Note: Descriptions are shown in the official language in which they were submitted.
~ WO94/12530 2 15 0 4 7 0 PCT~93/00065
POLYUNSATURATED FATTY ACYL-PEPTIDE COMPOSITIOM
Field of the Invention
The present invention relates to polyunsaturated fatty
acyl-peptide composition useful in inhibiting cell
proliferation.
Reference~
Ahn, N. G., et al ., (1991) J. Biol . Chem. 266:4220-
4227.
Bellot, F. (1990) J. Cell Biology, 110:491-502.
Berridge, M. J. (1987) Ann. Rev. Biochem. 56:159-193.
Calabrese, P. and Chabner, B.A. In: The Pharma-
coloaical Basis of Thera~eutics, Gilman, A.G. et al. (eds.)
Pergamon Press, New York.
Casneillie, J. E. (1982) Proc. Natl. Acad. Sci.
7g:2&2-286.
Chabner, A. B., et al., (1990) In: Cancer Chemo-
thera~v: Princi~les and Practice, Chabner, A. B. et al.
(eds.) W. B. Saunders Co., pages 154-179.
Hashida, T. and Yasumoto, S. (1990) Biochem. Biophys .
Res . Co~. 172:958-964.
Kano, J., et al., (1991) ~iochem. ~iophys. Res. Comm.
179:97-101.
Keri, G., et al., (19gl) ~umor Biol. 12: 61-67.
Kumagai, H., et al., (1991) Biochem. Biophys. Res.
Comm. 177:7~-82.
Meyers, C.A. (1980) Proc. Natl . Acad. Sci. 77:6171-
6174).
Shaw, B. R. et al. (1976) Proc. Natl. Acad. Sci. USA
73:505-509.
,35 Schally, A. V et al. (1986) In: Neural and
Endc,crine Pe~tides and Rece~tors, pages 73-88.
WO94/12530 . ~ : 2 PCTnHU93/00065 ~
2~0~
Stewart, et al. (1984) In: Solid Phase Pe~tide
Svnthesis, 2nd Edition, Pierce Chemical Company, Rockford,
Illinois.
weinstein, I.s. (1988) Cancer Res. 48:4135-4143.
Yarden, Y. and Ullrich, A. (1988) Annual Rev. Bioc~em.
57:4~3-47~.
sackground of the Invention
Uncontrolled cell proliferation is a characteristic of
a number of diseased states. Such growth is observed, for
example, in benign and malignant tumors, certain virally-
induced diseases and psoriasis. Generally, drugs used to
treat cellular abnormalities characterized by uncontrolled
cell growth ta-get important biochemical steps or processes
that are part of the cell growth cycle. However, such
drugs lack selectivity and inhibit the growth of both
diseased and healthy cells. Therefore, development of
chemotherapeutic agents having relatively high selectivity
for the diseased cells would be advantageous.
Agents based on peptide hormones, steroid hormones,
hormone-releasing factors, and their respective antagonists
and agonists are relatively specific to their target cells.
Proliferative cells, such as neoplastic cells or tumors,
which arise from hormone-sensitive tissues generally are
found to have hormonal requirements that are similar to
those of their healthy counterparts. By altering the amount
of hormone in the blood circulation it may be possible to
selectively restrict the growth of these cells. However,
due to the relatively high levels of hormone required for
such treatment, use of hormonal chemotherapeutic agents is
still limited in vivo by toxic side effects to normal
cells.
Likewise, cell proliferation may also be inhibited by
targeting one or more of the cellular signal transduction
systems implicated in the regulation of cell division.
These include a) the tyrosine kinase signal transduction
WO94/12530 215 ~ ~ 7 0 PCTnHU93/00065
3
pathway, b) the phospholipid metabolism/protein kinase C
signal transduction pathway, and c) the cAMP protein kinase
A signal transduction pathway. These pathways are
activated by endogenous ligands which initiate a cascade of
signalling events that eventually results in cell division.
Protein kinases have been found to be particularly
important regulators of these pathways. For example,
tyrosine kinases are known to play a critical role in the
regulation of cell division. High levels of tyrosine kinase
activity have been measured in highly proliferative cells,
such as neoplastic cells. Inhibition of such
phosphorylation activity can be correlated with a reduction
in cell division, in some cases.
