Note: Descriptions are shown in the official language in which they were submitted.
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IlVIPROVED FORMULATIONS OF ANTI-ANGIOGENIC PEPTIDES
This application claims priority from U.S. Provisional Application 60/648,391,
filed
01 February 2005.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention is directed to compositions comprising improved
formulations of the
Cys-containing anti-angiogenic peptide Pro-His-Ser-Cys-Asn that prevent
degradation and
spontaneous oxidative dimerization or oligomerization of the peptide.
Description of the Background Art
Most forms of cancer are manifest as, or derived from, solid tumors (Shockley
et al., Ann.
N.Y. Acad. Sci. 1991, 617:367-82). These types of tumors have generally proven
resistant in the
clinic to biological therapeutics such as monoclonal antibodies and
immunotoxins. Anti-angiogenic
therapy for the treatment of cancer developed from the recognition that solid
tumors require
angiogenesis (i.e., new blood vessel formation) for sustained growth (Folkman,
Ann. Surg. 1972,
175:409-16; Folkman, Mol. Med. 1995, 1:120-22; Folkman, Breast Cancer Res.
Treat. 1995,
36:109-18; Hanahan et al., Cell 1996, 86:353-64). Efficacy of anti-angiogenic
therapy in animal
models has been demonstrated in many studies, for example, Millauer et al.,
Cancer Res. 1996,
56:1615-20; Borgstrom et al., Prostate 1998, 35:1-10; Benjamin et al., J.
Clin. Invest. 1999,
103:159-65; Brewer GJ et al., Integr Cancer Tlier. 2002, 1:327-37; van Golen
KL et al., Neoplasia
2002, 4:373-9; Cox C et al., Arch Otolaryngol Head Neck Surg. 2003, 129:781-5.
In the absence of
angiogenesis, internal cell layers of solid tumors are inadequately nourished.
Further, angiogenesis
(i.e., aberrant vascularization) has now also been shown to be required for
the growth of non-solid,
hematological tumors and has been implicated in numerous other diseases,
including ocular
neovascular disease, macular degeneration, rheumatoid arthritis, etc.
In contrast, normal tissue does not require angiogenesis except under
specialized
circumstances such as wound repair, the proliferation of the uterine internal
lining during the
menstrual cycle, etc. Accordingly, a requirement for angiogenesis is a
significant difference
between tumor/cancerous tissue and normal tissue. Importantly, the dependence
of tumor cells on
angiogenesis, when compared to normal cells, is quantitatively greater than
differences in cell
replication and cell death. The latter differences are typically exploited in
cancer therapy.
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Tumor angiogenesis can be initiated by cytokines such as vascular endothelial
growth factor
(VEGF) and/or a fibroblast growth factor (FGF), which bind to specific
receptors on endothelial
cells ("ECs") in the local vasculature, under hypoxic conditions. The
activated ECs secrete enzymes
which remodel the associated tissue matrix and modulate expression of adhesion
molecules such as
integrins. Following matrix degradation, ECs proliferate and migrate toward
the tumor, which
results in the generation and maturation of new blood vessels.
Interestingly, protein fragments, such as endostatin, kringle 5 and PEX, which
inhibit
angiogenesis, are produced by degradation of matrix proteins (O'Reilly et al.,
Cell 1997, 88:277-85;
O'Reilly et al., Cell, 1994, 79:315-28; Brooks et al., Cell, 1998, 92:391-
400). Accordingly, these
protein fragments may inhibit neoangiogenesis, thus preventing tumor growth
and metastasis.
However, protein fragments have significant drawbacks associated with their
use. They are difficult
and expensive to produce in large quantities, show poor pharmacological
properties, are susceptible
to degradation, etc. One approach has been to identify short peptide fragments
of these larger
proteins or of the longer fragments or subunits, which shorter peptides retain
a significant portion of
the anti-angiogenic activity of the parent protein.
Although the search for peptides that inhibit angiogenesis has provided
compounds with
significant effectiveness in preventing growth of new blood vessels, molecules
with superior activity
profiles are still needed. Accordingly, novel peptides are needed to fully
explore the potential of
peptides in preventing angiogenesis and detecting aberrant vascularization.
The novel peptides may
have longer plasma half-lives, be more resistant to degradation, have
increased bio-availability,
higher affinity, greater selectivity, etc. in comparison to peptides described
in the art (Livant, U.S.
Pats. No. 6,001,965 and 6,472,369). Such novel peptides may be effective in
inhibiting cell
migration, invasion and proliferation and treating or preventing various
diseases associated with
undesired angiogenesis and aberrant vascularization. Examples of such
peptides, primarily
derivatives of the capped pentapeptide Ac-PHSCN-NH2 (also referred to herein
as ATN-161) are
described in commonly assigned U.S. Patent applications Ser. No. 10/723,144
filed 25 November
2003 (published as US20040162239A1, Allan et al. ) and Ser. No. 10/722,843,
filed 25 November
2003 (published as US 20050020810A1, Ternansky et al.), which are hereby
incorporated by
reference in their entirety
What is needed in the art is a method of preventing degradation of Ac-PHSCN-
NH2 under
both solution phase and solid phase conditions. One approach to improving the
activity profile of an
anti-angiogenic peptide is to exploit the method of its formulation as a means
to extend shelf life and
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enhance the resistance to degradation. In the case of peptides that include
Cys, it is also important
to prevent spontaneous oxidative dimerization or higher degrees of
oligomerization or
polymerization through disulfide bond formation. The present invention is
directed to such
improved formulations for anti-angiogenic peptide Ac-PHSCN-NH2 and its anti-
angiogenic
derivatives.
SUMMARY OF THE INVENTION
The present inventors and their colleagues have developed compositions
comprising
improved formulations for Cys-containing peptides and salts thereof that
preserve the
biological/biochemical potency of the peptides, particularly by inhibiting
disulfide bond formation
that leads to undesired dimerization.
Acid addition salts of Ac-PHSCN-NH2 useful in the formulations of the present
invention
are described in co-pending applications by Trenansky et al., both entitled
"Acid Addition Salts of
Ac-PHSCN-NH2", United States Provisional Application Serial No. 60/649,308,
filed February 1,
2005, and International Application No. (to be assigned), filed February 1,
2006 (Attorney Docket
No. 9715-022-228), each of which is incorporated by reference herein in its
entirety. The above
application discloses acid addition salts of Ac-PHSCN-NH2, methods of making
acid addition salts
of Ac-PHSCN-NH2, pharmaceutical compositions of acid addition salts of Ac-
PHSCN-NH2,
methods of using acid addition salts of Ac-PHSCN-NH2 and pharmaceutical
compositions thereof to
treat diseases associated with angiogenesis and aberrant vascularization and
methods of preventing
degradation of Ac-PHSCN-NH2 by salt formation. Thus, the novel formulations of
the present
application include those using the new salts described by Ternansky et al.,
supra.
The present invention is thus directed to a formulation comprising a peptide
Pro-His-Ser-
Cys-Asn (PHSCN), an analogue thereof of a salt of the peptide or analogue, and
at least one
additional compound that stabilizes the peptide or analogue against
spontaneous tandem
dimerization or higher oligomerization. Preferably, the peptide is capped at
both termini, with an
acetyl group at the N-terminus and amide group at the C-terminus. The
additional compound
inhibits, prevents or reverses disulfide bond formation between sulfhydryl
groups of Cys residues.
Also provided is the above formulation wherein the additional compound is a
biocompatible
acid buffer with a pK of about 5.
Preferably, in the presence of the buffer, the pH of the solution is greater
than about 3.0 and
less than about, or equal to, 7.5. A preferred acid buffer is citrate, acetate
or 2-(N-morpho-
lino)ethanesulfonic acid (MES).
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In the above formulation, the acid buffer is preferably citrate at a
concentration of about 25
mM. The buffer may be supplemented with glycine as an excipient and bulking
agent, preferably at
a concentration of about 50 mg/ml. The buffer may comprise citrate and acetate
and may also
comprise Tris.
In a preferred embodiment, the above formulation comprises (i) the peptide,
capped peptide
or analogue, (ii) about 50 mM citrate, and (iii) about 50 mg/ml glycine.
A preferred embodiment of the formulation is in a container or vial in
lyophilized form
having 100 mg peptide, capped peptide, salt or analogue, 50 mM citrate, 50
mg/ml glycine
lyophilized from 2 mL of a pH 5.0 solution.
The above formulation may further comprise or more reducing agents; preferred
agents are
dithiothreitol (3-mercaptoethanol, or glutathione. Preferably, the
concentration of the reducing
agent or agents does not exceed about 10 mM.
The above formulation may comprise a non-thiol biocompatible anti-oxidant.
The formulation may comprise a lyoprotectant present in an lyoprotecting
amount, for
example, about 50-600 mole lyoprotectant:l mole peptide. The lyoprotectant is
one or more sugars,
one or more amino acids, one or more methylamine, one or more lyotropic salts,
and/or one or more
polyols. In a preferred formulation above,
(a) the sugar is sucrose or trehalose; (b) the amino acid is monosodium
glutamate or histidine;
(c) the methylamine is betaine; (d) the lyotropic salt is magnesium sulfate;
and (e) the polyol is a
trihydric or higher sugar alcohol.
The formulation above may comprise one or more polyols selected from the group
consisting of glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol,
mannitol, propylene glycol, and
a combination thereof. In one formulation, the lyoprotectant is a non-reducing
sugar, preferably
trehalose or sucrose.
In all the above formulations the peptide is preferably capped at its N- and C-
termini, most
preferably the N-terminal cap is an acyl group and the C terminal cap is an
amide group such that
the peptide terminates in CO-NH2.
The formulation above is preferably sterile and formulated for in vivo
administration.
The present invention includes an article of manufacture or kit comprising
(a) a container which contains a formulation as above, in solution or in
lyophilized form;
(b) optionally a second container containing a diluent or reconstituting
solution for the
lyophilized formulation; and
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(c) optionally, instructions for (i) use of the solution or (ii)
reconstitution and/or use of the
lyophilized formulation.
The article or kit may further comprise one or more of (i) another buffer,
(ii) a diluent, (iii) a
filter, (iv) a needle, or (v) a syringe. The container is preferably a bottle,
a vial, a syringe or test
tube; it may be a multi-use container. the formulation is preferably
lyophilized.
