U.S. patent application number 17/365443 was filed with the patent office on 2022-01-27 for methods and compositions for treating cystic fibrosis.
The applicant listed for this patent is PhaseBio Pharmaceuticals, Inc.. Invention is credited to Susan ARNOLD, David James BALLANCE.
Application Number | 20220023390 17/365443 |
Document ID | / |
Family ID | 1000005898247 |
Filed Date | 2022-01-27 |
United States Patent
Application |
20220023390 |
Kind Code |
A1 |
ARNOLD; Susan ; et
al. |
January 27, 2022 |
METHODS AND COMPOSITIONS FOR TREATING CYSTIC FIBROSIS
Abstract
The present disclosure provides a method of treating diseases or
disorders associated with CFTR protein dysfunction, including
Cystic Fibrosis, by administering stable, long-lasting vasoactive
intestinal peptide therapeutic agents. These agents include one or
more elastin-like peptides and can be administered at a
low-dose.
Inventors: |
ARNOLD; Susan; (Malvern,
PA) ; BALLANCE; David James; (Malvern, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PhaseBio Pharmaceuticals, Inc. |
Malvem |
PA |
US |
|
|
Family ID: |
1000005898247 |
Appl. No.: |
17/365443 |
Filed: |
July 1, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15308626 |
Nov 3, 2016 |
11052132 |
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PCT/US2015/029926 |
May 8, 2015 |
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17365443 |
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61990425 |
May 8, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/443 20130101;
A61K 38/2278 20130101; A61K 31/47 20130101; A61K 38/39 20130101;
A61K 31/404 20130101; A61K 45/06 20130101 |
International
Class: |
A61K 38/22 20060101
A61K038/22; A61K 45/06 20060101 A61K045/06; A61P 11/00 20060101
A61P011/00 |
Claims
1-21. (canceled)
22. A method for treating cystic fibrosis comprising subcutaneously
administering to a patient in need thereof a pharmaceutical
composition comprising the amino acid sequence of SEQ ID NO:
15.
23. The method of claim 22, wherein the pharmaceutical composition
is formulated for systemic delivery.
24. The method of claim 22, wherein the pharmaceutical composition
is formulated for sustained release.
25. The method of claim 22, wherein the pharmaceutical composition
is administered with one or more additional cystic fibrosis
therapies or therapies to treat disorders associated with CFTR
protein dysfunction.
26. The method of claim 25, wherein the one or more additional
cystic fibrosis therapies is selected from the group consisting of
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
potentiators, CFTR correctors, nonsense mutation readthrough
agents, CFTR production correctors, read-through agents, small
molecule ion channel agents, osmotic agents, gene therapy, RNA
repair, soluble guanylate cyclase stimulators, S-nitrosoglutathione
reductase inhibitors, DNase, antibiotics, antifungals, mucolytics,
bronchodilators, nitric oxide, anticholinergics, nonsteroidal
anti-inflammatory drugs (NSAIDs), membrane stabilizers,
corticosteroids, and enzyme replacement therapy.
27. The method of claim 26, wherein the CFTR potentiator is
selected from the group consisting of ivacaftor (VX-770) and
QBW251.
28. The method of claim 26, wherein the CFTR corrector is selected
from the group consisting of lumacaftor (VX-809) and VX-661.
29. The method of claim 22, wherein the patient has at least one or
more mutations in a CFTR gene.
30. The method of claim 29, wherein the patient is homozygous for
the one or more mutations in the CFTR gene.
31. The method of claim 29, wherein the patient is heterozygous for
the one or more mutations in the CFTR gene.
32. The method of claim 29, wherein the patient has an F508del
mutation.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Utility
application Ser. No. 15/308,626, filed Nov. 3, 2016, which is a
U.S. National Phase of International Application No.:
PCT/US2015/029926, filed May 8, 2015, entitled "Methods and
Compositions For Treating Cystic Fibrosis" and claims priority
under 35 U.S.C. 119(c) to U.S. Provisional Application Ser. No.
61/990,425, filed May 8, 2014, entitled "Method For Treating Cystic
Fibrosis" the contents of which are herein incorporated by
reference in their entireties.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
[0002] The contents of the text file submitted electronically
herewith are incorporated herein by reference in their entirety: a
computer readable format copy of the sequence listing (filename:
PHAS_030/02US_SeqList_ST25.txt, date recorded: Jul. 1, 2021, file
size 21 kilobytes).
BACKGROUND
[0003] Cystic Fibrosis is a chronic, progressive, and fatal genetic
disorder afflicting approximately 1 in 2, 500 people worldwide.
This disease is caused by loss of function mutations in the Cystic
Fibrosis Transmembrane Conductance Regulator (CFTR) gene which
codes for a cAMP-regulated anion channel expressed primarily at the
apical plasma membrane of secretory epithelial cells in the
airways, pancreas, intestine, and other tissues. Nearly 2000
mutations in the CFTR gene have been identified that produce the
loss of function phenotype by impairing translation, cellular
processing, and/or chloride channel gating. (Rowe and Verkman
(2013)).
[0004] In addition to inherited mutations in the CFTR gene,
environmental factors, such as cigarette smoke, can lead to
acquired CFTR protein defects. The loss of function CFTR phenotype
leads to impaired ion and water transport across the cell membrane.
Consequently, the affected cells produce abnormally thick mucus
which obstructs the airways and glands, leading to difficulty
breathing, increased infection, infertility, tissue damage, and
death.
[0005] Current therapies focus on alleviating the symptoms of CFTR
protein dysfunction. However, therapies that correct the underlying
CFTR protein defect are needed.
SUMMARY OF THE INVENTION
[0006] The present disclosure provides Vasoactive Intestinal
Peptide (VIP) therapeutics to treat, delay, or ameliorate symptoms
of CFTR protein dysfunction. The buildup of thick, sticky mucus in
afflicted patients results in permanent tissue damage, including
the formation of scar tissue (fibrosis). This tissue damage leads
to severe patient impairment and death. Preventing, delaying, or
ameliorating the formation of this thick, sticky mucus can treat
CFTR protein dysfunction.
[0007] In some aspects, the present disclosure provides a method
for treating cystic fibrosis comprising administering to a patient
in need thereof a pharmaceutical composition comprising a
Vasoactive Intestinal Peptide (VIP) and one or more elastin-like
peptides (ELP).
[0008] In some aspects, the present disclosure provides a method
for treating symptoms of CFTR protein dysfunction comprising
administering to a patient in need thereof a pharmaceutical
composition comprising a Vasoactive Intestinal Peptide (VIP) and
one or more elastin-like peptides (ELP).
[0009] In some aspects, the present disclosure provides a method
for increasing CFTR protein function in a patient in need thereof
comprising administering a pharmaceutical composition comprising a
Vasoactive Intestinal Peptide (VIP) and one or more elastin-like
peptides (ELP).
[0010] In some aspects, the present disclosure provides a method
for increasing CFTR function comprising administering to a patient
with an acquired defect in CFTR function a pharmaceutical
composition comprising a Vasoactive Intestinal Peptide (VIP) and
one or more elastin-like peptides (ELP). In some aspects, the
patient acquired a defect in CFTR function through smoking. In some
aspects, the patient with an acquired defect in CFTR function has
chronic obstructive pulmonary disease (COPD).
[0011] In some aspects, the present disclosure provides a method
for increasing ion efflux rates in a the cells of a subject with
CFTR protein dysfunction comprising administering to the patient a
pharmaceutical composition comprising a Vasoactive Intestinal
Peptide (VIP) and one or more elastin-like peptides (ELP).
[0012] In some aspects, the present disclosure provides a method
for increasing respiratory rates in a subject with CFTR protein
dysfunction comprising administering to the patient a
pharmaceutical composition comprising a Vasoactive Intestinal
Peptide (VIP) and one or more elastin-like peptides (ELP).
[0013] In some aspects, the present disclosure provides a method
for decreasing sweat chloride concentration in a subject with CFTR
protein dysfunction comprising administering to the patient a
pharmaceutical composition comprising a Vasoactive Intestinal
Peptide (VIP) and one or more elastin-like peptides (ELP).
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 is a schematic depicting the mechanism whereby VIP
increases CFTR protein membrane density. (1) The dissociation of
CFTR from CAL into the cytoplasm to promote CFTR membrane
insertion; (2) Activation of the PKC.epsilon. signaling cascade
that potentiates NHERF1/PERMs complex interaction with membrane
CFTR to mediate its surface stability (Alshafie (2014)).
[0015] FIG. 2A-D shows the iodide efflux rates of VIP (Panel B),
PB1046 (Panel C), and PB1120 (Panel D). Cells were treated with the
indicated concentrations for 2 hours before stimulation with a cAMP
activator cocktail. Rescued F508-delCFTR were stimulated by a cAMP
activator cocktail added to the efflux buffer from time 3 to 15
minutes. Panel A shows the iodide efflux rates from JME/CF15 cells
maintained at 37.degree. C. in the absence of correctors. EC50 and
plateau concentrations (n=3-5) are indicated for each compound.
[0016] FIG. 3A-C shows the iodide efflux rates of JME/CF15 cells
maintained at 37.degree. C. and incubated with VIP, PB1046, or
PB1120 as indicated for 2 to 24 hours before stimulation with a
cAMP activator cocktail. Rescued F508-delCFTR were stimulated by a
cAMP activator cocktail added to the efflux buffer from time 3 to
15 minutes. The lower panels show the effect of addition of the
CFTR inhibitor CFTR.sub.inh172 (20 .mu.M) 30 minutes before and
during the entire efflux experiments.
[0017] FIG. 4A-B shows the correction of F508del-CFTR maturation
and membrane expression. JME/CF15 cells were immunostained for CFTR
(A). Panel B shows an immunoblott of lysates from cells maintained
at 37.degree. C. and incubated with each compound for 24 hours.
[0018] FIG. 5 shows iodide efflux rates measured on JME/CF15 cells
treated with the following conditions before stimulation with a
cAMP activator cocktail: (27C) 24 hours at 27.degree. C.; (VIP)
37.degree. C.+treatment with 900 nM VIP for 24 hours; (PB1120)
37.degree. C.+treatment with 1 .mu.M PB1120 for 24 hours; (PB1046)
37.degree. C.+treatment with 1.2 .mu.M PB1046 for 18 hours; (VX809)
37.degree. C.+treatment with 1 .mu.M VX809 for 24 hours; (VX661)
37.degree. C.+treatment with 3 .mu.M VX661 for 24 hours. Rescued
F508del-CFTR was stimulated by a cAMP activator cocktail.
[0019] FIG. 6A-C demonstrates iodide efflux rates measured on
JME/CF15 cells maintained at 37.degree. C. Panel A shows acute
treatment with 1 .mu.M VX770 at 37.degree. C. did not produce any
significant stimulation compared to basal levels (p>0.7). Panel
B shows treatment with 350 nM PB1046 for 18 hours alone or in
combination with acute treatment with 1 .mu.M VX770. Panel C shows
treatment with 140 nM PB1120 for 24 hours, alone or in combination
with acute treatment with 1 .mu.M VX770. Rescued F508del-CFTR was
stimulated by a cAMP activator cocktail. Administration of agents
together resulted in a synergistic effect on iodide efflux.
[0020] FIG. 7 demonstrates iodide efflux rates measured on JME/CF15
cells maintained at 37.degree. C. Cells were treated with 1 .mu.M
PB1120 for 24 hours alone or in combination with 1 .mu.M VX809 for
24 hours. Administration of the agents together resulted in a
synergistic effect on iodide efflux.
DETAILED DESCRIPTION
[0021] Cystic Fibrosis (CF) is a recessive genetic disorder
characterized by the buildup of thick, sticky mucus that leads to
increased incidence of infections and tissue damage in afflicted
patients. The disorder's most common symptoms include progressive
damage to the respiratory system and chronic digestive problems.
Cystic Fibrosis is caused by mutations in the Cystic Fibrosis
Transmembrane Conductance Regulator (CFTR) gene that reduce or
abolish the activity of the resulting protein. The CFTR protein is
a transmembrane chloride channel primarily localized to the
luminal, or apical membranes of epithelial cells in a variety of
different tissues and organs including airway tissues, intestine,
pancreas, kidney, vas deferens, and sweat duct.
[0022] Currently nearly 2,000 mutations in the CFTR gene have been
identified that lead to a loss of function phenotype. For example,
the F508del mutation, which is present in at least one allele in
about 90% of Cystic Fibrosis patients, impairs CFTR folding,
stability at the endoplasmic reticulum, and chloride channel gating
(Rowe and Verkman (2013). Other identified mutations alter, for
example, channel gating (e.g. G551D), conductance (e.g. R117H), or
translation (e.g. G542X).
