acceptable carriers or excipients, in either
single or multiple doses. Accordingly, pharmaceutical compositions
are provided comprising a therapeutically effective amount of at
least one NMX peptide, FNX peptide, or NMX receptor agonist
compound, or analog or derivative thereof, or a pharmaceutically
acceptable salt thereof, together with pharmaceutically acceptable
diluents, preservatives, solubilizers, emulsifiers, adjuvants
and/or carriers useful in the delivery of the compounds provided.
Conventional description and preparation techniques for
formulations are disclosed, for example, 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.
[0095] The compounds provided may be formulated for peripheral
administration, including formulation for injection, oral
administration, nasal administration, pulmonary administration,
topical administration, or other types of administration as one
skilled in the art will recognize. Administration of the
pharmaceutical compositions described herein may be via any common
route so long as the target tissue is available via that route. In
one embodiment, the pharmaceutical compositions may be administered
via a conventional central method, e.g., intracerebroventricular.
In one embodiment, the pharmaceutical compositions may be
introduced into the subject by any conventional peripheral method,
e.g., by intravenous, intradermal, intramuscular, intramammary,
intraarticular, intraperitoneal, intrathecal, retrobulbar,
intrapulmonary (e.g., term release); by oral, sublingual, nasal,
buccal, anal, vaginal, or transdermal delivery, or by surgical
implantation at a particular site. In some embodiments, a
subcutaneous route of administration is of use. In some
embodiments, mucosal delivery is exemplary. In some embodiments,
the pharmaceutical compositions provided are formulated so as to be
suitable for parenteral administration, e.g., via injection or
infusion. In some embodiments, the compounds provided are
administered in liquid, semi-solid, or solid form. The treatment
may consist of a single dose or a plurality of doses over a period
of time. Controlled continual release of the compounds provided is
also contemplated. Parenteral administration may be carried out
with an initial bolus followed by continuous infusion to maintain
therapeutic circulating levels of drug product.
[0096] A form of repository or "depot" slow release preparation may
be used so that therapeutically effective amounts of the
preparation are delivered into the bloodstream over many hours or
days following transdermal injection or delivery. Parenteral
controlled release delivery can be achieved by forming polymeric
microcapsules, matrices, solutions, implants and devices and
administering the same 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. These dosage forms may have a
lower bioavailability due to entrapment of some of the conjugates
in the polymer matrix or device. See e.g., U.S. Pat. Nos.
6,379,704, 6,379,703, and 6,296,842.
[0097] When the compounds provided are delivered by inhalation, the
peptides may follow the air-flow to the alveoli. Such delivery of
the compounds provided may include delivery as low or ultra-low
density particles, such as TECHNOSPHERES.TM. or as described for
example in U.S. Patent Application Publication No. 2004-0170568 and
U.S. Pat. No. 6,630,169.
[0098] In general, the compounds may be formulated into a stable,
safe pharmaceutical composition for administration to a patient.
The pharmaceutical formulations may be composed in various forms,
e.g., solid, liquid, semisolid or gel. 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, for example
for reconstitution. Aqueous compositions generally comprise an
effective amount of the compounds provided, dissolved or dispersed
in a pharmaceutically acceptable carrier or aqueous medium. The
phrase "pharmaceutically or pharmacologically acceptable" refers to
molecular entities and compositions that do not produce adverse,
allergic, or other untoward reactions when administered to an
animal or a human. As used herein, "pharmaceutically acceptable
carrier" includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in therapeutic compositions is
contemplated.
[0099] In some embodiments, the NMX peptides, FNX peptides, or NMX
receptor agonists, or analogs or derivatives thereof contemplated
herein may be prepared for administration as solutions of free
base, or pharmacologically acceptable salts in water suitably mixed
with a surfactant, such as hydroxypropylcellulose.
Pharmaceutically-acceptable salts include the acid addition salts
(formed with the free amino groups of the peptide) and which are
formed with inorganic acids such as, for example without
limitation, hydrochloric or phosphoric acids, or such organic acids
as acetic, oxalic, tartaric, mandelic, and the like. Salts formed
with the free carboxyl groups also can be derived from inorganic
bases such as, for example without limitation, sodium, potassium,
ammonium, calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the like.
Other pharmaceutically acceptable salts include, but are not
limited to, sulfuric, citric, maleic, hydrobromide, hydroiodide,
nitrate, sulfate, bisulfate, isonicotinate, lactate, salicylate,
citrate, oleate, tannate, pantothenate, bitartrate, ascorbate,
succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,
saccharate, formate, benzoate, glutamate, methanesulfonate,
ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate
(i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Such
products are readily prepared by procedures well known to those
skilled in the art. Dispersions also can be prepared in glycerol,
liquid polyethylene glycols, and mixtures thereof and in oils.
Typically, these preparations contain a preservative to prevent the
growth of microorganisms.