The current invention is directed to fatty acyl-
peptide compositions having enhanced biological activity,
compared to the peptide alone. Peptides used to form
compositions directed to the inhibition of cell
proliferation include peptide hormones, peptide hormone
analogues, and protein kinase peptide substrates or peptide
inhibitors. In experiments in support of the current
invention, it has been found that linking such peptides to
the polyunsaturated fatty acids lowers the concentration at
least several-fold of such peptides required to inhibit
cell proliferation. Such low che~otherapeutic drug
concentrations confer the advantage of reducing toxicity to
healthy cells.
Summary of the Invention
One general object of the invention is to provide a
fatty acyl-peptide composition which is useful in
inhibiting cell proliferation, such as neoplastic cell
proliferation. The composition includes a peptide having
antiproliferative activity and conjugated to the peptide, a
polyunsaturated fatty acyl moiety. The composition is
35 characterized by a cell proliferative inhibitory activity
WO94/12530 PCT~HU93/00065
215~470
which is several-fold greater than that of the
antiproliferative peptide alone.
In one embodiment the fatty acyl moiety of the
composition is a docosahexaenoyl or an eicosapentaenoyl
moiety. In a preferred embodiment, the fatty acyl moiety
is a cis-4,7,10,13,16,19-docosahexaenoyl(DHA) or cis-
5,8,11,14,17-eicosapentaenoyl(EPA) moiety. The fatty acyl
moiety is preferably conjugated to the peptide through an
amide linkage.
In another embodiment, the peptide portion of the
composition is a peptide hormone, and in a preferred
embodiment, the peptide hormone is a somatostatin analog or
a gonadotropin releasing hormone (GnRH) analog. In yet
another preferred embodiment, the peptide hormone has the
sequence of SEQ ID NO: 4, and in another preferred
embodiment, the peptide hormone of the composition has the
peptide sequence of SEQ ID NO: 5.
The peptide used in forming the fatty acyl-peptide
antiproliferative composition can also be a protein kinase
modulatory peptide. In a preferred embodiment, the protein
kinase modulatory peptide has a sequence selected from the
group consisting of SEQ ID NO: 1, S~Q ID NO: 2, SEQ ID NO:
3, and SEQ ID NO : 6 .
Another general object of the invention is to provide
a method for producing enhanced biological activity of a
peptide. In one aspect, the invention is used for
producing a several-fold enhancement of cell proliferative
inhibitory activity in a peptide composition. According to
the invention, this enhancement is achieved by conjugating
the peptide to a polyunsaturated fatty acid. Preferred
peptides and fatty acyl moieties in the method are
described above.
In a more general aspect, the invention provides fatty
acyl-peptide compositions having enhanced biological
activity, compared to the activity of the peptide alone.
Such enhanced biological activity includes such activities
~ WO94/12530 2 ~ S ~ 4 7 0 PCTnHU93/00065
as enhanced hormone activity, anti-tumor activity, enhanced
immunogenic activity and other peptide-specific activities.
These and other objects and features of the present
invention will become more fully apparent when the
following detailed description of the invention is read in
conjunction with the accompanying drawings.
.
srief Descri~tion of the Figures
Figure 1 shows exemplary peptide sequences, designated
sequences 1-31 and identified by SEQ ID NO: 1-31,
respectively, used in forming the novel peptide-fatty acyl
compositions of the invention: STKS (sequence 1, SEQ. ID
NO: 1), STKSI tsequence 2, SEQ ID NO: 2), SPKCS (sequence
3, SEQ. ID NO: 3), somatostatin analogue (sequence 4, SEQ.