The present invention is directed to a method of inhibiting angiogenesis in a
subject,
comprising administering to the subject a formulation as above, wherein the
peptide, analogue or
salt is administered in an anti-angiogenic effective amount. Such inhibition
of angiogenesis is
exploited in a method for the treatment of cancer and or Crohn's disease. The
invention provides
use of a composition as above in a medicament for administration to a subject
to inhibit undesired
angiogenesis, such as in the treatment of cancer or Crohn's disease. Also
included is use of a
composition as above in the manufacture of a medicament for administration to
a subject to inhibit
undesired angiogenesis and, thereby, to treat cancer and Crohn's disease.
DESCRIPTION OF THE PREFERRED EMBODIlVIENTS
The present inventors have devised an improved formulation for a Cys-
containing peptide,
PHSCN, most preferably, the terminally capped pentapeptide, Acetyl-PHSCN-NH2,
and various
salts thereof, and or analogues of capped or uncapped PHSCN and salts of the
derivatives and
analogues. Various other capping functions are discussed below. An "analogue"
of PHSCN refers
to a molecule, natural or non- natural, that is structurally and/or
functionally substantially similar to
PHSCN. Examples include conservative amino acid substitution variants,
addition variants of not
more than about 20 residues (to the exclusion of native polypeptides or their
structural domains),
and chemically modified peptides. Other analogues are peptidomimetics and
aptamers.
The peptide to be formulated is preferably pure, or essentially pure and,
desirably,
essentially homogeneous (i.e., free from contaminating peptides or proteins,
etc.) "Essentially
pure" means a peptide preparation wherein at least 90% by weight is the
peptide based on total
weight of the preparation, preferably at least 95% by weight. An "essentially
homogeneous"
preparation means a peptide preparation comprising at least 99% by weight of
peptide, based on
total weight of the peptide in the preparation.
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CAPPING GROUPS
As noted above, the PHSCN peptide is preferably capped at its N and C termini
with an acyl
(abbreviated "Ac") -and an amido (abbreviated "Am") group, respectively, for
example acetyl
(CH3CO-) at the N terminus and amido (CO-NH2) at the C terminus.
N-terminal Capping Groups
A broad range of N-terminal capping functions, preferably in a linkage to the
terminal amino
group, is contemplated, for example:
formyl;
C1_10 alkanoyl, such as acetyl, propionyl, butyryl;
C1_10 alkenoyl, such as hex-3-enoyl;
C1_10 alkynoyl, having from 1 to 10 carbon atoms, such as hex-5-ynoyl;
aroyl, such as benzoyl or 1-naphthoyl;
heteroaroyl, such as 3-pyrroyl or 4-quinoloyl;
alkylsulfonyl, such as methanesulfonyl;
arylsulfonyl, such as benzenesulfonyl or sulfanilyl;
heteroarylsulfonyl, such as pyridine-4-sulfonyl;
Cl_lo substituted alkanoyl, such as 4-aminobutyryl;
Cl_io substituted alkenoyl, such as 6-hydroxy-hex-3-enoyl;
C1_10 substituted alkynoyl, such as 3-hydroxy-hex-5-ynoyl;
substituted aroyl, such as 4-chlorobenzoyl or 8-hydroxy-naphth-2-oyl;
substituted heteroaroyl, such as 2,4-dioxo-1,2,3,4-tetrahydro-3-methyl-
quinazolin-6-oyl;
substituted alkylsulfonyl, such as 2-aminoethanesulfonyl;
substituted arylsulfonyl, such as 5-dimethylamino-l-naphthalenesulfonyl;
substituted heteroarylsulfonyl, such as 1-methoxy-6-isoquinolinesulfonyl;
carbamoyl or thiocarbamoyl;
substituted carbamoyl (R'-NH-CO) or substituted thiocarbamoyl (R'-NH-CS)
wherein R' is
alkyl, alkenyl, alkynyl, aryl, heteroaryl, substituted alkyl, substituted
alkenyl, substituted alkynyl,
substituted aryl, or substituted heteroaryl;
substituted carbamoyl (R'-NH-CO) and substituted thiocarbamoyl (R'-NH-CS)
wherein R'
is alkanoyl, alkenoyl, alkynoyl, aroyl, heteroaroyl, substituted alkanoyl,
substituted alkenoyl,
substituted alkynoyl, substituted aroyl, or substituted heteroaroyl, all as
above defined.
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C-terfninal Canninz Groups
The C-terminal capping function can either be in an amide or ester bond with
the terminal
carboxyl. Capping functions that provide for an amide bond are designated as
NR1R2 wherein R'
and R2 may be independently drawn from the following group:
hydrogen;
C1_10 alkyl, such as methyl, ethyl, isopropyl;
C1_10 alkenyl, such as prop-2-enyl;
Cl-lo alkynyl, such as prop-2-ynyl;
Cl-lo substituted alkyl such as hydroxyalkyl, alkoxyalkyl, mercaptoalkyl,
alkylthioalkyl,
halogenoalkyl, cyanoalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,
alkanoylalkyl,
carboxyalkyl, carbamoylalkyl;
Cl-lo substituted alkenyl such as hydroxyalkenyl, alkoxyalkenyl,
mercaptoalkenyl,
alkylthioalkenyl, halogenoalkenyl, cyanoalkenyl, aminoalkenyl,
alkylaminoalkenyl,
dialkylaminoalkenyl, alkanoylalkenyl, carboxyalkenyl, carbamoylalkenyl;
Cl-lo substituted alkynyl such as hydroxyalkynyl, alkoxyalkynyl,
mercaptoalkynyl,
alkylthioalkynyl, halogenoalkynyl, cyanoalkynyl, aminoalkynyl,
alkylaminoalkynyl,
dialkylaminoalkynyl, alkanoylalkynyl, carboxyalkynyl, carbamoylalkynyl;
C1_10 aroylalkyl such as phenacyl or 2-benzoylethyl;
aryl, such as phenyl or 1-naphthyl;
heteroaryl, such as 4-quinolyl;
Cl-lo alkanoyl such as acetyl or butyryl;
aroyl, such as benzoyl;
heteroaroyl, such as 3-quinoloyl;
OR' or NR'R" where R' and R" are independently hydrogen, alkyl, aryl,
heteroaryl, acyl,
aroyl, sulfonyl, sulfinyl, or S02-R"' or SO-R"' where R"' is substituted or
unsubstituted alkyl,
aryl, heteroaryl, alkenyl, or alkynyl.
Capping functions that provide for an ester bond are designated as OR, wherein
R may be:
alkoxy; aryloxy; heteroaryloxy; aralkyloxy; heteroaralkyloxy; substituted
alkoxy; substituted
aryloxy; substituted heteroaryloxy; substituted aralkyloxy; or substituted
heteroaralkyloxy.
Either the N-terminal or the C-terminal capping function, or both, may be of
such structure
that the capped molecule functions as a prodrug (a pharmacologically inactive
derivative of the
parent drug molecule) that undergoes spontaneous or enzymatic transformation
within the body in
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order to release the active drug and that has improved delivery properties
over the parent drug
molecule (Bundgaard H, Ed: Design of Prodrugs, Elsevier, Amsterdam, 1985).
Judicious choice of capping groups allows the addition of other activities on
the peptide.
For example, the presence of a sulfhydryl group linked to the N- or C-terminal
cap will permit
conjugation of the derivatized peptide to other molecules.
A "lyoprotectant" is a molecule which, when combined with a protein or peptide
of interest,
significantly prevents or reduces chemical and/or physical instability of the
protein or peptide upon
lyophilization and subsequent storage. Exemplary lyoprotectants include sugars
such as sucrose or
trehalose; an amino acid such as monosodium glutamate or histidine; a
methylamine such as
betaine; a lyotropic salt such as magnesium sulfate; a polyol such as
trihydric or higher sugar
alcohols, e.g., glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol,
and mannitol; propylene
glycol; polyethylene glycol; pluronics; and combinations thereof. The
preferred lyoprotectant is a
non-reducing sugar, such as trehalose or sucrose. The lyoprotectant is added
to the pre-lyophilized
formulation in a "lyoprotecting amount" which means that, following
lyophilization of the peptide in
the presence of the lyoprotecting amount of the lyoprotectant, the peptide
essentially retains its
physical and chemical stability and integrity upon lyophilization and storage.
The "diluent" of interest is one which is pharmaceutically acceptable (safe
and non-toxic for
administration to a human) and is useful for the preparation of a
reconstituted formulation.
Exemplary diluents include sterile water, bacteriostatic water for injection
(BWFT), a pH buffered
solution (e.g. phosphate-buffered saline), sterile saline solution, Ringer's
solution or dextrose
solution.
A"preservative" is a compound which can be added to the diluent to essentially
reduce
bacterial action in the reconstituted formulation, thus facilitating the
production of a multi-use
reconstituted formulation, for example. Examples of potential preservatives
include
octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,
benzalkonium chloride (a
mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are
long-chain
compounds), and benzethonium chloride. Other types of preservatives include
aromatic alcohols
such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or
propyl paraben, catechol,
resorcinol, cyclohexanol, 3-pentanol, and m-cresol. The most preferred
preservative herein is benzyl
alcohol.
A "bulking agent" is a compound which adds mass to the lyophilized mixture and
contributes to the physical structure of the lyophilized cake (e.g.,
facilitates the production of an
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essentially uniform lyophilized cake which maintains an open pore structure).
Exemplary bulking
agents include mannitol, glycine, polyethylene glycol and xorbitol.
One important purpose of the present formulation is to stabilize these peptide
or analogue
against spontaneous dimerization.. This is preferably accomplished by
formulating the peptides at
low pH that will prevent disulfide bond formation between two Cys residues.
Such acid buffers are
preferably biocompatible. Examples include citrate, acetate, 2-(N-
morpholino)ethanesulfonic acid
(MES), or any similar buffer with a pK of about 5, wherein in the presence of
the buffer, the pH of
the solution is :0.5 but preferably not below 3Ø A preferred acidic
formulation comprises citrate,
preferably at about 25mM. The buffer is preferably supplemented with glycine
(Gly) as an
excipient and bulking agent. A preferred concentration is about 50 mg/ml Gly.