[0100] 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. In some
embodiments, the compound provided is suspended in an aqueous
carrier, for example, in an isotonic buffer solution at a pH of
about 3.0 to about 8.0, at a pH of about 3.5 to about 7.4, at a pH
of about 3.5 to about 6.0, or at a pH of about 3.5 to about 5.0. In
certain embodiments, the pH of the formulation is maintained in the
range of about 3.5 to 6.5, in some embodiments from about 3.7 to
about 4.3 or about 3.8 to about 4.2. In some embodiments, pH may be
about 4.0.
[0101] Useful buffers include sodium citrate/citric acid, and
sodium phosphate/phosphoric acid, and sodium acetate/acetic acid
buffers. In certain embodiments, the buffer with the compound
provided herein is an acetate buffer (for example, at a final
formulation concentration of from about 1-5 to about 60 mM), a
phosphate buffer (for example, at a final formulation concentration
of from about 1-5 to about to about 30 mM), a glutamate buffer (for
example, at a final formulation concentration of from about 1-5 to
about to about 60 mM), or a citrate buffer (for example, at a final
formulation concentration of from about 1-5 to about 60 mM). In
some embodiments, the buffer is acetate (for example, at a final
formulation concentration of from about 5 to about 30 mM).
[0102] The pharmaceutically-acceptable carrier may be a solvent or
dispersion medium containing, for example, water, ethanol, polyol
(for example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating, such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants.
[0103] A stabilizer may be included in the formulations of
compounds provided but, and importantly, is not necessarily needed.
If included, however, a stabilizer useful in the provided
compositions is a carbohydrate or a polyhydric alcohol. An
exemplary suitable stabilizer 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 proteins. The
polyhydric alcohols include such compounds as sorbitol, mannitol,
glycerol, and polyethylene glycols (PEGs), which 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.
Suitable carbohydrates include without limitation galactose,
arabinose, or lactose. Should the formulation be for administration
to an individual with diabetes, the carbohydrate used should be one
which does not have an adverse affect on the 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 conjugates in non-diabetic
applications (e.g., treating obesity).
[0104] In certain embodiments, if a stabilizer is included, the
compound provided 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/or 8000.
Mannitol is an exemplary polyhydric alcohol in some
embodiments.
[0105] Another useful feature of the lyophilized formulations
provided herein is the maintenance of the tonicity of the
lyophilized formulations described herein with the same formulation
component that serves to maintain their stability. In some
embodiments, mannitol is an exemplary polyhydric alcohol used for
this purpose. In many cases, isotonic agents may be included (e.g.,
sugars or sodium chloride). In some cases, excipients are useful in
maintenance of the overall tonicity of the compound. 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, for example,
between 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. Exemplary
parenteral formulations may be isotonic or substantially
isotonic.
[0106] 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. The prevention of the action
of microorganisms can be brought about by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. While the preservative for
use in the pharmaceutical compositions can range from 0.005 to 1.0%
(w/v), in some embodiments the 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.
[0107] Surfactants frequently can 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 NMX peptide, FNX peptide, or NMX receptor
agonist, or analog or derivative thereof, 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.
[0108] An exemplary 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 typically the aqueous vehicle for use in the pharmaceutical
formulations.
[0109] 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)
and a zwitterion (e.g., an amino acid such as betaine, taurine,
arginine, glycine, lysine, histidine). Additionally, polymer
solutions, or mixtures with polymers provide the opportunity for
controlled release of the peptide. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption (e.g., aluminum
monostearate, gelatin). Such additional ingredients, of course,
should not adversely affect the overall stability of the provided
pharmaceutical formulation.
[0110] In some embodiments, a pharmaceutical formulation provided
may contain a range of concentrations of the compound provided,
e.g., between about 0.01% to about 98% (w/w), or between about 1 to
about 98% (w/w), or between 80% and 90% (w/w), or between about
0.01% to about 50% (w/w), or between about 10% to about 25% (w/w).
A sufficient amount of water for injection may be used to obtain
the desired concentration of solution.
[0111] Exemplary pharmaceutical formulations contemplated may
comprise approximately 0.01 to 1.0% (w/v), in certain cases 0.05 to
1.0% (w/v), of the compound provided, 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.
[0112] The pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions or dispersions and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases, the form should be sterile
and should be fluid to the extent that easy syringability exists.
It is generally desirable for the compounds provided to be stable
under the conditions of manufacture and storage and preserved
against the contaminating action of microorganisms, such as
bacteria and fungi.
[0113] Sterile injectable solutions may be prepared by
incorporating the active compounds in the required amount in the
appropriate solvent with various of the other ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the various
sterilized active ingredients into a sterile vehicle that contains
the basic dispersion medium and the required other ingredients from
those enumerated herein. In the case of sterile powders for the
preparation of sterile injectable solutions, exemplary methods of
preparation are vacuum-drying and freeze-drying techniques that
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof. In
certain embodiments, the compounding procedure involves dissolution
of ingredients in a specific order (e.g., preservative followed by
stabilizer/tonicity agents, buffers and peptide) or dissolving at
the same time. In some cases, the NMX peptide, FNX peptide, or NMX
receptor agonist, or analog or derivative thereof, can be
lyophilized into vials, syringes or cartridges for subsequent
reconstitution. Liquid formulations provided can be filled into one
or two chambered cartridges, or one or two chamber syringes.
