Note: Descriptions are shown in the official language in which they were submitted.
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VASOACTIVE INTESTINAL POLYPEPTIDE PHARMACEUTICALS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of and claims benefit of the
following applications: U.S.
application 11/245,499 filed October 7, 2005, International Application No.
PCT/US2005/036235 filed October
7, 2005, and U.S. application 11/279,238 filed April 10, 2006. The contents of
these applications are herein
incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] The invention relates to polypeptide analogs and their synthesis and
uses. More particularly, the
invention relates to synthetic polypeptide analogs related to vasoactive
intestinal polypeptide, and
pharmaceutical compositions thereof.
BACKGROUND
[0003] When food is present in the alimentary canal, cells in the gut secrete
a hormonal signal (an
"incretin"), which sensitizes the pancreas to the presence of glucose and
results in a potentiated glucose-
dependent insulin secretory response. Such a synergistic response to provide
glucose-dependent insulin release
(Kieffer TJ and Habener, JR., Endocr. Rev. 20, 876-913 (1999)) is seen for the
incretin signals, Glucagon-like
Peptide 1(GLP1) and Glucose-dependent Insulinotropic Peptide (GIP). These
incretin signals typically exhibit
short duration of action in the body, with GLP1 exhibiting a t1i2 of
approximately 1-2 minutes (Knudsen, LB., J.
Med. Chem. 47, 4128-34 (2004)). GLP 1 and GIP are cleaved by an amino
peptidase, dipeptidyl peptidase IV
(DPPIV) and thus, the naturally occurring native hormone is not generally used
in medicinal formulations. A
peptide found in the saliva of the Gila Monster (exendin 4, Exenatide, BYETTA
, Amylin Pharmaceuticals,
San Diego, California) was shown to bind to the GLP 1 receptor and exhibit
potent agonistic activity (Young,
AA, et al., Diabetes, 48: 1026-34 (1999)), thereby imparting a desirable
glucose-dependent insulin secretory
response (Nielsen LL, Young, AA, Parkes, DG., Regul. Peptides, 117, 77-88
(2004)). Exenatide and analogs of
GLP 1 have been administered to patients in need of treatment for type 2
diabetes.
[0004] Pituitary Adenylate Cyclase-Activating Peptide (PACAP) is a
neuromodulatory peptide which
stimulates PAC1, VPAC1, and VPAC2 receptors, and is emitted from nerve endings
in the pancreas. Receptors
of this general class reside in multiple tissues in the body, including in the
pancreas (Vaudry D, et al. Pharmacol
Rev 52: 269-324 (2000)). PACAP is believed to participate in the physiological
response to food in the gut and
thus appears to be complementary to the hormonal, incretin response
(Filipsson, K., et al., Am. J. Physiol.
Regulatory Integrative Comp. Physiol. 279: R424-32 (2000)). Administration
(infusion) of PACAP to human
volunteers or to rodents causes potentiated glucose-dependent insulin
secretion, but also results in
hyperglycemia (Filipsson K, Tomoe K, Holst J and Ahren B., J Clin Endocrinol
Metab 82: 3093-8 (1997)). In
contrast, Vasoactive Intestinal Polypeptide (VIP) activates only the VPACI and
VPAC2 receptors. In the
pancreas, stimulation of the VPAC2 receptors has been shown to provide a
potentiated, glucose-dependent
insulin release in response to elevated blood glucose levels similar to that
of GLP 1 or exenatide (Tsutsumi, M.,
et al,, Diabetes 51, 1453-60 (2002)). FurTliermore, VPAC2 receptors are
present on human pancreatic beta
cells. Thus, in view of the complementary physiological role of PACAP, such a
stimulus (from PACAP or
VPAC agonistic analogs) could be synergistic or alternative to incretin-like
signals in stimulating glucose-
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dependent insulin release, since a similar profile of potentiated insulin
secretion results from activation of a
second class of receptor. Such an effect would be beneficial in the treatment
of metabolic disorders, including
Type 2 Diabetes Mellitus (T2DM), metabolic acidosis, insulin resistance and
obesity. However, the lack of
blood glucose lowering by PACAP in vivo is thought to be related to its
ability to cause gluconeogenesis in the
liver and release of glucagon. These activities, as well as several side
effects (watery diarrhea, hypotension,
hepatic gluconeogenesis), are believed to be caused by activation of PAC1 and
VPAC1 receptors (Tsutsumi, M.,
et al., Diabetes 51, 1453-60 (2002)). It was therefore determined that a VPAC2
modulatory ligand could have
beneficial effects in the treatment of T2DM and have a reduced side effect
profile. In addition, the naturally
occurring native sequence of PACAP and its analogs also are typically short-
lived in the body. Therefore there
is an important medical need for selective VPAC2 modulators. VPAC2 modulators
can be either VPAC2
agonists or antagonists.
[0005] Another reptile hormone-like molecule, Heliodermin (SEQ ID NO: 80),
exhibits great selectivity for
the VPAC2 rather than for the VPAC1 receptor (Gourlet, P., et al. Ann. NY
Acad. Sci. 865: 247-52 (1998)).
Certain substitutions, such as Gln at positions 8 and 9, as well as Leu-Ala-
Lys at postions 14 through 16 may
have particular significance for receptor selectivity. However the reptile
peptides, being foreign to the human
body, can be highly antigenic in man. Although the reptile GLP 1 like molecule
is longer acting than the
mammalian incretins, synthetic exendin 4(BYETTA Amylin Pharmaceuticals, Inc.,
San Diego, CA) remains
a relatively short acting peptide (t1J2 2 hr in man) and there is a medical
need for longer-acting peptides that can
modulate glucose-dependent insulin secretion.
[0006] Treatment of preconstricted smooth muscle preparations from the lungs
of animals and humans with
VPAC2 agonists results in prompt relaxation (O'Donnell, K., et al., J.
Pharmacol. Exptl. Therapeut. 270: 1282-8
(1994)). Similarly, treatment of asthma patients with a VPAC2 agonist has been
reported to result in prompt
bronchodilatation (Linden, A., et al. Thorax 58: 217-21 (2003)).
SUMMARY OF THE INVENTION
[0007] The invention provides synthetic polypeptide analogs of PACAP and
Vasoactive Intestinal
Polypeptide (VIP), and salts thereof, in which the C-terminus comprises amino
acid residues that form an
amphipathic a -helix, said residues selected from hydrophilic amino acids
(Haa) and lipophilic amino acids
(Laa) ordered in the sequence:
(Laa Laa Haa Haa)n Laa.
wherein n=1-5. In an embodiment, n= 1 or 2.
[0008] In another embodiment of the invention, said residues selected from
hydrophilic amino acids (Haa)
and lipophilic amino acids (Laa) are ordered in the sequence:
Haa (Laa Laa Haa Haa)n Laa.
wherein n=1-5. In an embodiment, n=1 or 2.
[0009] Modifications introduced in the present polypeptide analogs of PACAP
and VIP facilitate increased
duration of action of therapeutics which activate the PACAP and VIP family of
receptors, preferably the
VPAC2 receptor. Without being bound to any particular theory, it is believed
that an increase in duration of
action may be due to the ability of the amphipathic helix in the C-terminal
region to interact with the
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phospholipids of the cell membranes in the body and thereby have a "depoting"
effect. Thus, the present
peptide analogs are thought to bind to cell membranes and then slowly re-
release to the plasma to impart its
effect distally. In contrast, if a peptide such as PACAP, VIP or GLP1 is free
in the plasma it is rapidly acted
upon by proteases or cleared by glomerular filtration into the urine (Nestor
JJ Jr., Improved Duration of Action
of Peptide Drugs. In Peptide-based Drug Design: Taylor MD, Amidon GL, Eds.;
American Chemical Society
Washington DC, 1995: 449-471).
[0010] Therefore, one aspect of the invention provides analogs to PACAP and/or
VIP, and the
physiologically active truncated analogs and homologs of same, or salts
thereof, in which the C-terminus
preferably comprises amino acid residues that form an amphipathic a -helix,
the sequence of said residues
selected from the native amino acids or selected unnatural amino acids having
the ability to stabilize said a-
helix.
[0011] Also provided are pharmaceutical compositions for the delivery of an
effective glucose-dependant
insulin releasing amount of a polypeptide analog of PACAP and/or VIP, and the
physiologically active
truncated analogs and homologs of same, or a salt thereof, in which the C-
terninus preferably comprises amino
acid residues that form an amphipathic cx helix, said residues selected from
hydrophilic amino acids (Haa) and
lipophilic amino acids (Laa) ordered in the sequence:
(Laa Laa Haa Haa)n Laa;
wherein n=1-5. In an embodiment, n= 1 or 2.
[0012] In another embodiment of the invention, said residues selected from
hydrophilic aniino acids (Haa)
and lipophilic aniino acids (Laa) are ordered in the sequence:
Haa (Laa Laa Haa Haa)n Laa.
wherein n=1-5. In an embodiment, n=1 or 2.
[0013] The invention further provides methods for treating mammalian
conditions characterized by high
blood glucose, which methods comprise administering to a mammal in need
thereof an effective glucose-
dependant insulin releasing amount of a polypeptide analog of PACAP and/or
VIP, and the physiologically
active truncated analogs and homologs of same, or a salt thereof, in which the
C-terminus preferably comprises
aniino acid that form an amphipathic a-helix, said residues selected from
hydrophilic amino acids (Haa) and
lipophilic amino acids (Laa) ordered in the following sequence:
(Laa Laa Haa Haa)n Laa;
wherein n=1-5. In an embodiment, n=1 or 2.
[0014] In another embodiment of the invention, said residues selected from
hydrophilic amino acids (Haa)
and lipophilic amino acids (Laa) are ordered in the sequence:
Haa (Laa Laa Haa Haa)n Laa.
wherein n=1-5. In an embodiment, n= 1 or 2.
[0015] The invention further provides methods for treating mammalian
conditions affected by VPAC
receptor activation, which methods comprise administering to a mammal in need
thereof an effective glucose-
dependant insulin releasing amount of a polypeptide analog of PACAP and/or
VIP, and the physiologically
active truncated analogs and homologs of same, or a salt thereof, in which the
C-terminus preferably comprises
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amino acid that form an amphipathic a-helix, said residues selected from
hydrophilic amino acids (Haa) and
lipophilic amino acids (Laa) ordered in the following sequence:
(Laa Laa Haa Haa)n Laa;
wherein n=1-5. In an embodiment, n= 1 or 2.
[0016] In another embodiment of the invention, said residues selected from
hydrophilic amino acids (Haa)
and lipophilic amino acids (Laa) are ordered in the sequence:
Haa (Laa Laa Haa Haa)n Laa.
wherein n=1-5. In an embodiment, n= 1 or 2.
[0017] The invention also includes processes for the solid phase synthesis of
polypeptide analogs of PACAP
and/or VIP, and the physiologically active truncated analogs and homologs of
same, or a salt thereof, in which
the C-terniinus preferably comprises amino acid residues that form an
amphipathic a -helix, said residues
selected from hydrophilic amino acids (Haa), and lipophilic amino acids (Laa)
ordered in the following
sequence:
(Laa Laa Haa Haa)n Laa;
wherein n=1-5. In an embodiment, n= 1 or 2.
[0018] In another embodiment of the invention, said residues selected from
hydrophilic amino acids (Haa)
and lipophilic amino acids (Laa) are ordered in the sequence:
Haa (Laa Laa Haa Haa)n Laa.
wherein n=1-5. In an embodiment, n= 1 or 2.
[0019] Processes presented herein for preparing polypeptide analogs comprise
sequentially coupling
protected amino acids on a suitable resin support, removing the side chain and
Na-protecting groups, and
cleaving the polypeptide from the resin.
[0020] In fiuther or alternative embodixnents of the invention, the method
further comprising the step of
using microwave assistance. In fnrther or alternative embodiments of the
invention, the microwave assistance
is used for synthesizing polypeptides containing at least one amino acid which
is not one of the twenty standard
amino acids.
[0021] The invention also provides DNA sequences, vectors, and plasmids for
the recombinant synthesis of
polypeptide analogs of PACAP and/or VIP, and the physiologically active
tntncated analogs and homologs of
same, or a salt thereof, in which the C-terminus comprises amino acid residues
that form an amphipathic a -
helix, said residues selected from hydrophilic amino acids (Haa) and
lipophilic amino acids (Laa) ordered in the
sequence:
(Laa Laa Haa Haa)õ Laa;
wherein n=1-5. In an embodiment, n= 1 or 2.
[0022] In another embodiment of the invention, said residues selected from
hydrophilic amino acids (Haa)
and lipophilic amino acids (Laa) are ordered in the sequence:
Haa (Laa Laa Haa Haa)n Laa.
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wherein n=1-5. In an embodiment, n= 1 or 2.
[0023] In addition, the invention provides pharmaceutical compositions and
methods for the prevention and
treatment of a variety of metabolic disorders, including diabetes, insulin
resistance, hyperglycemia, metabolic
acidosis and obesity, which are rnanifested by elevated blood glucose levels,
comprising an effective amount of
the polypeptide(s) of the invention, or salt thereof, and a pharmaceutically
acceptable carrier. In other aspects of
the invention, therapeutically effective amounts of metabolic disorder
compounds, including insulin, insulin
analogs, incretin, incretin analogs, glucagon-like peptide, glucagon-like
peptide analogs, glucose dependent
insulinotropic peptide analogs, exendin, exendin analogs, sulfonylureas,
meglitinides, biguanides, cx glucosidase
inhibitors, thiazolidinediones, peroxisome proliferator activated receptor
(PPAR) agonists, PPAR antagonists
and PPAR partial agonists may be administered in combination with the
polypeptides of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. lA, 1B, 1C, ID, and 1E are lists of exemplary polypeptide analogs
according to the invention.
Standard nomenclature using single letter abbreviations for aniino acids are
used. In certain embodiments, the
letter "X" refers to a polyethylene glycol chain or PEG having Clo-C3ooo
chain. Preferred polyethylene glycol
chains may be linear or branched and will have a molecular weight above 20
kiloDalton. In another
embodiment, the polyethylene glycol chain will have a molecular weight of 250
to 5,000 Da, preferably from
500 to 2,000 Da. The term "acyl" refers to a C2-C3o acyl chain. This chain may
comprise a linear aliphatic
chain, a branched aliphatic chain, an aralkyl chain, or an aryl chain
containing an acyl moiety. The letter "Z"
refers to lysine having a long acyl chain at the epsilon position. The term
"hex" refers to hexanoyl. The term
"pen" refers to pentanoyl. The terms "lau" refers to lauroyl. The term "myr"
refers to myristoyl. The term
"ste" refers to stearoyl. The term "pr" refers to propionyl. Arachidoyl refers
to a linear C20 saturated fatty acid
substituent (i.e. 20:0). The term "Be" refers to behenoyl (22:0), "Er" to
erucoyl (22:1), and "Ner" to nervonyl
(24:1).
[0025] FIG. 2 lists other polypeptide and polypeptide analogs.
[0026] FIG. 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3J, 3K, 3L, 3M, 3N, 3P, 3Q and 3R
list additional exemplary
polypeptide analogs according to the invention.
[0027] FIG. 4 shows a HPLC trace of crude product V2449 (SEQ ID NO: 96), from
normal solid phase
synthesis (product at retention time 14 min).
[0028] FIG. 5 shows a HPLC trace of crude product TP-135 (SEQ ID NO: 60) from
microwave-assisted
solid phase peptide synthesis (product at retention time 26.73 min).
[0029] FIG. 6A and 6B list preferred compounds of the present invention. FIG.
6C, 6D, 6E, 6F, 6G, and 6H
list additional exemplary polypeptide analogs according to the invention.
DETAILED DESCRIPTION OF THE INVENTION
Abbreviations and Defmitions
[0030] The one- and three-letter abbreviations for the various common
nucleotide bases and amino acids are
as recommended in Pure Appl. Chem. 31, 639-645 (1972) and 40, 277-290 (1974)
and comply with 37 CFR
1.822 (55 FR 18245, May 1, 1990). The abbreviations represent L-amino acids
unless otherwise designated as
D- or DL. Certain amino acids, both natural and non-natural, are achiral,
e.g., glycine. All peptide sequences
are presented with the N-terminal amino acid on the left and the C-terniinal
amino acid on the right.
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[0031] "Hydrophilic amino acid (Haa)" refers to an amino acid having at least
one hydrophilic functional
group in addition to those required for peptide bond formation, such as, but
not limited to, arginine, asparagine,
aspartic acid, glutamic acid, glutamine, histidine, lysine, serine, threonine,
and their homologs.
[0032] "Lipophilic amino acid (Laa)" refers to an uncharged, aliphatic or
aromatic ami.no acid, such as, but
not limited to, isoleucine, leucine, methionine, phenylalanine, tryptophan,
tyrosine, valine, and their homologs.
[0033] In the invention, alanine is classified as "ambiphilic" i.e., capable
of acting as either hydrophilic or
lipophilic.
[0034] "Homolog of PACAP or VIP" refers to a polypeptide comprising amino
acids in a sequence that is
substantially similar to the native sequence of PACAP or VIP, such as at least
50, 60, 70, 80, 85, 90, 91, 92, 93,
94, 95, 96, 97, 98, or 99% amino acid sequence identity. Homologs presented
herein may comprise aniino acid
substitutions, deletions, and/or insertions relative to the native sequence of
PACAP or VIP. Exemplary
homologs comprise a span of at least 5, 10, 15, 20, 25, 30, or 35 contiguous
amino acids that is identical or
substantially similar to the native sequence of PACAP or VIP.