Another advantage
of Gly is that it is an accepted excipient for intravenous infusion or
injection in humans.
Other amino acids or compounds can be used in place of Gly. Examples of
desirable acids
or combinations are citrate + acetate, acetate and Tris, and the like. The
goal is to keep the pH low
once the peptide is in lyophilized form. The lyophilized powder is then
reconstituted with water.
The present invention includes, in addition to lyophilized compositions,
stabilized liquid
pharmaceutical compositions comprising a peptide as disclosed herein,
preferably capped, whose
effectiveness as a therapeutically active component is normally compromised
during storage in
liquid formulations as a result of dimerization and higher order
oligomerization, aggregation, etc.
The stabilized liquid pharmaceutical composition comprises an amount of an
amino acid base
sufficient to decrease aggregate formation during storage, where the amino
acid base is an amino
acid or a combination of amino acids, where any given amino acid is present
either in its free base
form or in its salt form. It is understood that the composition comprises a
buffering agent to maintain
pH of the liquid composition within an acceptable range for stability of the
peptide, where the
buffering agent is an acid substantially free of its salt form, an acid in its
salt form, or a mixture of
an acid and its salt form. The amino acid base serves to stabilize the peptide
against aggregate
formation during storage of the liquid pharmaceutical composition, while use
of an acid buffering
agent (in either of it forms) results in a liquid composition having an
osmolarity that is nearly
isotonic. The liquid phannaceutical composition may additionally incorporate
other stabilizing
agents, more particularly methionine, a nonionic surfactant such as
polysorbate 80, and EDTA, to
further increase stability of the peptide. Such liquid pharmaceutical
compositions are said to be
stabilized, as addition of amino acid base in combination with an acid
substantially free of its salt
form, an acid in its salt form, or a mixture of an acid and its salt form,
results in the increased
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storage stability relative to liquid compositions formulated in the absence of
the combination of
these two components. Methods for increasing the stability of the peptide in a
liquid pharmaceutical
composition (and for increasing storage stability of such a composition)
comprise incorporating into
the liquid composition an amount of an amino acid base sufficient to decrease
aggregate formation
of the polypeptide during storage, and a buffering agent that is an acid
substantially free of its salt
form, an acid in its salt form, or a mixture of an acid and its salt form.
The acid addition salts of Ac-PHSCN-NH2 (see Ternansky et al., supra) may be
formed
from both organic and inorganic acids. Exemplary organic acids include
generally, carboxylic acids
and sulfonic acids, such as acetic acid, propionic acid, hexanoic acid,
cyclopentanepropionic acid,
glycolic acid, pyruvic acid, lactic acid, glycolic acid, malonic acid,
succinic acid, malic acid, maleic
acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-
hydroxybenzoyl) benzoic acid,
cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-
ethane-disulfonic acid,
2-hydroxyethanesulfonic acid, benzenesulfonic acid, fumaric acid, oxalic acid,
lactic acid, 4-
chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic
acid, camphorsulfonic
acid, 4-methylbicyclo[2.2.2]-oct-2-ene-l-carboxylic acid, glucoheptonic acid,
3-phenylpropionic
acid, trimethylacetic acid, t-butylacetic acid, lauryl sulfuric acid, gluconic
acid, glutamic acid,
hydroxynaphthoic acid, salicylic acid, stearic acid and muconic acid. Other
organic acids are also
known to the skilled artisan. In some embodiments, the acid addition salt of
Ac-PHSCN-NH2 is
formed from methanesulfonic acid, acetic acid, glycolic acid, (+)
camphorsulfonic acid, mandeleic
acid, salicyclic acid, succinic acid or combinations thereof.
Exemplary inorganic acids include hydrofluoric acid, perchloric acid,
hydrochloric acid,
hydrobromic acid, nitric acid, phosphoric acid, phosphorous acid, hydroiodic
acid, chloric acid,
thiocyanic acid, hypophosphorous acid, nitrous acid, cyanic acid, chromic
acid, sulfurous acid, and
hydrazoic acid. Other inorganic acids are known to those of skill in the art.
In some embodiments,
the acid addition salt of Ac-PHSCN-NH2 is formed from hydrobromic acid, nitric
acid, hydrochloric
acid, phosphoric acid or combinations thereof. In other embodiments, the acid
addition salt of
Ac-PHSCN-NH2 is formed from hydrochloric acid.
Generally, the acid addition salts of Ac-PHSCN-NH2 may be made any
conventional method
known to those of skill in the art. These methods include saturating solutions
of Ac-PHSCN-NH2
with gaseous acids, adding solutions of acids to solutions of Ac-PHSCN-NH2,
etc. In some
embodiments, an acid addition salt of Ac-PHSCN-NH2 is made by adding slightly
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equivalent (e.g., 1.05 equivalent) of the acid to a solution of Ac-PHSCN-NH2
dissolved in distilled
water. The acid addition salt is typically isolated as a solid from the
aqueous mixture.
The acid addition salt of Ac-PHSCN-NH2 is generally, considerably more stable
than the
free base in both the solid and solution phase. The acid addition salt is
believed to prevent oxidative
dimerization of Ac-PHSCN-NH2 mediated by cysteine. Acid addition salts which
prevent
degradation of Ac-PHSCN-NH2 are formed from, for example, methanesulfonic
acid, acetic acid,
glycolic acid, sulfuric acid, (+) camphorsulfonic acid, mandeleic acid,
salicyclic acid, succinic acid,
hydrobromic acid, hydrochloric acid, nitric acid and phosphoric acid.
The formulation of the present invention preferably comprises one or more
reducing agent,
such as dithiothreitol (DTT) at a low concentration, preferably not exceeding
about 10 mM,
(3-mercaptoethanol, glutathione (GSH) or other Cys-containing reducing agents.
Other non-thiol
containing anti-oxidants such as metal binding compounds (EDTA, EGTA) that are
biocompatible
may also be used.
Another embodiment of the invention is directed to formulations that increase
half-life of the
peptide after parenteral injection. Examples of these include formulation of
Ac-PHSCN-NH2 with a
cyclodextrins, cremaphor or liposomal formulations.
"Cyclodextrin" refers to a cyclic molecule containing six or more a-D-
glucopyranose units
linked at the 1,4 positions by a linkages as in amylose. (3-Cyclodextrin or
cycloheptaamylose
contains seven a-D-glucopyranose units. As used herein, the term
"cyclodextrin" also includes
cyclodextrin derivatives such as hydroxypropyl and sulfobutyl ether
cyclodextrins. Such derivatives
are described for example, in U.S. Patents No. 4,727,064 and 5,376,645. One
preferred cyclodextrin
is hydroxypropyl (3-cyclodextrin (H(3CD) having a degree of substitution of
from about 4.1-5.1 as
measured by Fourier Transform InfTared Spectroscopy. Such a cyclodextrin is
available from
Cerestar (Hammond, Ind., USA) under the name Cavitron 82003TM.
In one preferred embodiment, the Ac-PHSCN-NH2 or derivative is formulated in
an aqueous
solution containing a cyclodextrin. In another embodiment, the peptide of this
invention is
formulated as a lyophilized powder containing a cyclodextrin or as a sterile
powder containing a
cyclodextrin. Preferably, the cyclodextrin is (H(3CD) or sulfobutyl ether (3-
cyclodextrin; more
preferably, the cyclodextrin is hydroxypropyl-(3-cyclodextrin. Typically, in
an injectable solution,
the cyclodextrin will comprise about 1 - 25% (w/w), preferably, about 2 - 10%,
more preferably,
about 4 - 6%, of the formulation. Additionally, the weight ratio of the
cyclodextrin to the peptide
will preferably be from about 1:1 to about 10:1.
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In another embodiment of the invention, Ac-PHSCN-NH2 or its derivative is
formulated to
render the peptide orally or intranasally bioavailable.
Other useful components in the present formulation include polyols such as
mannitol, which
may act as stabilizers, and further help the bulk powder form a cake.
Preferably, the polyol has at
least three hydroxyl groups, and may be in the form of a mixture of two or
more polyols.
The formulations to be used for in vivo administration must be sterile. This
is readily
accomplished by filtration through sterile filtration membranes, prior to, or
following, lyophilization
and reconstitution. Alternatively, sterility, of the entire mixture may be
accomplished by autoclaving
the ingredients prior to addition of the peptide at about 120 C for about 30
minutes, for example.
After the peptide, lyoprotectant and other optional components are mixed
together, the
formulation is lyophilized. Many different freeze-drivers are available for
this purpose such as
Hu1150TM (Hull, USA) or GT20TM (Leybold-Heraeus, Germany) freeze-dryers.
Freeze-drying is
accomplished by freezing the formulation and subsequently subliming ice from
the frozen content at
a temperature suitable for primary drying. Under this condition, the product
temperature is below
the eutectic point or the collapse temperature of the formulation. Typically,
the shelf temperature
for the primary drying will range from about -30 to 25 C (provided the product
remains frozen
during primary dying) at a suitable pressure, ranging typically from about 50
to 250 mTorr. The
formulation, size and type of the container holding the sample (e.g., glass
vial) and the volume of
liquid will mainly dictate the time required for drying, which can range from
a few hours to several
days (e.g., 40-60 hrs). A secondary drying stage may be carried out at about 0-
40 C, depending
primarily on the type and size of container and nature of the peptide. For
example, the shelf
temperature throughout the entire water removal phase of lyophilization may be
from about 15-
C. (e.g., about 20 C.). The time and pressure required for secondary drying
will be that which
produces a suitable lyophilized cake, dependent e.g., on the temperature and
other parameters. The
25 secondary drying time is dictated by the desired residual moisture level in
the product and typically
takes at least about 5 hours (e.Q., 10-15 hours). The pressure may be the same
as that employed
during the primary drying step. Freeze-drying conditions can be varied
depending on the
formulation and vial size.
In some instances, it may be desirable to lyophilize the peptide formulation
in the container
30 in which reconstitution of the peptide is to be carried out in order to
avoid a transfer step. The
container in this instance may, for example, be a 3, 5, 10, 20, 50 or 100 cc
vial.
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As a general proposition, lyophilization will result in a lyophilized
formulation in which the
moisture content thereof is less than about 5%, preferably less than about 3%.