[0114] 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
exemplary method of sterilization for provided liquid formulations.
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.
[0115] The compounds provided herein 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 effect(s) 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.
Appropriate dosages may be ascertained through the use of
established assays for determining level of the psychiatric
disorder in conjunction with relevant dose-response data. The final
dosage regimen will be determined by the attending physician,
considering factors that modify the action of drugs, e.g., the
drug's specific activity, severity of the damage and the
responsiveness of the patient, the age, condition, body weight, sex
and diet of the patient, dosages of other concomitantly
administered drugs, time of administration and other clinical
factors. Those of ordinary skill in the art will readily optimize
effective dosages and administration regimens as determined by good
medical practice and the clinical condition of the individual
patient.
[0116] Typically, a dosage of between about 0.001 .mu.g/kg body
weight/day to about 1000 .mu.g/kg body weight/day may be used, but
more or less, as a skilled practitioner will recognize, may be
used, for example 1000 .mu.g/kg body weight/day to 10 mg/kg body
weight/day. Typical doses may contain from a lower limit of about
0.5 .mu.g, 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, 100 mg or even 200 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, provided continuously in a 24
hour period or any portion of that the 24 hours. The number of
doses per day may be from 1 to about 4 per day, although it could
be more. Dosing may be one or more times daily, or less frequently,
such as one or more times weekly or one or more times monthly, and
may be in conjunction with other compositions as described herein.
It should be noted that the present methods and compositions are
not limited to the dosages recited herein.
[0117] In some embodiments, an effective dose will typically be in
the range of about 1 to 30 .mu.g to about 5 mg/day, about 10 to 30
.mu.g to about 2 mg/day, about 5 to 100 .mu.g to about 1 mg/day, or
about 5 .mu.g to about 500 .mu.g/day, for a 50 kg patient,
administered in a single or divided doses. In some embodiments,
dosages are between about 0.01 .mu.g/kg/dose to about 100
.mu.g/kg/dose. In other embodiments, the composition is formulation
so as to deliver a dose of compound provided ranging from 1
.mu.g/kg to 100 mg/kg body weight/day or at doses ranging from 0.1
mg/kg to about 50 mg/kg body weight/day. Dosages for certain
routes, for example oral administration, may be increased to
account for decreased bioavailability, for example, by about 5-100
fold.
[0118] 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.
[0119] The frequency of dosing will depend in part on the
pharmacokinetic parameters of the agents and the routes of
administration. Pharmaceutical formulations may influence the
physical state, stability, rate of in vivo release and rate of in
vivo clearance of the administered agents. Depending on the route
of administration, a suitable dose may be calculated according to
body weight, body surface areas or organ size. Further refinement
of the calculations necessary to determine the appropriate
treatment dose is routinely made by those of ordinary skill in the
art without undue experimentation, especially in light of the
dosage information and assays disclosed herein, as well as
pharmacokinetic data observed in animals or human clinical
trials.
[0120] The following Examples are provided to illustrate, but not
limit, the invention.
EXAMPLES
Example 1
[0121] NMX peptides described herein were made by following
standard polypeptide synthesis methods. Unless indicated otherwise,
all exemplary NMX peptides, FNX peptides, NMX receptor agonists, or
analog or derivatives thereof described herein are C-terminal
amidated.
[0122] Polypeptides were synthesized on a Pioneer.TM. continuous
flow peptide synthesizer (Applied Biosystems, Foster City, Calif.)
using PAL-PEG-PS.TM. resin (Applied Biosystems) with a loading of
0.2 mmol/g (0.25 mmole scale). Fmoc amino acid (4.0 eq, 1.0 mmol)
residues were activated using 4.0 eq HBTU, 4.0 eq of HOBT, 8.0 eq
DIEA and coupled to the resin for 1 hour. The Fmoc group was
removed by treatment with 20% (v/v) piperidine in
dimethylformamide. Final deprotection and cleavage of the peptide
from the solid support was performed by treatment of the resin with
reagent B (93% trifluoroacetic acid (TFA), 3% phenol, 3% water and
1% triisopropylsilane) for 2-3 hours. The cleaved peptide was
precipitated using tert-butyl methyl ether, pelleted by
centrifugation and lyophilized. The pellet was re-dissolved in
water (10-15 mL), filtered and purified via reverse phase HPLC
using a C-18 column and an acetonitrile/water gradient containing
0.1% TFA. The purified product was lyophilized and analyzed by
ESI-LC/MS and analytical HPLC and were demonstrated to be pure
(>98%). Mass results all agreed with calculated values.