ID NO: 4 ), where lower case "dn signifies the prçsence of
a D-amino acid residue and "NH2" at the C-terminal
signifies the amidation of the C-terminal end, GnRH
(sequence 5, DHA-SEQ. ID NO: 5), where UGlp" signifies the
presence of pyroglutamate, EGFA (sequence 6, SEQ ID NO: 6);
an N-terminal sequence of human PTH (sequence 7, SEQ ID NO:
7), a polypeptide fragment derived from fibronectin
(sequence 8, SEQ ID NO: 8), and a T cell epitope modula~ory
peptide (sequence ~, SEQ ID NO: g) suitable for use in
forming compositions of the present invention; the
sequences of protein kinase modulatory peptides, such as
tyrosine kinase inhibitory peptides (sequence 10, SEQ ID
NO: 10), (sequence 11, SEQ ID NO: 11), (sequence 12, SEQ
ID NO: 12) where "ACM" signifies that the cysteine
sulfhydryl group has been modified by zn acetamidomethyl
group, (sequence 13, SEQ ID NO: 13) and (sequence 14, SEQ
ID NO : 14 ), calmodulin-dependent protein kinase III
inhibitory peptide (sequence 15, SEQ ID NO: 15), dsDNA-
dependent kinase inhibitory peptide (sequence 16, SEQ ID
NO: 16), protein kinase C modulatory peptide (sequence 17,
SEQ ID NO: 17), and other protein kinase modulatory pep-
tides, such as for cAMP dependent kinase (sequence 18, SEQ
.
W094/~530 i 6 pcTnlu93looo65
21504~ ~
ID NO:18), (sequence 19, SEQ ID NO: 19), and (sequence 20,
SEQ ID NO: 20), phosphorylase kinase (sequence 21, SEQ ID
NO: 21), calmodulin-dependent kinase I and II (sequence 22,
SEQ ID NO: 22 and sequence 23, SEQ ID NO: 23), dsRNA-
dependent kinase (sequence 24, SEQ ID NO: 24), proline-
dependent kinase (sequence 25, SEQ ID NO: 25 and sequence
26, SEQ ID NO:26), growth factor-regulated kinase (sequence
27, SEQ ID NO: 27), casein kinase I and II (sequence 28,
SEQ ID No:28 and sequence 29, SEQ ID NO: 29, AMP-activated
protein kinase (sequence 30, SEQ ID No:30), and S6 kinase
II (sequence 31, SEQ ID NO:31);
Figure 2 illustrates coupling of cis-4,7,10,13,16,19-
docosahexaenoicacid (DHA) to a peptide (sequence 1, SEQ ID
NO: 1) through an N-terminal amine group to form the fatty
acyl-peptide composition (composition I, DHA-SEQ ID NO: l);
and
Figure 3 shows sequences of exemplzry fatty acyl-pep-
tide compositions of the invention: DHA-STKS (composition
I, DHA-SEQ. ID NO.: 1), D~-STKSI (composition II, DHA-SEQ
ID NO: 2), DHA-SPKCS (composition III, DHA-SEQ. ID NO.: 3),
DHA-somatostatin analogue (composition IV, DHA-SEQ. ID NO.:
4 ), D-Lys6(DHA)-GnRH (composition V, Dr~-SEQ. ID NO. 5),
and DHA-EGFA (composition VI, DH~-SEQ ID NO:6), where D~
is a cis-~7~lo~l3~l6~l9-docosahexaenoyl moiety, Glp
represents pyroglutamate, a~d lower case ~d~ signifies the
presence of a D-amino acid residue, and "NH2" at the C-
terminal signifies the amidation of the C-terminal end.
Detailed De~cription of the Invention
I. Definitior.s
The term "polyunsaturated fatty acid'~ refers to a com-
pound having a carboxylic acid moiety and a long unbranched
carbon chain, usually containing between about 8 and 24
carbon atoms, and containing two or more carbon-carbon
double bonds. When a fatty acid is conjugated to a peptide
through an amide linkage, a fatty acyl-peptide derivative
is formed. The disclosed invention includes fatty acyl-
~WO94/12530 ~1 5 ~ 4 7 0 PCTnHU93/0006
peptide derivatives, and, more generally, peptides
linked to long unbranched, polyunsaturated carbon chains.
The term ~peptide hormone~ refers to a peptide that
elicits a biological response in a target cell. Peptide
hormones are generally low molecular weight proteins (<
50,000). Such hormones can be isolated from biological
sources, chemically synthesized or produced by recombinant
methods. Generally, in their natural setting, peptide hor-
mones are secreted from specific cells and produce
biological effects in other cells. Analogues of such
naturally occurring hormones are encompassed by the term
"peptide hormone~ and are produced recombinantly or
synthetically.