Reconstitution of the Lophilized Formulation
At the desired stage, typically when it is time to administer the peptide to
the patient, the
lyophilized formulation may be reconstituted with a diluent such that the
peptide concentration in
the reconstituted formulation is at least 10 mg/mL, for example from about 10
mg/mL to about 1000
mg/mL, more preferably from about 50 mg/mL to about 500 mg/mL, and most
preferably from
about 100 mg/mL to about 500 mg/mL. Such high peptide concentrations in the
reconstituted
formulation are considered to be particularly useful where subcutaneous
delivery of the reconstituted
formulation is intended. However, for other routes of administration, such as
intravenous (i.v.)
administration, lower concentrations of the peptide in the reconstituted
formulation may be desired
(for example from about 1-100 mg/mL, or from about 5-50 mg/mL peptide in the
reconstituted
formulation). In certain embodiments, the peptide concentration in the
reconstituted formulation is
significantly higher than that in the pre-lyophilized formulation. For
example, the peptide
concentration in the reconstituted formulation may be about 2-40 times,
preferably 3-10 times and
most preferably 3-6 times (e.g., at least three fold or at least four fold)
that of the pre-lyophilized
formulation.
Reconstitution generally takes place at a temperature of about 25 C to ensure
complete
hydration, although other temperatures may be employed as desired. The time
required for
reconstitution will depend, for example, on the type of diluent, amount of
excipient(s) and peptide.
Exemplary diluents include sterile water, bacteriostatic water for injection
(BWFI), a pH buffered
solution (e.g. phosphate-buffered saline, PBS), sterile saline solution,
Ringer's solution or dextrose
solution. The diluent optionally contains a preservative. Exemplary
preservatives have been
described above, with aromatic alcohols such as benzyl or phenol alcohol being
the preferred
preservatives. The amount of preservative employed is determined by assessing
different
preservative concentrations for compatibility with the peptide and
preservative efficacy testing. For
example, if the preservative is an aromatic alcohol (such as benzyl alcohol),
it can be present in an
amount from about 0.1-2.0% and preferably from about 0.5-1.5%, but most
preferably about
1.0-1.2%.
Preferably, the reconstituted formulation has less than 6000 particles per
vial which are
_10 m in size.
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WO 2006/083906 PCT/US2006/003461
Articles of Manufacture
In another embodiment of the invention, an article of manufacture is provided
which
contains the lyophilized formulation of the present invention and provides
instructions for its
reconstitution and/or use. The article of manufacture comprises a container.
Suitable containers
include, for example, bottles, vials (e.g. dual chamber vials), syringes (such
as dual chamber
syringes) and test tubes. The container may be formed from a variety of
materials such as glass or
plastic. The container holds the lyophilized formulation and a label on, or
associated with, the
container may indicate directions for reconstitution and/or use. For example,
the label may indicate
that the lyophilized formulation is reconstituted to peptide concentrations as
described above. The
label may further indicate that the formulation is useful or intended for
subcutaneous administration.
The container holding the formulation may be a multi-use vial, which allows
for repeat
administrations (e.g., from 2-6 administrations) of the reconstituted
formulation. The article of
manufacture may further comprise a second container comprising a suitable
diluent (e.g., BWFI).
Upon mixing of the diluent and the lyophilized formulation, the final protein
concentration in the
reconstituted formulation will generally be at least 50 mg/mL. The article of
manufacture may
further include other materials desirable from a commercial and user
standpoint, including other
buffers, diluents, filters, needles, syringes, and package inserts with
instructions for use.
Therapeutic kits may have a single container which contains the formulation of
the
Ac-PHSCN-NH2 pharmaceutical compositions with or without other components
(e.g., other
compounds or pharmaceutical compositions of these other compounds) or may have
distinct
container for each component. Preferably, therapeutic kits of the invention
include a formulation of
Ac-PHSCN-NH2 or an acid addition salt thereof as disclosed herein packaged for
use in combination
with the co-administration of a second compound (such as a chemotherapeutic
agent, a natural
product, a hormone or antagonist, a anti-angiogenesis agent or inhibitor, a
apoptosis-inducing agent
or a chelator) or a pharmaceutical composition thereof. The components of the
kit may be
pre-complexed or each component may be in a separate distinct container prior
to administration to
a patient. The components of the kit may be provided in one or more liquid
solutions, preferably, an
aqueous solution, more preferably, a sterile aqueous solution. The components
of the kit may also
be provided as solids, which may be converted into liquids by addition of
suitable solvents, which
are preferably provided in another distinct container.
The container of a therapeutic kit may be a vial, test tube, flask, bottle,
syringe, or any other
means of enclosing a solid or liquid. Usually, when there is more than one
component, the kit will
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WO 2006/083906 PCT/US2006/003461
contain a second vial or other container, which allows for separate dosing.
The kit may also contain
another container for a pharmaceutically acceptable liquid. Preferably, a
therapeutic kit will contain
apparatus (e.g., one or more needles, syringes, eye droppers, pipette, etc.),
which enables
administration of the agents of the invention which are components of the
present kit.
The present formulation is one that is suitable for administration of the
peptide by any
acceptable route such a oral (enteral), subcutaneous, intramuscular,
intravenous transdermal,
Administration may be by infusion pump. All modes and routes of administration
disclosed by
Ternansky et al., supra, are understood to be useful with the present
formulations and are
incorporated by reference as if written here in full.
The present invention is also directed to formulations of the peptide suitable
for
administration by inhalation. Many drugs currently administered by inhalation
come primarily as
liquid or solid aerosol particles of respirable size. For biotherapeutic
drugs, this may present a
problem, as many of these medicaments are unstable in aqueous environments for
extended periods
of time and are rapidly denatured if micronized by high shear grinding or
other comminution
methods when presented as dry powders. Additionally, a number of these
medicaments do not
survive long enough in the lung as they are extracted quickly from the lung
environment after they
are administered as inhalation aerosols. Significant drug loss could also
occur by deactivation either
as a result of reactivity of the medicament with device and container
surfaces, or during
aerosolization, particularly in high shear, energy intensive, nebulized
systems (Mumenthaler, M, et
al., Pharm. Res., 11:12-20 (1994). To overcome these instability problems,
many drug and
excipient systems contain biodegradable carriers, such as poly(lactide-co-
glycolides, have been
developed for biotherapeutic proteins and peptides (Liu, R. et al.,
Biotechnol. Bioeng., 37:177-184
(1991). Most therapeutic peptides are poorly absorbed through biologic
membranes even upon
formulation with penetration enhancer. In numerous therapies, drug dosimetry
is increased by
orders of magnitude to achieve minimum systemic concentrations required for
efficacy. In other
cases the drug product is formulated with exotic absorption promoters in order
to improve
permeability across an absorption barrier, often leading to toxic
consequences. The mode of drug
administration to the body has also gradually expanded from oral and
parenteral to transdermal,
rectal and the pulmonary routes of administration, i.e., nose and lung.
Success with these drug
delivery approaches have been mixed due to lack of acceptance of the newer,
complex molecules
that must be used for treating difficult diseases, e.g., infections,
malignancies, cardiovascular,
endocrine, neurologic diseases, and a variety of diseases of immunological
compromise.
CA 02596255 2007-07-27
WO 2006/083906 PCT/US2006/003461
Thus, the present invention exploits the existence of a fluid propelled
formulation system
comprising the peptide (drug) that is stable and protected by a rate-limiting
carrier, easily
manufactured, and therapeutically effective when administered as fluid
dispersed particles to the
lung of a patient. As noted above, the present invention includes formulations
of the peptide for oral
or nasal inhalation. Such formulations include, but are not limited to,
modulated release aerosol
particles, and to medicinal, respirable aerosol particles comprising
polysaccharide vesicles which
are associated with, and, may form a part of, a construct with or entrap a
selected medicament, here,
the peptide, and provide slow release thereof, as disclosed in U.S. Pat.
6,551,578, incorporated by
reference in its entirety. In this approach the peptide is formulated so that
it is suitable for
administration by oral and nasal inhalation. A stable, colloidal dispersion of
the peptide in a fluid,
e.g., air, hydrocarbon gases, chlorofluorocarbon (CFC) propellants or non-CFC
propellants, such as
tetrafluoroethane (HFA-134a) and heptafluoropropane (HFA-227) are intended.
For purposes of the formulations of this invention that are intended for
inhalation into the
lungs, the peptide is associated with the naturally occurring polysaccharide
polymer to which it is
destined to be combined. By "associate" or "associated" in this context is
meant that the peptide is
present as a matrix or a part of a polymeric construct along with the
polysaccharide polymer or is
encapsulated as a microsphere in a polysaccharide polymer or in polysaccharide
polymeric construct
particle, or is on a surface of such particle, whereby a therapeutically
effective amount or fraction
(e.g., 95% or more) of the peptide is particulate. Typically, the construct
particles have a diameter
of less than about 10 m, and preferably less than about 5 m, in order that
the particles can be
inhaled into the respiratory tract and/or lungs of the patient being treated.
A suitable polymeric construct is one which will incorporate therein or
encapsulate the
selected peptide and provide a controlled or modulated release of the
medicament therefrom to the
sites of action or application of the patient's body, e.g., from the lung to
the local surrounding
environment of the subject.
A suitable polysaccharide is a polymer selected from the group of an alginate
salt, where the
cation is, e.g., Li+, Na+, K+, Ca++, NH3+, NHa+ etc., such as sodium alginate,
calcium alginate,
sodium-calcium alginate, ammonium alginate, sodium-ammonium alginate, or
calcium-ammonium
alginate. A preferred alginate modulating releasing agent is ammonium calcium
alginate. These
materials are typically used in injectable implants and microsphere
preparations for controlled
release. A commercial form of ammonium calcium alginate is KeltoseTM,
manufactured and
distributed by International Specialty Products (Wayne, NJ). As used herein,
"alginate" means
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WO 2006/083906 PCT/US2006/003461
alginic acid, or any of its salts; or other naturally occurring polysaccharide
or carbohydrate based
polymers such as gum arabic, pectin, galacturonic acid, gum karaya; gum
Benjamin, plantago ovata
gum; agar; carrageenan; cellulose; gelatin; or a mixture of any of the
foregoing polymers. Alginates
are pharmaceutical excipients generally regarded as safe and used to prepare a
variety of well
documented pharmaceutical systems (U.S. Pats. No. 6,166,084; 6,166,043;
6,166,042; 6,166,004;
and 6,165,615). Alginates are naturally occurring polymers comprising
polysaccharide chains.