[0023] Subjects can also acquire a defect in CFTR protein function
(e.g. through smoking). For example, cigarette smoking inhibits
chloride transport in cultured bronchial epithelial cells, and
reduced CFTR activity is observed in smokers without mutations in
the CFTR gene (Sloane (2012). A number of extrapulmonary disorders
associated with CFTR dysfunction are also found in smokers,
including idiopathic pancreatitis, male infertility, cachexia, and
diabetes mellitus (Raju (2013)).
[0024] The loss of function CFTR phenotype decreases the movement
of chloride ions across the cell membrane, leading to aberrant ion
and fluid homeostasis at epithelial surfaces, and damage to
numerous organs and tissue systems. For example, in the lung, the
defect in chloride transport is coupled with hyperabsorption of
sodium, leading to the generation of thick and dehydrated mucus
which allows chronic bacterial infections, and causes
bronchiectasis and progressive airway destruction, eventually
leading to the loss of pulmonary function. In the pancreas, the
altered transport of electrolytes leads to decreased production of
sodium bicarbonate and a build-up of mucus which blocks the
pancreatic ducts. This blockage prevents digestive enzymes from
exiting the pancreas causing digestive issues, and also tissue
damage and fibrosis in the pancreas itself. In Cystic Fibrosis
patients, pancreatic fibrosis can decrease the production of
insulin, leading to Cystic Fibrosis-related diabetes mellitus. In
the intestines, the altered ion and water transport leads to
chronic digestive problems, diarrhea, and distal intestinal
obstruction syndrome.
[0025] Vasoactive Intestinal Peptide (VIP) stimulates water and
chloride transport across epithelial surfaces (Heinz-Erian (1985))
and was recently discovered to play a role in regulating CFTR
protein stability (Chappe and Said (2012)). Prolonged VIP exposure
can rescue F508delta-CFTR trafficking to the apical cell membrane
and restore protein function (Chappe and Said (2012)). In the
airway submucosal gland epithelial cell line Calu-3, VIP binding to
one of its receptors, VPAC1, stimulates CFTR-dependent chloride
secretion through activation of both PKA- and PKC-dependent
signaling pathways (Chappe (2008); Derand (2004)). This signaling
cascade results in CFTR protein being anchored to the actin
cytoskeleton, thereby maintaining the protein at the membrane and
reducing its endocytosis (Chappe and Said (2012)). As a protein
that has effects on correcting CFTR function, VIP is an attractive
therapeutic to treat diseases or disorders associated with CFTR
protein dysfunction, however, VIP's poor stability after systemic
administration (e.g. half-life of .ltoreq.1 minute in circulation)
has limited its clinical application.
[0026] The present disclosure provides a method of preventing,
delaying, or ameliorating the onset or progression of symptoms of
CFTR protein dysfunction in subjects by administering Vasoactive
Intestinal Peptide (VIP) therapeutics.
Vasoactive Intestinal Peptides
[0027] Vasoactive intestinal peptide (VIP) is a 28 amino acid
neuropeptide which binds to two receptors, VPAC1 and VPAC2, found
in a variety of tissues including the airway, small intestine,
testes, and pancreas. VIP and its functionally and structurally
related analogs are known to have many physiological functions,
including, relaxing airway smooth muscle thereby acting as a
bronchodilator, stimulating fluid secretion in airway submucosal
glands, and regulating water and electrolyte secretion in the
intestines and pancreas (Wine (2007); Wu (2011); Derand
(2004)).
[0028] VIP-producing nerve fibers are co-localized with
acetylcholine secreting neurons surrounding exocrine glands
(Lundberg (1980); Heinz-Erian (1986)). In glands from subjects with
functional CFTR protein, VIP induces fluid secretion, but this
induction is impaired or absent in Cystic Fibrosis patients (Joo
(2002); Joo (2012)). Further, in human and pig airway glands,
administration of low concentrations of both VIP and acetylcholine
stimulates the secretion mucus, but this synergism is lost in
cystic fibrosis patients (Choi (2007)).
[0029] As shown in FIG. 1, VIP increases CFTR membrane insertion,
stability, and function in human airway epithelial cells (Alshafie
(2014)). In a murine VIP knockout model CFTR does not localize to
the apical cell membrane, but instead remains mainly intracellular
(Chappe and Said (2012)). The absence of CFTR from the apical
membrane is associated with a lung pathology similar to that seen
in Cystic Fibrosis patients, with inflammatory cell infiltration,
thickening of the alveolar wall and the bronchiolar mucosa, and
goblet cell hyperplasia. Administration of VIP intraperitoneally
for three weeks restores CFTR apical membrane localization, and
prolonged VIP stimulation increases the number of CFTR channels at
the cell membrane (Chappe (2008)). This increase in apical CFTR
density, which occurs via stabilization of CFTR at the membrane, is
associated with an increase in CFTR-dependent function as measured
by iodide efflux assays (Chappe (2008)).
[0030] In some aspects the disclosure provides therapeutic
compositions that may include one or more various VIP peptides. For
example, the VIP peptide may comprise or consist of a polypeptide
having SEQ ID NO: 14, SEQ ID NO: 17, or SEQ ID NO: 19. In some
embodiments, the present disclosure provides a VIP without the
N-terminal Methionine (e.g. SEQ ID NO: 17). In some embodiments,
the present disclosure provides a VIP with the N-terminal
Methionine (e.g. SEQ ID NO: 14).
[0031] Mature human VIP has 28 amino acid residues with the
following sequence: HSDAVFTDNYTRLRKQMAVKKYLNSILN (SEQ ID NO: 17).
VIP results from processing of the 170-amino acid precursor
molecule prepro-VIP. Structures of VIP and exemplary analogs have
been described in U.S. Pat. Nos. 4,835,252, 4,939,224, 5,141,924,
4,734,400, 4,605,641, 6,080,837, 6,316,593, 5,677,419, 5,972,883,
6,489,297, 7,094,755, and 6,608,174.
[0032] A number of mutations to improve peptide stability against
proteases etc. are detailed in the literature (see Onune et al
Physicochemical and pharmacological characterization of novel
vasoactive intestinal peptide derivatives with improved stability,
Eur. J. Pharm. Biopharm. 2009). For example, modified VIP peptides
include the sequences of SEQ ID NOs: 14-19. In some aspects, the
present disclosure provides modified VIP peptides that include one
or more of these modifications. In some embodiments, the present
disclosure provides modified VIP peptides that include one or more
of these modifications and further include additional VIP
modifications described herein.
[0033] In various embodiments, the present disclosure provides a
modified VIP (e.g., comprising SEQ ID NO: 14) or a functional
analog as described herein. Generally, functional analogs of VIP,
include functional fragments truncated at the N- or C-terminus by
from 1 to 10 amino acids, including by 1, 2, 3, or up to about 5
amino acids (with respect to SEQ ID NO: 14). Such functional
analogs may contain from 1 to 5 amino acid insertions, deletions,
and/or substitutions (collectively) with respect to the native
sequence (e.g., SEQ ID NO: 17), and in each case retain the
activity of the native peptide (e.g., through VPAC2 and/or VPAC1
binding). Such activity may be confirmed or assayed using any
available assay, including an assay described herein, and including
any suitable assay to determine or quantify an activity described
in Delgado et al., The Significance of Vasoactive Intestinal
Peptide in Immunomodulation, Pharmacol. Reviews 56(2):249-290
(2004). In these or other embodiments, the VIP component of the
modified VIP has at least about 50%, 75%, 80%, 85%, 90%, 95%, or
97% identity with the native mature sequence (SEQ ID NO: 17). The
determination of sequence identity between two sequences (e.g.,
between a native sequence and a functional analog) can be
accomplished using any alignment tool, including for example, that
disclosed in Tatusova et al., Blast 2 sequences--a new tool for
comparing protein and nucleotide sequences, FEMS Microbiol Lett.
174:247-250 (1999).
[0034] In various aspects, the present disclosure provides a
modified VIP molecule having receptor preference for VPAC2 or
VPAC1, as compared to unmodified VIP (e.g., a peptide consisting of
the amino acid sequence of SEQ ID NO: 14). For example, the
modified VIP may have a relative binding preference for VPAC2 over
VPAC1 of at least about 2:1, about 5:1, about 10:1, about 25:1,
about 50:1, about 100:1, about 500:1 or more. In other embodiments,
the modified VIP may have a relative binding preference for VPAC1
over VPAC2 of at least about 2:1, about 5:1, about 10:1, about
25:1, about 50:1, about 100:1, about 500:1, or more. For example,
in certain embodiments, the modified VIP activates the VPAC2
receptor with an EC50 within a factor of about 2 of mature,
unmodified, human VIP (SEQ ID NO: 17). However, this same modified
VIP is 50- or 100-fold or more less potent than mature, unmodified,
human VIP in activating the VPAC1 receptor. In some embodiments,
the modified VIP may have relatively equipotent binding preferences
for VPAC1 and VPAC2.
[0035] Such modified VIP molecules may contain modified N-terminal
regions, such as an addition of from 1 to about 500 amino acids to
the N-terminal histidine of VIP, which may include heterologous
mammalian amino acid sequences. For example, the modified VIP may
contain a single methionine at the N-terminal side of the natural
N-terminal histidine of mature VIP. This can be prepared in E. coli
or other bacterial expression system, since the methionine will not
be removed by E coli when the adjacent amino acid is histidine.
Alternatively, the N-terminal amino acid may be any of the
naturally-occurring amino acids, namely alanine, arginine,
asparagine, aspartic acid, cysteine, glutamic acid, glutamine,
glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, serine, threonine, tryptophan, tyrosine, valine, and
proline.
[0036] The additional sequence added to the N-terminus of VIP may
be of any sequence, including biologically active and biologically
inert sequences of from 1 to about 100, 1 to about 50, 1 to about
20, 1 to about 10, and 1 to about 5 amino acids.
[0037] The N-terminus of the modified VIP may have the structure
M-N, where M is methionine, and N is the N-terminus of the VIP
molecule (e.g., SEQ ID NO. 14). This methionine supports
translation of the protein in a bacterial or eukaryotic host cell.
Thus, the modified VIP can be made in a biological system,
including bacterial and yeast expression systems (e.g., E. coli).
While methionine can sometimes be removed by methionine
aminopeptidase (MA) in bacterial expression systems, histidine (H)
is one of the least favored residues at position 2 for MA.
[0038] The half-life of protein therapeutics can be extended by a
variety of means, including increasing the size and thus the
hydrodynamic volume of the protein therapeutic, adding modified or
unnatural amino acids, conjugation of moieties (e.g. pegylation),
the addition of synthetic sequences (e.g. XTEN.RTM. sequences,
PASylation.RTM.), carboxy-terminal extension from hCG (CTP),
addition of albumin-binding sequences (e.g. AlbudAb.RTM.),
conjugation of albumin-binding fatty acids, and post-translational
modifications such as N-glycosylation and fusion to other peptides.
In still other embodiments, VIP is modified by fusion with a
mammalian heterologous protein, such as a mammalian protein
effective for extending half-life of therapeutic molecules. Such
sequences may be mammalian sequences, such as albumin, transferrin,
or antibody Fc sequences. Such sequences are described in See U.S.
Pat. No. 7,238,667 (particularly with respect to albumin
conjugates), U.S. Pat. No. 7,176,278 (particularly with respect to
transferrin conjugates), and U.S. Pat. No. 5,766,883. In some
embodiments, the VIP is modified at the N-terminus. In some
embodiments, the VIP is modified at the C-terminus.
[0039] In other embodiments, VIP is activatable by a peptidase or
protease, such as an endogenous peptidase or protease. Such
activatable sequences are described in International Application
No. PCT/US2009/068656. As used herein, the terms "peptidase" and
"protease" are interchangeable. For example, the VIP may be
designed to be activatable by a dipeptidyl peptidase. Exemplary
dipeptidyl peptidases include dipeptidyl peptidase-1 (DPP-I),
dipeptidyl peptidase-3 (DPP-III), dipeptidyl peptidase-4 (DPP-IV),
dipeptidyl peptidase-6 (DPP-VI), dipeptidyl peptidase-7 (DPP-VII),
dipeptidyl peptidase-8 (DPP-VIII), dipeptidyl peptidase-9 (DPP-IX),
dipeptidyl peptidase-10 (DPP-X). Substrate sequences for such
dipeptidases are known.
[0040] In some embodiments, the N-terminus of an activatable VIP
may have the structure Z-N, where Z is a substrate for a
dipeptidase (e.g., Z is removed by dipeptidase exposure), and N is
the N-terminus of VIP. The activatable VIP may have an N-terminal
sequence with the formula M-X-N where M is methionine, X is Pro,
Ala, or Ser, and N is the N-terminal of VIP or VIP analog. In this
manner, M and X will be sensitive to, and removed by a host cell
(e.g., E. coli), and/or a dipeptidase (e.g., DPP-IV), subsequently.