[0035] "Analogs of PACAP or VIP" refers to a polypeptide comprising: (i)
PACAP, VIP, and/or homologs
of PACAP or VIP; and (ii) at least one functionality not present in naturally
occurring native PACAP and/or
VIP. For example, analogs can optionally comprise a functionality within the
sidechain of an amino acid or at
the amino or carboxyl terminal of the polypeptide. Exemplary functionalities
include alkyl-, aryl-, acyl-, keto-,
azido-, hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl,
ether, thiol, seleno-, sulfonyl-, borate,
boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde,
ester, thioacid, hydroxylamine,
amino group, or the like or any combination thereof. Other exemplary
functionalities that can be introduced
include, but are not limited to, amino acids comprising a photoactivatable
cross-linker, spin-labeled amino acids,
fluorescent amino acids, metal binding amino acids, metal-containing amino
acids, radioactive amino acids,
amino acids with novel functional groups, amino acids that covalently or
noncovalently interact with other
molecules, photocaged and/or photoisomerizable amino acids, amino acids
comprising biotin or a biotin
analogue, glycosylated aniino acids such as a sugar substituted serine, other
carbohydrate modified amino acids,
keto containing amino acids, amino acids comprising polyethylene glycol or
polyether, heavy atom substituted
amino acids; chemically cleavable and/or photocleavable amino acids, amino
acids with an elongated side
chains as compared to natural amino acids, e.g., polyethers or long chain
hydrocarbons, e.g., greater than about
5 or greater than about 10 carbons, carbon-linked sugar-containing amino
acids, redox-active amino acids,
amino thioacid containing amino acids, and amino acids comprising one or more
toxic moiety.
[0036] Analogs presented herein may comprise non-natural amino acids based on
natural amino acids, such
as tyrosine analogs include para-substituted tyrosines, ortho-substituted
tyrosines, and meta substituted
tyrosines, wherein the substituted tyrosine comprises an acetyl group, a
benzoyl group, an amino group, a
hydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropyl
group, a methyl group, a C6-C20
straight chain or branched hydrocarbon, a saturated or unsaturated
hydrocarbon, an 0-methyl group, a polyether
group, a nitro group, or the like. Glutamine analogs include, but are not
limited to, a-hydroxy derivatives, ,6-
substituted derivatives, cyclic derivatives, and amide substituted glutamine
derivatives. Examples of
phenylalanine analogs include, but are not limited to, meta-substituted
phenylalanines, wherein the substituent
comprises a hydroxy group, a methoxy group, a methyl group, an allyl group, an
acetyl group, or the like.
Specific examples include, but are not limited to, 0-methyl-L-tyrosine, an L-3-
(2-naphthyl)alanine, a 3-methyl-
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phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-
G1cNAc.beta.-serine, an L-Dopa, a
fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-
phenylalanine, a p-acyl-L-phenylalanine,
a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a
phosphonotyrosine, a p-iodo-
phenylalanine, a p-bromophenylalanine, a p-amino-L-phenylalanine, and an
isopropyl-L-phenylalanine, and the
like.
[0037] Generally, analogs are optionally designed or selected to modify the
biological properties of the
polypeptide, such as to modulate toxicity, biodistribution, solubility,
stability, e.g., thermal, hydrolytic,
oxidative, resistance to enzymatic degradation, and the like, facility of
purification and processing, structural
properties, spectroscopic properties, chemical and/or photochemical
properties, catalytic activity, redox
potential, half-life, ability to react with other molecules, e.g., covalently
or noncovalently, and the like.
[0038] One type of modification is designed to block proteolysis in the
tissues. For example, it is known
that the proteolytic pattern for VIP administered to inflamed lungs shows
rapid cleavage by a trypsin-like
enzyme at the Arg residue at position Arg 14 to give largely VIP 1-14 (Lilly,
C.M., et al., J. Clin. Invest. 93: 2667-
74 (1994)). Thus substitution by a non-basic amino acid at this position would
block this principal clearance
route. The use of portions of the sequence found in Heliodermin in this region
(Leu13-Leu-Ala-Lys-Leu-Ala-
Leu-G1n20) is therefore a desirable modification, especially for development
of treatments for inflammatory lung
diseases like asthma and COPD. Particularly preferred is the use of Leu at
position 14.
[0039] "Physiologically active trancated homolog or analog of PACAP or VIP"
refers to a polypeptide
having a sequence comprising less than the full complement of amino acids
found in PACAP or VIP which,
however, elicits a similar physiological response. Representative truncated
homologs and/or analogs presented
herein comprise at least 5, 10, 15, 20, 25, 30, or 35 contiguous amino acids
found in the native sequence of
PACAP or VIP. The truncated PACAP or VIP need not be fully homologous with
PACAP or VIP to elicit a
similar physiological response. PACAP or VIP are preferred, but not exclusive,
representatives of this group.
[0040] "PEG" refers to polyethylene glycol, polypropylene glycol, or
polyoxyalkylenes attached to the
peptide or protein through a linker fanctional group (see reviews - Veronese,
F.M., et al., Drug Disc.Today 10:
1451-8 (2005); Greenwald, R.B., et al., Adv. Drug Deliv. Rev. 55: 217-50
(2003); Roberts, M.J., et al., Adv.
Drug Deliv. Rev., 54: 459-76 (2002)). PEG-modified (PEGylated) proteins or
peptides can exhibit very
beneficial characteristics such as very prolonged duration of action and
reduced antigenicity, following
parenteral delivery. These beneficial characteristics are believed to be due
in part to a decreased recognition by
proteases and the reticuloendothelial system due to a shielding effect by the
PEG chain. Another very
important mechanism is by increasing the apparent molecular weight so that it
becomes greater than the cutoff
for filtration through the glomerular barrier in the kidney and into the
urine. This cutoff size is near that of
serum albumin (circa 60kDa). The highly hydrated character of the PEG chain
causes it to have an "effective
molecular weight" with respect to glomerular filtration like that of a
globular protein more than three times
larger than its true molecular weight. Thus for prolongation of duration of
action following parenteral
administration, preferred forms of PEG in the instant invention have a
molecular weight of greater than 10,000
Da and most preferred forms have a molecular weight of 20,000 Da or greater.
PEG chains may be linear or
branched molecules.
[0041] Another type of PEG chain is modified to be amphiphilic in nature. That
is it has both the
hydrophilic PEG structure but is modified to contain hydrophobic regions such
as fatty acid esters and other
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hydrophobic components (see for example Miller, M.A., et al., Bioconjug. Chem.
17: 267-74 (2006); Ekwuribe,
et al. US 6,309,633; Ekwuribe, et al. US 6,815,530; Ekwuribe, et al. US
6,835,802). Although these amphiphilic
PEG conjugates to proteins were originally developed to increase oral
bioavailability they were relatively
ineffective in this role. However the use of such amphiphilic PEG conjugates
with the amphipathic peptides of
the invention will give significantly prolonged residence in the lung to
extend the useful biological activity of
these pharmaceuticals. The preferred PEG chains are in the molecular weight
range of 500 to 3000Da. Detailed
descriptions of the methods of synthesis of these conjugates is given in the
references above, the full content of
which is incorporated herein.
[00421 PEGylation of a protein can have potentially negative effects as well.
Thus PEGylation can cause a
substantial loss of biological activity for some proteins and this may relate
to ligands for specific classes of
receptors. In such instances there is a benefit to reversible PEGylation
(Peleg-Shulman, T., et al., J. Med.
Chem. 47: 4897-4904; Greenwald, R.B., et al. Adv. Drag Del. Rev., 55: 217-
50)).
[0043] In addition, the increased molecular mass may prevent penetration of
physiological barriers other
than the glomerular membrane barrier. For example, it has been suggested that
high molecular weight forms of
PEGylation may prevent penetration to some tissues and thereby reduce
therapeutic efficacy. In addition, high
molecular weight may prevent uptake across mucosal membrane barriers (nasal,
buccal, vaginal, oral, rectal,
lung delivery). However delayed uptake may be highly advantageous for
administration of stable molecules to
the lung, substantially prolonging the duration of action.
[0044] An important aspect of the invention is the use of not just long chain
PEG polymers, but the use of
short chain versions as well. Administration of treatments for diabetes by
inhalation is an important new
approach for drug delivery and the lung has a highly permeable barrier (e.g.
Exubera). For this application,
delayed penetration of the lung barrier, preferred forns of PEGylation are in
the lower molecular weight range
of Clo to C400 (roughly 250 to 10,000Da). Thus while a primary route to
prolongation by PEG is the
achievement of an "effective molecular weight" above the glomerular filtration
cut-off (greater than 60kDa), use
of shorter chains may be an important route for prolongation of residence in
the lung for treatment of lung
diseases and other respiratory conditions. Thus PEG chains of about 500 to
3000 Da are of sufficient size to
slow the entry into the peripheral circulation, but insufficient to cause them
to have a very prolonged circulation
time, and are preferred in certain embodiments of the invnetion. Thus, in
thses embodiments of the invention,
PEGylation may be applied to give increased local efficacy to the lung tissue
with reduced potential for systemic
side effects for the compounds of the invention. For purposes of this
invention, those PEG chains in the range
from about 750 to about 1500 Da are referred collectively as "PG1K."
[0045] Polyethylene glycol chains are functionalized to allow their
conjugation to reactive groups on the
polypeptide or protein chain. Typical functional groups allow reaction with
amino, carboxy or sulfhdryl groups
on the peptide through the corresponding carboxy, amino or maleimido groups
(and the like) on the
polyethylene glycol chain. In an embodiment, PEG comprises a Clo-C30oo chain.
In another embodiment, PEG
has a molecular weight above 40,000 Daltons. In yet another embodiment, PEG
has a molecular weight below
10,000 Daltons. PEG as a protein modification is well known in the art and its
use is described, for example, in
U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and
4,179,337.
[0046] "Amphipathic a -helix" refers to the secondary structure exhibited by
certain polypeptides in which
the amino acids assume an a-helical configuration having opposing polar and
nonpolar faces oriented along the
8
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long axis of the helix. Various authors use the terms amphipathic or
amphiphilic a-helix interchangeably in
that one face is polar and one is nonpolar, and both terms are used to mean
the same type of structure herein.
[0047] The possibility of a -helical structure in the polypeptide of interest
may be explored to some extent
by the construction of a "Schiffer-Edmundson wheel" (Schiffer, M.and
Edmundson, A. B., Biophys. J. 7, 121
(1967), incorporated herein by reference), of the appropriate pitch and noting
the segregation of the hydrophilic
and lipophilic residues on opposite faces of the cylinder circumscribing the
helix. Alternatively, empirical
evidence, such as circular dichroism or x-ray diffraction data, may be
available indicating the presence of an a-
helical region in a given polypeptide. An ideal a-helix has 3.6 amino acid
residues per turn with adjacent side
chains separated by 100 of arc.
[0048] Another aspect of protein structure relevant to the invention, and in
particular those compounds of
the structure corresponding to Formula II, is the use of a polyproline type II
helix (Stapley, B.J. and Creamer,
T.P., Protein Sci 8: 587-95 (1999)) to faciLitate the development of the
amphipathic alpha helix described above.
Polyproline helices increasingly are recognized as being an important element
in protein structure and an
important aspect of that helix is its amphiphilic character. Here we make use
of such a polyproline type II helix
to facilitate that formation of the amphipathic alpha helix described above to
yield potent VPAC2 ligands. A
prominent feature of polyproline helices is the very strong preference for Pro
residues within the helix and
specific amino acids as capping residues at the N-terminus. Some examples of
favored capping residues are
Gln, Ser, Gly, Asp, Ala, Arg, Lys, Glu (Rucker AL, et al., Proteins 53: 68-75
(2003)).
[0049] Another aspect of the polyproline helix is the resistance to
proteolysis that it affords. A number of
naturally occurring peptides and proteins have polyproline regions or Pro
residues at their C-terminus, where
they may also prevent proteolytic digestion. Examples that bind to the GLP 1
receptor are Exendin-4,
heliodermin, and heliospectin.
[0050] Unless stated otherwise, standard nomenclature using single letter
abbreviations for amino acids are
used. The letter "X" refers to a polyethylene glycol chain having C10-C3000
chain. Preferred polyethylene glycol
chains may be linear or branched and will have a molecular weight above 20
kiloDalton_ In another
embodiment, the polyethylene chain will have a molecular weight of from about
250 to about 5,000 Da,
preferably from about 500 to about 2,000 Da. The term "acyl" refers to a C2-
C30 acyl chain. This chain may
comprise a linear aliphatic chain, a branched aliphatic chain, an aralkyl
chain, or an aryl chain containing an
acyl moiety. The letter "Z" refers to lysine having a long acyl chain at the
epsilon position. The term "hex"
refers to hexanoyl. The term "pen" refers to pentanoyl. The terms "lau" refers
to lauroyl. The term "myr"
refers to myristoyl. The term "ste" refers to stearoyl. The term "pr" refers
to propionyl. Arachidoyl refers to a
linear C20 saturated fatty acid substituent (i.e. 20:0). The term "Be" refers
to behenoyl (22:0), "Er" to eracoyl
(22:1), and "Ner" to nervonyl (24:1). For example, in SEQ ID NO: 25, the "Z
myr" represents "Lys(epsilon
myristoyl)," making the sequence end Leu-Lys(epsilon myristoyl)-Pro-Pro-Pro.
[0051] Although it may be apparent to an ordinary person skilled in the art, a
PEG entitiy itself does not
have a fanctional group to be attached to a target molecular, such as a
peptide. Therefore, to create PEG
attachment, a PEG entity must be functionalized first, then a functionalized
attachment is used to attach the PEG
entity to a target molecule, such as a peptide (Veronese, F.M., et al., Drug
Disc.Today 10: 1451-8 (2005);
Greenwald, R.B., et al., Adv. Drug Deliv. Rev. 55: 217-50 (2003); Roberts,
M.J., et al., Adv. Drug Deliv. Rev.,
54: 459-76 (2002)). In one embodiment, site-specific PEGylation can be
achieved through Cys substitution on a
9
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peptide molecule. The target peptide can be synthesized by solid phase
synthesis, recombinant means, other
means, as described herein. The invention discloses the combination concept of
using acylation on a Lys
residue and specific PEGylation on at least one Cys residue. Certain Lys
residues in disclosed peptide
sequences can be substutited to Cys for site-specific PEGylation.
[0052] In another embodiment of the invention, a Lys or other residue residue
with a nucleophilic side chain
may be used for incorporation of a PEG residue. This may be accomplished
through the use of an amide or
carbamate linkage to a PEG-carboxyl or PEG-carbonate chain (for example, as
described in Veronese, F.M., et
al. Drug Disc.Today 10: 1451-8 (2005)). An alternative approach for use with
the invention is to modify the
Lys side chain amino function through attachment of an SH containing residue,
such as mercaptoacetyl,
mercaptopropionyl (CO-CH2-CH2-CH2-SH), and the like. Additional methods for
attaching PEG chains utilize
reaction with the side chains of His and Trp. Other similar methods of
modifying the peptide chain to allow
attachment of a PEG chain are known in the art and are incorporated herein by
reference.
[0053] Aside from the twenty standard amino acids, there are a vast number of
"nonstandard amino acids"
which exist in various life forms that may be incorporated in the compounds of
the present invention.
Examples of nonstandard amino acids include the sulfur-containing taurine and
the neurotransmitters GABA
and dopamine. Other examples are lanthionine, 2-Aminoisobutyric acid (Aib),
and dehydroalanine.
Nonstandard amino acids often occur in the metabolic pathways for standard
aniino acids - for example
ornithine (Orn) and citrulline (Cit) occur in the urea cycle, part of amino
acid breakdown.
[0054] The term "naturally occurring amino acid" as used herein incudes both
twenty standard amino acids
and other nonstandard amino acid, including, but not limited to, Aib, Orn, and
Cit.
Polypeptides
[00551 In an embodiment, polypeptides presented herein comprise truncated
portions of PACAP and/or VIP
having at least 5, 10, 15, 20, 25, 30, or 35 contiguous amino acids of the
native sequence of PACAP or VIP. In
another embodiment, the present polypeptides share at least 50, 60, 70, 80,
85, 90, 95, or 99% amino acid
sequence identity to the native sequence of PACAP or VIP. In yet another
embodiment, the present
polypeptidescomprise a span of at least 5, 10, 15, 20, 25, 30, or 35
contiguous amino acids of PACAP and/or
VIP having at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99, or 100% amino acid sequence
identity to the native sequence of PACAP or VIP.
[0056] One type of modification is designed to block proteolysis in the
tissues. For example, it is known
that the proteolytic pattern for VIP administered to inflamed lungs shows
rapid cleavage by a trypsin-like
enzyme at the Arg residue at position Arg14 to give largely VIP 1-14 (Lilly,
C.M., et al., J. Clin: Invest. 93: 2667-
74 (1994)). Thus substitution by a non-basic amino acid at this position would
block this principal clearance
route. The use of portions of the sequence found in Heliodermin in this region
(Leu13-Leu-Ala-Lys-Leu-Ala-
Leu-G1n20) is therefore a desirable modification, especially for development
of treatments for inflammatory lung
diseases like asthma and COPD. Particularly preferred is the use of Leu at
position 14. Certain substitutions,
such as Gln at positions 8 and 9, as well as Leu-Ala-Lys at postions 14
through 16 may have particular
significance for receptor selectivity.
[0057] Polypeptides presented herein optionally comprise modifications,
functionalities, and/or amino acid
substitutions which modulate VPAC2 selectivity. Exemplary modifications,
functionalities, and/or substitutions
include, but are not limited to, C-terminal cationic extensions and/or
mutations ( Gourlet et al., Peptides 18, 403-
CA 02638968 2008-08-01
WO 2007/044591 PCT/US2006/039267
8; (1997) ; Xia M, et al., J. Pharmacol. Exp. Ther. 281: 629-33 (1997); the
contents of both of which are
incorporated herein by reference).
[0058] Modifications at the amino or carboxyl terminus may optionally be
introduced into the present
polypeptides. For example, the present polypeptides, such as analogs of VIP,
can be acylated on the N-terminus
by long chain fatty acids to yield polypeptides exhibiting low efficacy,
partial agonist and antagonist activity
(Gourlet et al., Eur. J. Pharmacol. 354: 105-111 (1998)), the contents of
which is incorporated herein by
reference). Modification of the peptides of this invention with longer chain
fatty acids at the N-terminus,
similarly will yield antagonists with a prolonged duration of action (Moreno
D, et al., Peptides 21: 1543-9
(2000)). Other modifications to the N-terminus, such as deletions or
incorporation of D-amino acids such as D-
Phe also give potent and long acting antagonists when substituted into the
compounds of Formulas 1-3. Such
antagonists also have commercial utility and are within the scope of this
invention.