[0123] Alternatively, polypeptides were assembled on a
Symphony.RTM. peptide synthesizer (Protein Technologies, Inc.,
Woburn, Mass.) using Rink amide resin (Novabiochem, San Diego,
Calif.) with a loading of 0.43-0.49 mmol/g at 0.050-0.100 mmol Fmoc
amino acid (Applied Biosystems; 5.0 eq, 0.250-0.500 mmol) residues
were dissolved at a concentration of 0.10 M in
1-methyl-2-pyrrolidinone. All other reagents (HBTU,
1-hydroxybenzotriazole hydrate and N,N-diisopropylethylamine) were
prepared as 0.55 M dimethylformamide solutions. The Fmoc protected
amino acids were then coupled to the resin-bound amino acid using,
HBTU (2.0 eq, 0.100-0.200 mmol), 1-hydroxybenzotriazole hydrate
(1.8 eq, 0.090-0.18 mmol), N,N-diisopropylethylamine (2.4 eq,
0.120-0.240 mmol) for 2 hours. Following the last amino acid
coupling, the peptide was deprotected using 20% (v/v) piperidine in
dimethylformamide for 1 hour. Once peptide sequence is completed,
the Symphony.RTM. peptide synthesizer is programmed to cleave the
resin. Trifluoroacetic acid (TFA) cleavage of the peptide from
resin was carried out using a reagent mixture composed of 93% TFA,
3% phenol, 3% water and 1% triisopropylsilane. The cleaved peptide
was precipitated using tert-butyl methyl ether, pelleted by
centifugation and lyophilized. The pellet was dissolved in acetic
acid, lyophilized and then dissolved in water, filtered and
purified via reverse phase HPLC using a C.sub.18 column and an
acetonitrile/water gradient containing 0.1% TFA. Analytical HPLC
was used to assess purity of peptide and identity was confirmed by
LC/MS and MALDI-MS.
Example 2
[0124] Animal behavior assays were performed to test for anxiolytic
and antipsychotic effects of FN-38 (SEQ ID NO:5) administration.
The behavioral assays performed use art-accepted, animal models
that demonstrate properties characteristic of the respective
clinical condition (e.g., anxiety, schizophrenia,
obsessive-compulsive disorder) and hence, show face validity. These
specific behavioral tests are known to be sensitive to anxiolytic
or antipsychotic drugs. For these assays, FN-38 was administered to
mice at doses ranging from 0.1 to 10 mg/kg, intraperitoneally, and
their performance in the assay was assessed.
Stress-Induced Hyperthermia
[0125] Body temperature and emotionality are correlated in humans
and stress commonly induces an increase in body temperature
(hyperthermic response) in rodents. The thermic response to stress
is commonly used as an indication of enhanced emotionality or
anxiety in rodents 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 SIH 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. Buspirone is a partial 5-HT1A
agonist and a known anxiolytic agent. The animals were treated with
FN-38 (0.1, 1.0 or 10 mg/kg) or control agents (vehicle (water) or
15 mg/kg buspirone) 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 (Delta
Temperature) was the stress-induced hyperthermia. Results of this
assay are shown in FIG. 1 where * is P<0.05 vs. vehicle control.
As shown in FIG. 1, administration FN-38 at 10 mg/kg, like that of
the anxiolytic positive control, buspirone, blunted the SIH
response. The SIH test results demonstrate the anxiolytic activity
of FN-38 administration.
Marble Burying
[0126] 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. Benzodiazepine chlordiazepoxide (CDP) is a
known anxiolytic agent. Mice were injected with the test agent
(FN-38 (SEQ ID NO:5) at 0.1, 1.0 or 10 mg/kg, 15 mg/kg CDP, or
vehicle (water)) 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. 2 where * is P<0.05 vs. vehicle control. As
shown in FIG. 2, administration of FN-38 at 10 mg/kg, like that of
the anxiolytic positive control, CDP at 15 mg/kg, reduced the
number of marbles buried. These reductions in marble burying were
statistically significant. The marble burying assay results
demonstrate the anxiolytic activity and the anti-obsessive
compulsive activity of FN-38 administration.
Phencyclidine (PCP)-Induced Locomotion
[0127] The PCP-induced locomotion test is used with 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 (FN-38 (SEQ ID NO:5) at 0.1, 1.0 or 10
mg/kg, or vehicle (water)) 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 with FN-38 at 10 mg/kg are shown in FIG. 3. As shown in FIG.
3, PCP induced a characteristic response of hyper-locomotion in
vehicle pre-treated animals. Administration of FN-38 significantly
reduced this hyper-locomotion response as seen by a reduction in
the total distance traveled across all types assessed (total,
central, and peripheral) in the PCP-induced locomotion test. The
PCP-induced locomotion test results demonstrate the antipsychotic
activity of FN-38 administration.
Prepulse Inhibition
[0128] 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.