Alternatively, the N-terminal sequence of the activatable VIP may
be X1-X2-N, where X1 is Gly, Ala, Ser, Cys, Thr, Val, or Pro; X2 is
Pro, Ala, or Ser; and N is the N-terminal of VIP. X1-X2 is a
substrate for dipeptidase (e.g., DPP-IV), and dipeptidase digestion
will expose N, the desired N-terminus of the VIP or the VIP analog
(e.g., SEQ ID NO. 16). In such embodiments, the protein may be
produced by expression of a construct encoding M-X1-X2-N (where M
is methionine) in a host cell (e.g., E. coli), since Gly, Ala, Ser,
Cys, Thr, Val, or Pro at the second position will signal the
removal of the Met, thereby leaving X1-X2 on the N-terminus, which
can be activated by a dipeptidase (e.g., DPP-IV) in vivo. In some
embodiments, the peptidase may be present in the body and act on
the activatable VIP after injection.
[0041] In other embodiments, the N-terminus of the modified
activatable VIP has the structure M-Z-N, where M is methionine, Z
is a substrate for a dipeptidase (e.g., Z is removed by dipeptidase
exposure), and N is a non-His N-terminal of an active VIP (modified
VIP). For example, the modified activatable VIP may have an
N-terminal sequence with the formula M-X-N where M is methionine; X
is Pro, Ala, or Ser; and N is a non-His N-terminal of the active
VIP. In this manner, M and X will be sensitive to, and removed by a
host cell (e.g., E. coli), and/or a dipeptidase (e.g., DPP-IV),
subsequently. Alternatively, the N-terminal sequence of the
activatable VIP may be X1-X2-N, where X1 is Gly, Ala, Ser, Cys,
Thr, Val, or Pro; X2 is Pro, Ala, or Ser; and N is a non-His
N-terminal of the active VIP. X1-X2 is a substrate for dipeptidase
(e.g., DPP-IV), and dipeptidase digestion will expose N, the
desired non-His N-terminus of the VIP.
[0042] Still other embodiments, the N-terminus of a modified
activatable VIP has the structure M-Z-S-N, where M is methionine; Z
is a substrate for a dipeptidase (e.g., Z is removed by dipeptidase
exposure); N is the N-terminus of mature VIP (His); and S is one or
more amino acids which will be exposed after dipeptidase digestion,
and which provide a modified VIP as previously described. For
example, the modified activatable VIP may have an N-terminal
sequence with the formula M-X-S-N where M is methionine, X is Pro,
Ala, or Ser; N is the N-terminal of mature VIP; and S is one or
more amino acids which will be exposed after dipeptidase digestion,
and will provide receptor preference. Alternatively, the N-terminal
sequence of the activatable VIP may be X1-X2-S-N, where X1 is Gly,
Ala, Ser, Cys, Thr, Val, or Pro; X2 is Pro, Ala, or Ser; N is a
non-His N-terminal of VIP; and S is one or more amino acids which
will be exposed after dipeptidase digestion. X1-X2 is a substrate
for dipeptidase (e.g., DPP-IV), and dipeptidase digestion will
expose S.
[0043] In some embodiments, N-terminal chemical modifications to
the VIP N-terminus provides receptor preference. Chemical
modification of proteins and methods thereof are well known in the
art. Non-limiting exemplary chemical modifications are PEGylation,
methylglyoxalation, reductive alkylation, performic acid oxidation,
succinylation, aminoethylation, and lipidation (Clifton, New
Protein Techniques, New Jersey: Humana Press, 1985. ISBX.
0-89603-126-8. Volume. 3 of. Methods in Molecular Biology).
Chemical groups, such as PEGylation, may be attached by
modifications of cysteine, methionine, histidine, lysine, arginine,
tryptophan, tyrosine, carboxyl groups have been described
previously (see Lundblad, Techniques in Protein Modification, CRC
Press, 1995).
Elastin-Like Peptides
[0044] In some aspects the disclosure provides therapeutic
compositions that include a Vasoactive Intestinal Peptide and one
or more elastin-like peptides (ELP). In some embodiments, a VIP and
one or more ELPs are fused together. In some embodiments, a VIP and
one or more ELPs are produced as a recombinant fusion polypeptide.
In some embodiments, the therapeutic composition includes a
Vasoactive Intestinal Peptide and one or more ELPs as separate
molecules. In yet other embodiments, the compositions include a
VIP-ELP fusion protein and ELPs as separate molecules. In some
embodiments, the compositions include SEQ ID NO: 15 (PB1046). In
some embodiments, the compositions include SEQ ID NO: 20
(PB1120).
[0045] The ELP sequence includes structural peptide units or
sequences that are related to, or mimics of, the elastin protein.
The ELP sequence is constructed from structural units of from three
to about twenty amino acids, or in some embodiments, from four to
ten amino acids, such as four, five or six amino acids. The length
of the individual structural units may vary or may be uniform. For
example, structural units include units defined by SEQ ID NOS:
1-13, which may be employed as repeating structural units,
including tandem-repeating units, or may be employed in some
combination. Thus, the ELP includes essentially structural unit(s)
selected from SEQ ID NOS: 1-13.
[0046] In some embodiments, the amino acid sequence of the ELP unit
is from about 1 to about 500 structural units, or in certain
embodiments about 9 to about 200 structural units, or in certain
embodiments about 10 to 200 structural units, or in certain
embodiments about 50 to about 200 structural units, or in certain
embodiments from about 80 to about 200 structural units, or from
about 80 to about 150 structural units, such as one or a
combination of units defined by SEQ ID NOS: 1-13. Thus, the
structural units collectively may have a length of from about 50 to
about 2000 amino acid residues, or from about 100 to about 800
amino acid residues, or from about 200 to about 700 amino acid
residues, or from about 400 to about 600 amino acid residues, or
from about 500 to about 700 amino acid residues. In exemplary
embodiments, the amino acid sequence of the ELP structural unit
includes about 3 structural units, about 7 structural units, about
9 structural units, about 10 structural units, about 15 structural
units, about 18 structural units, about 20 structural units, about
40 structural units, about 80 structural units, about 100
structural units, about 120 structural units, about 140 structural
units, about 144 structural units, about 160 structural units,
about 180 structural units, about 200 structural units, or about
500 structural units. In exemplary embodiments, the structural
units collectively have a length of about 45 amino acid residues,
of about 90 amino acid residues, of about 100 amino acid residues,
of about 200 amino acid residues, of about 300 amino acid residues,
of about 400 amino acid residues, of about 500 amino acid residues,
of about 600 amino acid residues, of about 700 amino acid residues,
of about 800 amino acid residues, or of about 1000 amino acid
residues.
[0047] The ELP amino acid sequence may exhibit a visible and
reversible inverse phase transition with the selected formulation.
That is, the amino acid sequence may be structurally disordered and
highly soluble in the formulation below a transition temperature
(Tt), but exhibit a sharp (2-3.degree. C. range) disorder-to-order
phase transition when the temperature of the formulation is raised
above the Tt. In addition to temperature, length of the amino acid
polymer, amino acid composition, ionic strength, pH, pressure,
temperature, selected solvents, presence of organic solutes, and
protein concentration may also affect the transition properties,
and these may be tailored in the formulation for the desired
absorption profile. Absorption profile can be easily tested by
determining plasma concentration or activity of the active agent
over time.
[0048] In certain embodiments, the ELP component(s) may be formed
of multipeptide structural units (e.g. tetrapeptides,
pentapeptides, hexapeptides, octapeptides, or nonapeptides),
including but not limited to: [0049] (a) the tetrapeptide
Val-Pro-Gly-Gly, or VPGG (SEQ ID NO: 1); [0050] (b) the
tetrapeptide Ile-Pro-Gly-Gly, or IPGG (SEQ ID NO: 2); [0051] (c)
the pentapeptide Val-Pro-Gly-X-Gly (SEQ ID NO: 3), or VPGXG, where
X is any natural or non-natural amino acid residue, and where X
optionally varies among polymeric or oligomeric repeats; [0052] (d)
the pentapeptide Ala-Val-Gly-Val-Pro, or AVGVP (SEQ ID NO: 4);
[0053] (e) the pentapeptide Ile-Pro-Gly-X-Gly, or IPGXG (SEQ ID NO:
5), where X is any natural or non-natural amino acid residue, and
where X optionally varies among polymeric or oligomeric repeats;
[0054] (e) the pentapeptide Ile-Pro-Gly-Val-Gly, or IPGVG (SEQ ID
NO: 6); [0055] (f) the pentapeptide Leu-Pro-Gly-X-Gly, or LPGXG
(SEQ ID NO: 7), where X is any natural or non-natural amino acid
residue, and where X optionally varies among polymeric or
oligomeric repeats; [0056] (g) the pentapeptide
Leu-Pro-Gly-Val-Gly, or LPGVG (SEQ ID NO: 8); [0057] (h) the
hexapeptide Val-Ala-Pro-Gly-Val-Gly, or VAPGVG (SEQ ID NO: 9);
[0058] (i) the octapeptide Gly-Val-Gly-Val-Pro-Gly-Val-Gly, or
GVGVPGVG (SEQ ID NO: 10); [0059] (j) the nonapeptide
Val-Pro-Gly-Phe-Gly-Val-Gly-Ala-Gly, or VPGFGVGAG (SEQ ID NO: 11);
[0060] (k) the nonapeptides Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Gly, or
VPGVGVPGG (SEQ ID NO: 12); and [0061] (l) the pentapeptide
Xaa-Pro-Gly-Val-Gly, or XPGVG (SEQ ID NO:13) where X is any natural
or non-natural amino acid residue, and where X optionally varies
among polymeric or oligomeric repeats.
[0062] The multipeptide structural units as defined in SEQ ID NOs:
1-13 form the elastin-like peptide component. In some embodiments,
the ELP includes more than one structural unit. In some
embodiments, the ELP includes two or more structural units of any
of SEQ ID NOs: 1-13, which may be in any combination. In some
embodiments, the two or more structural units are the same and are
repeated tandemly. In some embodiments, the two or more structural
units are different and are repeated alternately. In some
embodiments, the ELP includes structural units repeated tandemly
for one or more portions of sequence, and also different structural
units repeated alternately for other portions of the sequence. In
some embodiments, the ELP component is formed entirely (or almost
entirely) of one or a combination of (e.g., 2, 3 or 4) structural
units selected from SEQ ID NOS: 1-13. In other embodiments, at
least 75%, or at least 80%, or at least 90% of the ELP component is
formed from one or a combination of structural units selected from
SEQ ID NOS: 1-13. In certain embodiments, the ELP contains repeat
units, including tandem repeating units, of Val-Pro-Gly-X-Gly (SEQ
ID NO: 3), where X is as defined above, and where the percentage of
Val-Pro-Gly-X-Gly (SEQ ID NO: 3) units taken with respect to the
entire ELP component (which may comprise structural units other
than VPGXG) is greater than about 50%, or greater than about 75%,
or greater than about 85%, or greater than about 95% of the ELP.
The ELP may contain motifs of 5 to 15 structural units (e.g. about
10 structural units) of SEQ ID NO: 3, with the guest residue X
varying among at least 2 or at least 3 of the units in the motif.
The guest residues may be independently selected, such as from
non-polar or hydrophobic residues, such as the amino acids V, I, L,
A, G, and W (and may be selected so as to retain a desired inverse
phase transition property). In certain embodiments, the guest
residues are selected from V, G, and A. In some embodiments, the
ELP includes the ELP 1 series (VPGXG: V5A2G3). In some embodiments,
the ELP includes the ELP 4 series (VPGXG: V-5). In some
embodiments, the ELP includes a combination of the ELP1 and ELP4
series. Without being bound by theory, the differences in the ELP
polymer hydrophobicity is determined by the guest residues and
their ratios, with the ELP 4 series being more hydrophobic than the
ELP1 series.
[0063] In certain embodiments, the ELP contains repeat units,
including tandem repeating units, of Xaa-Pro-Gly-Val-Gly (SEQ ID
NO: 13), where X is as defined above, and where the percentage of
Xaa-Pro-Gly-Val-Gly units taken with respect to the entire ELP
component (which may include structural units other than XPGVG) is
greater than about 50%, or greater than about 75%, or greater than
about 85%, or greater than about 95% of the ELP. The ELP may
contain motifs of 5 to 15 structural units (e.g. about 9 structural
units) of SEQ ID NO: 13, with the guest residue X varying among at
least 2 or at least 3 of the units in the motif. The guest residues
may be independently selected, such as from non-polar or
hydrophobic residues, such as the amino acids V, I, L, A, G, and W
(and may be selected so as to retain a desired inverse phase
transition property). In certain embodiments, the guest residues
are selected from V and A.
[0064] In certain embodiments, the ELP contains repeat units,
including tandem repeating units of any of SEQ ID NOs: 1-13 either
alone or in combination. In one embodiment, the ELP contains
repeats of two or more of any of SEQ ID NOs: 1-13 in combination.