Peptide hormones, as described herein, are divided
into two main categories, according to their known acti-
vities in vivo. Generally,~hormones act either directly or
indirectly to produce the desired biological effect. In the
context of the present invention, the desired effect is
inhibition of cell proliferation. Peptide hormones that
act directly on a cell to inhibit division of the cell are
referred to herein as direct peptide hormones; those pep-
tides that act to,stimulate or inhibit synthesis or
secretion of endogenous peptide growth regulatory hormones
are referred to as indirect peptide hormones.
Generally, it will be seen that an indirect peptide
hormone effects a change (increase or decrease) in the
extracellular levels of a naturally occurring direct pep-
tide hormone. Two exemplary types of indirect peptide hor-
mones are peptide hormone-releasing hormones and peptide
hormone release-inhibiting hormones. Naturally occurring
indirect peptide hormones are generally short polypeptides,
usually under 20 amino acids in length.
Peptide hormone analogs are synthetically or
recombinantly prepared peptides which are structurally
similar to naturally occurring peptide hormones. For the
purposes of the present invention, such peptide analogs are
WO94/12530 8 PCTnlU93/00065 ~
21S~
included by the term 'Ipeptide hormone.~ Generally, to be
useful in the present invention, such analogs have
essentially a similar or a higher biological activity than
that of the endogenous peptide hormone.
Protein kinase modulatory peptides are peptides which
act as inhibitors of protein kinase activity. Such pep-
tides may act, for example, as protein kinase substrates,
residue
containing a phosphorylatable amino acid/in the sequence.
Alternatively, such kinase modulatory peptides may bind to
the kinase catalytic site, to effect inhibition of kinase
activity. Such kinase modulatory peptides act to produce
reduced phosphorylation of endogenous protein kinase
substrates.
Peptides and compositions that inhibit cell
proliferation are sometimes referred to herein as ~'anti-
proliferative" peptides or compositions.
II. Selection of Pe~tides for Use in the Fatty acvl-Pe~-
tide Comgosition
A. Selection of Pegtide Hormones
The present invention is directed to peptide
compositions having anti-proliferative activity. More
generally, it is the discovery of the invention that these
fatty acyl-peptide compositions have enhanced biological
activity in comparison to underivatized peptides. Anti-
proliferative fatty acyl-peptide compositions of the
invention are effective to inhibit proliferation of highly
proliferative cells, such as neoplastic cells or virally-
infected cells. For use in the compositions of the
invention, peptides known to have anti-proliferative
activity are linked to a fatty acyl moiety, such as a
docosahexaenoyl (DH~.) or eicosapentaenoyl (EPA) group, as
described below. Peptide hormones having cell
proliferative inhibitory activity are known in the art, and
may be direct or indirect hormones. Such anti-
proliferative peptide hormones are usually, but not
~ WO94/12~30 215 0 ~ 7 0 PCTnHU93/00065
necessarily, specific to a particular type of cell, such as
a cell with specific hormonal requirements.
1. Indirect Pe~tide Hormones. As noted above,
in the context of the present invention, indirect peptide
hormones include those peptides which act to effect a
decrease in the level of a direct cell proliferation
stimulatory hormone or to effect an increase in the level
of a direct cell proliferation inhibitory hormone.
~n example of an indirect peptide hormone which
stimulates release of an antiproliferative hormone in some
ma~malian species is gonadotropin releasing hormone (GnRH).
GnRH stimulates release of several gonadotropins, including
luteinizing hormone (LH). GnRH and GnRH analogues can also
inhibit the growth of hormone dependent carcinomas, such as
androgen-dependent prostate tumors (Schally). GnRH is also
effective against such tumors as colon and pancreatic
tumors. Figure 1 shows a sequence of a GnRH analog useful
in treating such tumors (5, SEQ I3 NO: 5). Figure 3 shows
a DHA-GnRH fatty acyl-peptide co~position of the invention
(v, SEQ ID NO: 5) having enhanced activity, as described
below.
GnRH analogues can be screened for potential anti-
proliferative activity, by assessing their abilities to
stimulate release of LH from anterior pituitary cells, as
detailed in Example 9. Active GnRH analogue peptides are
then used to form fatty acyl peptide co~positions of this
invention, and tested for enhanced biological activity, as
discussed below.