[0059] Other exemplary modifications of the present polypeptides, such as
analogs of VIP and/or PACAP,
include acylation with hexanoic acid to yield polypeptides that exhibit
increased selectivity towards VPAC2
(Langer et al., Peptides 25: 275-8 (2004)), the contents of which is
incorporated herein by reference). Thus the
length and positioning of such acylation is important since it can alter
efficacy, and could result in loss of
efficacy (antagonistic) or agonistic analogs. Contrary to this
unpredictability, polypeptides of the type presented
herein have been designed and tested to obtain desired efficacy and activity.
[0060] Another very favorable aspect of N-terminal acylation is the blockade
of rapid proteolysis by DPPN
seen for the parent peptide due to such acylation. Thus although PACAP and VIP
have very short duration of
action in vivo, the peptides of the invention preferably have a principal
proteolysis route blocked by this N-
terminal acylation.
[0061] Modifications may optionally be introduced within the side chain of at
least one amino acid within
the present polypeptides to increase duration of action and/or potency. For
example, the present polypeptides
can optionally comprise at least one amino acid acylated to a functionality in
the side chain (i.e., R group).
Representative modifications include fatty acid acylation, directly or through
linkers, of reactive side chains
(such as Lys) at various positions within the polypeptide. Similar
modifications have been reported in Kurtzhals
et al. where acylation of insulin on LysB29 resulted in insulin detemir
(Kurtzhals et al., Biochem J. 312, 725-31
(1995) and Kurtzhals, P., Int. J. Obesity 28: Supp12, S23-8 (2004)).
Similarly, acylation with long chain fatty
acids through linkers (preferably Glu) has resulted in potent and long-acting
analogs of GLP 1(Knudsen L.B., et
al., J. Med. Chem. 43: 1664-69 (2000)), but the acylation can result in loss
of activity or potent agonists,
depending on the length and positioning of the acyl chain(s). Contrary to the
unpredictable effects with the
introduction of long chain fatty acids, polypeptides presented herein have
been designed to incorporate an
optimal number, length and positioning of the acyl chains so as to obtain
desired activity. Such linkage is
demonstrated here for direct acylation to Lys, but linkage through other
linkers, such as Glu (Knudsen, LB, et
al., J. Med. Chem. 43: 1664-9 (2000)), is also within the scope of the present
invention.
[0062] Another type of modification that can optionally be introduced into the
present polypeptides (e.g.
within the polypeptide chain or at either the N- or C-terminal) to extend
duration of action is PEGylation or
incorporation of long-chain polyethylene glycol polymers (PEG). Introduction
of PEG or long chain polymers
of PEG increases the effective molecular weight of the present polypeptides to
prevent rapid filtration into the
urine. Any Lys residue in any peptide analog sequence may be conjugated to PEG
directly or through a linker
11
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WO 2007/044591 PCT/US2006/039267
to yield a potent and long acting analog. Such linker can be a Glu residue or
an acyl residue containing a thiol
functional group for linkage to the appropriately modified PEG chain. An
alternative method for introducing a
PEG chain is to first introduce a Cys residue at the C-terminus or at solvent
exposed residues such as
replacements for Arg or Lys residues. This Cys residue is then site-
specifically attached to a PEG chain
containing, for example, a maleimide function. Methods for incorporating PEG
or long chain polymers of PEG
are well known in the art (described, for example, in Veronese, F.M., et al.,
Drug Disc.Today 10: 1451-8 (2005);
Greenwald, R.B., et al., Adv. Drug Deliv. Rev. 55: 217-50 (2003); Roberts,
M.J., et al., Adv. Drug Deliv. Rev.,
54: 459-76 (2002)), the contents of which is incorporated herein by reference.
[00631 A more recently reported alternative approach for incorporating PEG or
PEG polymers through
incorporation of non-natural amino acids can be performed with the present
polypeptides. This approach
utilizes an evolved tRNA/ tRNA synthetase pair and is coded in the expression
plasmid by the amber suppressor
codon (Deiters, A, et al. (2004). Bio-org. Med. Chem. Left. 14, 5743-5). For
example, p-azidophenylalanine can
be incorporated into the present polypeptides and then reacted with a PEG
polymer having an acetylene moiety
in the presence of a reducing agent and copper ions to facilitate an organic
reaction known as "Huisgen [3+2]
cycloaddition."
Amphipathic Helix
[0064] Polypeptides of the present invention comprise amphipathic a -helix
corresponding to the formula:
(Laa Laa Haa Haa)n Laa
wherein n = 1-5, each Haa is independently selected from the group of
hydrophilic amino acids and each Laa is
independently selected from the group of lipophilic amino acids, as defined
above.
[0065] In another embodiment of the invention, said residues selected from
hydrophilic amino acids (Haa)
and lipophilic amino acids (Laa) are ordered in the sequence:
Haa (Laa Laa Haa Haa)II Laa.
wherein n=1-5. In an embodiment, n= 1 or 2.
[0066] Polypeptides of the present invention comprise a peptide region that is
an amphipathic alpha helix,
not merely an alpha helix. Without wishing to be bound by any particular
theory, the amphipathic alpha helix
is believed to facilitate increased interaction with cell membranes and assist
in proper placement of C-terminal
fatty acyl chain modifications for membrane interaction. In addition, and
without being bound to any particular
theory, it is believed that the amphipathic helix in the C-terminal region
imparts an increase in duration of action
of the present polypeptides by interacting with the phospholipids of the cell
membranes in the body and thereby
have a "depoting" effect. Further, addition of positive charge in this
amphipathic alpha helical region can
significantly increase the binding to the negatively charged phospholipid
membrane. Such a charged region
generates increased Guoy-Chapman forces that cause the peptide to accumulate
on the membrane. This can be
beneficial in further prolonging the duration of action and increasing the
amount of peptide in the biologically
active conformation in proximity to the VPAC2 receptors in the cell membranes.
[0067] Studies by Eisenberg et al. have combined a hydrophobicity scale with
the helical wheel to quantify
the concept of amphipatlii.c helices (Nature 299: 371-374 (1982) and Proc.
Nat. Acad. Sci. USA 81: 140-144
(1984); the disclosures of which are hereby incorporated by reference). The
mean hydrophobic moment is
defined as the vector sum of the hydrophobicities of the component aniino
acids making up the helix. The
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WO 2007/044591 PCT/US2006/039267
following hydrophobicities for the amino acids are those reported by Eisenberg
et al. as the "consensus" scale:
Ile 0.73; Phe 0.61; Va10.54; Leu 0.53; Trp 0.37; Met 0.26 Ala 0.25; Gly 0.16;
Cys 0.04; Tyr 0.02; Pro -0.07;
Thr -0.18; Ser -0.26; His -0.40; Glu -0.62; Asn -0.64; Gln -0.69; Asp -0.72;
Lys -1.10; Arg -1.76.
[0068] The hydrophobic moment, .H, for an ideal a-helix having 3.6 residues
per turn (or a 100 arc
(=360 /3.6) between side chains), may be calculated from:
H =[( EiN sine 5(N-1)2 +( EEIN cos 8(N-1))2]112,
where HN is the hydrophobicity value of the Nffi amino acid and the sums are
taken over the N amino acids in the
sequence with periodicity 5 =100 . The hydrophobic moment may be expressed as
the mean hydrophobic
moment per residue by dividing H by N to obtain < H >. A value of < H > at
100 +-.20 of about 0.20 or
greater is suggestive of amphipathic helix formation.
[0069] A study by Cornett et al. has further extended the study of
amphiphathic a-helices by introducing the
"amphipathic index" as a predictor of amphipathicity (J. Mol. Biol., 195: 659-
685 (1987); the disclosure of
which is hereby incorporated by reference). They concluded that approximately
half of all known a -helices are
amphipathic, and that the dorninant frequency is 97.5 rather than 100 , with
the number of residues per turn
being closer to 3.7 than 3.6. The basic approach of Eisenberg, et al. is
sufficient to classify a given sequence as
amphipathic, particularly when one is designing a sequence ab initio to form
an amphipathic a -helix.
[0070] A substitute amphipathic a -helical amino acid sequence may lack
homology with the sequence of a
given segment of a naturally occurring polypeptide but elicits a similar
secondary structure, i.e. an a -helix
having opposing polar and nonpolar faces, in the physiological environment.
Replacement of the naturally
occurring amino acid sequence with an alternative sequence may beneficially
affect the physiological activity,
stability, or other properties of the altered parent polypeptide. Exemplary
reports describing the design and
selection of such sequences is provided in J. L. Krstenansky, et al., FEBS
Letters 242: 2, 409-413 (1989), and J.
P. Segrest, et al. Proteins: Structure, Function, and Genetics 8: 103-117
(1990) among others.
[00711 Polypeptides of the present invention comprise amphipathic a -helix
corresponding to the forinula:
(Laa Laa Haa Haa)n Laa
wherein each Haa is independently selected from the group of hydrophilic amino
acids and each Laa is
independently selected from the group of lipophilic amino acids, as defmed
above.
[0072] In another embodiment of the invention, said residues selected from
hydrophilic amino acids (Haa)
and lipophilic amino acids (Laa) are ordered in the sequence:
Haa (Laa Laa Haa Haa)n Laa.
wherein n=1-5. In an embodiment, n=1 or 2.
[0073] Assuming an idealized a-helix in an embodiment where n=2, residues 1,
4, 5, 8, and 9 are
distributed along one face (A) of the helix within about a 140 arc of each
other, while residues 2, 3, 6, 7, and 10
occupy an opposing 140 arc on the other face (B) of the helix. In an
embodiment, all the residues on one face
are of the same polarity while all those on the other face are of the opposite
polarity, i.e., if face A is all
hydrophilic, face B is all lipophilic and vice versa. The skilled artisan will
recognize that while the helices of
the invention are described by
(Laa Laa Haa Haa)n Laa,
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WO 2007/044591 PCT/US2006/039267
the reverse sequence,
Laa (Haa Haa Laa Laa)n
will also meet the residue distribution criteria and is an equivalent
descriptor of the helices of the invention.
[0074] Accordingly, in another embodiment of the invention, the skilled
artisan will recognize that while the
helices of the invention are described by
Haa (Laa Laa Haa Haa)n Laa,
the reverse sequence,
Laa (Haa Haa Laa Laa)n Haa
will also meet the residue distribution criteria and is an equivalent
descriptor of the helices of the invention.
[0075] Alanine may be substituted for either hydrophilic or lipophilic amino
acids, since Ala can reside
readily on either face of an amphipathic a -helix, although Ala-10 does not
form an amphipathic a -helix.
Generally, proline, cysteine, and tyrosine are not used; however, their
presence and other random errors in the
sequence may be tolerated, e.g. a hydrophilic residue on the lipophilic face,
as long as the remaining amino
acids in the segment substantially conform to the hydrophilic face--lipophilic
face division. A convenient
method for determining if a sequence is sufficiently amphipathic to be a
sequence of this invention is to
calculate the mean hydrophobic moment, as defmed above. If the peak mean
moment per residue at 1000 +-20
exceeds about 0.20, then the sequence will form an amphipathic helix and is a
sequence of the invention.
[0076] In applying this concept to PACAP and VIP, it is hypothesized that
either or both regions (N-
terminal or C-terminal), preferably the C-terminal, may exhibit a-helical
secondary structure and could be
replaced with a non-homologous sequence having similar structural tendencies,
without loss of biological
activity or induction of immunoreaction.
Pharmaceutical Formulations
[0077] Polypeptides of the present invention may be administered in any amount
to impart beneficial
therapeutic effect. In a preferred embodiment, compounds of the present
invention are useful in the treatment
of elevated blood glucose levels, hyperglyceinia, diabetes, including Type 2
Diabetes Mellitus, insulin
resistance, metabolic acidosis and obesity. In an embodiment, compounds
presented herein impart beneficial
activity in the modulation of insulin and/or glucose levels. In an embodiment,
the present polypeptides are
adtninistered to a patient at concentrations higher or lower than that of
other forms of treatment which modulate
insulin and/or glucose secretion. In yet another embodiment, the present
polypeptides are administered with
other compounds to produce a synergistic therapeutic effect. For example,
polypeptides of the invention may
be administered in conjunction with exendin-4 or exendin analogs.
EXAMPLES
Example 1: Synthetic Analogs
[0078] Some of the exemplary synthetic polypeptide analogs illustrated in
FIGs. 1A-1E and 3A-3R are
derived from VPAC2 sel U1dB (see FIGs. 1 and 3). Other exemplary synthetic
polypeptide analogs illustrated
in FIGs. lA-lE and 3A-3R are truncated homologs of VIP (see FIGs. 1 and 3).
[0079] In one aspect, the present polypeptide analogs of the physiologically
active truncated homologs of
VIP, such as those shown in FIG. 1 as TP 1 to TP 6. Analogs TP 1 to TP 6 have
a long acyl residue comprising
C12-C24, preferably C16-C24. Analogs TP 7 to TP 12 shown in FIG. 1 have an
acyl residue on the N-terminus
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comprising C2-C16, preferably C6. Analogs SQNM 10-12 (corresponding to SEQ ID
NO: 76-78) shown in
FIG. 2 do not contain acylation at either the C or N-termini.
[00801 Other representative polypeptide analogs presented herein have amino
acid sequences corresponding
to general formula (I):
Acyl-His-Ser-Asp-Xaa4 -Xaa5 -Phe-Thr-Xaa8 -Xaa9 -Tyr-Xaa11-Arg-Xaa13 -Xaala -
Xaal5 -Xaa16 -Xaal7
-Ala-Xaa19 -XaaZo -Xaa21-Tyr-Leu- Xaa24 -Xaa25 -Xaa26 -Xaa27 -XaaZs- Xaa29 -
Xaa30 -Xaa31 -Xaa32
-(Laa-Laa-Haa-Haa)II Laa-Lys(s-long acyl)-X (SEQ ID NO: 81)
Forn-ula (I)
wherein:
n=1-5;
each Haa is independently a hydrophilic amino acid;
each Laa is independently a lipophilic amino acid;
acyl is a C2_I6 acyl chain;
long acyl is a C12_30 acyl chain;
X is selected from the group consisting of OH, Cys(PEG), PEG, and NHRI,
wherein Rl is selected
from H, lower alkyl, or haloalkyl;
PEG is a functionalized polyethylene glycol chain of CIo-C3ooo chain;
Xaa4 is Gly or Ala;
Xaa5 is Val, Ile, or Leu;
Xaa8 is Asp, Arg, Gln, or Glu;
Xaa9 is Ser, Asn, Gin, Asp or Glu;
Xaa11 is Ser or Thr;
Xaa13 is Leu or Tyr;
Xaa14 is Arg or Leu;
Xaa15 is Lys, Leu, or Arg;
Xaa16 is Gln, Lys or Ala;
Xaa17 is Met, Leu, Val or Ala;
Xaa19 is Ala or Val;
Xaa20 is Lys, Arg or Gln;
Xaa21 is Lys, Arg or Gln;
Xaa24 is Asn, Gln, Ala or Glu;
Xaa25 is Trp, Ala, or Ser;
Xaa26 is Ile, Val or Trp;
Xaa27 is Leu, Lys, Arg or Gln;
XaaZ$ is Lys, Arg, Asn, Gln, or Gly;
Xaa29 is Ala, Gly, Gln, Lys or Arg;
Xaa3o is Lys, Arg, Leu, Ala or absent;
Xaa31 is Lys, Arg, Leu, Ala or absent; and
Xaa32 is any naturally occurring amino acid or absent;
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WO 2007/044591 PCT/US2006/039267
provided that if any of Xaa30i Xaa31, or Xaa32 is absent, the next amino acid
present downstream is the next
amino acid in the peptide agonist sequence. In a preferred embodiment, acyl is
a C2_8 acyl chain and long acyl is
a C12.30 acyl chain. In certain embodiments, Xaa32 is a hydrophilic amino acid
(Haa).
[0081] Other representative polypeptide analogs presented herein have amino
acid sequences corresponding
to general formula (II):
Acyl-His-Ser-Asp-Xaa4 -Xaa5 -Phe-Thr-Xaa8 -Xaay -Tyr-Xaall -Arg-Xaa13 -Xaa14 -
Xaa1s -Xaa16 -Xaal7
-Ala-Xaa19 -Xaa20 -Xaa21 -Tyr-Leu-Xaa24 -Xaa25 -Xaa26 -Xaa27 -Xaa28 -Xaa2g -
Xaa30 -Xaa31 -Xaa32
-(Laa-Laa-Haa-Haa)Il Laa-Xaa54 -Pro-Pro-Pro-Lys(s-long acyl)-X (SEQ ID NO: 82)
Formula (II)
wherein:
each Haa is independently a hydrophilic amino acid;
each Laa is independently a lipophilic amino acid;
acyl is a C2_16 acyl chain;
long acyl is a C12_30 acyl chain;
X is selected from the group consisting of OH, Cys(PEG), PEG, and NHRI,
wherein RI is selected
from H, lower alkyl, or haloalkyl;
PEG is a functionalized polyethylene glycol chain of Clo-C3ooo chain;
Xaa4 is Gly or Ala;
Xaa5 is Val, Ile, or Leu;
Xaa$ is Asp, Arg, Gln, or Glu;
Xaa9 is Ser, Asn, Gln, Asp or Glu;
Xaall is Ser or Thr;
Xaa13 is Leu or Tyr;
Xaa14 is Arg or Leu;
Xaa15 is Lys, Leu, or Arg;
Xaa16 is Gln, Lys or Ala;
Xaa17 is Met, Leu, Val or Ala;
Xaa19 is Ala or Val;
XaaZo is Lys, Arg or Gln;
XaaZl is Lys, Arg or Gln;
Xaa24 is Asn, Gln, Ala or Glu;
Xaa25 is Trp, Ala, or Ser;
Xaa26 is Ile, Val or Trp;
Xaa27 is Leu, Lys, Arg or Gln;
Xaa28 is Lys, Arg, Asn, Gln, or Gly;
Xaa29 is Ala, Gly, Gln, Lys or Arg;
Xaa3o is Lys, Arg, Leu, Ala or absent;
Xaa31 is Lys, Arg, Leu, Ala or absent;
Xaa32 is any naturally occurring amino acid or absent; and
Xaa54 is Gln, Ser, Gly, Asp, Ala, Arg, Lys, Glu, Pro, Asn, Leu, or absent;
16
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provided that if any of Xaa30i Xaa31, Xaa32, or Xaa54 is absent, the next
amino acid present downstream is the
next amino acid in the peptide agonist sequence. In a preferred embodiment,
acyl is a C2_8 acyl chain and long
acyl is a C12_30 acyl chain. In certain embodiments, Xaa32 is a hydrophilic
am.ino acid (Haa).