These polymers have the propensity to absorb water thus swelling to become gel-
like structures in
solution. Upon inhalation of the resultant core formulation by a patient being
treated, the gel
dissolves in the body of such patient, thus releasing its drug payloads in a
dissolution controlled
manner. Such a polymer system forms a construct or a matrix when formed in
situ with a selected
medicament or medicaments whereby such medicament or medicaments forms part of
the matrix or
is encapsulated within the matrix. Upon such formation or encapsulation, the
medicament is time-
released or modulated from the site of action in the body, e.g., the lungs,
the respiratory tract, ear,
etc., to the surrounding environment or tissues of the subject's body. The
polysaccharide polymer,
e.g., an alginate salt, is typically present in the resultant controlled-
release formulation in an amount
ranging from about 0.000001 % to about 10% by weight of the total weight of
the formulation.
The therapeutic peptide is present in the inventive polymer construct in a
therapeutically
effective amount, that is, an amount such that the peptide can be incorporated
into an aerosol
formulation such as a dispersion aerosol, via oral or nasal inhalation, and
cause its desired
therapeutic effect, preferably with one dose, or through several doses.
Other formulations for prolonged administration are also contemplated,
including use of
liposomes, either multilamellar or unilamellar, the preparation of which is
well known to those
skilled in the art. Liposomes may be phospholipid or non-phospholipid based.
Assays
Those of skill in the art will appreciate that the in vitro and in vivo assays
useful for
measuring the activity of the present formulation of Ac-PHSCN-NH2 and its
salts, described herein,
are illustrative rather than comprehensive. These can be found, for example in
Ternansky et al.,
supra, and in commonly assigned co-pending applications USSN 10/074, 225 and
10/661,784), all
of which are incorporated by reference in their entirety. Examples of
preferred assays are set forth
below.
Assay for Endothelial Cell Mi agr tion
For EC migration, transwells are coated with type I collagen (50 g/mL) by
adding 200 L
of the collagen solution per transwell, then incubating overnight at 37 C. The
transwells are
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WO 2006/083906 PCT/US2006/003461
assembled in a 24-well plate and a chemoattractant (e.g., FGF-2) is added to
the bottom chamber in
a total volume of 0.8 mL media. ECs, such as human umbilical vein endothelial
cells (HUVEC),
which have been detached from monolayer culture using trypsin, are diluted to
a final concentration
of about 106 cells/mL with serum-free media and 0.2 mL of this cell suspension
is added to the
upper chamber of each transwell. Salts of Ac-PHSCN-NH2 may be added to both
the upper and
lower chambers and the migration is allowed to proceed for 5 hrs in a
humidified atmosphere at
37 C. The transwells are removed from the plate stained using DiffQuik . Cells
which did not
migrate are removed from the upper chamber by scraping with a cotton swab and
the membranes are
detached, mounted on slides, and counted under a high-power field (400x) to
determine the number
of cells migrated.
Bioloizical Assay of Anti-Invasive ActivitX
The ability of cells such as ECs or tumor cells (e.g., PC-3 human prostatic
carcinoma) cells
to invade through a reconstituted basement membrane (Matrigel ) in an assay
known as a
Matrigel invasion assay system has been described in detail in the art
(Kleinman et al.,
Biochemistry 1986, 25: 312-318; Parish et al., 1992, Int. J. Cancer 52:378-
383). Matrigel is a
reconstituted basement membrane containing type IV collagen, laminin, heparan
sulfate
proteoglycans such as perlecan, which bind to and localize bFGF, vitronectin
as well as
transforming growth factor-(3 (TGF(3, urokinase-type plasminogen activator
(uPA), tissue
plasminogen activator (tPA) and the serpin known as plasminogen activator
inhibitor type 1(PAI-1)
(Chambers et al., Canc. Res. 1995, 55:1578-1585,). It is accepted in the art
that results obtained in
this assay for compounds which target extracellular receptors or enzymes are
predictive of the
efficacy of these compounds in vivo (Rabbani et al., Int. T. Cancer 1995, 63:
840-845).
Such assays employ transwell tissue culture inserts. Invasive cells are
defined as cells which
are able to traverse through the Matrigel and upper aspect of a polycarbonate
membrane and
adhere to the bottom of the membrane. Transwells (Costar) containing
polycarbonate membranes
(8.0 m pore size) are coated with Matrigel (Collaborative Research), which
has been diluted in
sterile PBS to a final concentration of 75 g/mL (60 L of diluted Matrigel
per insert), and placed
in the wells of a 24-well plate. The membranes are dried overnight in a
biological safety cabinet,
then rehydrated by adding 100 L of DMEM containing antibiotics for 1 hour on
a shaker table.
The DMEM is removed from each insert by aspiration and 0.8 mL of DMEM/10 %
FBS/antibiotics
is added to each well of the 24-well plate such that it surrounds the outside
of the transwell ("lower
chamber"). Fresh DMEM/ antibiotics (100 L), human Glu-plasminogen (5 g/mL),
and any
inhibitors to be tested are added to the top, inside of the transwell ("upper
chamber"). The cells
which are to be tested are trypsinized and resuspended in DMEM/antibiotics,
then added to the top
chamber of the transwell at a final concentration of 800,000 cells/mL. The
final volume of the
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CA 02596255 2007-07-27
WO 2006/083906 PCT/US2006/003461
upper chamber is adjusted to 200 A. The assembled plate is then incubated in a
humid 5% CO2
atmosphere for 72 hours. After incubation, the cells are fixed and stained
using DiffQuik (Giemsa
stain) and the upper chamber is then scraped using a cotton swab to remove the
Matrigel and any
cells which did not invade through the membrane. The membranes are detached
from the transwell,
e.g., using an X-acto ' blade, mounted on slides using Permount and cover-
slips, then counted
under a high-powered (400x) field. An average of the cells invaded is
determined from 5-10 fields
counted and plotted as a function of peptide concentration.
Tube-Formation Assays of Anti-Anaiogenic Activity
Endothelial cells, for example, human umbilical vein endothelial cells (HUVEC)
or human
microvascular endothelial cells (HMVEC) which can be prepared or obtained
commercially, are
mixed at a concentration of 2 x 105 cells/mL with fibrinogen (5mg/mL in
phosphate buffered saline
(PBS) in a 1:1 (v/v) ratio. Thrombin is added (5 units/ mL final
concentration) and the mixture is
immediately transferred to a 24-well plate (0.5 mL per well). The fibrin gel
is allowed to form and
then VEGF and bFGF are added to the wells (each at 5 ng/mL final
concentration) along with the
test compound. The cells are incubated at 37 C in 5% CO2 for 4 days at which
time the cells in each
well are counted and classified as either rounded, elongated with no branches,
elongated with one
branch, or elongated with 2 or more branches. Results are expressed as the
average of 5 different
wells for each concentration of compound. Typically, in the presence of
angiogenic inhibitors, cells
remain either rounded or form undifferentiated tubes (e.g. 0 or 1 branch).
This assay is recognized
in the art to be predictive of angiogenic (or anti-angiogenic) efficacy in
vivo (Min et al., Cancer Res.
1996, 56: 2428-2433,).
In an alternate assay, endothelial cell tube formation is observed when
endothelial cells are
cultured on Matrigel (Schnaper et al., J. Cell. Playsiol. 1995, 165:107-118).
Endothelial cells (104
cells/well) are transferred onto Matrigel -coated 24-well plates and tube
formation is quantitated
after 48 hrs. Inhibitors are tested by adding them either at the same time as
the endothelial cells or
at various time points thereafter. Tube formation can also be stimulated by
adding (a) angiogenic
growth factors such as bFGF or VEGF, (b) differentiation stimulating agents
(e.g.,. PMA) or (c) a
combination of these.
While not wishing to be bound by theory, this assay models angiogenesis by
presenting to
the endothelial cells a particular type of basement membrane, namely the layer
of matrix which
migrating and differentiating endothelial cells might be expected to first
encounter. In addition to
bound growth factors, the matrix components found in Matrigel (and in
basement membranes in
situ) or proteolytic products thereof may also be stimulatory for endothelial
cell tube formation
which makes this model complementary to the fibrin gel angiogenesis model
previously described
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WO 2006/083906 PCT/US2006/003461
(Blood et al., Biochim. Biophys. Acta 1990, 1032:89-118; Odedrat al.,
Plaarmac. Ther-. 1991,
49:111-124,).
Assays for Inhibition of Proliferation
The ability of the compounds of the invention to inhibit the proliferation of
EC's may be
determined in a 96-well format. Type I collagen (gelatin) is used to coat the
wells of the plate (0.1-1
mg/mL in PBS, 0.1 mL per well for 30 minutes at room temperature). After
washing the plate (3x
w/PBS), 3-6,000 cells are plated per well and allowed to attach for 4 hrs (37
C/5% C02) in
Endothelial Growth Medium (EGM; Clonetics) or M199 media containing 0.1-2%
FBS. The media
and any unattached cells are removed at the end of 4 hrs and fresh media
containing bFGF (1-10
ng/mL) or VEGF (1-10 ng/mL) is added to each well. Compounds to be tested are
added last and
the plate is allowed to incubate (37 C/5% C02) for 24-48 hrs. MTS (Promega) is
added to each well
and allowed to incubate from 1-4 hrs. The absorbance at 490nm, which is
proportional to the cell
number, is then measured to determine the differences in proliferation between
control wells and
those containing test compounds.
A similar assay system can be set up with cultured adherent tumor cells.
However, collagen
may be omitted in this format. Tumor cells (e.g., 3,000-10,000/well) are
plated and allowed to
attach overnight. Serum free medium is then added to the wells,, and the cells
are synchronized for
24 hrs. Medium containing 10% FBS is then added to each well to stimulate
proliferation.
Compounds to be tested are included in some of the wells. After 24 hrs, MTS is
added to the plate
and the assay developed and read as described above.