[0129] Mice were injected with the test agent (FN-38 (SEQ ID NO:5)
at 0.1, 1.0 or 10 mg/kg, or vehicle (water)) 15 prior to the test
or with haloperidol at 1 mg/kg or vehicle (10% DMSO) 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 of 120 dB. The
amount of the animal's "reaction" to the strong stimulus was
recorded. Results of the PPI assay with FN-38 at 10 mg/kg are shown
in FIG. 4. As shown in FIG. 4, administration of FN-38 at 10 mg/kg,
like that of the antipsychotic positive control halperidol,
increased the percent of prepulse inhibition at the 78 and 82 dB
prepulse stimulatory intensity levels. Halperidol is a dopamine
receptor antagonist and a first generation antipsychotic agent. The
PPI test results support the antipsychotic effects of FN-38
administration.
[0130] All patents and other references cited herein are indicative
of the level of skill of those skilled in the art to which the
references pertain, and are incorporated by reference in their
entireties and for all purposes, including any tables and figures,
to the same extent as if each reference had been incorporated by
reference in its entirety individually.
[0131] One skilled in the art would readily appreciate that the
present invention is well adapted to obtain the ends and advantages
mentioned, as well as those inherent therein. The methods,
variances, and compositions described herein as presently
representative embodiments are exemplary and are not intended as
limitations on the scope of the invention. Changes therein and
other uses will occur to those skilled in the art, which are
encompassed within the spirit of the invention, are defined by the
scope of the claims.
[0132] It will be readily apparent to one skilled in the art that
varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention.
[0133] The invention illustratively described herein suitably may
be practiced in the absence of any element or elements, limitation
or limitations which is not specifically disclosed herein. Thus,
for example, in each instance herein any of the terms "comprising",
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms to describe distinct subject
matter. The terms and expressions which have been employed are used
as terms of description and not of limitation, and there is no
intention that in the use of such terms and expressions of
excluding any equivalents of the features shown and described or
portions thereof, but it is recognized that various modifications
are possible within the scope of the invention claimed. Thus, it
should be understood that although the present invention has been
specifically disclosed by embodiments and optional features,
modification and variation of the concepts herein disclosed may be
resorted to by those skilled in the art, and that such
modifications and variations are considered to be within the scope
of this invention as defined by the claims.
[0134] In addition, where features or aspects of the invention are
described in terms of Markush groups or other grouping of
alternatives, those skilled in the art will recognize that the
invention is also thereby described in terms of any individual
member or subgroup of members of the Markush group or other group.
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.
[0135] Also, unless indicated to the contrary, where various
numerical values are provided for embodiments, additional
embodiments are described by taking any two different values as the
endpoints of a range. Such ranges are also within the scope of the
described invention.
Sequence CWU 1
1
76145PRTHomo sapiens 1Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu
Gly Lys Ser Asn Val 1 5 10 15 Val Glu Glu Phe Gln Ser Pro Phe Ala
Ser Gln Ser Arg Gly Tyr Phe 20 25 30 Leu Phe Arg Pro Arg Asn Gly
Arg Arg Ser Ala Gly Phe 35 40 45236PRTRattus sp. 2Phe Leu Phe His
Tyr Ser Lys Thr Gln Lys Leu Gly Asn Ser Asn Val 1 5 10 15 Val Glu
Tyr Gln Gly Pro Val Ala Pro Ser Gly Gly Phe Phe Leu Phe 20 25 30
Arg Pro Arg Asn 35 338PRTLitoria sp. 3Phe Leu Phe His Tyr Ser Lys
Ser His Asp Ser Gly Asn Ser Asp Ile 1 5 10 15 Thr Glu Glu Val Gln
Val Pro Gly Gly Val Ile Ser Asn Gly Tyr Phe 20 25 30 Leu Phe Arg
Pro Arg Asn 35 438PRTGallus sp. 4Phe Leu Phe His Tyr Ser Lys Thr
His Asp Ser Gly Asn Ser Asp Val 1 5 10 15 Arg Glu Asp Leu Gln Gly