[00821 Other representative polypeptide analogs presented herein have amino
acid sequences corresponding
to general formula (III):
Acyl-His-Ser-Asp-Xaa4 -Xaa5 -Phe-Thr-Xaas -Xaay -Tyr-Xaa11-Arg-Xaa13 -Xaald -
Xaa15 -Xaa16 -Xaal7
-Ala-Xaa19 -Xaa20 -Xaa21 -Tyr-Leu-Xaa24 -Xaa25 -Xaa26 -Xaa27 -Xaa28 -Xaa29 -
Xaa30 -Xaa3 l -Xaa32
-(Laa-Laa-Haa-Haa)n Laa-Xaa54 -Lys(E-long acyl)-PEG (SEQ ID NO: 83)
Formula (III)
wherein:
each Haa is independently a hydrophilic amino acid;
each Laa is independently a lipophilic amino acid;
acyl is a C2_16 acyl chain;
long acyl is a C12_30 acyl chain;
PEG is a functionalized polyethylene glycol chain of C10-C3000 chain;
Xaa4 is Gly or Ala;
Xaa5 is Val, Ile, or Leu;
Xaa8 is Asp, Arg, Gln, or Glu;
Xaa9 is Ser, Asn, Gln, Asp or Glu;
Xaall is Ser or Thr;
Xaa13 is Leu or Tyr;
Xaa14 is Arg or Leu;
Xaals is Lys, Leu, or Arg;
Xaa16 is Gln, Lys or Ala;
Xaa17 is Met, Leu, Val or Ala;
Xaal9 is Ala or Val;
Xaa20 is Lys, Arg or Ghi;
Xaa21 is Lys, Arg or Gln;
Xaa24 is Asn, Gln, Ala or Glu;
Xaa25 is Trp, Ala, or Ser;
Xaa26 is Ile, Val or Trp;
Xaa27 is Leu, Lys, Arg dr Gln;
Xaa28 is Lys, Arg, Asn, Gln, or Gly;
Xaa29 is Ala, Gly, Gln, Lys or Arg;
Xaa30 is Lys, Arg, Leu, Ala or absent;
Xaa31 is Lys, Arg, Leu, Ala or absent;
Xaa32 is any naturally occurring amino acid or absent; and
Xaa54 is Gln, Ser, Gly, Asp, Ala, Arg, Lys, Glu, Pro, Asn, Leu, or absent.
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provided that if any of Xaa30i Xaa31, Xaa32, or Xaa54 is absent, the next
amino acid present downstream is the
next amino acid in the peptide agonist sequence. In a preferred embodiment,
acyl is a Ca_$ acyl chain and long
acyl is a C12_30 acyl chain. In certain embodiments, Xaa32 is a hydrophilic
amino acid (Haa).
[0083] The slailled artisan will appreciate that numerous permutations of the
polypeptide analogs may be
synthesized which will possess the desirable attributes of those described
herein provided that an amino acid
sequence having a mean hydrophobic moment per residue at 100 f 20 greater
than about 0.20 is inserted at
positions inthe C-terminal region.
Example 2: Additional Analogs
[0084] In some embodiments of the invention, representative polypeptide
analogs presented herein have the
following amino acid sequences:
Acyl-Xaa1-Xaa2 -Xaa3 -Xaa4 -Xaas -Xaa6 -Thr-Xaa$ -Xaay -Xaalp -Thr-Xaa12 -
Xaa13 -Xaa14
-Xaa15 -Xaa16 -Xaa17 -Ala-Xaa19 -Xaa20 -XaaZl -Xaa22 -Xaa23 -Xaa24 -Xaa25 -
Xaa26 -Xaa27
-Xaa28 -Xaa29 -Xaa30 -Xaa31 -Xaa32 -Xaa33 -Xaa34 -Xaa35 -Xaa36 -Xaa37 -Xaa38 -
Xaa39 -Xaa40
(SEQ ID NO: 84)
Formula (IV)
wherein:
Xaal is: any naturally occurring amino acid, dH, or is absent;
Xaa2 is: any naturally occurring amino acid, dA, or dS;
Xaa3 is: Asp or Glu;
Xaa4 is: any naturally occurring amino acid, dA, or NMeA;
Xaa5 is: any naturally occurring amino acid, or dV;
Xaa6 is: any naturally occurring amino acid;
Xaag is: Asp, Glu, Ala, Lys, Leu, Arg, or Tyr;
Xaay is: Asn, Gin, Asp, or Glu;
Xaalo is: any naturally occurring aromatic amino acid, or Tyr (OMe);
Xaa12 is: hR, Lys (isopropyl), or any naturally occurring amino acid except
Pro;
Xaa13 is: any naturally occurring amino acid except Pro;
Xaa14 is: hR, Lys (isopropyl), or any naturally occurring amino acid except
Pro;
Xaa15 is: hR, Lys (isopropyl), K (Ac), or any naturally occurring amino acid
except Pro;
Xaa16 is: hR, Lys (isopropyl), or any naturally occurring amino acid except
Pro;
Xaa17 is: Nle, or any naturally occurring amino acid except Pro;
Xaa19 is: any naturally occurri.ng amino acid except Pro;
Xaa20 is: hR, Lys (isopropyl), Aib, K(Ac), or any naturally occurring amino
acid except Pro;
Xaa21 is: hR, K(Ac), or any naturally occurring amino acid except Pro;
Xaa22 is: Tyr (OMe), or any naturally occurring amino acid except Pro;
Xaa23 is: any naturally occurring amino acid except Pro;
Xaa24 is: any naturally occurring amino acid except Pro;
Xaa25 is: any naturally occurring aniino acid except Pro;
Xaa26 is: any naturally occurring amino acid except Pro;
Xaa27 is: hR, Lys (isopropyl), dK, or any naturally occurring amino acid
except Pro;
Xaa28 is: any naturally occurring amino acid, hR, dK, or is absent;
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Xaa29 is: any naturally occurring amino acid, hR, or is absent;
Xaa30 is: any naturally occurring amino acid, hR, or is absent; and
each of Xaa31 to Xaa40 is independently any naturally occurring amino acid or
absent;
and a C-terminal sequence selected from the group consisting of:
(a) -Xaa41-(Laa-Laa-Haa-Haa)õLaa-Lys(s-long acyl)-X;
(b) -Xaa4t -(Laa-Laa-Haa-Haa)a Laa-Xaa55 -Pro-Pro-Pro-Lys(E-long acyl)-X (SEQ
ID NO: 84);
(c) -Xaaal -(Laa-Laa-Haa-Haa)n Laa-Xaa55 -Lys(s-long acyl)-PEG; and
(d) a polyproline type II helix;
wherein:
n is an integer number from 1 to 3;
each Haa is independently a hydrophilic amino acid;
each Laa is independently a lipophilic amino acid;
acyl is a C2_16 acyl chain;
long acyl is a C12_30 acyl chain;
X is selected from the group consisting of OH, Cys(PEG), PEG, and NHRI,
wherein R' is selected
from H, lower alkyl, or haloalkyl; and
each of Xaa41 and Xaa55 is independently any naturally occurring amino acid or
absent;
provided that if any of Xaa1, Xaa28, Xaa29, Xaa30, Xaa31, Xaa32, Xaa33, Xaa34,
Xaa35, Xaa36, Xaa37, Xaa38, Xaa39,
or Xaa40, Xaa41, or Xaa55 is absent, the next aniino acid present downstream
is the next aniino acid in the peptide
agonist sequence.
[0085] In certain embodiments, PEG is a functionalized polyethylene glycol
chain of C10-C3000 chain. In
certain embodiments, PEG is a functionalized polyethylene glycol chain of Cloo-
C3ooo chain. In certain
embodiments, Xaa41 is a hydrophilic amino acid (Haa).
[0086] In some embodiments of the invention, representative polypeptide
analogs presented herein have the
following amino acid sequences:
Acyl-Xaa1 -Xaa2 -Xaa3 -Xaa4 -Xaa5 -Xaa6 -Thr-Xaa8 -Xaa9 -Xaa10 -Thr-Xaa12 -
Xaa13 -Xaa14
-Xaa15 -Xaa16 -Xaal7 -Ala-Xaa19 -Xaa20 -Xaa21-Xaa22 -Xaa23 -Xaa24 -Xaa25 -
Xaa26 -Xaa27
-Xaa28 -Xaa29 -Xaa30 -Xaa31 -Xaa32 -Xaa33 -Xaa34 -Xaa35 -Xaa36 -Xaa37 -Xaa38 -
Xaa3y -Xaa4o
Formula (V)
wherein:
Xaal is: His, dH, or is absent;
Xaa2 is: dA, Ser, Val, Gly, Thr, Leu, dS, Pro, or Aib;
Xaa3 is: Asp or Glu;
Xaa4 is: Ala, Ile, Tyr, Phe, Val, Thr, Leu, Trp, Gly, dA, Aib, or NMeA;
Xaa5 is: Val, Leu, Phe, Ile, Thr, Trp, Tyr, dV, Aib, or NMeV;
Xaa6 is: Phe, Ile, Leu, Thr, Val, Trp, or Tyr;
Xaas is: Asp, Glu, Ala, Lys, Leu, Arg, or Tyr;
Xaa9 is: Asn, Gln, Asp, or Glu;
Xaalo is: Tyr, Trp, or Tyr(OMe);
Xaa12 is: Arg, Lys, Glu, hR. Om, Lys (isopropyl), Aib, Cit, or Ala;
Xaa13 is: Leu, Phe, Glu, Ala, or Aib;
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Xaa14 is: Arg, Leu, Lys, Ala, hR, Orn, Lys (isopropyl), Phe, Gln, Aib, or Cit;
Xaa15 is: Lys, Ala, Arg, Glu, Leu, hR. Orn, Lys (isopropyl), Phe, Gln, Aib,
K(Ac), or Cit;
Xaa16 is: Gln, Lys, Glu, Ala, hR. Om, Lys (isopropyl), or Cit;
Xaa17 is: Val, Ala, Leu, Ile, Met, Nle, Lys, or Aib;
Xaa19 is: Val, Ala, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,
Arg, Ser, Thr, Trp, Tyr, Cys,
or Asp;
Xaa20 is: Lys, Gln, hR, Arg, Ser, His, Orn, Lys (isopropyl), Ala, Aib, Trp,
Thr, Leu, Ile, Phe, Tyr, Val,
K(Ac), or Cit;
Xaa21 is: Lys, His, Arg, Ala, Phe, Aib, Leu, Gln, Orn, hR, K(Ac) or Cit;
Xaa22 is: Tyr, Trp, Phe, Thr, Leu, Ile, Val, Tyr(OMe), Ala, or Aib;
Xaa2,3 is: Leu, Phe, Ile, Ala, Trp, Thr, Val, or Aib;
Xaa24 is: Gln, Glu, or Asn;
Xaa25 is: Ser, Asp, Phe, Ile, Leu, Thr, Val, Trp, Gln, Asn, Tyr, Aib, or Glu;
Xaa26 is: Ile, Leu, Thr, Val, Trp, Tyr, Phe or Aib;
Xaa27 is: Lys, hR. Arg, Gln, Ala, Asp, Glu, Phe, Gly, His, Ile, Met, Asn, Pro,
Ser, Thr, Val, Trp, Tyr,
Lys (isopropyl), Cys, Leu, Orn, or dK;
Xaa28 is: Asn, Asp, Gln, Lys, Arg, Aib, Orn, hR, Cit, Pro, dK, or is absent;
Xaa29 is: Lys, Ser, Aig, Asn, hR, Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met,
Pro, Gln, Thr, Val, Trp,
Tyr, Cys, Orn, Cit, Aib or is absent;
Xaa30 is: Arg, Lys, Ile, Ala, Asp, Glu, Phe, Gly, His, Leu, Met, Asn, Pro,
Gln, Ser, Thr, Val, Trp, Tyr,
Cys, hR. Cit, Aib, Orn, or is absent;
Xaa31 is: Tyr, His, Phe, Thr, Cys, or is absent;
Xaa32 is: Ser, Cys, or is absent;
Xaa33 is: Trp or is absent;
Xaa34 is: Cys or is absent;
Xaa35 is: Glu or is absent;
Xaa36 is: Pro or is absent;
Xaa37 is: Gly or is absent;
Xaa38 is: Trp or is absent;
Xaa39 is: Cys or is absent; and
Xaa40 is: Arg or is absent;
and a C-terminal sequence selected from the group consisting of:
(a) -Xaa41 -(Laa-Laa-Haa-Haa)Il Laa-Lys(E-long acyl)-X;
(b) -Xaa41 -(Laa-Laa-Haa-Haa),i-Laa-Xaa55 -Pro-Pro-Pro-Lys(E-long acyl)-X (SEQ
ID NO: 85);
(c) -Xaa41 -(Laa-Laa-Haa-Haa)n Laa-Xaa55 -Lys(s-long acyl)-PEG; and
(d) a polyproline type II helix;
wherein:
n is an integer number from 1 to 3;
each Haa is independently a hydrophilic amino acid;
each Laa is independently a lipophilic amino acid;
CA 02638968 2008-08-01
WO 2007/044591 PCT/US2006/039267
acyl is a C2_I6 acyl chain;
long acyl is a C12_3o acyl chain;
X is selected from the group consisting of OH, Cys(PEG), PEG, and NHRI,
wherein Rl is selected
from H, lower alkyl, or haloalkyl; and
each of Xaa41 and Xaa55 is independently any naturally occurring amino acid or
absent;
provided that if any of Xaa1, Xaa28, Xaa29, Xaa30i Xaa31i Xaa32, Xaa33, Xaa34,
Xaa35, Xaa36, Xaa37, Xaa38, Xaa39,
Xaa40, Xaadl, or Xaa55 is absent, the next amino acid present downstream is
the next amino acid in the peptide
agonist sequence.
[0087] In certain embodiments, PEG is a functionalized polyethylene glycol
chain of C10-C3000 chain. In
certain embodiments, PEG is a functionalized polyethylene glycol chain of Cloo-
C3ooo chain. In certain
embodiments, Xaa41 is a hydrophilic amino acid (Haa).
Example 3: Methods for Synthesizing Polypeptides
[0088] The polypeptides of the invention may be synthesized by methods such as
those set forth in J. M.
Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce
Chemical Co., Rockford, Ill. (1984) and
J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, Academic Press, New
York, (1973) for solid phase
synthesis and E. Schroder and K. Lubke, The Peptides, Vol. 1, Academic Press,
New York, (1965) for solution
synthesis and Houben-Weyl, Synthesis of Peptides and Peptidonai aetics. 4th
ed. Vol E22; M. Goodman, A.
Felix, L. Moroder, C. Toniolo, Eds., Thieme: New York, 2004 for general
synthesis techniques. The disclosures
of the foregoing treatises are incorporated by reference herein.
[0089] Microwave assisted peptide synthesis is an attractive method and will
be a particularly effective
method of synthesis for the peptides of the invention (Erdelyi M, et al.,
Synthesis 1592-6 (2002)). We have
demonstrated that use of microwave-assisted synthesis has achieved large
increases in purity and yield for the
peptides of the invention, relative to standard synthesis techniques. For
example, FIG. 4 shows a typical HPLC
trace for a crude peptide synthesized by standard solid phase procedures, for
which the yield of pure peptide is
approximately 2% from crude. In contrast, FIG. 5 shows the HPLC trace for a
typical microwave-assisted solid
phase synthesis of a VPAC2 selective ana.log. The yield in the latter case is
18% of pure peptide from the
crude. In other instances yields of 30% of pure peptide from crude have been
achieved. Thus this method has
important advantages for the synthesis of peptides of this class and size.
[0090] VIP and/or PACAP analogs, especially those of the invention, are
expected to have a high degree of
structure due to their inherent helical preference and to the amphiphilic a-
helical character designed into them.
Peptides with high propensity to adopt structure in solution may be prone to
synthetic difficulties due to the
reduced ability of reagents to penetrate their structure and therefore reduced
reactivity. The ability of
microwave assistance to put energy into these chains may be of increased
importance for the structures of the
invention, or other VIP and/or PACAP analogs, because of their inherent
helical conformational propensity.
Increases in yield from 2% to roughly 20% or more can have ixnportant
commercial consequences, since the
former renders preparation of commercial quantities very difficult.
[0091] In further or alternative embodiments of the invention, the microwave
assistance is used for
synthesizing polypeptides containing at least one amino acid which is not one
of the twenty standard aniino
acids.
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[0092] Thus our process for the synthesis of VIP and/or PACAP analogs is
useful for the synthesis of the
compounds of the invention, but also for other VIP and/or PACAP analogs known
in the art. Examples of the
latter structures are:
C6-HSDAVFTDNYTRLRKQVAAKKYLQSIKNSRTSPPPK(E-16)-NH2 (P81; SEQ ID NO: 316);
C6-HSDAVFTDNYTRLRAibQVAAAibKYLQSIKNSRTSPPP-NH2 (P309; SEQ ID NO: 317);
C6-HSDAVFTDNYTRLLLKVAAKKYLQSIKNSRTSPPP-NH2 (P156; SEQ ID NO: 318).
[0093] Even if these structures do not have the amphiphilic helical character
of the peptides of the invention,
they are expected to have some helical potential and engender synthetic
difficulties that can be remedied using
the microwave-assisted synthesis techniques disclosed herein. Thus, in certain
embodiments of the invention,
the microwave assistance is used for synthesizing VIP and/or PACAP analogs
having helical potential.