In certain embodiments, the ELP contains repeats of SEQ ID NO: 3
and SEQ ID NO: 13. In some embodiments, the ELP contains repeats of
SEQ ID NO: 3 and SEQ ID NO: 13, wherein the guest residues are
independently selected, such as from non-polar or hydrophobic
residues, such as the amino acids V, I, L, A, G, and W (and may be
selected so as to retain a desired inverse phase transition
property). In certain embodiments, the guest residues are selected
from V and A. In some embodiments, the ELP comprises 9mers
comprising five copies of a pentapeptide disclosed herein. In some
embodiments, the ELP comprises 9mers comprising SEQ ID NOs: 3 and
13 in any combination. In some embodiments, the ELP comprises a
sequence alternating between SEQ ID NOs: 3 and 13.
[0065] In some embodiments, the ELP may form a (3-turn structure.
Exemplary peptide sequences suitable for creating a (3-turn
structure are described in International Patent Application
PCT/US96/05186. For example, the fourth residue (X) in the sequence
VPGXG, can be altered without eliminating the formation of a
(3-turn.
[0066] The structure of exemplary ELPs may be described using the
notation ELPk [XiYj-n], where k designates a particular ELP repeat
unit, the bracketed capital letters are single letter amino acid
codes and their corresponding subscripts designate the relative
ratio of each guest residue X in the structural units (where
applicable), and n describes the total length of the ELP in number
of the structural repeats. For example, ELP1
[V.sub.5A.sub.2G.sub.3-10] designates an ELP component containing
10 repeating units of the pentapeptide VPGXG, where X is valine,
alanine, and glycine at a relative ratio of about 5:2:3; ELP1
[K.sub.1V.sub.2F.sub.1-4] designates an ELP component containing 4
repeating units of the pentapeptide VPGXG, where X is lysine,
valine, and phenylalanine at a relative ratio of about 1:2:1; ELP1
[K.sub.1V.sub.7F.sub.1-9] designates a polypeptide containing 9
repeating units of the pentapeptide VPGXG, where X is lysine,
valine, and phenylalanine at a relative ratio of about 1:7:1; ELP1
[V-5] designates a polypeptide containing 5 repeating units of the
pentapeptide VPGXG, where X is valine; ELP1 [V-20] designates a
polypeptide containing 20 repeating units of the pentapeptide
VPGXG, where X is valine; ELP2 [5] designates a polypeptide
containing 5 repeating units of the pentapeptide AVGVP (SEQ ID NO:
4); ELP3 [V-5] designates a polypeptide containing 5 repeating
units of the pentapeptide IPGXG (SEQ ID NO: 5), where X is valine;
ELP4 [V-5] designates a polypeptide containing 5 repeating units of
the pentapeptide LPGXG (SEQ ID NO: 7), where X is valine.
[0067] With respect to ELP, the Tt is a function of the
hydrophobicity of the guest residue. Thus, by varying the identity
of the guest residue(s) and their mole fraction(s), ELPs can be
synthesized that exhibit an inverse transition over a broad range.
Thus, the Tt at a given ELP length may be decreased by
incorporating a larger fraction of hydrophobic guest residues in
the ELP sequence. Examples of suitable hydrophobic guest residues
include valine, leucine, isoleucine, phenylalanine, tryptophan and
methionine. Tyrosine, which is moderately hydrophobic, may also be
used. Conversely, the Tt may be increased by incorporating
residues, such as those selected from: glutamic acid, cysteine,
lysine, aspartate, alanine, asparagine, serine, threonine, glycine,
arginine, and glutamine.
[0068] For polypeptides having a molecular weight >100,000 Da,
the hydrophobicity scale disclosed in PCT/US96/05186 provides one
means for predicting the approximate Tt of a specific ELP sequence.
For polypeptides having a molecular weight <100,000 Da, the Tt
may be predicted or determined by the following quadratic function:
Tt=M0+M1X+M2X2 where X is the MW of the fusion protein, and
M0=116.21; M1=-1.7499; M2=0.010349.
[0069] The ELP in some embodiments is selected or designed to
provide a Tt ranging from about 10 to about 37.degree. C. at
formulation conditions, such as from about 20 to about 37.degree.
C., or from about 25 to about 37.degree. C. In some embodiments,
the transition temperature at physiological conditions (e.g., 0.9%
saline) is from about 34 to 36.degree. C., to take into account a
slightly lower peripheral temperature.
[0070] In certain embodiments, the amino acid sequence capable of
forming the hydrogen-bonded matrix at body temperature is the ELP-1
series which includes [VPGXG].sub.m, where m is any number from 1
to 200, each X is selected from V, G, and A, and wherein the ratio
of V:G:A may be about 5:3:2. In certain embodiments, the amino acid
sequence capable of forming the hydrogen-bonded matrix at body
temperature includes [VPGXG].sub.90, where each X is selected from
V, G, and A, and wherein the ratio of V:G:A may be about 5:3:2. In
certain embodiments, the amino acid sequence capable of forming the
hydrogen-bonded matrix at body temperature includes
[VPGXG].sub.120, where each X is selected from V, G, and A, and
wherein the ratio of V:G:A may be about 5:3:2.
[0071] In certain embodiments, the amino acid sequence capable of
forming the hydrogen-bonded matrix at body temperature includes
[VPGXG].sub.144, where each X is selected from V, G, and A, and
wherein the ratio of V:G:A is about 7:2:0. In certain embodiments,
the amino acid sequence capable of forming the hydrogen-bonded
matrix at body temperature includes [VPGXG].sub.144, where each X
is selected from V, G, and A, and wherein the ratio of V:G:A is
about 7:0:2. In certain embodiments, the amino acid sequence
capable of forming the hydrogen-bonded matrix at body temperature
includes [VPGXG].sub.144, where each X is selected from V, G, and
A, and wherein the ratio of V:G:A is about 6:0:3. In certain
embodiments, the amino acid sequence capable of forming the
hydrogen-bonded matrix at body temperature includes
[VPGXG].sub.144, where each X is selected from V, G, and A, and
wherein the ratio of V:G:A is about 5:2:2.
[0072] In certain embodiments, the amino acid sequence capable of
forming the hydrogen-bonded matrix at body temperature includes
[XPGVG]m, where m is any number from 1 to 200, each X is selected
from V, G, and A. In certain embodiments, the amino acid sequence
capable of forming the hydrogen-bonded matrix at body temperature
includes [XPGVG].sub.144, where m is any number from 1 to 200, each
X is selected from V, G, and A and wherein the ratio of V:G:A is
about 5:0:4. In certain embodiments, the amino acid sequence
capable of forming the hydrogen-bonded matrix at body temperature
includes [XPGVG].sub.144, where each X is selected from V, G, and
A, and wherein the ratio of V:G:A is about 5:0:4.
[0073] Alternatively, the amino acid sequence capable of forming
the matrix at body temperature is the ELP-4 series which includes
[VPGVG].sub.90, or [VPGVG].sub.120. 120 structural units of this
ELP can provide a transition temperature at about 37.degree. C.
with about 0.005 to about 0.05 mg/ml (e.g., about 0.01 mg/ml) of
protein. Alternatively, the amino acid sequence capable of forming
the matrix at body temperature includes [VPGXG].sub.144 or
[XPGVG].sub.144. For example, 144 structural units of either of
these ELPs can provide a transition temperature at between about
28.degree. C. and 35.degree. C.
[0074] Elastin-like-peptide (ELP) protein polymers and recombinant
fusion proteins can be prepared as described in U.S. Patent
Publication No. 2010/0022455. In some embodiments, the ELP protein
polymers are constructed through recursive ligation to rapidly
clone highly repetitive polypeptides of any sequence and specified
length over a large range of molecular weights. In a single cycle,
two halves of a parent plasmid, each containing a copy of an
oligomer, are ligated together, thereby dimerizing the oligomer and
reconstituting a functional plasmid. This process is carried out
recursively to assemble an oligomeric gene with the desired number
of repeats. For example, one ELP structural subunit (e.g. a
pentapeptide or a 9mer of pentapeptides) is inserted into a vector.
The vector is digested, and another ELP structural unit (e.g. a
pentapeptide or a 9mer of pentapeptides) is inserted. Each
subsequent round of digestion and ligation doubles the number of
ELP structural units contained in the resulting vector until the
ELP polymer is the desired length.
[0075] In other embodiments, the amino acid sequence capable of
forming the matrix at body temperature includes a random coil or
non-globular extended structure. For example, the amino acid
sequence capable of forming the matrix at body temperature includes
an amino acid sequence disclosed in U.S. Patent Publication No.
2008/0286808, WIPO Patent Publication No. 2008/155134, and U.S.
Patent Publication No. 2011/0123487.
[0076] For example, in some embodiments the amino acid sequence
includes an unstructured recombinant polymer of at least 40 amino
acids. For example, the unstructured polymer may be defined where
the sum of glycine (G), aspartate (D), alanine (A), serine (S),
threonine (T), glutamate (E) and proline (P) residues contained in
the unstructured polymer, constitutes more than about 80% of the
total amino acids. In some embodiments, at least 50% of the amino
acids are devoid of secondary structure as determined by the
Chou-Fasman algorithm. The unstructured polymer includes more than
about 100, 150, 200 or more contiguous amino acids. In some
embodiments, the amino acid sequence forms a random coil domain. In
particular, a polypeptide or amino acid polymer having or forming
"random coil conformation" substantially lacks a defined secondary
and tertiary structure.
[0077] In various embodiments, the intended subject is human, and
the body temperature is about 37.degree. C., and thus the
therapeutic agent is designed to provide a sustained release at or
near this temperature (e.g. between about 28.degree. C. to about
37.degree. C.). A slow release into the circulation with reversal
of hydrogen bonding and/or hydrophobic interactions is driven by a
drop in concentration as the product diffuses at the injection
site, even though body temperature remains constant. In other
embodiments, the subject is a non-human mammal, and the therapeutic
agent is designed to exhibit a sustained release at the body
temperature of the mammal, which may be from about 30 to about
40.degree. C. in some embodiments, such as for certain domesticated
pets (e.g., dog or cat) or livestock (e.g., cow, horse, sheep, or
pig). Generally, the Tt is higher than the storage conditions of
the formulation (which may be from about 2 to about 30.degree. C.,
or about 10 to about 25.degree. C., or from about 15 to about
22.degree. C., or from about 2 to about 8.degree. C.), such that
the therapeutic agent remains in solution for injection.
Alternatively, the therapeutic agent may be stored frozen, such as
from about -80.degree. C. to about -20.degree. C.
Disorders Associated with CFTR Protein Dysfunction and Methods of
Treatment
[0078] Dysfunction of the CFTR protein occurs in various diseases
or disorders, including but not limited to Cystic Fibrosis, Chronic
Obstructive Pulmonary Disease (COPD), exocrine organ disorders,
non-Cystic Fibrosis bronchiectasis, recurrent pancreatitis,
congenital bilateral absence of vas deferens and disorders
associated with an acquired defect in CFTR protein function (e.g.
caused by smoking or other environmental factors). In an exemplary
embodiment, CFTR dysfunction is associated with Cystic Fibrosis. In
another exemplary embodiment, CFTR dysfunction is associated with
smoking-related lung damage.
[0079] As used herein, the term "CFTR protein dysfunction" refers
to any decrease in CFTR protein function compared to a healthy
subject. The CFTR protein may exhibit a total loss of function, or
it may exhibit some residual or partial function compared to CFTR
function in a healthy subject. In some embodiments, CFTR function
is decreased by about 1%, about 5%, about 10%, about 20%, about
30%, about 40%, about 50%, about 60%, about 70%, about 80%, about
90%, about 95%, or about 99% compared with CFTR protein function in
a healthy subject.
[0080] In some embodiments, CFTR protein dysfunction is
characterized by a loss of chloride ion transport across the cell
membrane. In some embodiments, CFTR protein dysfunction is
characterized by a decrease in water transport across the cell
membrane. In some embodiments, the CFTR protein is mis-localized in
the cell. In some embodiments, the CFTR protein is not localized to
the apical membrane of the cell.
[0081] CFTR protein dysfunction symptoms vary among individual
patients, but in general, CFTR protein dysfunction is characterized
by production of thick mucus that for example, clogs respiratory
airways, obstructs the intestines, blocks pancreatic and bile
ducts, interferes with liver function, and damages tissue. The
tissue damage observed in subjects with CFTR protein dysfunction
may affect a variety of tissues and organs including, but not
limited to, the lungs, the pancreas, the liver, the intestines, the
reproductive system, the airway system, and/or the digestive
system.
[0082] In some embodiments, CFTR protein dysfunction is
characterized by an increased salinity in sweat or other body
fluids. In some embodiments, CFTR protein dysfunction is
characterized by defective excretion of bicarbonate in the gut. In
some embodiments, CFTR protein dysfunction is characterized by
increased incidence of infections, including but not limited to
infections of the airway. In some embodiments, CFTR protein
dysfunction is characterized by an inflammatory lung phenotype. In
some embodiments, CFTR protein dysfunction is characterized by
impaired respiratory activity. In some embodiments, CFTR protein
dysfunction is characterized by chronic digestive problems. In some
embodiments, Cystic Fibrosis is characterized by male infertility.