Caspase-3 Activity
The ability of the compounds of the invention to promote apoptosis of EC's may
be
determined by measuring activation of caspase-3. Type I collagen (gelatin) is
used to coat a P100
plate and 5x105 ECs are seeded in EGM containing 10% FBS. After 24 hours (at
37 C in5% C02)
the medium is replaced by EGM containing 2% FBS, 10 ng/ml bFGF and the desired
test
compound. The cells are harvested after 6 hours, cell lysates prepared in 1%
Triton and assayed
using the EnzChek Caspase-3 Assay Kit #1 (Molecular Probes) according to the
manufactures'
instructions.
Corneal Angiogenesis Model
The protocol used is essentially identical to that described by Volpert et
al., J. Clin. Ifavest.
1996, 98:671-679. Briefly, female Fischer rats (120-140g) are anesthetized and
pellets (5 l)
comprised of Hydron , bFGF (150nM), and the compounds to be tested are
implanted into tiny
incisions made in the cornea 1.0-1.5mm from the limbus. Neovascularization is
assessed at 5 and 7
days after implantation. On day 7, animals are anesthetized and infused with a
dye such as colloidal
carbon to stain the vessels. The animals are then euthanized, the corneas
fixed with formalin, and
CA 02596255 2007-07-27
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the corneas flattened and photographed to assess the degree of
neovascularization. Neovessels may
be quantitated by imaging the total vessel area or length or simply by
counting vessels.
Matri elg Plug AssaX
This assay is performed essentially as described by Passaniti et al., 1992,
Lab Iravest. 67:519-
528. Ice-cold Matrigel (e.g., 500 L) (Collaborative Biomedical Products,
Inc., Bedford, MA) is
mixed with heparin (e.g., 50 g/ml), FGF-2 (e.g., 400 ng/ml) and the compound
to be tested. In
some assays, bFGF may be substituted with tumor cells as the angiogenic
stimulus. The Matrigel
mixture is injected subcutaneously into 4-8 week-old athymic nude mice at
sites near the abdominal
midline, preferably 3 injections per mouse. The injected Matrigel forms a
palpable solid gel.
Injection sites are chosen such that each animal receives a positive control
plug (such as FGF-2 +
heparin), a negative control plug (e.g., buffer + heparin) and a plug that
includes the compound
being tested for its effect on angiogenesis, e.g., (FGF-2 + heparin +
compound). All treatments are
preferably run in triplicate. Animals are sacrificed by cervical dislocation
at about 7 days post
injection or another time that may be optimal for observing angiogenesis. The
mouse skin is
detached along the abdominal midline, and the Matrigel plugs are recovered
and scanned
immediately at high resolution. Plugs are then dispersed in water and
incubated at 37 C overnight.
Hemoglobin (Hb) levels are determined using Drabkin's solution (e.g., obtained
from Sigma)
according to the manufacturers' instructions. The amount of Hb in the plug is
an indirect measure
of angiogenesis as it reflects the amount of blood in the sample. In addition,
or alternatively,
animals may be injected prior to sacrifice with a 0.1 ml buffer (preferably
PBS) containing a high
molecular weight dextran to which is conjugated a fluorophore. The amount of
fluorescence in the
dispersed plug, determined fluorimetrically, also serves as a measure of
angiogenesis in the plug.
Staining with mAb anti-CD31 (CD31 is "platelet-endothelial cell adhesion
molecule or PECAM")
may also be used to confirm neovessel formation and microvessel density in the
plugs.
Chick Chorioallantoic Membrane (CAM) Angiogenesis Assay
This assay is performed essentially as described by Nguyen et al.,
Microvascular Res. 1994,
47:31-40. A mesh containing either angiogenic factors (bFGF) or tumor cells
plus inhibitors is
placed onto the CAM of an 8-day old chick embryo and the CAM observed for 3-9
days after
implantation of the sample. Angiogenesis is quantitated by determining the
percentage of squares in
the mesh which contain blood vessels.
In Vivo Assessment of Angiogenesis Inhibition and Anti-Tumor Effects Using the
Matrigel Plug Assay with Tumor Cells
In this assay, tumor cells, for example 1-5 x 106 cells of the 3LL Lewis lung
carcinoma or
the rat prostate cell line MatLyLu, are mixed with Matrigel and then injected
into the flank of a
mouse following the protocol described in Sec. B., above. A mass of tumor
cells and a powerful
21
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angiogenic response can be observed in the plugs after about 5 to 7 days. The
anti-tumor and anti-
angiogenic action of a compound in an actual tumor environment can be
evaluated by including it in
the plug. Measurement is then made of tumor weight, Hb levels or fluorescence
levels (of a
dextran-fluorophore conjugate injected prior to sacrifice). To measure Hb or
fluorescence, the plugs
are first homogenize with a tissue homogenizer.
Xenograft Model of Subcutaneous (s.c.) Tumor Growth
Nude mice are inoculated with MDA-MB-231 cells (human breast carcinoma) and
Matrigel (106 cells in 0.2mL) s.c. in the right flank of the animals. The
tumors are staged to 200
mm3 and then treatment with a test composition is initiated (100 g/animaUday
given q.d. IP).
Tumor volumes are obtained every other day and the animals are sacrificed
after 2 weeks of
treatment. The tumors are excised, weighed and paraffin embedded. Histological
sections of the
tumors are analyzed by H and E, anti-CD3 1, Ki-67, TUNEL, and CD68 staining.
Xenograft Model of Metastasis
The compounds of the invention are also tested for inhibition of late
metastasis using an
experimental metastasis model (Crowley et al., Proc. Natl. Acad. Sci. USA
1993, 90 5021-5025).
Late metastasis involves the steps of attachment and extravasation of tumor
cells, local invasion,
seeding, proliferation and angiogenesis. Human prostatic carcinoma cells (PC-
3) transfected with a
reporter gene, preferably the green fluorescent protein (GFP) gene, but as an
alternative with a gene
encoding the enzymes chloramphenicol acetyl-transferase (CAT), luciferase or
LacZ, are inoculated
into nude mice. This approach permits utilization of either of these markers
(fluorescence detection
of GFP or histochemical colorimetric detection of enzymatic activity) to
follow the fate of these
cells. Cells are injected, preferably iv, and metastases identified after
about 14 days, particularly in
the lungs but also in regional lymph nodes, femurs and brain. This mimics the
organ tropism of
naturally occurring metastases in human prostate cancer. For example, GFP-
expressing PC-3 cells
(106 cells per mouse) are injected iv into the tail veins of nude (nu/nu)
mice. Animals are treated
with a test composition at 100 g/animal/day given q.d. IP. Single metastatic
cells and foci are
visualized and quantitated by fluorescence microscopy or light microscopic
histochemistry or by
grinding the tissue and quantitative colorimetric assay of the detectable
label.
Inhibition of Spontaneous Metastasis In Vivo by PHSCN and Functional
Derivatives
The rat syngeneic breast cancer system employs Mat BIII rat breast cancer
cells (Xing et al.,
Int. J. Cancer 1996, 67:423-429). Tumor cells, for example, about 106
suspended in 0.1 mL PBS,
are inoculated into the mammary fat pads of female Fisher rats. At the time of
inoculation, a 14-day
Alza osmotic mini-pump is implanted intraperitoneally to dispense the test
compound. The
compound is dissolved in PBS (e.g., 200 mM stock), sterile filtered and placed
in the minipump to
achieve a release rate of about 4 mg/kg/day. Control animals receive vehicle
(PBS) alone or a
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vehicle control peptide in the minipump. Animals are sacrificed at about day
14. In the rats treated
with the compounds of the present invention, significant reductions in the
size of the primary tumor
and in the number of metastases in the spleen, lungs, liver, kidney and lymph
nodes (enumerated as
discrete foci) may be observed. Histological and immunohistochemical analysis
reveal increased
necrosis and signs of apoptosis in tumors in treated animals. Large necrotic
areas are seen in tumor
regions lacking neovascularization. Human or rabbit PHSCN and their
derivatives to which 131I is
conjugated (either 1 or 2 I atoms per molecule of peptide) are effective
radiotherapeutics and are
found to be at least two-fold more potent than the unconjugated polypeptides.
In contrast, treatment
with control peptides fails to cause a significant change in tumor size or
metastasis.
3LL Lewis Lung Carcinoma: Primary Tumor Growth
This tumor line arose spontaneously as carcinoma of the lung in a C57BL/6
mouse (Malave
et al., J. Nat'l. Canc. Inst. 1979, 62:83-88). It is propagated by passage in
C57BL/6 mice by
subcutaneous (sc) inoculation and is tested in semiallogeneic C57BL/6 x DBA/2
Fl mice or in
allogeneic C3H mice. Typically six animals per group for subcutaneously (sc)
implant, or ten for
intramuscular (im) implant are used. Tumor may be implanted sc as a 2-4 mm
fragment, or im or sc
as an inoculum of suspended cells of about 0.5-2 x 106-cells. Treatment begins
24 hours after
implant or is delayed until a tumor of specified size (usually approximately
400 mg) can be
palpated. The test compound is administered ip daily for 11 days
Animals are followed by weighing, palpation, and measurement of tumor size.
Typical
tumor weight in untreated control recipients on day 12 after im inoculation is
500-2500 mg. Typical
median survival time is 18-28 days. A positive control compound, for example
cyclophosphamide
at 20 mg/kg/injection per day on days 1-11 is used. Results computed include
mean animal weight,
tumor size, tumor weight, survival time. For confirmed therapeutic activity,
the test composition
should be tested in two multi-dose assays.
3LL Lewis Lung Carcinoma: Primary Growth and Metastasis Model
This assay is well known in the art (Gorelik et al., J. Nat'l. Canc. Inst.
1980, 65:1257-1264;
Gorelik et al., Rec. Results Canc. Res. 1980, 75:20-28; Isakov et al.,
Invasion Metas. 2:12-32
(1982); Talmadge et al., J. Nat'l. Canc. Inst. 1982, 69:975-980; Hilgard et
al., Br. J. Cancer 1977,
35:78-86). Test mice are male C57BL/6 mice, 2-3 months old. Following sc, im,
or intra-footpad
implantation, this tumor produces metastases, preferentially in the lungs.
With some lines of the
tumor, the primary tumor exerts anti-metastatic effects and must first be
excised before study of the
metastatic phase (see also U.S. Patent No. 5,639,725).