Thr Gly Gly Ile Gln Ser Arg Gly Tyr Phe 20 25 30 Phe Phe Arg Pro
Arg Asn 35 538PRTHomo sapiens 5Phe Leu Phe His Tyr Ser Lys Thr Gln
Lys Leu Gly Lys Ser Asn Val 1 5 10 15 Val Glu Glu Leu Gln Ser Pro
Phe Ala Ser Gln Ser Arg Gly Tyr Phe 20 25 30 Leu Phe Arg Pro Arg
Asn 35 628PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 6Phe Leu Phe His Tyr Ser Lys
Thr Gln Lys Leu Gly Lys Ser Asn Val 1 5 10 15 Val Glu Glu Leu Gln
Ser Pro Phe Ala Ser Gln Ser 20 25 715PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 7Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser
Asn 1 5 10 15833PRTHomo sapiens 8Ile Leu Gln Arg Gly Ser Gly Thr
Ala Ala Val Asp Phe Thr Lys Lys 1 5 10 15 Asp His Thr Ala Thr Trp
Gly Arg Pro Phe Phe Leu Phe Arg Pro Arg 20 25 30 Asn 936PRTRattus
sp. 9Leu Pro Arg Leu Leu His Thr Asp Ser Arg Met Ala Thr Ile Asp
Phe 1 5 10 15 Pro Lys Lys Asp Pro Thr Thr Ser Leu Gly Arg Pro Phe
Phe Leu Phe 20 25 30 Arg Pro Arg Asn 35 108PRTPorcine sp. 10Tyr Phe
Leu Phe Arg Pro Arg Asn 1 5 1123PRTRattus sp. 11Tyr Lys Val Asn Glu
Tyr Gln Gly Pro Val Ala Pro Ser Gly Gly Phe 1 5 10 15 Phe Leu Phe
Arg Pro Arg Asn 20 129PRTHomo sapiens 12Gly Tyr Phe Leu Phe Arg Pro
Arg Asn 1 5 1323PRTLitoria sp. 13Ser Asp Glu Glu Val Gln Val Pro
Gly Gly Val Ile Ser Asn Gly Tyr 1 5 10 15 Phe Leu Phe Arg Pro Arg
Asn 20 1438PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 14Phe Leu Phe His Tyr Ser
Lys Thr Gln Lys Leu Gly Lys Ser Asn Ser 1 5 10 15 Asp Glu Glu Val
Gln Val Pro Gly Gly Val Ile Ser Asn Gly Tyr Phe 20 25 30 Leu Phe
Arg Pro Arg Asn 35 1530PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 15Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser
Asn Ser 1 5 10 15 Asp Glu Glu Val Gln Val Pro Gly Gly Val Ile Ser
Asn Gly 20 25 301630PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 16Phe Leu Phe His Tyr Ser
Lys Thr Gln Lys Leu Gly Lys Ser Asn Val 1 5 10 15 Val Glu Glu Leu
Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly 20 25 301725PRTHomo sapiens
17Phe Arg Val Asp Glu Glu Phe Gln Ser Pro Phe Ala Ser Gln Ser Arg 1
5 10 15 Gly Tyr Phe Leu Phe Arg Pro Arg Asn 20 25188PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 18Tyr Phe Leu Phe Arg Pro Arg Asn 1 5 198PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 19Tyr Phe Leu Tyr Arg Pro Arg Asn 1 5 208PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 20Tyr Phe Val Phe Arg Pro Arg Asn 1 5 218PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 21Phe Phe Leu Phe Arg Pro Arg Asn 1 5 228PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 22Tyr Phe Leu Val Arg Pro Arg Asn 1 5 238PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 23Tyr Phe Phe Phe Arg Pro Arg Asn 1 5 248PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 24Tyr Phe Leu Phe His Pro Arg Asn 1 5 258PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 25Tyr Phe Leu Phe Arg Pro His Asn 1 5 265PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 26Tyr Phe Leu Phe Arg 1 5278PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 27Phe Phe Phe Tyr His Pro His Asn 1 5 288PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 28Phe Phe Phe Phe Arg Pro Arg Asn 1 5 298PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 29Phe Phe Phe Phe Lys His His Asn 1 5 305PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 30Phe Phe Phe Phe Lys 1 53138PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 31Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser
Asn Val 1 5 10 15 Val Glu Glu Leu Gln Ser Pro Phe Ala Ser Gln Ser
Arg Gly Phe Phe 20 25 30 Leu Phe Arg Pro Arg Asn 35
3238PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 32Phe Leu Phe His Tyr Ser Lys Thr Gln
Lys Leu Gly Lys Ser Asn Val 1 5 10 15 Val Glu Glu Leu Gln Ser Pro
Phe Ala Ser Gln Ser Arg Gly Tyr Phe 20 25 30 Leu Val Arg Pro Arg
Asn 35 3338PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 33Phe Leu Phe His Tyr Ser
Lys Thr Gln Lys Leu Gly Lys Ser Asn Val 1 5 10 15 Val Glu Glu Leu
Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly Tyr Phe 20 25 30 Phe Phe
Arg Pro Arg Asn 35 3438PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 34Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser
Asn Val 1 5 10 15 Val Glu Glu Leu Gln Ser Pro Phe Ala Ser Gln Ser
Arg Gly Tyr Phe 20 25 30 Leu Phe His Pro Arg Asn 35
3538PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 35Phe Leu Phe His Tyr Ser Lys Thr Gln