A patient will not necessarily present with all of these symptoms,
some of which might be absent in milder cases of CFTR protein
dysfunction, or earlier during disease progression.
[0083] In some aspects, the present disclosure provides a method of
treating, delaying, or ameliorating symptoms of CFTR protein
dysfunction comprising administering pharmaceutical compositions of
a vasoactive intestinal peptide and one or more ELPs to a subject
in need.
[0084] Cystic Fibrosis caused by any one or more mutations in the
CFTR gene may be treated, delayed, or ameliorated by the
pharmaceutical compositions disclosed herein. In some embodiments,
the subject is homozygous for one or more mutations in the CFTR
gene. In some embodiments, the subject is heterozygous for one or
more mutations in the CFTR gene. In some embodiments, the one or
more mutations are nonsense mutations. In some embodiments, the one
or more mutations are gating mutations. In some embodiments, the
one or more mutations are protein processing mutations. In some
embodiments, the one or more mutations are conductance mutations.
In some embodiments, the one or more mutations are translation
mutations. Examples of CFTR mutations include, but are not limited
to, F508del, G542X, G85E, R334W, Y122X, G551D, R117H, A455E, S549R,
R553X, V520F, R1162X, R347H, N1203K, S549N, R347P, R560T, S1255X,
Add9T, Y1092X, M1191K, W1282X, 3659de;C, 394delTT, 3905insT,
1078delT, delta 1507, 3876delA, 2184delA, 2307insA, 711+1G>T,
1717-1G>A, 2789+5G>A, 1898+5G>T, 3120+1G>A,
621+1G>T, 3849+10kbC>T, 1898+1G>A, 2183 AA>G, and/or
5/7/9T. In a preferred embodiment, the mutation is F508del.
[0085] In some embodiments, the CFTR protein dysfunction is
acquired (e.g. through smoking or by exposure to environmental
damage). In some embodiments, the CFTR protein dysfunction is not
associated with a mutation in the CFTR gene.
[0086] The treatment, delay, or amelioration of CFTR protein
dysfunction symptoms may be measured by any means known in the art.
For example, tests used to evaluate patients with CFTR protein
dysfunction include, but are not limited to, a sweat chloride test,
an immunoreactive trypsinogen test (IRT), a blood test (e.g. to
test pancreatic function), chest X-rays, lung function tests, a
nasal potential difference test, CFTR protein function assays (e.g.
testing the efflux of ions in the cells), cellular current
measurement test, forced expiratory volume in 1 second (FEV1),
and/or immunofluorescence to detect the localization of the CFTR
protein in the cell.
[0087] The effects of administration of the pharmaceutical
compositions disclosed herein may be measured in any relevant
tissue and/or organ, including but not limited to epithelial cells
(e.g. nasal epithelial cells), lungs, pancreas, the digestive
system, the reproductive system and/or the airway system.
[0088] In some embodiments, administration of the pharmaceutical
compositions disclosed herein prevents, delays, or ameliorates one
or more CFTR protein dysfunction symptoms in a subject. In some
embodiments, one or more CFTR protein dysfunction symptoms are
prevented, delayed, or ameliorated for about 1 week, about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months,
about 6 months, about 8 months, about 1 year, about 2 years, about
5 years, and/or about 10 years compared with the one or more CFTR
protein dysfunction symptoms in an untreated subject with CFTR
dysfunction. In some embodiments, one or more CFTR protein
dysfunction symptoms are prevented, delayed, or ameliorated by
about 1%, about 5%, about 10%, about 20%, about 30%, about 40%,
about 50%, about 60%, about 70%, about 80%, about 90%, about 95%,
or about 99% compared with the one or more CFTR protein dysfunction
symptoms in an untreated subject with CFTR dysfunction. In some
embodiments, this prevention, delay, or amelioration of one or more
CFTR protein dysfunction symptoms is observed at the time points
disclosed herein.
[0089] In some embodiments, administration of the pharmaceutical
compositions disclosed herein decrease sweat chloride levels in a
subject compared to an untreated subject with CFTR protein
dysfunction. In some embodiments, sweat chloride levels are
decreased for about 1 week, about 1 month, about 2 months, about 3
months, about 4 months, about 5 months, about 6 months, about 8
months, about 1 year, about 2 years, about 5 years, and/or about 10
years compared with the mucus viscosity of an untreated subject
with CFTR protein dysfunction. In some embodiments, sweat chloride
levels are decreased by about 1%, about 5%, about 10%, about 20%,
about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,
about 90%, about 95%, or about 99% compared with the mucus
viscosity of an untreated subject with CFTR protein dysfunction. In
some embodiments, this decrease in sweat chloride levels is
observed at the time points disclosed herein.
[0090] In some embodiments, administration of the pharmaceutical
compositions disclosed herein improves mucus viscosity in a subject
compared to an untreated subject with CFTR protein dysfunction. In
some embodiments, mucus viscosity is improved for about 1 week,
about 1 month, about 2 months, about 3 months, about 4 months,
about 5 months, about 6 months, about 8 months, about 1 year, about
2 years, about 5 years, and/or about 10 years compared with the
mucus viscosity of an untreated subject with CFTR protein
dysfunction. In some embodiments, mucus viscosity is improved by
about 1%, about 5%, about 10%, about 20%, about 30%, about 40%,
about 50%, about 60%, about 70%, about 80%, about 90%, about 95%,
or about 99% compared with the mucus viscosity of an untreated
subject with CFTR protein dysfunction. In some embodiments, this
improvement in mucus viscosity is observed at the time points
disclosed herein.
[0091] In some embodiments, administration of the pharmaceutical
compositions disclosed herein prevents, delays, or ameliorates the
development of fibrosis in a subject. In some embodiments,
development of fibrosis is prevented, delayed, or ameliorated for
about 1 week, about 1 month, about 2 months, about 3 months, about
4 months, about 5 months, about 6 months, about 8 months, about 1
year, about 2 years, about 5 years, and/or about 10 years compared
with the development of fibrosis in an untreated subject with CFTR
protein dysfunction. In some embodiments, the development of
fibrosis is prevented, delayed, or ameliorated by about 1%, about
5%, about 10%, about 20%, about 30%, about 40%, about 50%, about
60%, about 70%, about 80%, about 90%, about 95%, or about 99%
compared with the development of fibrosis in an untreated subject
with CFTR protein dysfunction. In some embodiments, this
prevention, delay, or amelioration of fibrosis is observed at the
time points disclosed herein.
[0092] In some embodiments, administration of the pharmaceutical
compositions disclosed herein prevents, delays, or ameliorates
tissue damage in one or more organs and/or tissues in a subject. In
some embodiments, the tissue damage is caused by inflammation. In
preferred embodiments, administration of the pharmaceutical
compositions disclosed herein prevents, delays, or ameliorates
tissue damage in the digestive tract. In some embodiments,
preventing, delaying, or ameliorating tissue damage in the
digestive tract alleviates digestive problems in the subject. In
preferred embodiments, administration of the pharmaceutical
compositions disclosed herein prevents, delays, or ameliorates
tissue damage in the lungs in a subject. In some embodiments,
tissue damage is prevented, delayed, or ameliorated for about 1
week, about 1 month, about 2 months, about 3 months, about 4
months, about 5 months, about 6 months, about 8 months, about 1
year, about 2 years, about 5 years, or about 10 years compared with
the tissue damage in an untreated subject with CFTR protein
dysfunction. In some embodiments, tissue damage is prevented,
delayed, or ameliorated by about 1%, about 5%, about 10%, about
20%, about 30%, about 40%, about 50%, about 60%, about 70%, about
80%, about 90%, about 95%, or about 99% compared with the tissue
damage in an untreated subject with CFTR protein dysfunction. In
some embodiments, this prevention, delay, or amelioration of tissue
damage is observed at the time points disclosed herein.
[0093] In some embodiments, administration of the pharmaceutical
compositions disclosed herein improves respiratory function in a
subject. In some embodiments the improvement in respiratory
function is determined by measuring the forced expiratory volume in
1 second (FEV1) using methods well known in the art. In some
embodiments, respiratory function is improved by about 20%, about
30%, about 40%, about 50%, about 60%, about 70%, about 80%, about
90%, about 95%, or about 99% of the respiratory function of an
untreated subject with CFTR protein dysfunction. In some
embodiments, administration of the pharmaceutical compositions
disclosed herein improves respiratory function in a subject for
about 1 week, about 1 month, about 2 months, about 3 months, about
4 months, about 5 months, about 6 months, about 8 months, about 1
year, about 2 years, about 5 years, and/or about 10 years compared
to the respiratory function of an untreated subject with CFTR
protein dysfunction. In some embodiments, the respiratory function
is improved in the subject at the time points disclosed herein.
[0094] In some embodiments, administration of the pharmaceutical
compositions disclosed herein improves water transport across cell
membranes in a subject. In some embodiments, the water transport is
improved by about 20%, about 30%, about 40%, about 50%, about 60%,
about 70%, about 80%, about 90%, about 95%, or about 99% of the
rate of water transport of an untreated subject with CFTR protein
dysfunction. In some embodiments, administration of the
pharmaceutical compositions disclosed herein improves water
transport across cell membranes in a subject for about 1 week,
about 1 month, about 2 months, about 3 months, about 4 months,
about 5 months, about 6 months, about 8 months, about 1 year, about
2 years, about 5 years, and/or about 10 years compared to the rate
of water transport of an untreated subject with CFTR protein
dysfunction. In some embodiments, the rate of water transport
across cell membranes is improved in the subject at the time points
disclosed herein.
[0095] In some embodiments, administration of the pharmaceutical
compositions disclosed herein increases CFTR protein function in a
subject. In some embodiments, the CFTR protein function is the
transport of chloride ions across the cell membrane. In some
embodiments, administration of the pharmaceutical compositions
disclosed herein increases CFTR protein function by about 20%,
about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,
about 90%, about 95%, or about 99% compared with the CFTR protein
function in an untreated subject with CFTR protein dysfunction. In
some embodiments, administration of the pharmaceutical compositions
disclosed herein increases CFTR protein function in a subject for
about 1 week, about 1 month, about 2 months, about 3 months, about
4 months, about 5 months, about 6 months, about 8 months, about 1
year, about 2 years, about 5 years, and/or about 10 years compared
with CFTR protein function in an untreated subject with CFTR
protein dysfunction. In some embodiments, the degree of increased
CFTR protein function is observed in the subject at the time points
disclosed herein.
[0096] In some embodiments, administration of the pharmaceutical
compositions disclosed herein increases CFTR protein function more
than other CFTR protein dysfunction treatments. In some
embodiments, the CFTR protein function is the transport of chloride
ions across the cell membrane. In some embodiments, the other CFTR
protein dysfunction treatments are CFTR correctors, CFTR
potentiators, and/or nonsense mutation suppressors (e.g. ataluren).
In some embodiments, the other Cystic Fibrosis treatment is a
combination treatment. In some embodiments, the combination
treatment is a combination of a CFTR corrector and a CFTR
potentiator. In some embodiments, the other CFTR protein
dysfunction treatments are VX770, VX809, or VX661. In some
embodiments, administration of the pharmaceutical compositions
disclosed herein increases CFTR protein function by about 20%,
about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,
about 90%, about 95%, or about 99% compared with the CFTR protein
function in a CFTR protein dysfunction subject treated with the
other CFTR protein dysfunction treatment. In some embodiments,
administration of the pharmaceutical compositions disclosed herein
increases CFTR protein function in a subject for about 1 week,
about 1 month, about 2 months, about 3 months, about 4 months,
about 5 months, about 6 months, about 8 months, about 1 year, about
2 years, about 5 years, and/or about 10 years compared with the
CFTR protein function in a subject with CFTR protein dysfunction
treated with the other CFTR protein dysfunction treatment. In some
embodiments, the degree of increased CFTR protein function is
observed in the subject at the time points disclosed herein.
[0097] In some embodiments, administration of the pharmaceutical
compositions disclosed herein increases the density of the CFTR
protein at the apical cell membrane in a subject. In some
embodiments, administration of the pharmaceutical compositions
disclosed herein increases the density of the CFTR protein at the
apical cell membrane in a subject as measured by immunoblotting. In
some embodiments, the CFTR protein is not recycled as quickly as in
an untreated subject with CFTR protein dysfunction. In some
embodiments, administration of the pharmaceutical compositions
disclosed herein increases the density of the CFTR protein at the
apical cell membrane by about 20%, about 30%, about 40%, about 50%,
about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%
compared to the density of the CFTR protein at the apical cell
membrane in an untreated subject with CFTR protein dysfunction. In
some embodiments, administration of the pharmaceutical compositions
disclosed herein increases the density of the CFTR protein at the
apical cell membrane in a subject for about 1 week, about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months,
about 6 months, about 8 months, about 1 year, about 2 years, about
5 years, and/or about 10 years compared with the density of the
CFTR protein at the apical cell membrane in an untreated subject
with CFTR protein dysfunction. In some embodiments, the degree of
increased localization of the CFTR protein in the cell membrane is
observed in the subject at the time points disclosed herein.