[0094] The present invention provides methods for producing the polypeptide of
VIP and/or PACAP
analogs, said method comprising synthesizing the polypeptide by the sequential
addition of protected amino
acids to a peptide chain, removing the protecting groups, desalting and
purifying the polypeptide. In certain
embodiments, the method fiuther comprises the step of using microwave
assistance. In a preferred embodiment,
the method with rnicrowave assistance produces a yield of polypepetides from
about 10% to about 50%. In a
more preferred embodiment, the method with microwave assistance produces a
yield of polypepetides from
about 12% to about 40%. In the most preferred embodiment, the method with
microwave assistance produces a
yield of polypepetides from about 15% to about 35%. In other embodiments, the
method with microwave
assistance provides a yield of polyeptides of at least two-fold increase, or
between two-fold and five-fold
increase as compared with a similar method without using microwave assistance.
[0095] In general, peptide synthesis methods involve the sequential addition
of protected amino acids to a
growing peptide chain. Normally, either the amino or carboxyl group of the
first amino acid and any reactive
side chain group are protected. This protected amino acid is then either
attached to an inert solid support, or
utilized in solution, and the next amino acid in the sequence, also suitably
protected, is added under conditions
amenable to formation of the amide linkage. After all the desired amino acids
have been linked in the proper
sequence, protecting groups and any solid support are removed to afford the
crude polypeptide. The
polypeptide is desalted and purified, preferably chromatographically, to yield
the fmal product.
[0096] A preferred method of preparing the analogs of the physiologically
active truncated polypeptides,
having fewer than about forty amino acids, involves solid phase peptide
synthesis. In this method the a-amino
(Na) functions and any reactive side chains are protected by acid- or base-
sensitive groups. The protecting
group should be stable to the conditions of peptide linkage formation, while
being readily removable without
affecting the extant polypeptide chain. Suitable a -amino protecting groups
include, but are not limited to t-
butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), o-chlorobenzyloxycarbonyl,
biphenylisopropyloxycarbonyl, t-
amyloxycarbonyl (Amoc), isobornyloxycarbonyl, a, a-dimethyl-3,5-
dimethoxybenzyloxy-carbonyl, o-
nitrophenylsulfenyl, 2-cyano-t-butoxycarbonyl, 9-fluorenyl-methoxycarbonyl
(Fmoc) and the like, preferably
Boc or more preferably, Fmoc. Suitable side chain protecting groups include,
but are not limited to: acetyl,
benzyl (Bzl), benzyloxymethyl (Bom), Boc, t-butyl, o-bromobenzyloxycarbonyl, t-
butyl, t-butyldimethylsilyl, 2-
chlorobenzyl (Cl-z), 2,6-dichlorobenzyl, cyclohexyl, cyclopentyl, isopropyl,
pivalyl, tetrahydropyran-2-yl, tosyl
(Tos), 2,2,4,6,7-pentamethyldihydrobenzofitran-5-sulfonyl (Pbf),
trimethylsilyl and trityl. A preferred Na-
protecting group for synthesis of the compounds of the invention is the Fmoc
group. Preferred side chain
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protecting groups are O-t-Butyl group for Glu, Tyr, Thr, Asp and Ser; Boc
group for Lys and Trp side chains;
Pbf group for Arg; Trt group for Asn, Gln, and His. For selective modification
of a Lys residue, orthogonal
protection with a protecting group not removed by reagents that cleave the
Fmoc or t-butyl based protecting
groups is preferred. Preferred examples for modification of the Lys side chain
include, but are not limited to,
those removed by hydrazine but not piperidine; for example 1-(4,4-dimethyl-2,6-
dioxocyclohex-l-ylidene)-3-
methylbutyl (ivDde) or 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidee)ethyl (Dde).
[0097] In solid phase synthesis, the C-terminal amino acid is first attached
to a suitable resin support.
Suitable resin supports are those materials which are inert to the reagents
and reaction conditions of the stepwise
condensation and deprotection reactions, as well as being insoluble in the
media used. Examples of
conunercially available resins include styrene/divinylbenzene resins modified
with a reactive group, e.g.,
chloromethylated co-poly-(styrene-divinylbenzene), hydroxymethylated co-poly-
(styrene-divinylbenzene), and
the like. Benzylated, hydroxymethylated phenylacetamidomethyl (PAM) resin is
preferred for the preparation
of peptide acids. When the C-terminus of the compound is an amide, a preferred
resin is p-
methylbenzhydrylamino-co-poly(styrene-divinyl-benzene) resin, a 2,4
dimethoxybenzhydrylamino-based resin
("Rink amide"), and the like. An especially preferred support for the
synthesis of larger peptides are
commericially available resins containing PEG sequences grafted onto other
polymeric matricies, such as the
Rink Amide-PEG and PAL-PEG-PS resins (Applied Biosystems) or similar resins
designed for peptide amide
synthesis using the Fmoc protocol.
[0098] Attachment to the PAM resin may be accomplished by reaction of the Na
protected amino acid, for
example the Boc-amino acid, as its ammonium, cesium, triethylammonium, 1,5-
diazabicyclo-[5.4.0]undec-5-
ene, tetramethylammoniuxn, or similar salt in ethanol, acetonitrile, N,N-
dimethylformamide (DMF), and the
like, preferably the cesium salt in DMF, with the resin at an elevated
temperature, for example between about
400 and
60 C, preferably about 50 C, for from about 12 to 72 hours, preferably about
48 hours. This will eventually
yield the peptide acid product following acid cleavage or an amide following
aminolysis.
10099] The Na -Boc-amino acid may be attached to the benzhydrylaniine resin by
means of, for example, an
N,N'-diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) mediated
coupling for from about 2 to
about 24 hours, preferably about 2 hours at a temperature of between about 10
and 50 C, preferably 25 C in a
solvent such as dichloromethane or dimethylformamide, preferably
dichloromethane.
[00100] For Boc-based protocols, the successive coupling of protected amino
acids may be carried out by
methods well known in the art, typically in an automated peptide synthesizer.
Following neutralization with
triethylamine, N,N-di-isopropylethylamine (DIEA), N-methylmorpholine (NMM),
collidine, or similar base,
each protected amino acid is preferably introduced in approximately 1.5 to 2.5
fold molar excess and the
coupling carried out in an inert, nonaqueous, polar solvent such as
dichloromethane, DMF, N-methylpyrrolidone
(NMP), N,N-dimethylacetamide (DMA), or mixtures thereof, preferably in
dichloromethane at ambient
temperature. For Fmoc-based protocols no acid is used for deprotection but a
base, preferably DIEA or NMM,
is usually incorporated into the coupling mixture. Couplings are typically
done in DMF, NMP, DMA or mixed
solvents, preferably DMF. Representative coupling agents are N,N'-
dicyclohexylcarbodiimide (DCC), N,N'-
diisopropyl-carbodiimide (DIC) or other carbodiiniide, either alone or in the
presence of HOBt, 0-acyl ureas,
benzotriazol-1-yl-oxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBop),
N-hydroxysuccinimide,
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WO 2007/044591 PCT/US2006/039267
other N-hydroxyimides, or oximes. Altematively, protected amino acid active
esters (e.g. p-nitrophenyl,
pentafluorophenyl and the like) or symmetrical anhydrides may be used.
Preferred coupling agents are of the
aminium/uronium (alternative nomenclatures used by suppliers) class such as 2-
(1H-benzotriazole-l-yl)-1,1,3,3-
tetramethylaminium hexafluorophosphate (HBTU), O-(7-azabenzotraiazole-l-yl)-
1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU), 2-(6-Chloro-lH-benzotraiazole-l-yl)-1,1,3,3-
tetramethylaminium
hexafluorophosphate (HCTU), and the like.
[00101] A preferred method of attachment to the Fmoc-PAL-PEG-PS resin may be
accomplished by
deprotection of the resin linker with 20% piperidine in DMF, followed by
reaction of the N-a-Fmoc protected
amino acid, preferably a 5 fold molar excess of the N-a-Fmoc-amino acid, using
HBTU: di-isopropylethylamine
(DIEA) (1:2) in DMF in a microwave-assisted peptide synthesizer with a 5min,
750 max coupling cycle.
[00102] For this Fmoc-based protocol in the microwave-assisted peptide
synthesizer, the N-a-Fmoc amino
acid protecting groups are removed with 20% piperadine in DMF containing 0.1M
1-hydroxybenzotriazole
(HOBt), in a double deprotection protocol for 30 sec and then for 3min with a
temperature maximum set at
75 C. HOBt is added to the deprotection solution to reduce aspartimide
formation. Coupling of the next
amino acid then employs a five fold molar excess using HBTU:DIEA (1:2) with a
5min, 75 max. double-
coupling cycle.
[00103] At the end of the solid phase synthesis the fully protected peptide is
removed from the resin. When
the linkage to the resin support is of the benzyl ester type, cleavage ma.y be
effected by means of aminolysis
with an alkylamine or fluoroalkylamine for peptides with an alkylamide C-
terminus, or by ammonolysis with,
for example, ammonia/methanol or ammonia/ethanol for peptides with an
unsubstituted amide C-terminus, at a
temperature between about -10 and 50 C, preferably about 25 C, for between
about 12 and 24 hours,
preferably about 18 hours. Peptides with a hydroxy C-terminus may be cleaved
by HF or other strongly acidic
deprotection regimen or by saponification. Alternatively, the peptide may be
removed from the resin by
transesterification, e.g., with niethanol, followed by aminolysis or
saponification. The protected peptide may be
purified by silica gel or reverse-phase HPLC.
[00104] The side chain protecting groups may be removed from the peptide by
treating the aminolysis
product with, for example, anhydrous liquid hydrogen fluoride in the presence
of anisole or other carbonium ion
scavenger, treatment with hydrogen fluoride/pyridine complex, treatment with
tris(trifluoroacetyl)boron and
trifluoroacetic acid, by reduction with hydrogen and palladium on carbon or
polyvinylpyrrolidone, or by
reduction with sodium in liquid ammonia, preferably with liquid hydrogen
fluoride and anisole at a temperature
between about -10 and +10 C, preferably at about 0 C, for between about 15
minutes and 2 hours, preferably
about 1.5 hours.
[00105] For peptides on the benzhydrylamine type resins, the resin cleavage
and deprotection steps may be
combined in a single step utilizing liquid hydrogen fluoride and anisole as
described above or preferably
through the use of milder cleavage cocktails. For example, for the PAL-PEG-PS
resin, a preferred method is
through the use of a double deprotection protocol in the microwave-assisted
peptide synthesizer using one of the
mild cleavage cocktails known in the art, such as TFA/water/tri-iso-
propylsilane/3,6-dioxa-1,8-octanedithiol
(DODT) (92.5/2.5/2.5/2.5) for 18min at 38 C each time. Typically the fitlly
deprotected product is precipitated
and washed with cold (-70 to 4 C) diethylether, dissolved in deionized water
and lyophilized to yield the crude
product as a white powder.
24
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[00106] The peptide solution may be desalted (e.g. with BioRad AG-3® anion
exchange resin)'and the
peptide purified by a sequence of chromatographic steps employing any or all
of the following types: ion
exchange on a weakly basic resin in the acetate form; hydrophobic adsorption
chromatography on underivatized
co-poly(styrene-divinylbenzene), e.g. Amberlite® XAD; silica gel
adsorption chrorima.tography; ion
exchange chromatography on carboxymethylcellulose; partition chromatography,
e.g. on Sephadex® G-25;
counter-current distribution; or HPLC, especially reversed-phase HPLC on octyl-
or octadecylsilylsilica (ODS)
bonded phase column packing.
[00107] Thus, another aspect of the present invention relates to processes for
preparing polypeptides and
pharmaceutically acceptable salts thereof, which processes comprise
sequentially condensing protected amino
acids on a suitable resin support, removing the protecting groups and resin
support, and purifying the product, to
afford analogs of the physiologically active truncated homologs and analogs of
PACAP and VIP, preferably of
PACAP and VIP in which the amino acids at the C-terminus form an amphipathic a-
helical peptide sequence,
as defined above.
[00108] Another aspect of the present invention relates to processes for
preparing polypeptides and
pharmaceutically acceptable salts thereof, which processes comprise the use of
microwave-assisted solid phase
synthesis-based processes to sequentially condense protected amino acids on a
suitable resin support, removing
the protecting groups and resin support, and purifying the product, to afford
analogs of the physiologically
active truncated homologs and analogs of PACAP and VIP, preferably of PACAP
and VIP in which the amino
acids at the C-terminus form an amphipathic a-helical peptide sequence, as
defined above.
Example 4: Exemplary Synthesis and Purification Protocol for a Representative
Polypeptide Analog
[001091 Representative polypeptide analog corresponding to SEQ ID NO: 1 is
prepared using the synthetic
and purification methods described below.
Pentanoyl-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Val-
Ala-Ala-Lys-
Lys-Tyr-Leu-Asn-Trp-Ile-Lys-Lys-Ala-Lys-Arg-Glu-Leu-Leu-Glu-Lys-Leu-
Lys(epsilon stearoyl)-NHZ
(SEQ ID NO: 1)
[00110] Generally, the peptide is synthesized on Fmoc-Rink-Amide-PEG resin via
Fmoc chemistry.
Protecting groups used for amino acid side chain fanctional groups are: t-
Butyl group for Glu, Tyr, Thr, Asp and
Ser; Boc group for Lys and Trp; Pbf group for Arg; Trt group for Asn and His.
N-a-Fmoc protected amino acids
are purchased from EMD Biosciences (San Diego, CA). Reagents for coupling and
cleavage are purchased
from Aldrich (St. Louis, MO). Solvents are purchased from Fisher Scientific
(Fairlawn, NJ).
[00111] Generally, the synthetic protocol involved assembly of the peptide
chain on resin by repetitive
removal of the Fmoc protecting group and coupling of protected amino acid. For
the synthesis, Dde-
Lys(Fmoc)-OH is coupled onto the deprotected Rink Amide resin first. The side
chain Fmoc protecting group is
then removed by 20% piperidine in DMF. Stearic acid is coupled onto the side
chain of Lys using HBTU,
HOBt and NMM. The Dde group is removed by 2% hydrazine in DMF and the next
Fmoc protected amino acid
is coupled. HBTU and HOBt are used as coupling reagent and NMM is used as
base. After removal of last
Fmoc protecting group, valeric acid (4 equivalents) is coupled to the amino
ternvnus with DIC (4 equivalents)
and HOBt (4 equivalents). The peptide resin is treated with cocktail 1 for
cleavage and removal of the side chain
protecting groups. Crude peptide is precipitated from cold ether and collected
by filtration.
CA 02638968 2008-08-01
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[00112] Purification of crude peptide is achieved via RP-HPLC using 20mm x
250mm column from Waters
(Milford, MA). Peptide is purified using TFA Buffer. A linear gradient of 35%
to 55% acetonitrile in 60 minutes
is used. Pooled fractions are lyophilized. The peptide identity is verified by
mass spectrometry analysis and
amino acid analysis. The peptide purity is deterniined by analytical HPLC
column (C1 8 column, 4.6 x 250mm,
manufactured by Supelco (St. Louis, MO)) chromatography.
[00113] The above procedure can be summarized in the following step wise
protocol:
Step 1. Resin swelling: Fmoc-Rink-Amide-PEG resin is swelled in DCM for 30
minutes (10 ml/g
resin)
Step 2. Deprotection:
a. 20% piperidine/DMF solution (10 nil/g resin) is added to the resin;
b. Solution stirred for 30 minutes (timing is started when all the resin is
free floating in the
reaction vessel); and
c. Solution is drained.
Step 3. Washing: Resin is washed with DMF (10 ml/g resin) five times. The
ninhydrin test is
performed and appeared positive.
Step 4. Coupling:
a. Fmoc,-AA-OH (3 equivalents calculated relative to resin loading) and HOBt
(3
equivalents relative to resin loading) is weighed into a plastic bottle.
b. Solids are dissolved with DMF (5 ml/g resin).
c. HBTU (3 equivalents relative to resin loading) is added to the mixture,
followed by the
addition of NMM (6 equivalents relative to resin loading).
d. Mixture is added to the resin.
e. Mixture is bubbled (or stirred) gently for 10 - 60 minutes until a negative
ninhydrin test
on a small sample of resin is obtained.
Step 5. Washing: Resin is washed three times with DMF.
Step 6. Steps 2-5 are repeated until the peptide is assembled.
Step 7. N-terminal Fmoc Deprotection: Step 2 is repeated.
Step 8. Washing and Drying:
a. After the final coupling, resin is washed three times with DMF, one time
with MeOH,
tbree times with DCM, and three times with MeOH.
b. Resin is dried under vacuum (e.g., water aspirator) for 2 hours and high
vacuum (oil
pump) for a minimum of 12 hours.
Step 9. Cleavage:
a. Dry resin is placed in a plastic bottle and the cleavage cocktail is added.
The mixture is
shaken at room temperature for 2.5 hours.
b. The resin is removed by filtration under reduced pressure. The resin is
washed twice with
TFA. Filtrates are combined and an 8-10 fold volume of cold ether is added to
obtain a
precipitate.
26
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WO 2007/044591 PCT/US2006/039267
c. Crude peptides are isolated by filtration and then washed twice with cold
ether. FIG. 4
shows an HPLC trace. of a typical crude peptide which typically yields
purified peptide on
scale of 5% or less from the crude material.
[00114] The following chen-ucals and solvents are used in the synthetic
protocol described above: NMM (N-
Methylmorpholine); HBTU (2-(1H-Benzotriazole-l-yl)-1,1,3,3-
tetramethyluroniumHexafluorophosphate);
HOBt (1-Hydroxybenzotriazole); DMF (Dimethylformamide); DCM (Dichloromethane);
Methanol;
Diethylether; Piperidine; Tis (Triisopropylsilane); Thioanisole; Phenol; EDT
(1,2-Ethanedithiol); Trifluoroacetic
acid Cocktail 1: TFA/Thioanisole/I'henol/ H20/EDT (87.5/5/2.5/2.5/2.5 v/v/);
TFA buffer: A(0.1 % TFA in
water); and TFA buffer B(0.1% TFA in Acetonitrile).
[001151 Other representative polypeptide analogs are prepared in a manner
similar to that described above.
Listed below in TABLE 1 are chemical properties of exemplary polypeptide
analogs of the invention.