Lys Leu Gly Lys Ser Asn Val 1 5 10 15 Val Glu Glu Leu Gln Ser Pro
Phe Ala Ser Gln Ser Arg Gly Tyr Phe 20 25 30 Leu Phe Arg Pro His
Asn 35 3635PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 36Phe Leu Phe His Tyr Ser
Lys Thr Gln Lys Leu Gly Lys Ser Asn Val 1 5 10 15 Val Glu Glu Leu
Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly Tyr Phe 20 25 30 Leu Phe
Arg 353738PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 37Phe Leu Phe His Tyr Ser
Lys Thr Gln Lys Leu Gly Lys Ser Asn Val 1 5 10 15 Val Glu Glu Leu
Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly Phe Phe 20 25 30 Phe Tyr
His Pro His Asn 35 3838PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 38Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser
Asn Val 1 5 10 15 Val Glu Glu Leu Gln Ser Pro Phe Ala Ser Gln Ser
Arg Gly Phe Phe 20 25 30 Phe Phe Arg Pro Arg Asn 35
3938PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 39Phe Leu Phe His Tyr Ser Lys Thr Gln
Lys Leu Gly Lys Ser Asn Val 1 5 10 15 Val Glu Glu Leu Gln Ser Pro
Phe Ala Ser Gln Ser Arg Gly Phe Phe 20 25 30 Phe Phe Lys His His
Asn 35 4035PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 40Phe Leu Phe His Tyr Ser
Lys Thr Gln Lys Leu Gly Lys Ser Asn Val 1 5 10 15 Val Glu Glu Leu
Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly Phe Phe 20 25 30 Phe Phe
Lys 354138PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 41Phe Leu Phe His Tyr Ser
Lys Thr Gln Lys Leu Gly Lys Ser Asn Ser 1 5 10 15 Asp Glu Glu Val
Gln Val Pro Gly Gly Val Ile Ser Asn Gly Phe Phe 20 25 30 Leu Phe
Arg Pro Arg Asn 35 4238PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 42Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser
Asn Ser 1 5 10 15 Asp Glu Glu Val Gln Val Pro Gly Gly Val Ile Ser
Asn Gly Tyr Phe 20 25 30 Leu Val Arg Pro Arg Asn 35
4338PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 43Phe Leu Phe His Tyr Ser Lys Thr Gln
Lys Leu Gly Lys Ser Asn Ser 1 5 10 15 Asp Glu Glu Val Gln Val Pro
Gly Gly Val Ile Ser Asn Gly Tyr Phe 20 25 30 Phe Phe Arg Pro Arg
Asn 35 4438PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 44Phe Leu Phe His Tyr Ser
Lys Thr Gln Lys Leu Gly Lys Ser Asn Ser 1 5 10 15 Asp Glu Glu Val
Gln Val Pro Gly Gly Val Ile Ser Asn Gly Tyr Phe 20 25 30 Leu Phe
His Pro Arg Asn 35 4538PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 45Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser
Asn Ser 1 5 10 15 Asp Glu Glu Val Gln Val Pro Gly Gly Val Ile Ser
Asn Gly Tyr Phe 20 25 30 Leu Phe Arg Pro His Asn 35
4635PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 46Phe Leu Phe His Tyr Ser Lys Thr Gln
Lys Leu Gly Lys Ser Asn Ser 1 5 10 15 Asp Glu Glu Val Gln Val Pro
Gly Gly Val Ile Ser Asn Gly Tyr Phe 20 25 30 Leu Phe Arg
354738PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 47Phe Leu Phe His Tyr Ser Lys Thr Gln
Lys Leu Gly Lys Ser Asn Ser 1 5 10 15 Asp Glu Glu Val Gln Val Pro
Gly Gly Val Ile Ser Asn Gly Phe Phe 20 25 30 Phe Tyr His Pro His
Asn 35 4838PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 48Phe Leu Phe His Tyr Ser
Lys Thr Gln Lys Leu Gly Lys Ser Asn Ser 1 5 10 15 Asp Glu Glu Val
Gln Val Pro Gly Gly Val Ile Ser Asn Gly Phe Phe 20 25 30 Phe Phe
Arg Pro Arg Asn 35 4938PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 49Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser
Asn Ser 1 5 10 15 Asp Glu Glu Val Gln Val Pro Gly Gly Val Ile Ser
Asn Gly Phe Phe 20 25 30 Phe Phe Lys His His Asn 35
5035PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 50Phe Leu Phe His Tyr Ser Lys Thr Gln
Lys Leu Gly Lys Ser Asn Ser 1 5 10 15 Asp Glu Glu Val Gln Val Pro
Gly Gly Val Ile Ser Asn Gly Phe Phe 20 25 30 Phe Phe Lys
355136PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 51Phe Leu Phe His Tyr Ser Lys Thr Gln
Lys Leu Gly Lys Ser Asn Glu 1 5 10 15 Glu Leu Gln Ser Pro Phe Ala
Ser Gln Ser Arg Gly Tyr Phe Leu Phe 20 25 30 Arg Pro Arg Asn 35
5234PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 52Phe Leu Phe His Tyr Ser Lys Thr Gln
Lys Leu Gly Lys Ser Asn Val 1 5 10 15 Val Glu Glu Leu Gln Ser Pro
Ser Arg Gly Tyr Phe Leu Phe Arg Pro 20 25 30 Arg Asn
5332PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 53Phe Leu Phe His Tyr Ser Lys Thr Gln
Lys Leu Gly Lys Ser Asn Glu 1 5 10 15 Glu Leu Gln Ser Pro Ser Arg