[0098] In some embodiments, administration of the pharmaceutical
compositions disclosed herein corrects CFTR protein maturation and
membrane expression in a subject. In some embodiments, this
correction of CFTR protein maturation and membrane expression is
measured by immunoblotting or immunostaining with molecules that
bind to CFTR. In some embodiments, administration of the
pharmaceutical compositions disclosed herein increases the CFTR
immunoblot or immunostain signal by about 20%, about 30%, about
40%, about 50%, about 60%, about 70%, about 80%, about 90%, about
95%, or about 99% compared to the CFTR immunoblot or immunostain
signal in an untreated subject with CFTR protein dysfunction. In
some embodiments, administration of the pharmaceutical compositions
disclosed herein increases the CFTR immunoblot or immunostain
signal for about 1 week, about 1 month, about 2 months, about 3
months, about 4 months, about 5 months, about 6 months, about 8
months, about 1 year, about 2 years, about 5 years, and/or about 10
years compared with the CFTR immunoblot or immunostain signal in an
untreated subject with CFTR protein dysfunction. In some
embodiments, the degree of increased CFTR immunoblot or immunostain
signal is observed in the subject at the time points disclosed
herein.
Pharmaceutical Compositions and Administration
[0099] The present disclosure provides pharmaceutical compositions
including a Vasoactive Intestinal Peptide and one or more ELPs with
one or more pharmaceutically acceptable excipients and/or diluents.
For example, such excipients include salts, and other excipients
that may act to stabilize hydrogen bonding. Exemplary salts include
alkaline earth metal salts such as sodium, potassium, and calcium.
Counter ions include chloride and phosphate. Exemplary salts
include sodium chloride, potassium chloride, magnesium chloride,
calcium chloride, and potassium phosphate.
[0100] The protein concentration in the formulation is tailored to
drive the formation of the matrix at the temperature of
administration. For example, higher protein concentrations help
drive the formation of the matrix, and the protein concentration
needed for this purpose varies depending on the ELP series used.
For example, in embodiments using an ELP1-120, or amino acid
sequences with comparable transition temperatures, the protein is
present in the range of about 1 mg/mL to about 200 mg/mL, or is
present in the range of about 5 mg/mL to about 125 mg/mL. The
vasoactive intestinal peptide portion of the fusion protein in the
therapeutic composition may be present in the range of about 10
mg/mL to about 50 mg/mL, or about 15 mg/mL to about 30 mg/mL, or
about 10-20 mg/ml, or about 5-15 mg/ml, or about 1-10 mg/ml. In
embodiments using an ELP4-120, or amino acid sequences with
comparable transition temperatures, the protein is present in the
range of about 0.005 mg/mL to about 10 mg/mL, or is present in the
range of about 0.01 mg/mL to about 5 mg/mL. In some embodiments,
the vasoactive intestinal peptide is present in the range of about
0.5 mg/mL to about 200 mg/mL, or is present in the range of about 5
mg/mL to about 125 mg/mL. In some embodiments, vasoactive
intestinal peptide is present in the range of about 10 mg/mL to
about 50 mg/mL, or the range of about 15 mg/mL to about 30
mg/mL.
[0101] The pharmaceutical composition is generally prepared such
that it does not form the matrix at storage conditions. Storage
conditions are generally less than the transition temperature of
the formulation, such as less than about 32.degree. C., or less
than about 30.degree. C., or less than about 27.degree. C., or less
than about 25.degree. C., or less than about 20.degree. C., or less
than about 15.degree. C., or less than about 10.degree. C. The
storage condition may, alternatively, be below freezing, such as
less than about -10.degree. C., or less than about -20.degree. C.,
or less than about -40.degree. C., or less than about -70.degree.
C. For example, the formulation may be isotonic with blood or have
an ionic strength that mimics physiological conditions. For
example, the formulation may have an ionic strength of at least
that of 25 mM Sodium Chloride, or at least that of 30 mM Sodium
chloride, or at least that of 40 mM Sodium Chloride, or at least
that of 50 mM Sodium Chloride, or at least that of 75 mM Sodium
Chloride, or at least that of 100 mM Sodium Chloride, or at least
that of 150 mM Sodium Chloride. In certain embodiments, the
formulation has an ionic strength less than that of 0.9% saline. In
some embodiments, the pharmaceutical composition includes two or
more of calcium chloride, magnesium chloride, potassium chloride,
potassium dihydrogen phosphate, potassium hydrogen phosphate,
sodium chloride, sodium dihydrogen phosphate and disodium hydrogen
phosphate. The liquid pharmaceutical composition can be stored
frozen, refrigerated or at room temperature.
[0102] In exemplary embodiments, the disclosure provides a
sustained release pharmaceutical composition that includes a
vasoactive intestinal peptide or derivatives thereof (e.g. having
an N-terminal moiety such as a Methionine) and one or more amino
acid sequences including [VPGXG].sub.90, or [VPGXG].sub.120, where
each X is selected from V, G, and A. V, G, and A may be present at
a ratio of about 5:3:2, of about 7:2:0, of about 7:0:2, of about
6:0:3, or of about 5:2:2. Alternatively, the amino acid sequence
includes [VPGVG].sub.90 or [VPGVG].sub.120. In exemplary
embodiments, the disclosure provides a sustained release
pharmaceutical composition that includes a vasoactive intestinal
peptide or derivatives thereof (e.g. having an N-terminal moiety
such as a Methionine) and one or more amino acid sequences
including [XPGVG].sub.144, where each X is selected from V, G, and
A. V, G, and A may be present at a ratio of about 5:0:4.
Alternatively, the amino acid sequence includes [XPGVG].sub.144.
The formulation further includes one or more pharmaceutically
acceptable excipients and/or diluents for formation of a reversible
matrix from an aqueous form upon administration to a human subject.
VIP and derivatives thereof are disclosed in U.S. Patent
Publication No. 2011/0178017.
[0103] Other formulation components for achieving the desired
stability, for example, may also be employed. Such components
include one or more amino acids or sugar alcohol (e.g., mannitol),
surfactants (e.g. polysorbate 20, polysorbate 80), preservatives,
and buffering agents (e.g. histidine), and such ingredients are
well known in the art. In certain embodiments, the pharmaceutical
compositions disclosed herein have enhanced efficacy,
bioavailability, therapeutic half-life, persistence, degradation
assistance, etc.
[0104] Advantageously, the compositions provide for prolonged
pharmacokinetic exposure due to sustained release of the active
agent. In particular aspects, the maximal exposure level may be
achieved at about 10 hours, about 24 hours, about 48 hours or about
72 hours after administration; typically the maximum exposure rate
is achieved between about 10 hours and about 48 hours after
administration. After the maximal exposure rate is achieved the
compositions may achieve a sustained rate of release whereby a
substantial percentage of the maximal rate is obtained for a period
of time. For example, the sustained rate may about 50%, about 60%,
about 70%, about 80%, about 90% or about 100%. Exemplary periods of
time for maintaining the sustained rate are about 3 days, about 4
days, about 5 days, about 6 days, about 1 week, about 2 weeks,
about 4 weeks, about 6 weeks, or about 8 weeks, after the maximal
exposure rate is achieved. Subsequently, the sustained rate may
lower to a reduced exposure rate. Such reduced exposure rates may
be about 5%, about 10%, about 20%, about 30%, about 40%, about 50%
or about 60%.
[0105] In various embodiments, the plasma concentration of the
active agent does not change by more than a factor of 10, or a
factor of about 5, or a factor of about 3 over the course of a
plurality of administrations, such as at least 2, at least about 5,
or at least about 10 administrations of the formulation. The
administrations are substantially evenly spaced, such as, for
example, about daily, or about once per week, or from one to about
five times per month, or about once every two months, or about once
every three months.
[0106] In another aspect, the disclosure provides a method for
delivering a sustained release regimen of a vasoactive intestinal
peptide or analogues thereof. The method comprises administering
the pharmaceutical composition described herein to a subject in
need, wherein the pharmaceutical composition is administered from
about 1 to about 8 times per month. In some embodiments, the
pharmaceutical composition is administered about 1 time, about 2
times, about 3 times, and/or about 4 times per month. In some
embodiments, the pharmaceutical composition is administered weekly.
In some embodiments, the pharmaceutical composition is administered
daily. In some embodiments, the pharmaceutical composition is
administered from one to three times weekly. In some embodiments,
the pharmaceutical composition is administered once every two
weeks. In some embodiments, the pharmaceutical composition is
administered from one to two times a month. In particular
embodiments, the pharmaceutical composition is administered about 1
time per month. In some embodiments, the pharmaceutical composition
is administered about once every 2 months, about once every 3
months, about once every 4 months, about once every 5 months,
and/or about once every 6 months. In some embodiments, VIP may have
an additional moiety such as Methionine at the N-terminus to alter
the receptor binding profile, as described in U.S. Patent
Publication No. 2011/0178017. In some embodiments, VIP is fused to
ELP1 (having from about 90 to about 150 ELP units). In some
embodiments, VIP is fused to ELP4 (having from about (having from
about 90 to about 150 ELP units). The pharmaceutical composition
can be packaged in the form of pre-filled pens or syringes for
administration once per week, twice per week, or from one to eight
times per month, or alternatively filled in conventional vials and
the like.
[0107] In some embodiments, the pharmaceutical compositions
disclosed herein are administered chronically. In some embodiments,
the pharmaceutical compositions disclosed herein are administered
for about 6 months, for about 7 months, for about 8 months, for
about 9 months, for about 10 months, for about 11 months, for about
1 year, for about 2 years, for about 3 years, for about 4 years,
for about 5 years, for about 10 years or more. The pharmaceutical
compositions may be administered at any required dose and/or
frequency disclosed herein.
[0108] In some embodiments, the pharmaceutical compositions
disclosed herein are administered until CFTR protein dysfunction
symptoms improve. In some embodiments, the pharmaceutical
compositions disclosed herein are administered until CFTR protein
dysfunction symptoms are ameliorated, delayed, and/or cured.
[0109] In some embodiments, the pharmaceutical compositions
disclosed herein are administered before the patient begins to
exhibit one or more CFTR protein dysfunction symptoms. In some
embodiments, the pharmaceutical compositions disclosed herein are
administered immediately or shortly after diagnosis. In some
embodiments, the pharmaceutical compositions disclosed herein are
administered at the onset of CFTR protein dysfunction symptoms. In
some embodiments, the pharmaceutical compositions disclosed herein
are administered at the onset of an exacerbation of CFTR protein
dysfunction symptoms.
[0110] The therapeutic agent is generally for "systemic delivery,"
meaning that the agent is not delivered locally to a pathological
site or a site of action. Instead, the agent is absorbed into the
bloodstream from the injection site, where the agent acts
systemically or is transported to a site of action via the
circulation. The therapeutic agent may be administered by any known
route, such as for example, orally, intravenously, intramuscularly,
nasally, subcutaneously, intra-vaginally, and intra-rectally. In
one embodiment, the formulation is generally for subcutaneous
administration. In one embodiment, the pharmacokinetic (PK)
parameters are prolonged when the agent is administered
subcutaneously. In one embodiment, the half-life of the fusion
protein is prolonged. In one embodiment, the PK parameters when the
agent is administered subcutaneously are prolonged compared with
the agent administered by other means (e.g. intravenously). In one
embodiment, the depot of the agent is prolonged when the agent is
administered subcutaneously compared with the agent administered by
other means (e.g. intravenously).
[0111] In some embodiments, the formulation is administered about
monthly, and may be administered subcutaneously or intramuscularly.
In some embodiments, the formulation is administered about weekly,
and may be administered subcutaneously or intramuscularly. In some
embodiments, the site of administration is not a pathological site,
for example, is not the intended site of action.
[0112] The pharmaceutical compositions disclosed herein may be
administered in smaller doses and/or less frequently than unfused
or unconjugated counterparts. While one of skill in the art can
determine the desirable dose in each case, a suitable dose of the
therapeutic agent for achievement of therapeutic benefit, may, for
example, be in a range of about 1 microgram (.mu.g) to about 100
milligrams (mg) per kilogram body weight of the recipient per day,
preferably in a range of about 10 .mu.g to about 50 mg per kilogram
body weight per day and most preferably in a range of about 10
.mu.g to about 50 mg per kilogram body weight per day. In some
embodiments, the pharmaceutical composition is administered at a
low dose. In some embodiments, the pharmaceutical composition is
administered at a dose between 1 mg per kilogram per body weight
per day to about 9 mg per kilogram per body weight per day. In some
embodiments, the pharmaceutical composition is administered at
about 1 mg per kilogram body weight per day, about 3 mg per
kilogram body weight per day, and/or about 9 mg per kilogram body
weight per day. The desired dose may be presented as one dose or
two or more sub-doses administered at appropriate intervals
throughout the day. These sub-doses can be administered in unit
dosage forms, for example, containing from about 10 .mu.g to about
1000 mg, preferably from about 50 .mu.g to about 500 mg, and most
preferably from about 50 .mu.g to about 250 mg of active ingredient
per unit dosage form. Alternatively, if the condition of the
recipient so requires, the doses may be administered as a
continuous infusion.