TABLE 1. Properties of Exemplary Polypeptide Analogs
Name of Amino Acid Sequence Purity Based on RP- Molecular Weight Based on
Analog HPLC Chromatogram Electrospray Mass S ectrometr
TP-103 SEQ ID NO: 2 96.9% 5267.2 a.m.u.
TP-104 SEQ ID NO: 3 95.5% 4756.7 a.m.u.
TP-105 SEQ ID NO: 4 96.1% 5183.3 a.m.u.
TP-106 SEQ ID NO: 5 95.2% 4784.8 a.m.u.
TP-107 SEQ ID NO: 6 99.6% 4955.1 a.m.u.
TP-108 SEQ ID NO: 7 91.5% 5172.4 a.m.u.
Example 5: Exemplary Microwave-Assisted Synthesis and Purification Protocol
for a Representative
Polypeptide Analog.
[00116] Representative polypeptide analog corresponding to SEQ ID NO: 60 (TP-
135) is prepared using the
synthesis and purification methods described below.
Hexanoyl-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Gln-Tyr-Thr-Arg-Leu-Leu-Lys-Gln-Val-
Ala-Ala-Lys-
Lys-Tyr-Leu-Gln-Trp-Ile-Lys-Lys-Ala-Lys-Arg-Glu-Leu-Leu-Glu-Lys-Leu-
Lys(stearoyl)-NHZ
(SEQ ID NO: 60)
[00117] Generally, the peptide is synthesized on a CEM Liberty Automated
Peptide Synthesizer on 0.lmmol
scale. This synthesizer uses niicrowave-assisted synthesis and has the ability
to monitor internal reaction vessel
temperatures. Fmoc-PAL-PEG-PS resin (0.18mm.o1/gm nominal substitution) is
used as support with N-a-
Fmoc protecting group chemistry. Protecting groups used for amino acid side
chain functional groups are: O-t-
Butyl group for Glu, Tyr, Thr, Asp and Ser; Boc group for Lys and Tip side
chains, except for the C-terminal
Lys; Pbf group for Arg; Trt group for Asn, Gln, and His. Reagents for coupling
and cleavage, as well as N-alpha
Fmoc protected amino acids, are from CEM Corporation (Matthews, NC). N-a-Fmoc
deprotection is carried
out with 20% piperidine in DMF containing 0.1M HOBt. Double Fmoc deprotection
is carried out for 30 sec
and then for 3min with a temperature maximum set at 75 C. For the removal of
side chain ivDde protection
from the C-terminal Lys residue, a triple deprotection scheme with 2%
hydrazine in DMF is used:
3min/6min/6min, 75 C max. Amino acid activation is carried out on five fold
molar excess using
HBTU:DIEA (1:2) with a 5min, 75 max. double-coupling cycle on all residues,
except single coupling on
Fmoc-Lys(ivDde)-OH (initial step) and triple coupling of stearic acid (fmal
assembly step).
27
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WO 2007/044591 PCT/US2006/039267
[00118] The synthetic protocol generally involves assembly of the peptide
chain on resin by repetitive
removal of the Fmoc protecting group and coupling of protected amino acid,
similar to that described in
example 4 above, but with differences in side chain protection, molar excess,
etc. as described herein. For the
synthesis, Fmoc-Lys(ivDde)-OH is coupled onto the deprotected, conunercially
available Fmoc-PAL-PEG-PS
resin first. The Fmoc protecting group is then removed by 20% piperidine in
DMF. The peptide is assembled by
repetitive cycles of coupling, Fmoc deprotection and further coupling.
Following the last amino acid coupling,
the N-a-Fmoc group is removed from His(Trt) and it is coupled with hexanoic
acid (double coupling protocol).
At this point, preferavly approximately one half of the peptide resin is
removed and saved for other analog
syntheses.
[001191 Finally, the ivDde group is removed from the C-terminal Lys by 2%
hydrazine in DMF using a triple
deprotection protocol (3 min/ 6min/ 6min; 75 max) and stearic acid is coupled
using a triple coupling protocol.
Final cleavage and deprotection is carried out using two rounds of microwave
assisted cleavage with
TFA/Water/TIS/3,6-dioxa-l,8-octanedithiol (92.5/2.5/2.5/2.5) for 18 min at 38
C each time. The crude product
is precipitated and washed with cold diethylether, dissolved in distilled
water and lyophilized to yield the
product as a white powder. Yield: 140 mg crude yield of peptide product after
lyophilization. Purification of
the crude peptide is carried out by reverse-phase (C-18) HPLC using a gradient
from 10 to 40% Solvent B
(Solvent A: 0.1% TFA in water; Solvent B: 0.1% TFA in acetonitrile). Fractions
are cut for purity from the
major peak, pooled and lyophilized to yield the product as 25 mg of white
powder (18% yield by weight from
crude material). The purity is assessed by analytical reverse-phase HPLC as
described above and is shown to
be >95% (mass spee peak at M+1=4957/3 positive charge). FIG. 5 shows an HPLC
trace of a crude peptide
from a typical synthesis and pure peptide is typically obtained in 15 to 30%
yield from crude peptide.
[00120] Other representative polypeptide analogs are prepared in a manner
similar to that described above.
Listed below in TABLE 2 are chemical properties of exemplary polypeptide
analogs of the invention.
TABLE 2. Properties of Exemplary Polypeptide Analogs
Name of Aniino Acid Sequence Purity Based on RP- Molecular Weight Based on
Analog HPLC Chromatogram Electros ra Mass S ectrometr
TP-135 SEQ ID NO: 60 96.9% 4955.1 a.m.u.
V2448 SEQ ID NO: 95 >99% 5138 a.m.u.
Example 6: Recombinant Synthesis of the Polypeptides
[00121] Alternatively, the polypeptides of the present invention may be
prepared by cloning and expression
of a gene encoding for the desired polypeptide. In this process, a plasmid
containing the desired DNA sequence
is prepared and inserted into an appropriate host microorganism, typically a
bacteria, such as E. coli, or a yeast,
such as Saccharomyces cerevisiae, inducing the host microorganism to produce
multiple-copies of the plasmid,
and so of the cDNA encoding for the polypeptide analogs of the invention.
[001221 First, a synthetic gene coding for the selected PACAP or VIP analog is
designed with convenient
restriction enzyme cleavage sites to facilitate subsequent alterations.
Polymerase chain reaction (PCR), as taught
by Mullis in U.S. Pat. Nos. 4,683,195 and 4,683,202, incorporated herein by
reference, may be used to amplify
the sequence.
[00123] The amplified synthetic gene may be isolated and ligated to a suitable
plasmid, such as a Trp LE
plasniid, into which four copies of the gene may be inserted in tandem.
Preparation of Trp LE plasmids is
28
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WO 2007/044591 PCT/US2006/039267
described in U.S. Pat. No. 4,738,921 and European Patent Publication No.
0212532, incorporated herein by
reference. Trp LE plasmids generally produce 8-10 times more protein than Trp
E plasmids. The multi-copy
gene may then be expressed in an appropriate host, such as E. coli or S.
cerevisiae.
[00124] Trp LE 18 Prot (Ile3, Pro5) may be used as an expression vector in the
present invention. Trp LE 18
Prot (I1e3, Pro5) contains the following elements: a pBR322 fragment (EcoRI-
BamHI) containing the
ampicillin resistant gene and the plasmid origin of replication; an EcoRI-
Sacll fragment containing the trp
promoter and the trpE gene; an HIV protease (Ile3, Pro5) gene fragment (SacII-
HindIII); a bGRF gene fragment
(HindIII-BamHI); and a transcription terminator from E. coli rpoc gene. The
HIV protease and bGRF gene
fragments are not critical and may be replaced with other coding sequences, if
desired.
[00125] The expressed multimeric fusion proteins then accumulate
intracellularly into stable inclusion bodies
and may be separated by centrifugation from the rest of the cellular protein.
VIP and PACAP related peptides do
not denature so purification is straightforward through a combined ion
exchange concentration / purification
protocol followed by "polishing" on preparative reversed-phase high
performance chromatograpy using a
aqueous to aqueous-organic buffer gradient using 0.1% trifluoroacetic acid or
0.4M NH4OAc (pH 4) as the pH
modifier. The organic modifier used may be any of a number of water miscible
solvents, for example
acetonitrile, n-propanol, isopropanol, and the like, preferably n-propanol.
The isolated fusion protein is
converted to the monomeric PACAP or VIP analog by acylation with activated
fatty acids and may be purified
by cation exchange and/or reverse phase HPLC. The precise protocol is
dependent on the particular sequence
being synthesized. Typically the free amino terminus is less reactive than a
Lys side chain, so differential
acylation is straightforward. Alternatively, a fragment of the fmal sesquence
may be prepared in this way with
subsequent condensation with a synthetically produced fragment containing the
N- or C-terminal modifications.
Chemical or "native" conjugations may be used (Dawson, P. E.; Muir, T. W.;
Clark-Lewis, I.; Kent, S. B.
Science 1994, 266, (5186), 776-9; Nilsson, B. L.; Soellner, M. B.; Raines, R.
T. Annu Rev Biophys Biomol
Struct 2005, 34, 91-118.).
[00126] Alternative methods of cloning, amplification, expression, and
purification will be apparent to the
skilled artisan. Representative methods are disclosed in Maniatis, et al.,
Molecular Cloning, a Laboratory
Manual, 3rd Ed., Cold Spring Harbor Laboratory (2001), incorporated herein by
reference.
Example 7: In Vitro Bioassay with Islet Cell Static Cultures
[00127] The following exemplary in vitro bioassay was conducted to evaluate
the ability of representative
polypeptide analogs to modulate insulin secretion.
[00128] Islet isolation. Rat islets were harvested (Sweet IR, et al. (2004)
Biochem. Biophys. Res. Commun.
314, 976-983) from male Fisher rats weighing about 250 g and which were
anesthetized by intraperitoneal
injection of sodium pentobarbital (35 mg/230 g rat). Generally, the islets
were prepared by injecting collagenase
(l OmL of 0.23 mg/niL Liberase, Roche Molecular Biochernicals, Indianapolis,
IN) into the pancreatic duct of
the partially dissected pancreas and surgically removing it. All procedures
were approved by the Institutional
Animal Care and Use Committee at the University of Washington.
[00129] The pancreata were placed into 15mL conical tubes containing 5mL of
0.23 mg/mL Liberase and
incubated at 37 C for 30 min. The digestate was then filtered through a 400-
micrometer stainless steel screen,
rinsed with Hanks' buffered salt solution, and purified in a gradient solution
of Optiprep (Nycomed, Oslo,
Norway). Islets were cultured for 18-24 h prior to performing the assay in
RPMI Media 1640 supplemented
29
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WO 2007/044591 PCT/US2006/039267
with 10% (v/v) heat inactivated fetal bovine senim (FBS), antibiotic-
antimycotic (100U/mL penicillin, 100
lg/mL streptomycin, and 0.251g/mL amphotericin B), 2mM glutamine (all from
Gibco-BRL, Grand Island,
NY), and 1mM beta mercaptoethanol.
[00130] Bioassay. Islets were picked under a microscope and placed into 10 m13
mM Krebs Ringer Buffer
(KRB) solution for washing. Islets were incubated in 3mM glucose KRB for 60
min and then groups of 10 islets
per well were placed into 200 l media in a 96-well plate. The islets were
incubated for 120 min under control
or treatment conditions, and supernatants were collected. A typical set of
conditions tested 3mM glucose
(resting control), 16mM glucose (testing control), 16 mM glucose + 10 nm GLP1,
16 miVl glucose + 10 nM
Exendin-4, 16 mM glucose + 50 nM test peptide. The buffer conditions were KRB
with 0.1% BSA, 20 mM
HEPES and the assay is performed in quadruplicate. Supernatants were evaluated
for insulin content using a
commercial insulin enzyme-linked immunosorbent (ELISA) assay per
manufacturer's directions.
[00131] Results of Bioassay. TABLE 3 illustrates the insulin secretion
obtained in the above assay for analog
TP-106, which exhibited maximal activity in this assay at a concentration of
200nM. For comparison, Exendin
4 was tested in this assay and showed maximal activity at lOnM. TP-106 is a
highly hydrophobic analog,
designed to depot in the site of sc injection and therefore the effective
concentration of TP-106 is expected to be
much lower than the nominal concentration (200nM).
TABLE 3. Results of Islet Cell Static Culture Bioassay with TP-106
Insulin secreted (ng/100
islets/niin) Standard Deviation
3mM glucose 0.01 0.00
16mM glucose 1.38 0.17
Exendin 4 + 16mM glucose 4.82 0.20
50nM TP-106 + 16mM glucose 2.72 0.60
200nM TP-106 + 16mM glucose 5.20 0.50
16mM glucose + 16mM glucose 1.58 0.05
[00132] The islet cell static culture assay described above is performed on
additional exemplary polypeptide
analogs. TP-107 exhibited maximal activity in this assay at a concentration of
100nM. For comparison,
Exendin 4 is tested in this assay and showed maximal activity at l OnM.
Presented peptides are designed to bind
to serum albumin and thus, the concentration of free peptide to impart insulin
activity is expected to be much
lower and therefore the analog more potent than indicated in this in vitro
assay. Similar observations have been
reported during studies with the hydrophobic peptide, insulin detimir
(Kurtzhals, P., et al., Diabetes 49:999-
1005 (2000)).
TABLE 4. Results of Islet Cell Static Culture Bioassay with TP-107 and TP-108
Average Insulin secreted (ng/100
islets/nrin) Standard Deviation
3 mM glucose 0.14 0.00
16 mM glucose 3.65 0.80
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Average Insulin secreted (ng/100
islets/min) Standard Deviation
nM Exendin 4+ 16mM glucose 6.75 1.15
10 nM PACAP + 16mM glucose 6.07 1.67
10 nM TP-107 + 16mM glucose 2.89 0.21
100 nM TP-107 + 16mM glucose 6.10 1.55
1 uM TP-107 + 16mM glucose 6.07 0.90
100 nM TP-108 + 16mM glucose 4.10 1.21
1 uM TP-108 + 16mM glucose 5.65 0.13
Example 8: In vitro Flow assay
[00133] Static assays may suffer from feedback loop suppression of secretion
of insulin or other hormones.
Therefore in vitro flow assay conditions are useful in order to confirm the
results of static assays. Thus islets are
isolated as described in Example 7 and seeded into a flow apparatus as
described (Sweet, I., et al., Diabetes 53:
5 401-9 (2004)). The islet flow culture system (Sweet, I., et al., Diabetes
Technol Ther. 4: 67-76 (2002))
includes a pump, gas equilibrator, a glass islet perifusion chamber, detectors
for oxygen and cytochromes, and a
fraction collector. Islets are stabilized with Cytopore beads (Amersham
Biosciences, Piscataway, NJ) that are
layered into the chamber using a P200 pipette as follows: First, 0.4 mg of
beads in 20 1 media are allowed to
settle onto the porous polyethylene,frit at the chamber's bottom. A mixture of
600 islets and Cytodex beads
10 (0.12 mg; Amersham Biosciences) is added followed by another 0.4 mg
Cytopore beads and a top frit. Porous
frits are cored (0.3 cm) from polyethylene sheets (Small Parts, Miami Lakes,
FL). Typically 600 or 300 islets
are used but the number can be varied depending on the compounds being assayed
and the number of
supematant samples desired. Krebs Ringer or RPMI media at a flow rate of 200 L
per min. The islets are
challenged with 16mM glucose solution and then with test compound in 16mM
glucose containing buffer.
Samples are taken from the effluent from the chamber and assayed for insulin
content using an enzyme-linked
immunosorbent assay according to the manufacturer's instructions (ALPCO,
Windham, NH). Table 5
illustrates the substantial glucose-dependent insulin secretion stimulated by
test peptides that are within the
scope of and representative of the invention, i.e., TP-128 and V2449.
TABLE 5. Results of Islet Flow Culture Bioassay with TP-128 and V2449.
Insulin secreted (ng/100
islets/min)
3mM glucose 0.5
16mM glucose 1
lOOnM TP-128 + 16mM glucose 14
100nM V2449 + 16mM glucose 12
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Example 9: In Vivo Bioassay
[00134] The following exemplary in vivo assay was conducted to evaluate the
ability of representative
polypeptide analogs to modulate insulin secretion.
[00135] Tested Study Groups. Naive, 8 weeks old female db/db mice were
acclimated for one week, during
which period animals were handled periodically to allow them to be acclimated
to experiment procedures.
Study groups contained 6 mice per group and were administered with one of the
following by intraperitoneal
injection:
(1) Vehicle control;
(2) Positive control (exendin-4 or other standard treatment);
(3) Polypeptide Analog at high dose; or
(4) Polypeptide Analog at low dose.
A small volume of blood was taken from a cut at the tip of tail for blood
sampling. Blood glucose levels were
determined on a commercial, hand-held glucose meter. On Day 1, animals were
injected with polypeptide
analogs and controls in the morning. Blood samples were taken and analyzed
immediately before injection and
at 2, 4, 8, 14, and 24 hours after injection. Animals were allowed to feed, ad
libitem, throughout the assay
(Tsutsumi et al., Diabetes 51:1453-60 (2002)).
[001361 TABLE 6 lists a representative sampling of the data obtained from the
iri vivo assay described above.
As shown below, TP-106 exhibited statistically significant activity (e.g.,
reduced plasma glucose) at a high dose
2hr after injection and maintains activity at 4 hrs post dosing.
TABLE 6. Results of In Vivo Assay with TP-103 and TP-106
Mean Blood Glucose Levels mmol/L
0 hr 2hr 4hr 8hr 14hr 24hr
23.9 21.9 18.3 27.3 22.5 23.5
7ehicle s.d.*= 1.33 s.d.= 1.22 s.d.= 1.01 s.d.= 1.52 s.d.= 1.25 s.d.= 1.31
22.9 20.5 17.6 26.4 24.6 21.4
P-103 Low dose s.d.= 1.27 s.d.= 1.14 s.d.= 0.98 s.d.= 1.47 s.d.= 1.37 s.d.=
1.19
20.7 17.3 16.9 23.4 23.7 25.0
TP-103 High dose s.d.= 1.15 s.d: 0.96 s.d.= 0.94 s.d.=1.30 s.d.= 1.31
s.d.=1.39
23.9 20.5 16.1 24.0 28.2 23.2
TP-106 Low dose s.d.= 1.33 s.d.= 1.14 s.d.= 0.89 s.d.= 1.33 s.d.= 1.57 s.d.=
1.29
21.8 13.4 14.7 25.1 26.3 21.2
TP-106 High dose s.d = 1.21 s.d = 0.75 s.d.= 0.82 s.d.= 1.39 s.d.= 1.46 s.d.=
1.18
*s.d.= standard deviation
Example 10: Relaxation of guinea pig tracheal smooth muscle.