Single-cell suspensions prepared from solid tumors by trypsinization are
washed and
suspended in PBS. Viability of 3LL cells prepared in this way is generally
about 95-99%. Viable
tumor cells (3 x 104 - 5 x 106) suspended in 50 l PBS are injected sc, either
in the dorsal region or
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into one hind foot pad of C57BL/6 mice. Visible tumors appear 3-4 days after
dorsal sc injection of
106 cells. The day of tumor appearance and the diameters of established tumors
are measured every
two days. The treatment is given as one to five doses of peptide or analogue
per week. In another
embodiment, the peptide is delivered by osmotic minipump.
In experiments involving tumor excision of dorsal tumors, when tumors reach
about 1500
mm3 in size, mice are randomized into two groups: (1) primary tumor is
completely excised; or (2)
sham surgery is performed and the tumor is left intact. Although tumors from
500-3000 mm3
inhibit growth of metastases, 1500 mm3 is the largest size primary tumor that
can be safely resected
with high survival and without local regrowth. The phenomenon of acceleration
of metastatic
growth following excision of local tumors had been repeatedly observed (see,
for example, U. S.
Pat. No. 5,639,725). These observations have implications for the prognosis of
patients who
undergo cancer surgery. After 21 days, all mice are sacrificed and autopsied.
Lungs are removed,
weighed and fixed in Bouin's solution and the number of visible metastases is
recorded as is the
diameters of the metastases. On the basis of the recorded diameters, one
calculates the volume of
each metastasis. To determine the total volume of metastases per lung, the
mean number of visible
metastases is multiplied by the mean volume.
It is also possible to measure incorporation of 125IdUrd into lung cells
(Thakur et al., J. Lab.
Clin. Med. 1977, 89:217-228). Ten days following tumor amputation, 25gg of
fluorodeoxyuridine is
injected i.p. into tumor-bearing (and, if used, tumor-resected mice). After 30
min, mice are given
1 Ci of 125IdUrd (iododeoxyuridine). One day later, lungs and spleens are
removed and weighed,
and the degree of iz5IdUrd incorporation is measured using a gamma counter.
In mice with footpad tumors, when tumors reach about 8-10 mm in diameter, mice
are
randomized into two groups: (1) legs with tumors are amputated after ligation
above the knee joints;
or (2) mice are left intact as nonamputated tumor-bearing controls.
(Amputation of a tumor-free leg
in a tumor-bearing mouse has no known effect on subsequent metastasis, ruling
out possible effects
of anesthesia, stress or surgery.) Mice are killed 10-14 days after
amputation. Metastases are
evaluated as above.
Statistics: Values representing the incidence of metastases and their growth
in the lungs of
tumor-bearing mice are not normally distributed. Therefore, non-parametric
statistics such as the
Mann-Whitney U-Test may be used for analysis.
Use of PHSCN Peptides, Analogues and Salts in Therapy
An Ac-PHSCN-NH2 salt, or pharmaceutical compositions thereof, will generally
be used in
an amount effective to achieve the intended purpose. For use to treat or
prevent diseases or
disorders characterized by aberrant vascularization or aberrant angiogenesis,
the Ac-PHSCN-NH2
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WO 2006/083906 PCT/US2006/003461
salts which may be in pharmaceutical compositions, are administered or applied
in a therapeutically
effective amount. The amount of a Ac-PHSCN-NH2 salt that will be effective in
the treatment of a
particular disorder or condition disclosed herein will depend on the nature of
the disorder or
condition, and can be determined by standard clinical techniques known in the
art. In addition, in
vitro or in vivo assays may optionally be employed to help identify optimal
doses or dose ranges.
The amount of an Ac-PHSCN-NH2 salt administered will, of course, be dependent
on, among other
factors, the subject being treated, the weight of the subject, the severity of
the affliction, the manner
of administration and the judgment of the prescribing physician. For example,
the dosage may be
delivered in a pharmaceutical composition by a single or multiple
administrations, or by controlled
release. Dosing may be repeated intermittently, may be performed alone or in
combination with
other drugs and may be continued as long as required for effective treatment
of the disease or
disorder. Suitable dosage ranges for oral administration are dependent on the
potency of the drug,
but are generally 0.001 mg to 200 mg, preferably 0.01 mg to 50 mg, more
preferably, 0.1 to 50 mg,
of a compound of the invention per kilogram body weight. Dose ranges may be
readily determined
by methods known to those of ordinary skill in the art. Suitable dose ranges
for i.v. administration
are about 0.01 mg to about 100 mg per kg body weight. Suitable dosage ranges
for intranasal
administration are generally 0.01 mg/kg body weight to 50 mg/kg body weight or
0.10 mg/kg body
weight to 10 mg/kg body weight. Suppositories generally contain about 0.01 mg
to about 50 mg of
the compound of the invention per kg body weight and comprise about 0.5% to
about 10% by
weight the active ingredient. Recommended dosages for intradermal,
intramuscular, intraperitoneal,
subcutaneous, epidural, sublingual or intracerebral administration are in the
range of about 0.001 mg
to about 200 mg per kg body weight. Effective doses may be extrapolated from
dose-response
curves derived from in vitro or animal model test systems. Such animal models
and systems are
well-known in the art.
In a specific embodiment, the dose administered is not based on body weight,
but is an
absolute amount, for example, in the range of 1 mg to 1 g per dose. In another
specific embodiment,
the dose is 10 -750 mg per dose, e.g., 20 mg, 100 mg, or 600 mg per dose. In a
specific
embodiment, the dose is administered from one to several (e.g., 2, 3, 4, or 7)
times per week.
The Ac-PHSCN-NH2 salts are preferably assayed in vitro and in vivo, as
described above for
the desired therapeutic or prophylactic activity, prior to use in humans. For
example, in vitro assays
can be used to determine whether administration of an Ac-PHSCN-NH2 salt or a
combination of Ac-
PHSCN-NH2 salts is preferred for treating diseases characterized by aberrant
angiogenesis or
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vascularization. Safety and efficacy of the Ac-PHSCN-NHZ salts may be
demonstrated using animal
model systems. Preferably, a therapeutically effective dose of a Ac-PHSCN-NH2
salt described
herein will provide therapeutic benefit without causing substantial toxicity.
Toxicity of Ac-PHSCN-
NH2 salts may be determined using standard pharmaceutical procedures and may
be readily
ascertained by the skilled artisan. In the treatment of diseases or disorders,
the "therapeutic index"
(ratio between a toxic and a therapeutic dose) of an Ac-PHSCN-NH2 salt will
preferably be high.
The preferred dose of an Ac-PHSCN-NH2 salt described herein will preferably
result in a range of
circulating concentrations of the agent that are effective but with little or
no toxicity.
Having now generally described the invention, the same will be more readily
understood
through reference to the following examples which are provided by way of
illustration, and are not
intended to be limiting of the present invention, unless specified.
EXAMPLE I
Preparation of Ac-PHSCN-NH2, hydrochloride salt
1) 20% piperidine/DMF (3 x 3 min)
2) Fmoc-Asn(trt)-OH (3 eq),
HBTU (3 eq), HOBt (3 eq),
NMM (6 eq), DMF, lh
FmocHN Fmoc-His(trt)-Ser(trt)-Cys(trt)-Asn(trt
3) Repeat step 2
Rink Amide 4) Repeat steps 1, 2 & 3 for each
AM resin amino acid (Fmoc-Cys(trt)-OH,
Fmoc-Ser(trt)-OH, Fmoc-His(trt)-OH) 1) 20% piperidine/DMF (3 x 3 min)
2) Ac-Pro-OH (3 eq),
HBTU (3 eq), HOBt (3 eq),
NMM (6 eq), DMF, lh
TFA/TIS/H20
(95:2.5:2.5), 1 h
Ac-Pro-His-Ser-Cys-Asn-NH2 =TFA . Ac-Pro-His(trt)-Ser(trt)-Cys(trt)-Asn(trt
Amberlyst A-26
Ac-PHSCN-NH2 -TFA OH Resin Ac-PHSCN-NH2 1 eq HCI - Ac-PHSCN-NH2 = HCI
distilled water
5 min ATN-161.030
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EXAIVIPLE H
Preparation and Purification of Ac-Pro-His-Ser-Cvs-Asn-NH2, TFA salt
Rink Amide AM resin (Novabiochem) was treated with 20% piperidine in DMF (1 mL
per
100 mg of resin) for three minutes with nitrogen agitation and the reaction
mixture was filtered and
washed once with DMF. This step was repeated an additional two times. The
resin was washed
three times with DMF and three times with dichloromethane. Fmoc-Asn(trt)-OH (3
eq), HBTU (3
eq), and HOBt (3 eq) were dissolved in DMF (1 mL per 100 mg of resin) and
added to the above
resin, followed by the addition of N-methylmorpholine (NMM) (6 eq) and the
mixture was agitated
for 1 hour. The reaction mixture was filtered and the resin was washed three
times with DMF and
three times with dichloromethane. This coupling step was repeated. The Fmoc
deprotection and the
coupling steps described above were sequentially used with Fmoc-Cys(trt)-OH,
Fmoc-Ser(trt)-OH
and Fmoc-His(trt)-OH to afford Fmoc-His(trt)-Ser(trt)-Cys(trt)-Asn(trt) bound
to the resin. This
resin was treated with 20% piperidine in DMF (1 mL per 100 mg of resin) for
three minutes with
nitrogen agitation and the reaction mixture was filtered and washed once with
DMF. This step was
repeated an additional two times. The resin was washed three times with DMF
and three times with
dichloromethane. Ac-Pro-OH (3 eq), HBTU (3 eq), and HOBt (3 eq) were dissolved
in DMF (1 mL
per 100 mg of resin) and added to the above resin, followed by the addition of
N-methylmorpholine
(NMM) (6 eq) and the mixture was agitated for 1 hour. The reaction mixture was
filtered and the
resin was washed three times with DMF and three times with dichloromethane to
afford Rink Amide
AM resin bound Ac-Pro-His(trt)-Ser(trt)-Cys(trt)-Asn(trt). The resin was
treated with
TFA/TIS/water (95:2.5:2.5, 1 mL per 100 mg of resin) and agitated with
nitrogen for 2 hours. The
reaction mixture was filtered, and the resin was washed once with
TFA/TIS/water and three times
with dichloromethane. The solvent was removed in vacuo and the resulting
residue was triturated
three times with ether to afford crude Ac-Pro-His-Ser-Cys-Asn-NH2, TFA salt.