Gly Tyr Phe Leu Phe Arg Pro Arg Asn 20 25 30 5434PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 54Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser Asn Val Val Glu
Glu Leu 1 5 10 15 Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly Tyr Phe
Leu Phe Arg Pro 20 25 30 Arg Asn 5534PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 55Phe Leu Phe His Tyr Lys Leu Gly Lys Ser Asn Val Val Glu
Glu Leu 1 5 10 15 Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly Tyr Phe
Leu Phe Arg Pro 20 25 30 Arg Asn 5631PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 56Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Leu Gln
Ser Pro 1 5 10 15 Phe Ala Ser Gln Ser Arg Gly Tyr Phe Leu Phe Arg
Pro Arg Asn 20 25 30 5731PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 57Phe Gln Lys Leu Gly Lys Ser Asn Val Val Glu Glu Leu Gln
Ser Pro 1 5 10 15 Phe Ala Ser Gln Ser Arg Gly Tyr Phe Leu Phe Arg
Pro Arg Asn 20 25 30 5821PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 58Phe Leu Phe His Tyr Ser Phe Ala Ser Gln Ser Arg Gly Tyr
Phe Leu 1 5 10 15 Phe Arg Pro Arg Asn 20 5924PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 59Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser
Asn Gly 1 5 10 15 Tyr Phe Leu Phe Arg Pro Arg Asn 20
6026PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 60Phe Leu Phe His Tyr Ser Lys Thr Gln
Lys Leu Gly Lys Ser Asn Ser 1 5 10 15 Arg Gly Tyr Phe Leu Phe Arg
Pro Arg Asn 20 25 6132PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 61Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser
Asn Glu 1 5 10 15 Glu Leu Gln Ser Pro Ser Arg Gly Tyr Phe Leu Phe
Lys Pro Arg Asn 20 25 30 6215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 62Phe Leu Phe His Tyr Ser Gly Tyr Phe Leu Phe Arg Pro Arg
Asn 1 5 10 156329PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 63Lys Leu Gly Lys Ser Asn
Val Val Glu Glu Leu Gln Ser Pro Phe Ala 1 5 10 15 Ser Gln Ser Arg
Gly Tyr Phe Leu Phe Arg Pro Arg Asn 20 25 6428PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 64Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser Asn Glu Glu Leu
Gln Ser 1 5 10 15 Pro Ser Arg Gly Tyr Phe Leu Phe Arg Pro Arg Asn
20 25 6512PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 65Phe Leu Phe His Tyr Ser
Gly Tyr Phe Leu Phe Arg 1 5 10 6612PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 66Phe Leu Phe His Tyr Ser Gly Phe Phe Leu Phe Arg 1 5 10
6715PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 67Phe Leu Phe His Tyr Ser Gly Tyr Phe
Leu Phe Lys Pro Arg Asn 1 5 10 156815PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 68Phe Leu Phe His Tyr Ser Gly Phe Phe Leu Phe Arg Pro Arg
Asn 1 5 10 156915PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 69Phe Leu Phe His Tyr Ser
Gly Phe Phe Leu Phe Lys Pro Arg Asn 1 5 10 157036PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 70Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser
Asn Glu 1 5 10 15 Glu Val Gln Val Pro Gly Gly Val Ile Ser Asn Gly
Tyr Phe Leu Phe 20 25 30 Arg Pro Arg Asn 35 7134PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 71Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser
Asn Ser 1 5 10 15 Asp Glu Glu Val Gln Val Pro Ser Asn Gly Tyr Phe
Leu Phe Arg Pro 20 25 30 Arg Asn 7232PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 72Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser
Asn Glu 1 5 10 15 Glu Val Gln Val Pro Ser Asn Gly Tyr Phe Leu Phe
Arg Pro Arg Asn 20 25 30 7334PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 73Tyr Ser Lys Thr Gln Lys Leu Gly Lys Ser Asn Val Val Glu
Glu Leu 1 5 10 15 Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly Tyr Phe
Leu Phe Arg Pro 20 25 30 Arg Asn 7434PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 74Phe Leu Phe His Tyr Lys Leu Gly Lys Ser Asn Val Val Glu
Glu Leu 1 5 10 15 Gln Ser Pro Phe Ala Ser Gln Ser Arg Gly Tyr Phe
Leu Phe Arg Pro 20 25 30 Arg Asn 7531PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 75Phe Leu Phe His Tyr Ser Lys Thr Gln Lys Leu Gly Val Gln
Val Pro 1 5 10 15 Gly Gly Val Ile Ser Asn Gly Tyr Phe Leu Phe Arg
Pro Arg Asn 20 25 30 7631PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 76Phe Gln Lys Leu Gly Lys Ser Asn Ser Asp Glu Glu Val Gln
Val Pro 1 5 10 15 Gly Gly Val Ile Ser Asn Gly Tyr Phe Leu Phe Arg
Pro Arg Asn 20 25 30
* * * * *