[00137] Tracheal tissue is removed from Hartley guinea pigs (500-700g) after
sacrificing them with an
overdose of urethane (O'Donnell, M., et al. J. Pharmacol. Exptl. Therapeut.
270: 1282-8 (1994)). The trachea
is divided into four ring segments. Each ring is suspended by stainless steel
wires in a l OmL jacketed tissue
bath and attached to a Grass force displacement transducer for isometric
recording of tension. The smooth
muscle tissue is bathed in modified Kreb's-Hanseleit solution at 37.5 C with
constant bubbling of O2/CO2
(95:5). Tracheal rings are placed under a resting tension of 1.5 g and
readjusted as required. Tissues are
precontracted with carbachol (30nM) or KCI (10mM) and treated with the test
agent. The difference intension
32
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WO 2007/044591 PCT/US2006/039267
between the precontraction induced by carbachol and the level during a fmal
maximum theophyline-induced
relaxation (1mM) is regarded as 100% active tension.
[00138] Paired concentration response experiments are carried out for the test
peptide and standard VIP. The
concentration of the test peptide and the VIP strandard are increased
cumulatively as soon as the peak drug
response is observed. Relaxant responses are expressed as a percentage of
relaxation relative to the 100%
active tension and EC50 values are determined by linear regression.
Example 11: Selective PEGylation of a VPAC2 agonist to prepare P307
Hexanoyl-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Gln-Tyr-Thr-Arg-Leu-Leu-Lys-Gln-Val-
Ala-Ala-Lys-
Lys-Tyr-Leu-Asn-S er-Ile-Lys-Lys-Ala-Lys-Arg-Leu-Leu-Arg-Lys-Leu-Lys(stearoyl)-
Cys (PEG 1 K)-
NH2 (SEQ ID NO: 315)
[00139] The cysteine containing precursor to P307 is prepared in the free SH
form according to the
microwave-assisted synthesis procedure of Example 5. A sample of 55 mg of P307
precursor is dissolved in
100 mL of 100mM phosphate buffer at pH 7.5 (containing 15 mM disodium
ethylenediaminetetraacetic acid)
that is deaerated by argon bubbling, and treated with 70 mg of PEG1150 (MeO-
PEG-maleinimide;PEG-WM
750Da; IRIS Biotech) during a period of approximately 3 hr. The reaction is
monitored by Ellman reagent to
detect disappearance of SH functional groups and purified by size exclusion
chromatography on a 300mL
column of Sephadex 2000 swollen with phosphate buffer. The effluent is
followed by uv absorption and cut for
purity (early peaks) to remove unreacted PEG and srnaller molecular weight
impurities. Further purification by
ion exchange chromatography (for example carboxymethylcellulose, CM Sepharose,
or the like) or preparative
HPLC is available is preferred. The solution of product in elution buffer is
dialyzed (lkDa cut-off membrane;
Amersham) against a suitable buffer (e.g. acetate, pH5) and lyophilized to
yield the product as a white powder.
The protein conjugate is characterized by analysis on a Po1yCAT A column (Nest
Group).
Example 12: Uses of the Invention
[00140] The polypeptides of the present invention are useful for the
prevention and treatment of a variety of
metabolic disorders. In particular, the compounds of the present invention are
indicated for the prophylaxis and
therapeutic treatment of: elevated blood glucose levels, hyperglycemia,
dyslipidemia, hypertriglyceridemia,
diabetes, including Type 2 Diabetes Mellitus, Metabolic Syndrome (Grundy,
S.M., et al. Nature Rev. Drug Disc.
5: 295-309 (2006)), Maturity Onset Diabetes of the Young (MODY, Herman, W.H.,
et al, Diabetes 43:40-6
(1994); Fajans, S.S., et al. Diabet Med. 13 (9 suppl 6): s90-5 (1996)), Latent
Autoimmune Diabetes Adult
(LADA; Zimmet, P.Z., et al., Diabetes Med. 11:299-303 (1994); impaired glucose
tolerance (IGT); impaired
fasting glucose (IFG); gestational diabetes (Rumbold, A.R. and Crowther, C.A.,
Aust N. Z. J. Obstet. Gynaecol.
41: 86-90)); Syndrome X, insulin resistance, stimulate proliferation of beta
cells, improve beta cell function,
activate dormant beta cells, metabolic acidosis and obesity. The polypeptides
of the invention are useful for
prevention and treatment of secondary causes of diabetes and other metabolic
diseases such as glucocorticoid
excess, growth hormone excess, pheochromocytoma and drug-induced diabetes (for
example due to pyriminil,
nicotinic acid, glucocorticoids, phenytoin, thyroid hormone, (3-adrenergic
agents, a-interferon and drugs used to
treat HIV infection).
[00141] The polypeptides of the present invention are also useful for treating
complications caused by
diabetes and the metabolic syndrome such as atherosclerotic disease,
hyperlipidemia, hypercholesteremia, low
33,
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WO 2007/044591 PCT/US2006/039267
HDL levels, hypertension, cardiovascular disease (including atherosclerosis,
coronary heart disease, coronary
artery disease, and hypertension), cerebrovascular disease and peripheral
vessel disease; and for the treatment of
lupus, polycystic ovary syndrome, carcinogenesis, and hyperplasia, asthma,
male and female reproduction
problems, sexual disorders, ulcers, sleep disorders, disorders of lipid and
carbohydrate metabolism, circadian
dysfunction, growth disorders, disorders of energy homeostasis, immune
diseases including autoimmune
diseases (e.g., systemic lupus erythematosus), as well as acute and chronic
inflammatory diseases, rheumatoid
arthritis, and septic shock.
[00142] The polypeptides of the present invention are also useful for treating
physiological disorders related
to, for example, cell differentiation to produce lipid accumulating cells,
regulation of insulin sensitivity and
blood glucose levels, which are involved in, for example, abnorrnal pancreatic
beta-cell function, insulin
secreting tumors and/or autoimmune hypoglycemia due to autoantibodies to
insulin, autoantibodies to the
insulin receptor, or autoantibodies that are stimulatory to pancreatic beta-
cells, macrophage differentiation
which leads to the formation of atherosclerotic plaques, inflammatory
response, carcinogenesis, hyperplasia,
adipocyte gene expression, adipocyte differentiation, reduction in the
pancreatic beta-cell mass, insulin
secretion, tissue sensitivity to insulin, liposarcoma cell growth, polycystic
ovarian disease, chronic anovulation,
hyperandrogenism, progesterone production, steroidogenesis, redox potential
and oxidative stress in cells, nitric
oxide synthase (NOS) production, increased gamma glutamyl transpeptidase,
catalase, plasma triglycerides,
HDL, and LDL cholesterol levels, and the like.
[00143] The polypeptides of the present invention are useful for the
prevention and treatment of a variety of
inflammatory disorders, defined broadly. In particular the compounds of the
present invention are indicated for
the prophylaxis and therapeutic treatment of asthma (Linden A, et al. (2003).
Thorax 58: 217-21),
cardioprotection during ischemia (Kalfm, et al., J Pharma.col Exp Ther 1268:
952-8 (1994); Das, et al., Ann NY
Acad Sci 865: 297-308 (1998)), primary pulmonary hypertension (Petkov, V., et
al. J Clin Invest 111: 1339-46.
(2003)), and the like.
[00144] As indicated above, the lung is an iunportant new medical target for
treatment by VPAC2 agonists.
For example, asthma is a large and rapidly growing disease but the current
methods of treatment carry
substantial risk of serious side effects. Studies both in vitro and in vivo
with animal models showed that
VPAC2 selective agonists cause prompt relaxation of tracheal smooth muscle
preconstricted with carbachol,
histamine or KC1(O'Donnell, K., et al., J. Pharmacol. Exptl. Therapeut. 270:
1282-8 (1994) and Example 10) as
well as in sensitized guinea pigs (O'Donnell, K., et al., J. Pharma.col.
Exptl. Therapeut. 270: 1289-94 (1994)).
Human bronchial tissue responds similarly to PACAP analogs (Yoshihara, S., et
al., Regulatory Peptides 123:
161-5 (2004)). Treatment of asthma patients with a VPAC2 selective molecule
showed prompt
bronchodilatation and a similar maximal effect to that shown by a leading
beta2 adrenoceptor agonist,
formoterol (Linden, A., et al. Thorax 58: 217-21 (2003)). While beta2
adrenoceptor agonists are effective
bronchodilators, they have black box warnings for sudden death. In contrast,
no clinically significant side
effects are seen for the VPAC2 agonist. However it is short acting and
therefore could not be developed
commercially. In contrast, the compounds of the invention are designed to have
high VPAC2 selectivity, long
duration of action, and to be permeable into lung tissue thus making them
attractive drug development
candidates for treatment of asthrna and other obstructive diseases of the
lung.
34
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WO 2007/044591 PCT/US2006/039267
[00145] Another important activity of VPAC2 agonists is their ability to
suppress the proinflannnatory
response of mast cells in response to inflammatory signals like bacterial
lipopolysaccharide (Delgado, M. and
Ganea, D., J. Immunol. 167: 966-75 (2001)). Mast cells are thought to be
important effectors in asthma (Kraft,
M., et al., Chest 124: 42-50 (2003)) as well as in chronic obstructive
pulmonary disease (COPD), based on
recent research (Barnes, P.J., J. COPD 1: 59-70 (2004)). The compounds of the
present invention are novel,
disease modifying treatments for both of these important lung diseases, asthma
and COPD as well as for the
treatment of other respiratory conditions.
[00146] Pulmonary hypertension is an important disease caused by increased
vascular resistance in the
pulmonary arteries. This can be caused either by some common conditions -
congenital heart defects,
scleroderma, HIV infection, blood clots, liver disease, etc. (secondary
pulmonary hypertension; SPH) or by
unknown causes (primary pulmonary hypertension; PPH). While PPH is a rare
disease, SPH is a major disease
category with unmet medical needs (Benisty, J.I., Circulation 106: e192-4
(2002)). Research in PPH has
demonstrated that VIP has an important beneficial effect on exercise time
/distance (Petkov V, et al., J Clin
Invest 111: 1339-46 (2003)). The long acting VPAC2 analogs of the present
invention will have a similar
beneficial effect in the treatment of such diseases and disease and this
effect will be extended to SPH.
[00147] In another embodiment, the polypeptides of the invention may be
administered in combination with
other compounds usefnl in the treatment of metabolic disorders. For example,
the polypeptides of the invention
may be administered with one or more of the following compounds used in the
treatment of metabolic disorders,
including but not limited to insulin, insulin analogs, incretin, incretin
analogs, glucagon-like peptide, glucagon-
like peptide analogs, glucose dependent insulinotropic peptide analogs,
exendin, exendin analogs, sulfonylureas,
biguanides, a-glucosidase inhibitors, thiazolidinediones, peroxisome
proliferator activated receptor (PPAR, of
which includes agents acting on the a, 0, or y subtypes of PPAR receptors
and/or those agent acting on multiple
subtypes of the PPAR receptors) agonists, PPAR antagonists and PPAR partial
agonists may be administered in
combination with the polypeptides of the present invention. In order to
clarify the types of pharma.ceutical
agents mentioned by the general terms above, specific examples are given. For
example, Eli Lilly sells a fast-
acting insulin analog called "lispro" under the trade name Humalog and Novo
Nordisk sells another fast-
acting insulin analog called "aspart" under the trade name NovoLog . In
addition, Aventis sells a long-acting
insulin analog called "glargine" under the trade name Lantus and Novo Nordisk
sells another long-acting
insulin analog called "detemir" under the trade name Levemir . Examples of
incretin analogs (GLP 1 or GIP
analogs) are exendin-4 (BYETTA Amylin Pharmaceuticals, Inc., San Diego, CA),
liraglutide, ZP-10 (AVE-
010), albugon, and the like. Examples of sulfonylureas/ and the insulin
secretogoguese known as glinides are
Glipizide, Gliclazide, Glibenclamide (glyburide), Glimepiride, and the
glinides Repaglinide, and Nateglinide).
Examples of the "biguanides" are , metformin (Glucophage), buformin, and
phenformin. Examples of "a-
glucosidase inhibitors" are acarbose (Precose) and miglitol (Glycet). Examples
of currently marketed PPARy
pharmaceuticals are the thiazolidinediones pioglitizone (Actos) and
rosiglitazone (Avandia).
[00148] The term "insulin" as used herein includes, but not limited to,
insulin analogs, natural extracted
human insulin, recombinantly produced human insulin, insulin extracted from
bovine and/or porcine sources,
recombinantly produced porcine and bovine insulin and mixtures of any of these
insulin products, and likewise
include all the specific examples disclosed in the previous paragraphs. The
term is intended to encompass the
polypeptide normally used in the treatment of diabetics in a substantially
purified form but encompasses the use
CA 02638968 2008-08-01
WO 2007/044591 PCT/US2006/039267
of the term in its commercially available pharmaceutical form, which includes
additional excipients. The insulin
is preferably recombinantly produced and may be dehydrated (completely dried)
or in solution.
[00149] The terms "insulin analog," "monomeric insulin" and the like are used
interchangeably herein and are
intended to encompass any form of "insulin" as defined above, wherein one or
more of the amino acids within
the polypeptide chain has been replaced with an alternative amino acid and/or
wherein one or more of the amino
acids has been deleted or wherein one or more additional amino acids has been
added to the polypeptide chain
or amino acid sequences, which act as insulin in decreasing blood glucose
levels. In general, the term "insulin
analogs" of the present invention include "insulin lispro analogs," as
disclosed in U.S. Pat. No. 5,547,929,
incorporated hereinto by reference in its entirety; insulin analogs including
LysPro insulin and humalog insulin,
and other "super insulin analogs", wherein the ability of the insulin analog
to affect serum glucose levels is
substantially enhanced as compared with conventional insulin as well as
hepatoselective insulin analogs which
are more active in the liver than in adipose tissue. Preferred analogs are
monomeric insulin analogs, which are
insulin-like compounds used for the same general purpose as insulin, such as
insulin lispro, i.e., compounds
which are administered to reduce blood glucose levels.
[00150] "Insulin analogs" are well known compounds. Insulin analogs are known
to be divided into two
categories: animal insulin analogs and modified insulin analogs (pages 716-20,
chapter 41, Nolte M.S. and
Karam, J.H., "Pancreatic Hormones & Antidiabetic Drugs" In Basic & Clinical
Pharmacology, Katzung, B.G.,
Ed., Lange Medical Books, New York, 2001). Historically, animal insulin
analogs include porcine insulin
(having one amino acid different from human insulin) and bovine insulin
(having three amino acids different
from human insulin) which have been widely used for treatment of diabetes.
Since the development of genetic
engineering technology, modifications are made to create modified insulin
analogs, including fast-acting insulin
analogs or longer acting insulin analogs.
[00151] Several insulin analog molecules have been on the market prior to the
filing date of the subject
application. For example, Eli Lilly sells a fast-acting insulin analog called
"lispro" under the trade name
Humalog and Novo Nordisk sells another fast-acting insulin analog called
"aspart" under the trade name
NovoLog . In addition, Aventis sells a long-acting insulin analog called
"glargine" under the trade name
Lantusg and Novo Nordisk sells another long-acting insulin analog called
"detemir" under the trade name
Levemir . Table 41-4 of the article by Nolte and Karam (2001) referenced above
illustrates the wide range of
types of molecules generically referred to as insulin preparations.
[00152] The term "incretin analogs" refers to incretin hormones responsible
for the phenomenon of enhanced
insulin secretion in the presence of food in the gut and the this action (GLP-
1 and GIP) is widely known (e.g.
articles referenced in Creutzfeldt, W, "The [pre-] history of the incretin
concept". Regulatory Peptides 128: 87-
91 (2005). Examples of incretin analogs (GLP 1 or GIP analogs) are exendin-4
(BYETTA Amylin
Pharmaceuticals, Inc., San Diego, CA), liraglutide, ZP-10 (AVE-0 10), albugon,
and the like.
[00153] The term "glucagon-like peptide analogs" refers to well known analogs
of Glucagon-Like Peptide
(GLP 1) (e.g. Nourparvar, A., et al.. "Novel strategies for the
pharmacological management of type 2 diabetes"
Trends in Pharmacological Sciences 25, 86-91 (2004)), and reviews of the area
discussed their range of structure
and function in detail (cf Table 1 in Knudsen, L.B. "Glucagon-like Peptide-1:
The Basis of a New Class of
Treatment for Type 2 Diabetes ". J. Med. Chem. 47: 4128-4134 (2004) and
references therein). Examples of
"glucagon-like peptide analogs" include Liraglutide, Albugon, and BIM-51077.
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[00154] The term "exendin analogs" refers to exendin (also known as exendin-4,
exanetide, (BYETTA
Amylin Pharmaceuticals, Inc., San Diego, CA) and its analogs which have been
major diabetes research
objectives (c.f. Thorkildsen C. "Glucagozz-Like Peptide 1 Receptor Agonist
ZPIOA Increases Insulin znRNA
Expression and Prevents Diabetic Progression in db/db Mice ". J. Pharmacol.
Exptl. Therapeut. 307: 490-6
(2003)). Exendin is known to be a specific type of glucagon-like peptide-1
mimic. For example, ZP-10 (AVE-
010) is an exendin analog that binds to the GLP 1 receptor.
[00155] The term "sulfonylureas" refers to well known sulfonylureas used for
many years in the treatment of
type 2 diabetes. Extensive clinical trial literature and reviews of
sulfonylureas are available (c.f. Buse, J., et al.