Using this general procedure, 708 mg of crude Ac-PHSCN-NH2, TFA salt was
prepared
from 2 grams of Rink Amide AM resin (loading: 0.63 mmol/g).
Purification
The crude peptide, dissolved in a minimum amount of methanol and water, was
purified by
preparative reverse phase HPLC (Beckman) with a Phenomenex Synergi hydro-RP
C18 column
(250mm x 21.2 mm). The peptide was eluted using a gradient from 3-100% B over
30 min with a
flow rate of 20 mL/min, where solvent A was water containing 0.1%
trifluoroacetic acid and solvent
B was acetonitrile containing 0.1% trifluoroacetic acid. Detection was at 220
nm. Fractions >95%
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pure by analytical HPLC analysis (Phenomenex hydro RP (250mm x 4.6mm) using
gradient 3-100%
B) (Waters) were combined, concentrated to a volume of about 2-4 ml by rotary
evaporation, and
lyophilized. Samples were redissolved in water and transferred to a tared 2
dram vial and lyophilized
a second time.
Using this method, 140 mg of pure Ac-PHSCN-NH2, TFA salt was obtained from 338
mg of
crude material: ES MS fn/z (M+H)+ 598.2; HPLC: 99% pure.
EXAMPLE III
Preparation of Ac-Pro-His-Ser-Cys-Asn-NIi2
Ac-Pro-His-Ser-Cys-Asn-NH2, TFA salt (140 mg, 0.197 mmol) was dissolved in 2
mL of
distilled water and Amberlyst A-26 (OH) resin (4.2 meq/g, 273 mg, 5.8 eq) was
added. The reaction
mixture was stirred at room temperature for 5 minutes. The aqueous solution
was decanted, the resin
was washed twice with distilled water, and the combined aqueous layers were
lyophilized to afford
81 mg (69%) of Ac-PHSCN-NH2 as a fluffy, white solid: ES MS m/z (M+H)+ 598.2;
HPLC: 94%
monomer, 6% dimer.
EXAMPLE IV
Preparation of Ac-Pro-His-Ser-Cys-Asn-NH2, hydrochloride salt
Ac-Pro-His-Ser-Cys-Asn-NH2 (77 mg, 0.13 mmol) was dissolved in 3 mL of
distilled water
at room temperature and 1 M hydrochloric acid (0.13 mL, 0.13 mmol) was added
immediately. The
mixture was swirled once and then frozen and lyophilized to afford Ac-PHSCN-
NH2, hydrochloride
salt as a fluffy white solid: 'H NMR (300 MHz, DMSO-d6) 8 9.00 (s, 1H), 8.57-
8.26 (m, 2H), 8.21-
8.03 (m, 2H), 7.45-7.36 (m, 2H), 7.13 (s, 1H), 7.09 (s, 1H), 6.94 (s, 1H),
4.79-4.59 (m, 1H), 4.50-
4.25 (m, 4H), 3.74-3.56 (m, 3H, overlapping with water peak), 3.25-3.15 (m,
2H, overlapping with
water peak), 3.11-2.98 (m, 1H), 2.88-2.72 (m, 2H), 2.57-2.38 (m, 2H,
overlapping with DMSO
peak), 2.02 (s, 3H), 1.92-1.65 (m, 4H); HPLC: 93% monomer, 7% dimer.
EXAMPLE V
Materials and Methods in Formulating and Testin2
Formulations
Ac-PHSCN-NH2a 50 mg/ml, was formulated in solutions that included the 50mM
Citrate
and the ingredients shown in Table 1, below. The solutions were lyophilized
and dispensed into
vials for reconstitution in 1 mL water.
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TABLE 1
Soln# Glycine Mannitol Sugar (10 mg) pH
9 - 50 m - 5
- 50 mg sucrose 5
11 - 50 mg glucose 5
12 10m 50m - 5
13 50 m - - 5
14 50 mg - glucose 5
Testing of Stability
Accelerated Stability was tested by storage of the solution for 4 day at 40 C
and 34 days at
55 C. Accelerated stability experiments are performed using extreme conditions
of temperature
5 and/or humidity to force degradation and to quickly test the relative
stability of a particular
formulation (typical storage conditions are 22 , 4 , or -20 C for most drugs).
This data then
provides the ability to select a formulation that will most likely be the most
stable from a number of
test formulations. After reconstitution with water each formulation was
examined by reverse phase
HPLC using a standard mobile phases such as water/methanol or
water/acetonitrile. This
10 methodology allows the separation of Ac-PHSCN-NH2 from Ac-PHSCN-NH2
degradation products
such as fragments of Ac-PHSCN-NH2 or the disulfide-bonded dimer.
Calculation of Potency
Potency was calculated as the area under the Ac-PHSCN-NH2 peak in the HPLC
chromatogram vs. a standard curve.
Calculation of Purity
Purity was calculated as the area under the Ac-PHSCN-NH2 peak as a percentage
of the
integrated area of entire chromatogram.
Calculation of % Dimer
The relative amount of Ac-PHSCN-NH2 dimer was calculated as the area under the
dimer
peak as a percentage of the integrated area of entire chromatogram.
EXAMPLE VI
Stability of Various Formulations of Ac-PHSCN-NH2
The stability of the peptide formulations were expressed in three ways:
Potency (mg/mL)
Purity (% monomer and % dimer). The results are shown in Table 2 and Table 3
below. Table 3
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shows the potency normalized to that of the solution at t=0; the columns
indicated with an N show
the normalized values.
TABLE 2
POTENCY mg/mL
Solution # Prior to Lyo* t(O) 1 day 5 days 9 days 17 da s
1 50 50.4 48.9 49.5 48.3 48.5
2 50 49.9 48.7 49.7 50.8 47.0
3 50 49.8 50.0 49.8 49.8 46.6
4 50 50.8 50.7 50.1 49.9 45.6
50 50.4 50.9 50.4 50.1 47.4
6 50 50.2 50.2 49.9 49.7 46.2
PURITY %Monomer
Solution # Prior to Lyo* t(O) 1 day 5 days 9 days 17 days
1 95 95.2 94.6 94.5 94.2 94.1
2 95 95.1 95.0 95.0 94.5 94.5
3 95 94.8 94.9 94.7 95.0 94.6
4 95 95.0 94.9 95.2 94.7 94.5
5 95 95.0 95.0 95.2 94.6 94.3
6 95 95.3 94.8 94.8 94.6 93.2
PURITY %Dimer
Solution # Prior to Lyo* t(O) 1 day 5 days 9 days 17 days
1 0.9 0.7 0.8 0.9 1.1 1.3
2 0.9 0.9 0.7 0.7 1.0 1.2
3 0.9 1.0 0.8 0.8 0.8 1.1
4 0.9 0.9 0.8 0.7 1.0 1.4
5 0.9 0.8 0.8 0.8 1.0 1.3
6 0.9 0.7 0.8 0.7 1.0 1.8
* Lyo=lyophilization
5 TABLE 3
Time Formulation/Solution #
(days) 1 1 N* 2 2N 3 3N 4 4N 5 5N 6 6N
0 50.4 100.0 49.9 100.0 49.8 100.0 50.8 100.0 50.4 100.0 50.2 100.0
1 48.9 97.0 48.7 97.4 50.0 100.4 50.7 99.9 50.9 101.0 50.2 99.9
5 49.5 98.1 49.7 99.5 49.8 100.1 50.1 98.6 50.4 99.9 49.9 99.3
9 48.3 95.9 50.8 101.7 49.8 99.9 49.9 98.1 50.1 99.4 49.7 98.8
17 48.5 96.2 47.0 94.0 46.6 93.6 45.6 89.8 47.4 94.1 46.2 91.9
* = N=normalized
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EXAMPLE VII
Selection of Preferred Formulation(s)
The following summarizes the characteristics of the peptide formulations
A. The formulation in Solutions 1-4 were characterized as follows:
1. Inconsistent Cake shape, a majority collapsed in the lyophilizer
2. Loss of cake volume during accelerated stability.
3. Unreasonable reconstitution times for collapsed cakes.
B. The formulation in Solution 6 was characterized as follows:
Color changed to brown during accelerated stability most likely due to a
reaction between
glucose and glycine.
C. The formulation in Solution 5 was characterized as follows:
1. Good quality cake, white in color, rapid reconstitution and low moisture
content.
The composition of solution 5 is 50mg/mL of the peptide, 50mg/mL glycine, 50mM
citrate, pH 5.0, lyophilized (lmL).
2. Robust performance in the lyophilizer, uniform appearance with no collapse
in any
sample.
3. Maintained appearance and reconstitution ability during accelerated
stability.
4. Maintained stability that was equal to ( 2%) or better than other
formulations.
EXAMPLE VIII
Real time stability data indicated that the preferred intravenous formulation
(100 mg
Ac-PHSCN-NH2 (=ATN-161) 50 mM citrate 50 mg/niL glycine, pH 5.0, lyophilized
from a 2 mL
solution of 50 mg/mL ATN-161) remained within specifications (i.e., >90% ATN-
161) for 3 years
when stored at -20 C or at 2-8 C. It was decided based on these results that a
low pH formulation
with sufficient bulking agent (about 50 mg/ml glycine) and low moisture
content in the vial after
lyophilization would yield the desired quality and stability for the ATN-161
drug product. Moisture
content can be varied by optimizing the lyophilization cycle using methods
well-known in the art.
Similar parameters would be followed for developing additional formulations
for ATN-161 or any
of its acid addition salts or analogues.
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All the references cited above are incorporated herein by reference in their
entirety, whether
specifically incorporated or not. Citation of the above documents is not
intended as an admission
that any of the foregoing is pertinent prior art. All statements as to the
date or representation as to
the contents of these documents is based on the information available to the
applicant and does not
constitute any admission as to the correctness of the dates or contents of
these documents.
Having now fully described this invention, it will be appreciated by those
skilled in the art
that the same can be performed within a wide range of equivalent parameters,
concentrations, and
conditions without departing from the spirit and scope of the invention and
without undue
experimentation.
32