[0113] In certain embodiments, the subject is a human, but in other
embodiments may be a non-human mammal, such as a domesticated pet
(e.g., dog or cat), or livestock or farm animal (e.g., horse, cow,
sheep, or pig).
Combination Therapies
[0114] The pharmaceutical compositions disclosed herein may be
administered with various therapies used to treat, prevent, delay,
or ameliorate symptoms of CFTR protein dysfunction, including, but
not limited to, physical therapy, oxygen therapy, respiratory
therapy, gene therapy, bronchial or postural drainage, and/or
therapeutic agents. The pharmaceutical compositions disclosed
herein may be used alone or in combination with one or more
therapeutic agents. The one or more therapeutic agents may be any
compound, molecule, or substance that exerts therapeutic effect to
a subject in need thereof.
[0115] The one or more therapeutic agents may be "co-administered",
i.e., administered together in a coordinated fashion to a subject,
either as separate pharmaceutical compositions or admixed in a
single pharmaceutical composition. By "co-administered", the one or
more therapeutic agents may also be administered simultaneously
with the present pharmaceutical compositions, or be administered
separately, including at different times and with different
frequencies. The one or more therapeutic agents may be administered
by any known route, such as orally, intravenously, intramuscularly,
nasally, subcutaneously, intra-vaginally, intra-rectally, and the
like; and the therapeutic agent may also be administered by any
conventional route. In many embodiments, at least one therapeutic
agent may be administered subcutaneously.
[0116] These one or more therapeutic agents include, but are not
limited to, antibiotics, mucolytics, CFTR potentiators (e.g.
flavones, xanthines, benzimidazoles, ivacaftor (VX-770), QBW251,
PG-01, VR-532), CFTR correctors (e.g. lumacaftor (VX-809), VX-661,
curcumin, miglustat, sidenafil, 4-phenyl-butyrate, corr-4a,
glafanine, RDR1), nonsense mutation read-through agents (e.g.
ataluren), CFTR production correctors, read-through agents, small
molecule ion channel agents, osmotic agents, RNA repair, soluble
guanylate cyclase stimulators, S-nitrosoglutathione reductase
inhibitors, DNase, antifungals, bronchodilators, nitric oxide,
anticholinergics, nonsteroidal anti-inflammatory drugs (NSAIDs),
membrane stabilizers, corticosteroids, enzyme replacement therapy,
corticosteriods, glucocorticosteroids, decongestants, and/or
antifibrotic agents (e.g. halofuginone). In some embodiments, a
CFTR potentiator and a CFTR corrector are co-administered. In some
embodiments, the pharmaceutical compositions disclosed herein are
co-administered with one or more CFTR potentiators and/or CFTR
correctors. In preferred embodiments, the pharmaceutical
compositions disclosed herein are co-administered with VX770,
VX809, and/or VX661.
[0117] In some embodiments, the co-administration of the
pharmaceutical compositions disclosed herein with one or more
therapeutic agents has a synergistic effect. In some embodiments,
the synergistic effect is on CFTR protein function. In some
embodiments, the one or more therapeutic agents are CFTR
correctors. In some embodiments, the one or more therapeutic agents
are CFTR potentiators. In some embodiments, the one or more
therapeutic agents are VX770, VX809, and/or VX661.
[0118] When two or more therapeutic agents are used in combination,
the dosage of each therapeutic agent is commonly identical to the
dosage of the agent when used independently. However, when a
therapeutic agent interferes with the metabolism of others, the
dosage of each therapeutic agent is properly adjusted.
Alternatively, where the two or more therapeutic agents show
synergistic effects, the dose of one or more may be reduced. Each
therapeutic agent may be administered simultaneously or separately
in an appropriate time interval.
[0119] It should be understood that singular forms such as "a,"
"an," and "the" are used throughout this application for
convenience, however, except where context or an explicit statement
indicates otherwise, the singular forms are intended to include the
plural. All numerical ranges should be understood to include each
and every numerical point within the numerical range, and should be
interpreted as reciting each and every numerical point
individually. The endpoints of all ranges directed to the same
component or property are inclusive, and intended to be
independently combinable.
[0120] The term "about" when used in connection with a referenced
numeric indication means the referenced numeric indication plus or
minus up to 10% of that referenced numeric indication. For example,
the language "about 50" covers the range of 45 to 55.
[0121] As used herein, the word "include," and its variants, is
intended to be non-limiting, such that recitation of items in a
list is not to the exclusion of other like items that may also be
useful in the materials, compositions, devices, and methods of this
technology. Similarly, the terms "can" and "may" and their variants
are intended to be non-limiting, such that recitation that an
embodiment can or may comprise certain elements or features does
not exclude other embodiments of the present technology that do not
contain those elements or features. Although the open-ended term
"comprising," as a synonym of terms such as including, containing,
or having, is used herein to describe and claim the disclosure, the
present technology, or embodiments thereof, may alternatively be
described using more limiting terms such as "consisting of" or
"consisting essentially of" the recited ingredients.
[0122] Unless defined otherwise, all technical and scientific terms
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
Although any methods and materials, similar or equivalent to those
described herein, can be used in the practice or testing of the
present disclosure, the preferred methods and materials are
described herein.
[0123] This disclosure is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1: Administration of PB1046 or PB1120 Rescues F508del-CFTR
Function
[0124] Highly functional F508del-CFTR can be rescued in Cystic
Fibrosis epithelial cells by correcting its misfolding to promote
membrane targeting while increasing surface stability. While VIP
has been shown to rescue this mutation in Cystic Fibrosis cells,
the brief half-life of this protein in serum (.ltoreq.1 minute)
limits its therapeutic use. Disclosed herein are long lasting,
stable VIP therapeutics, PB1120 and PB1046 which include ELP
sequences. These data demonstrate PB1120 and PB1046 rescue
F508del-CFTR.
[0125] A human nasal epithelial cell line JME/CF15, derived from a
Cystic Fibrosis patient homozygous for the F508del mutation
(Jefferson (1990)) was used to evaluate the effect of VIP compounds
on CFTR protein function. Cells were cultured at 37.degree. C. with
at 5% CO.sub.2-95% humidity in DMEM-F12 with 10% FBS and
supplemented with transferrin (5 .mu.g/ml), triiodothyronine (2
nM), insulin (5 .mu.g/ml), hydrocortisone (1.1 .mu.M), EGF (1.64
nM), epinephrine (5.5 .mu.M), and adenine (18 .mu.M). Cells were
maintained at 37.degree. C. and incubated with 900 mM VIP, 1.2
.mu.M PB1046 (SEQ ID NO: 15), or 1 .mu.M PB1120 (SEQ ID NO: 20) for
18 or 24 hours before the assaying for iodide efflux. For
comparison, cells were incubated with the CFTR correctors VX-809
(lumacaftor) or VX-661.
[0126] To evaluate the effect of test compounds on CFTR protein
function, the activity of the CFTR chloride channel was determined
by studying the efflux of iodide ions using an iodide sensitive
electrode. Cells were incubated with NaI loading buffer (136 mM
NaI, 3 mM KNO.sub.3, 2 mM Ca(NO.sub.3), 11 mM glucose, 20 mM HEPES,
pH 7.4) for 1 hour at room temperature. Extracellular NaI solution
was then removed and replaced with efflux buffer in which NaI was
replaced with NaNO.sub.3. Samples were taken and replaced at 1
minute intervals. The first 3 samples taken before addition of the
CFTR activation cocktail (time 0-2 min) were used to establish a
stable baseline of ion efflux. CFTR activation cocktail (150 .mu.M
cpt-cAMP+1 mM IBMX+10 .mu.M forskolin) was included in the efflux
buffer from time 3 minutes. NaI concentration was then measured
using an iodide sensitive electrode moved over each sample by a
computerised autosampler and the NaI efflux rate constant k
(min.sup.-1) was calculated. Iodide efflux peaks (maximum efflux
rate during stimulation-basal level) were compared.
[0127] Iodide efflux rates were measured on JME/CF15 cells
maintained at 37.degree. C. and pre-incubated with VIP, PB1046, or
PB1120 at 30-3,500 nM for 2 hours. FIG. 2 shows the iodide efflux
rates for these agents, and Table 1 shows the EC50 and plateau
concentrations (n=3-5) for each. Rescued F508del-CFTR was
stimulated by a cAMP activator cocktail added to the efflux buffer.
FIG. 3 shows the time-course of the corrector effects of these VIP
therapeutics. The activity was significantly reduced when the CFTR
inhibitor compound CFTR.sub.inh172 (20 .mu.M) was included in the
incubation, confirming that the iodide efflux was mediated by
CFTR.
TABLE-US-00001 TABLE 1 VIP PB1120 (n = 3-5) PB1046 (n = 3-5)
EC.sub.50 65 nM 140 nM 355 nM plateau concentration 900 nM 1000 nM
1200 nM
[0128] Immunoblotting was performed to visualize the localization
of the F508del-CFTR in the epithelial cells treated with VIP,
PB1120, or PB1046. As shown in FIG. 4, treatment with PB1120 and
PB1046 corrected F508del-CFTR maturation and membrane
expression.
[0129] Incubation of JME/CF15 cells at 27.degree. C., rather than
37.degree. C. allows processing of F508del-CFTR and its expression
at the cell membrane. When cells are incubated at 37.degree. C.
essentially no iodide efflux is detected above background. FIG. 5
shows that VIP, PB1046 and PB1120 were all able to correct and/or
potentiate the activity of F508del-CFTR to an equivalent or greater
extent than VX-809 and VX-661.
Example 2: Co Administration of PB1120 or PB1046 with VX-770 or
VX-809 has a Synergistic Effect on Rescuing F508del-CFTR
Function
[0130] To study the effects of administration of PB1120 or PB1046
together with CFTR potentiators or CFTR correctors, iodide efflux
assays were performed as described in Example 1.
[0131] JME/CF15 ells were acutely treated with 1 .mu.M of the CFTR
potentiator VX-770 (ivacaftor) and then treated with 350 nM PB1046
for 18 hours or 140 nM PB1120 for 24 hours. As shown in FIG. 6A,
treatment with VX-770 alone demonstrated no effect on iodide
efflux. However, when PB1046 or PB1120 were administered in
combination with VX-770 a synergistic effect on iodide efflux was
observed (FIGS. 6B and C). This synergism is also seen when cells
are treated with both 1 .mu.M PB1120 and 1 .mu.M VX-809 for 24
hours (FIG. 7).
[0132] All publications, patents, and patent publications cited are
incorporated by reference herein in their entirety for all
purposes.
[0133] This application incorporates by reference the following
publications in their entireties for all purposes: US 2001/0034050;
US 2009/0220455; U.S. Pat. No. 8,334,257; US 2013/0310538; US
2013/0172274; US 2011/0236384; U.S. Pat. Nos. 6,582,926; 7,429,458;
7,364,859; 8,178,495; US 2013/0079277; US 2013/0085099; US
2013/0143802; US 2014/0024600; US 2011/0178017; U.S. Pat. No.
7,709,227; US 2011/0123487; U.S. Pat. No. 8,729,018; US
2014/0171370; US 2013/0150291; WO/2014/113434; US 2014/0213516;
U.S. Application No. 62/082,945 filed Nov. 21, 2014; U.S.
Application No. 62/113,943 filed Feb. 9, 2015; and U.S. Application
No. 62/145,770 filed Apr. 10, 2015.
REFERENCES
[0134] Aliakbari et al. (1978) Selective localization of vasoactive
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(2014). Am. J. Physiol-Cell Physiol. 307(1):C107-109. [0136] Chappe
et al. (2008) VIP increases CFTR levels in the apical membrane of
calu-3 cells through a PKC-dependent mechanism. J. Pharmacol Exp.
Ther. 327:226-38. [0137] Chappe and Said (2012) VIP as a Corrector
of CFTR Trafficking and membrane stability. In Cystic
Fibrosis--Renewed Hopes Through Research. D. Sriramulu (Ed.).
[0138] Choi et al. (2007) Synergistic airway gland mucus secretion
in response to vasoactive intestinal peptide and carbachol is lost
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et al. (1978) Release of vasoactive intestinal polypeptide in mast
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et al. (2004) Activation of VPAC1 receptors by VIP and PACAP-27 in
human bronchial epithelial cells induces CFTR-dependent chloride
secretion. Br. J. Pharmacol. 141:698-708. [0141] Heinz-Erian et al.