"Tlze effects of oral anti-hyperglycaemic medications on serum lipid profzles
in patients witlz type 2 diabetes ".
Diabetes Obesity Metabol. 6: 133-156 (2004)). In table 1 in the Buse
reference, the major
sulfonylureas/glinides are listed chronologically as Glipizide, Gliclazide,
Glibenclamide (glyburide),
Glimepiride. The last two members of the list (Repaglinide, and Nateglinide)
differ in their specific mechanism
of action (Meglitinides), but again are oral agents that stimulate insulin
secretion. The Buse reference focuses
on studies that are directed at lipid effects, but also illustrates classes of
compounds well known as
"sulfonylureas". For example, it is widely believed that only a few compounds
constitute the major market
share of "sulfonylureas," such as Dymelor, Diabinese, Amaryl, Glucotrol,
Micronase, Tolinase, Orinase and
their generic equivalents (see pgs 725-32, chapter 41, Nolte M.S. and Karam,
J.H., "Pancreatic Hornzones &
Antidiabetic Drugs " In Basic & Clinical Pharma.cology, Katzung, B.G., Ed.,
Lange Medical Books, New York,
2001).
[001561 Examples of sulfonylureas and the insulin secretogoguese known as
glinides are Glipizide,
Gliclazide, Glibenclamide (glyburide), Glimepiride, and the glinides
Repaglinide, and Nateglinide).
[00157] The term "biguanides" refers to well known biguanides compounds, such
as extensively reviewed
on pages 716-20, chapter 41, Nolte M.S. and Karam, J.H., "Pancreatic Hormones
& Azztidiabetic Drugs" In
Basic & Clinical Pharmacology, Katzung, B.G., Ed., Lange Medical Books, New
York, 2001. For example,
well known compounds that constitute the major market share of "biguanides"
include metformin (Glucophage),
buforinin, and phenformin (Buse, J., et al. "Tlze effects of oral anti-
lzyperglycaemic medications on serum lipid
profiles in patients with type 2 diabetes. " Diabetes Obesity Metabol. 6: 133-
156 (2004)).
[00158] Examples of the "biguanides" are metformin (Glucophage), buformin, and
phenforniin.
[00159] The term "a-glucosidase inhibitors" refers to well known compounds
having a-glucosidase inhibitors
activity which has been the subject of extensive clinical studies (pg 729-30,
chapter 41, Nolte M.S. and Karam,
J.H., "Pancreatic Hornzones & Antidiabetic Drugs" In Basic & Clinical
Pharmacology, Katzung, B.G., Ed.,
Lange Medical Books, New York, 2001; Buse, J., et al. "The effects of oral
anti-hyperglycaemic medicatiozzs on
serunz lipid profzles in patients with type 2 diabetes. " Diabetes Obesity
Metabol. 6: 133-156 (2004)).
Compounds that constitute the major market share of "a-glucosidase inhibitors"
include acarbose (Precose) and
miglitol (Glycet).
[00160] Examples of "a-glucosidase inhibitors" are acarbose (Precose) and
miglitol (Glycet).
[00161] The term "PPAR ligands" refers to compounds having Peroxisome
Proliferator-Activated Receptor
Ligand activity, also interchangeably referred to as thizolidinediones for the
predominant structural class, as
compounds active in the treatment of type 2 diabetes (c.f. pg 728, chapter 41,
Nolte M.S. and Karam, J.H.,
"Patzcreatic Hormones & Antidiabetic Drugs" In Basic & Clinical Pharmacology,
Katzung, B.G., Ed., Lange
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WO 2007/044591 PCT/US2006/039267
Medical Books, New York, 2001; Lee, et al. "Minir=eview: Lipid Metabolisnz,
Metabolic Diseases, and
Peroxisome Proliferator Activated Receptors ". Endocrinol. 144: 2201-7
(2003)). PPAR ligands such as
pioglitazone are known to have beneficial effects on protection of pancreatic
islets (Diani, A.R., et al.
"Pioglitazone preserves pancreatic islet structure and insulin secretory
function in three murine models of type
2 diabetes". Am. J. Physiol. Endocrinol. Metab. 286: E116-122 (2004).
Compounds that constitute the major
market share of "PPAR ligands" include pioglitizone (Actos) and rosiglitazone
(Avandia) (c.f pg 732 in Nolte,
M.S. and Karam, J.H. 2001, referenced above). Additional PPAR ligands are
undergoing clinical trials.
[00162] Examples of currently marketed PPARy pharmaceuticals are the
thiazolidinediones pioglitizone
(Actos) and rosiglitazone (Avandia).
[00163] The tenn DPPN inhibitor refers to compounds that that are intended to
potentiate the endogenous
incretin response by preventing the proteolysis of GLP 1 or GIP through the
inhibition of one or more of the
DPPN isoforms in the body (McIntosh, C.H.S., et al., Regulatory Peptides 128:
159-65 (2005)). A number of
such agents are in review at the FDA or in clinical development (Hunziker, D.,
et al., Curr. Top. Med. Chem. 5:
1623-37 (2005); Kim, D., et al., J. Med. Chem. 48: 141-51 (2005)), Some non-
limiting examples of such agents
are: Galvus (vildagliptin; LAF 237); Januvia (sitagliptin; MK-431);
saxagliptin; sulphostin; "P93/01"; "KRP-
104"; "PHX1149" (Phenomix Corp); and the like.
[00164] For combination treatment with more than one active agent, where the
active agents are in separate
dosage formulations, the active agents can be administered concurrently, or
they each can be administered at
separately staggered times.
[00165] The dosages of the compounds of the present invention are adjusted
when combined with other
therapeutic agents. Dosages of these various agents may be independently
optimized and combined to achieve a
synergistic result wherein the pathology is reduced more than it would be if
either agent were used alone. In
addition, co-administration or sequential administration of other agents may
be desirable.
[00166] In other contemplated disease applications, the peptides of the
invention can be used advantageously
in coordination with pharmaceuticals currently applied for that disease.
Particularly beneficial are combination
drug formulations containing mixtures of the active pharmaceutical ingredients
with excipients. For example, in
asthma and COPD, the VPAC2 agonists can used in combination with inhaled
formulations containing
bronchodilators, beta 2 adrenoceptor agonists such as salmeterol, terbutaline,
albuterol, bitolterol, pirbuterol,
salbutamol, formoterol, indacaterol and the like (Sears, M.R and Lotvall, J.,
Resp. Med. 99: 152-170 (2005));
inhaled corticosteroids such as fluticasone (Flovent), budesonide (Pulmicort),
triamcinolone acetonide,
beclomethasone, flunisolide, ciclesonide, mometasone and the like; anti-
inflammatory steroids; leukotriene
modifiers; leukotriene receptor antagonists such as zafirlukast (Accolate )
and montelukast (Singulair ); 5-
lipooxygenase inhibitors like zileuton; chemokine modifiers; chemokine
receptor antagonists; cromolyn;
nedocromil; xanthines such as theophylline; anticholinergic agents; immune
modulating agents; protease
inhibitors; other known anti-asthnia medications, and the like. We expect that
the additional agents in
development (Corry DB and Kheradmand F (2006) J Allergy Clin Inununol 117 (2
Suppl): S461-47) also will
be beneficial when used in combination with VPAC2 agonists.
[00167] VPAC2 combination treatments may make use of currently applied
therapeutics for treatment of
pulmonary hypertension, as well. Thus a VPAC2 agonist may be utilized in
combination with nitric oxide
donors, prostacyclins, endothelin antagonists, adrenoceptor blockers,
phosphodiesterases inhibitors, ion channel
38
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WO 2007/044591 PCT/US2006/039267
blockers and other vasodilators (as outlined in Levy JH Tex Heart Inst J 32:
467-71 (2005); Haj RM, et al., Curr
Opin Anesthesiol 19: 88-95 (2006)).
[00168] Non-limiting examples of particularly important classes of combination
treatments for diabetes are
VPAC2 Modulator plus Insulin Analog and VPAC2 Modulator plus Incretin Analog.
Since PACAP and the
"incretins"are complementary parts of the pancreatic beta cell response to a
meal (neuronal and hormonal,
respectively), use of the combination drug will be a more complete
physiological mimic and may reduce the
required dose of either, with expected beneficial effects. Specific, but non-
limiting, examples here are
BYETTA (Amylin Pharmaceuticals, Inc., San Diego, CA) plus VPAC2 Modulator or
liraglutide plus VPAC2
Modulator. Furthermore, being peptides of similar size, they can be delivered
together from the same
formulation. Similarly, insulin and the glucose-dependent insulin secretory
response caused by the PACAP
signal, can be complementary and, importantly, lead to better glucose control
with less risk of hypoglycemic
responses. Specific, but non-limiting, examples here are Levemir plus VPAC2
Modulator or Lantus plus
VPAC2 Modulator. Examples of combination treatments using DPPIV inhibitors are
VPAC2 Modulator plus
PHX1 149 (Phenomix Corp), VPAC2 Modulator plus Galvus, or VPAC2 Modulator plus
Januvia. Some
DPPIV inhibitors have poor oral bioavailability and would benfit from a
combination formulation for inhalation.
In each of these instances the formulation and route of administration can be
for use by injection or inhalation.
[00169] Similarly, important combination treatments for asthma are within the
scope of the invention.
Specific, but non-limiting, examples here relate to combinations with long-
acting beta2 adrenoceptor agonists
such as: VPAC2 Modulator plus formoterol, VPAC2 Modulator plus indacaterol,
and VPAC2 Modulator plus
salmeterol. Another class of combination treatment uses inhaled
corticosteroids with the VPAC2 Modulator.
Non-limiting examples here are VPAC2 Modulator plus fluticasone, VPAC2
Modulator plus mometasone,
VPAC2 Modulator plus beclomethasone, and VPAC2 Modulator plus Ciclesonide.
[00170] A particularly iinportant consequence of such combination treatments
is the potential for dose-
sparing of these agents with their significant side effects, i.e. the insulin,
incretin, beta2 adreoceptor agonist, or
corticosteroid analogs. This is particularly important in view of the severe
nature of these side effects: for
insulin, death from hypoglycemia; for incretin mimetics, emesis; for beta2
adrenoceptor agonists, heart rate
effects/sudden death; for corticosteroids, diminished growth in children. For
the inhaled corticosteroids, the
formulation of the agent with the very hydrophobic VPAC2 analog offers the
further benefit of delayed release
of the corticosteroid to prolong the relatively short duration of action of
such agents (Winkler, J, et al., Proc Am
Thorac Soc. 1: 356-63 (2004)). In each case the forrnulation of the
combination treatment for inhalation offers
significant commercial and medical benefits.
[00171] Representative delivery regimens include oral, parenteral (including
subcutaneous, intramuscular and
intravenous injection), rectal, buccal (including sublingual), transdermal,
inhalation and intranasal. An attractive
and widely used method for delivery of peptides entails subcutaneous injection
of a controlled release injectable
formulation. Preferred administration routes for the application of the
peptides of the invention are
subcutaneous, intranasal and inhalation administration.
[00172] The selection of the exact dose and composition and the most
appropriate delivery regimen will be
influenced by, inter alia, the pharmacological properties of the selected
polypeptide, the nature and severity of
the condition being treated, and the physical condition and mental acuity of
the recipient. Additionally, the route
of administration will result in differential amounts of absorbed material.
Bioavailabilities for administration of
39
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WO 2007/044591 PCT/US2006/039267
peptides through different routes are particularly variable, with amounts from
less than 1% to near 100% being
seen. Typically, bioavailability from routes other than intravenous injection
are 50% or less.
[00173] In general, the polypeptides of the invention, or salts thereof, are
administered in amounts between
about 0.1 and 60 g/kg body weight per day, preferably from about 0.1 to about
1 g/kg body weight per day,
by subcutaneous injection. For a 50 kg human female subject, the daily dose of
active ingredient is from about 5
to about 1000 g, preferably from about 5 to about 500 g by subcutaneous
injection. Different doses will be
needed, depending on the route of administration and the applicable
bioavailability observed. By inhalation, the
daily dose is from 100 to about 5,000 g, twice daily. In other mammals, such
as horses, dogs, and cattle,
higher doses may be required. This dosage may be delivered in a conventional
pharmaceutical composition by a
single administration, by multiple applications, or via controlled release, as
needed to achieve the most effective
results, preferably one or more times daily by injection.
[001741 Pharmaceutically acceptable salts retain the desired biological
activity of the parent polypeptide
without toxic side effects. Examples of such salts are (a) acid addition salts
formed with inorganic acids, for
example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,
nitric acid and the like; and salts
formed with organic acids such as, for example, acetic acid, oxalic acid,
tartaric acid, succinic acid, maleic acid,
fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic
acid, tannic acid, pamoic acid, alginic
acid, polyglutamic acid, naphthalenesulfonic acids, naphthalene disulfonic
acids, polygalacturonic acid and the
like; (b) base addition salts formed with polyvalent metal cations such as
zinc, calcium, bismuth, barium,
magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or with an
organic cation formed from
N,N'-dibenzylethylenediamine or ethylenediamine; or (c) combinations of (a)
and (b), e.g., a zinc tannate salt
and the like.
[00175] A further aspect of the present invention relates to pharma.ceutical
compositions comprising as an
active ingredient a polypeptide of the present invention, or pharmaceutically
acceptable salt thereof, in
admixture with a pharmaceutically acceptable, non-toxic carrier. As mentioned
above, such compositions may
be prepared for parenteral (subcutaneous, intramuscular or intravenous)
administration, particularly in the form
of liquid solutions or suspensions; for oral or buccal administration,
particularly in the form of tablets or
capsules; for intranasal administration, particularly in the form of powders,
nasal drops or aerosols; for
inhalation, particularly in the form of liquid solutions or dry powders with
excipients, defmed broadly; and for
rectal or transdermal administration.
[00176] The compositions may conveniently be administered in unit dosage form
and may be prepared by
any of the methods well-known in the pharmaceutical art, for example as
described in Remington's
Pharma.ceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,
(1985), incorporated herein by
reference. Formulations for parenteral administration may contain as
excipients sterile water or saline, alkylene
glycols such as propylene glycol, polyalkylene glycols such as polyethylene
glycol, oils of vegetable origin,
hydrogenated naphthalenes, serum albumin nanoparticles (as used in AbraxaneTM,
American Pharmaceutical
Partners, Inc. Schaumburg IL), and the like. For oral administration, the
formulation can be enhanced by the
addition of bile salts or acylcarnitines. Formulations for nasal
adnwiistration may be solid and may contain
excipients, for example, lactose or dextran, or may be aqueous or oily
solutions for use in the form of nasal
drops or metered spray. For buccal adniinistration typical excipients include
sugars, calcium stearate,
magnesium stearate, pregelatinated starch, and the like.
CA 02638968 2008-08-01
WO 2007/044591 PCT/US2006/039267
[00177] When formulated for nasal administration, the absorption across the
nasal mucous membrane may be
enhanced by surfactant acids, such as for example, glycocholic acid, cholic
acid, taurocholic acid, ethocholic
acid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid,
glycodeoxycholic acid, cyclodextrins and
the like in an amount in the range between about 0.2 and 15 weight percent,
preferably between about 0.5 and 4
weight percent, most preferably about 2 weight percent. An additional class of
absorption enhancers exhibiting
greater efficacy with decreased irritation is the class of alkyl maltosides,
such as tetradecylmaltoside (Arnold,
J.J., et al., J Pharm. Sci. 93, 2205-13 (2004) and references therein, all of
which are hereby incorporated by
reference).
[00178] When forinulated for delivery by inhalation, a number of formulations
offer advantages. Adsorption
of the active peptide to readily dispersed solids such as diketopiperazines
(for example Technosphere particles;
Pfatzner, A. and Forst, T., Expert Opin Drug Deliv 2: 1097-106 (2005) or
similar structures gives a formulation
which results in a rapid initial uptake of the therapeutic agent. Lyophylized
powders, especially glassy particles,
containing the active peptide and an excipient are useful for delivery to the
lung with good bioavailability, for
example, see Exubera (inhaled insulin by Pfizer and Aventis Pharmaceuticals
Inc.). Additional systems for
delivery of polypeptides by inhalation (Mandal, T.K., Am. J. Health Syst.
Pharrn. 62: 13 59-64 (2005)) are well
known in the art and are incorporated into this invention.
[00179] Delivery of the compounds of the present invention to the subject over
prolonged periods of time, for
example, for periods of one week to one year, may be accomplished by a single
administration of a controlled
release system containing sufficient active ingredient for the desired release
period. Various controlled release
systems, such as monolithic or reservoir-type microcapsules, depot implants,
osmotic pumps, vesicles, micelles,
liposomes, transdermal patches, iontophoretic devices and alternative
injectable dosage forms rna.y be utilized
for this purpose. Localization at the site to which delivery of the active
ingredient is desired is an additional
feature of some controlled release devices, which may prove beneficial in the
treatment of certain disorders.
[00180] One form of controlled release formulation contains the polypeptide or
its salt dispersed or
encapsulated in a slowly degrading, non-toxic, non-antigenic polymer such as
copoly(lactic/glycolic) acid, as
described in the pioneering work of Kent, Lewis, Sanders, and Tice, U.S. Pat.
No. 4,675,189, incorporated by
reference herein. The compounds or, preferably, their relatively insoluble
salts, may also be forxnulated in
cholesterol or other lipid matrix pellets, or silastomer matrix implants.
Additional slow release, depot implant or
injectable forrnulations will be apparent to the skilled artisan. See, for
example, Sustained and Controlled
Release Drug Delivery Systems, J. R. Robinson ed., Marcel Dekker, Inc., New
York, 1978, and R. W. Baker,
Controlled Release of Biologically Active Agents, John Wiley & Sons, New York,
1987, incorporated by
reference herein.
[00181] All publications and patent applications mentioned in this
specification are herein incorporated by
reference to the same extent as if each independent publication or patent
application is specifically and
individually indicated to be incorporated by reference.
[00182] While the examples and discussion given above are intended to
illustrate the synthesis and testing of
representative compounds of the invention, it will be understood that it is
capable of further modifications and
should not be construed as limiting the scope of the appended claims.
41