(1985) Deficient vasoactive intestinal peptide innervation in the
sweat glands of cystic fibrosis patients. Science 229:1407-8.
[0142] Heinz-Erian et al. (1986) Receptors for vasoactive
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Channel in the Apical Compartments: Spatiotemporal Coupling to its
Interacting Partners. Integr Biol (Camb) April 7; 2(4): 161-177.
[0147] Lundberg et al. (1980) Vasoactive intestinal polypeptide in
cholinergic neurons of exocrine glands: Functional significance of
coexisting transmitters for vasodilation and secretion. PNAS.
77(3): 1651-5. [0148] Raju et al. (2013) Cigarette smoke induces
systemic defects in Cystic Fibrosis Transmembrane Conductance
Regulator Function. Am. J. Respir. Crit. Care. Med. 188:1321-1330.
[0149] Riordan et al. (1989) Identification of the cystic fibrosis
gene: cloning and characterization of complementary DNA. Science.
245:1066-1073. [0150] Rowe and Verkman (2013) Cystic Fibrosis
Transmembrane Regulator Correctors and Potentiators. Cold Spring
Harb Perspect Med; 3a009761. [0151] Sloane et al. (2012) A
pharmacologic approach to acquired Cystic Fibrosis transmembrane
conductance regulator dysfunction in smoking related lung disease.
PLOS One. 7:e39809. [0152] Quinton P M. (1983) Chloride
impermeability in cystic fibrosis. Nature 301:421-422. [0153]
Therapeutic Proteins: Strategies to Modulate Their Plasma
Half-Life. Kontermann, R. (Ed.) Wiley-Blackwell Press. (2012) pp.
7-9 [0154] Wine et al. (2007) Parasympathetic control of airway
submucosal glands: Central reflexes and the airway intrinsic
nervous system. Autonomic Neuroscience: Basic & Clinical.
133:35-54. [0155] Wu et al. (2011) Prospect of vasoactive
intestinal peptide therapy for COPD/PAH and asthma: A review.
Respiratory Res. 12:45-9921-12-45.
Sequence CWU 1
1
2014PRTArtificial SequenceELP component sequence 1Val Pro Gly
Gly124PRTArtificial SequenceELP component sequence 2Ile Pro Gly
Gly135PRTArtificial SequenceELP component
sequencemisc_feature(4)..(4)Xaa can be any naturally occurring or
non-natural amino acid 3Val Pro Gly Xaa Gly1 545PRTArtificial
SequenceELP component sequence 4Ala Val Gly Val Pro1
555PRTArtificial SequenceELP component
sequencemisc_feature(4)..(4)Xaa can be any naturally occurring or
non-natural amino acid 5Ile Pro Gly Xaa Gly1 565PRTArtificial
SequenceELP component sequence 6Ile Pro Gly Val Gly1
575PRTArtificial SequenceELP component
sequencemisc_feature(4)..(4)Xaa can be any naturally occurring or
non-natural amino acid 7Leu Pro Gly Xaa Gly1 585PRTArtificial
SequenceELP component sequence 8Leu Pro Gly Val Gly1
596PRTArtificial SequenceELP component sequence 9Val Ala Pro Gly
Val Gly1 5108PRTArtificial SequenceELP component sequence 10Gly Val
Gly Val Pro Gly Val Gly1 5119PRTArtificial SequenceELP component
sequence 11Val Pro Gly Phe Gly Val Gly Ala Gly1 5129PRTArtificial
SequenceELP component sequence 12Val Pro Gly Val Gly Val Pro Gly
Gly1 5135PRTArtificial SequenceELP component
sequencemisc_feature(1)..(1)Xaa can be any naturally occurring or
non-natural amino acid 13Xaa Pro Gly Val Gly1 51429PRTArtificial
SequenceM-VIP 14Met His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg
Leu Arg Lys1 5 10 15Gln Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu
Asn 20 2515634PRTArtificial SequenceM-VIP ELP1-120 15Met His Ser
Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys1 5 10 15Gln Met
Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn Val Pro Gly 20 25 30Val
Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala 35 40
45Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
50 55 60Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly
Val65 70 75 80Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly
Gly Val Pro 85 90 95Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly 100 105 110Val Gly Val Pro Gly Gly Gly Val Pro Gly
Ala Gly Val Pro Gly Gly 115 120 125Gly Val Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly Gly Gly 130 135 140Val Pro Gly Ala Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly Val145 150 155 160Pro Gly Val
Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro 165 170 175Gly
Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180 185
190Gly Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val
195 200 205Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly
Ala Gly 210 215 220Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro
Gly Val Gly Val225 230 235 240Pro Gly Gly Gly Val Pro Gly Ala Gly
Val Pro Gly Val Gly Val Pro 245 250 255Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly Gly Gly Val Pro Gly 260 265 270Ala Gly Val Pro Gly
Gly Gly Val Pro Gly Val Gly Val Pro Gly Val 275 280 285Gly Val Pro
Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly 290 295 300Val
Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val305 310
315 320Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val
Pro 325 330 335Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly
Val Pro Gly 340 345 350Val Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly Gly 355 360 365Gly Val Pro Gly Ala Gly Val Pro Gly
Gly Gly Val Pro Gly Val Gly 370 375 380Val Pro Gly Val Gly Val Pro
Gly Gly Gly Val Pro Gly Ala Gly Val385 390 395 400Pro Gly Val Gly
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 405 410 415Gly Gly
Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly 420 425
430Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala
435 440 445Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
Val Gly 450 455 460Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro
Gly Gly Gly Val465 470 475 480Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro Gly Gly Gly Val Pro 485 490 495Gly Ala Gly Val Pro Gly Val
Gly Val Pro Gly Val Gly Val Pro Gly 500 505 510Val Gly Val Pro Gly
Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly 515 520 525Gly Val Pro
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly 530 535 540Val
Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val545 550
555 560Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val
Pro 565 570 575Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro Gly 580 585 590Gly Gly Val Pro Gly Ala Gly Val Pro Gly Val
Gly Val Pro Gly Val 595 600 605Gly Val Pro Gly Val Gly Val Pro Gly
Gly Gly Val Pro Gly Ala Gly 610 615 620Val Pro Gly Gly Gly Val Pro
Gly Trp Pro625 63016636PRTArtificial SequenceMAA-VIP ELP1-120 16Met
Ala Ala His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu1 5 10
15Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn Val
20 25 30Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val
Pro 35 40 45Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
Pro Gly 50 55 60Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val
Pro Gly Gly65 70 75 80Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro Gly Gly Gly 85 90 95Val Pro Gly Ala Gly Val Pro Gly Val Gly
Val Pro Gly Val Gly Val 100 105 110Pro Gly Val Gly Val Pro Gly Gly
Gly Val Pro Gly Ala Gly Val Pro 115 120 125Gly Gly Gly Val Pro Gly
Val Gly Val Pro Gly Val Gly Val Pro Gly 130 135 140Gly Gly Val Pro
Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val145 150 155 160Gly
Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly 165 170
175Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
180 185 190Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly
Val Pro 195 200 205Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly
Gly Val Pro Gly 210 215 220Ala Gly Val Pro Gly Gly Gly Val Pro Gly
Val Gly Val Pro Gly Val225 230 235 240Gly Val Pro Gly Gly Gly Val
Pro Gly Ala Gly Val Pro Gly Val Gly 245 250 255Val Pro Gly Val Gly
Val Pro Gly Val Gly Val Pro Gly Gly Gly Val 260 265 270Pro Gly Ala
Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro 275 280 285Gly
Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly 290 295
300Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
Gly305 310 315 320Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val
Pro Gly Val Gly 325 330 335Val Pro Gly Val Gly Val Pro Gly Gly Gly
Val Pro Gly Ala Gly Val 340 345 350Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly Val Gly Val Pro 355 360 365Gly Gly Gly Val Pro Gly
Ala Gly Val Pro Gly Gly Gly Val Pro Gly 370 375 380Val Gly Val Pro
Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala385 390 395 400Gly
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 405 410
415Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val
420 425 430Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly
Val Pro 435 440 445Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val
Gly Val Pro Gly 450 455 460Val Gly Val Pro Gly Gly Gly Val Pro Gly
Ala Gly Val Pro Gly Gly465 470 475 480Gly Val Pro Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly Gly Gly 485 490 495Val Pro Gly Ala Gly
Val Pro Gly Val Gly Val Pro Gly Val Gly Val 500 505 510Pro Gly Val
Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro 515 520 525Gly
Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 530 535
540Gly Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly
Val545 550 555 560Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val
Pro Gly Ala Gly 565 570 575Val Pro Gly Gly Gly Val Pro Gly Val Gly
Val Pro Gly Val Gly Val 580 585 590Pro Gly Gly Gly Val Pro Gly Ala
Gly Val Pro Gly Val Gly Val Pro 595 600 605Gly Val Gly Val Pro Gly
Val Gly Val Pro Gly Gly Gly Val Pro Gly 610 615 620Ala Gly Val Pro
Gly Gly Gly Val Pro Gly Trp Pro625 630 6351728PRTHomo sapiens 17His
Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln1 5 10
15Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Asn 20
251850PRTArtificial SequenceM-VIP-ELP1 18Met His Ser Asp Ala Val
Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys1 5 10 15Gln Met Ala Val Lys
Lys Tyr Leu Asn Ser Ile Leu Asn Val Pro Gly 20 25 30Val Gly Val Pro
Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala 35 40 45Gly Val
501950PRTArtificial SequenceMAA-VIP 19Met Ala Ala His Ser Asp Ala
Val Phe Thr Asp Asn Tyr Thr Arg Leu1 5 10 15Arg Lys Gln Met Ala Val
Lys Lys Tyr Leu Asn Ser Ile Leu Asn Val 20 25 30Pro Gly Val Gly Val
Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro 35 40 45Gly Ala
5020633PRTArtificial SequenceVIP ELP1-120 20His Ser Asp Ala Val Phe
Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln1 5 10 15Met Ala Val Lys Lys
Tyr Leu Asn Ser Ile Leu Asn Val Pro Gly Val 20 25 30Gly Val Pro Gly
Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly 35 40 45Val Pro Gly
Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 50 55 60Pro Gly
Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro65 70 75
80Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly
85 90 95Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
Val 100 105 110Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro
Gly Gly Gly 115 120 125Val Pro Gly Val Gly Val Pro Gly Val Gly Val
Pro Gly Gly Gly Val 130 135 140Pro Gly Ala Gly Val Pro Gly Val Gly
Val Pro Gly Val Gly Val Pro145 150 155 160Gly Val Gly Val Pro Gly
Gly Gly Val Pro Gly Ala Gly Val Pro Gly 165 170 175Gly Gly Val Pro
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly 180 185 190Gly Val
Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 195 200
205Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val
210 215 220Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly
Val Pro225 230 235 240Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly
Val Gly Val Pro Gly 245 250 255Val Gly Val Pro Gly Val Gly Val Pro
Gly Gly Gly Val Pro Gly Ala 260 265 270Gly Val Pro Gly Gly Gly Val
Pro Gly Val Gly Val Pro Gly Val Gly 275 280 285Val Pro Gly Gly Gly
Val Pro Gly Ala Gly Val Pro Gly Val Gly Val 290 295 300Pro Gly Val
Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro305 310 315
320Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly
325 330 335Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro
Gly Val 340 345 350Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
Pro Gly Gly Gly 355 360 365Val Pro Gly Ala Gly Val Pro Gly Gly Gly
Val Pro Gly Val Gly Val 370 375 380Pro Gly Val Gly Val Pro Gly Gly
Gly Val Pro Gly Ala Gly Val Pro385 390 395 400Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 405 410 415Gly Gly Val
Pro Gly Ala Gly Val Pro Gly Gly Gly Val Pro Gly Val 420 425 430Gly
Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly 435 440
445Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val
450 455 460Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Gly Gly
Val Pro465 470 475 480Gly Val Gly Val Pro Gly Val Gly Val Pro Gly
Gly Gly Val Pro Gly 485 490 495Ala Gly Val Pro Gly Val Gly Val Pro
Gly Val Gly Val Pro Gly Val 500 505 510Gly Val Pro Gly Gly Gly Val
Pro Gly Ala Gly Val Pro Gly Gly Gly 515 520 525Val Pro Gly Val Gly
Val Pro Gly Val Gly Val Pro Gly Gly Gly Val 530 535 540Pro Gly Ala
Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro545 550 555
560Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly
565 570 575Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro
Gly Gly 580 585 590Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val
Pro Gly Val Gly 595 600 605Val Pro Gly Val Gly Val Pro Gly Gly Gly
Val Pro Gly Ala Gly Val 610 615 620Pro Gly Gly Gly Val Pro Gly Trp
Pro625 630
* * * * *