Indirect peptide hormones also include peptide hormone
release-inhibiting hormones. An exemplary peptide hormone
of this type is somatostatin. Somatostatin is a 14 amino
acid peptide that inhibits the release of growth hormone
(GH). GH enhances the proliferation of its cellular
~35 targets. Native somatostatin peptide and analogs are used
to form fatty acyl peptide derivative compositions of the
2 ~g O ~ ~ 10 PCTnlU93/00065
invention, as described below. An analog of somatostatin
used to form fatty acyl peptide compositions in studies
described herein has the sequence ~ (SEQ ID NO: ~) in
Figure 1, and a fatty acyl-peptide composition which
S includes this peptide is shown as composition IV (DHA-SEQ
ID NO: 4) in Figure 3. The somatostatin fatty-acyl peptide
is about 150 times more potent than the parent molecule.
It can be appreciated that other indirect acting pep-
tide hormones can be used in forming fatty acyl-peptide
compositions of the invention. Preferably, peptides are
selected based on their known anti-proliferative activit~
in a cell proliferation assay, described in Section III
below. Selected peptides are then conjugated to fatty acyl
moieties, and are tested for enhanced potency in such
assays. A fatty-acyl peptide derivative composition is a
useful anti-tumor or anti-neoplastic cell agent, when it is
found to have at least a several-fold increase in potency,
in comparison to the unconjugated peptide.
2. Direct Pe~tide Hormones. Also used in
forming compositions of the invention are peptides which
are known to have direct antiproliferative effects on
cells. ~n exemplary direct peptide hormone is parathyroid
hormone (PTH). This peptide is 8~ amino acids in le~gth,
and can directly inhibit osteoblast division. Certain bone
cancers are characterized by hyperproliferating
osteoblasts. Structure-function studies indicate that an
N-terminal fragment of PTH is active in inhibiting
osteoblast proliferation (Kano). This se~uence 7 (SEQ ID
NO: 7) is shown in Figure 1.
Another example of a peptide which is defined as a
direct peptide hormone for purposes of this invention is a
peptide fragment of fibronectin having the sequence SEQ ID
NO: 8, shown in Figure 1 as sequence 8. This fragment
spans the recognition site within the fibronectin molecule
to which cells bind for attachment to fibronectin. This
215Q470
WO94/12530 11 PCT~HU93/00065
fibr ~ectin recognition site peptide and analogues the-eof
are used to inhibit fibronectin-mediated cell attachmen~
and spreading in vitro. They may also be important for
regulation of tumor cell proliferation and metastasis in
vivo (Kumagai). According to the present invention,
fibronectin recognition site peptides are coupled to fatty
acyl moieties to form compositions useful in inhibiting
tumor cell proliferation and metastasis.
Direct peptide hormones, as defined in the current
invention, also encompass peptides derived from immunogenic
polypeptides for modulating a response from specific irumune
cell populations. For example, a T-cell epitope peptide
(TCEP) is coupled to a fatty acyl moiety as described
herein to form a composition useful in modulating the T-
cell immune response. The seouence of TCEP (SEQ ID NO:9)
is shown in Figure 1 as sequence 9, and synthesis of a DHA
derivative of TCEP is detailed in Example 7.
B. Selection of Protein ~in2se Modulatorv Pe~tices
Elevated protein kinase C and tyrosine kinase
activities are associated with neoplastic cell prolif-
eration or transformation (weinstein, Yarden). Inhibition
of such kinase activities can be effected by the presence
of small peptide fragments which mimic protein substrate
phosphorylation sites and/or protein kinase modulatory
domains. Such protein kinase modulatory peptides are
effective to compete with endogenous protein kinase
substrates. Selection of specific protein kinase
modulatory peptides for use in forming the fatty acyl-pep-
tide derivatives of the invention is described below.
1. Tyrosine Kinase Substrates and Inhibitors.
Receptors for a number of growth factors, including
epidermal growth factor (EGF), insulin growth factor (IGF),
and platelet derived growth factor (PDGF), contain tyrosine
kinase catalytic domains which phosphorylate specific
WO94/12530 PCTnHU93/00065
21~7~ `~ 12
intracellular protein substrates, including the receptor
itself, in some cases, at tyrosine residues. Cellular
transformation by a virus, such as the Rous sarcoma virus,
can result from expression of a single viral protein, which
functions as a tyrosine kinase.
Tyrosine kinases may also play a role in the
uncontrolled growth of keratinocytes which can result in
psoriasis or other highly proliferative skin disorders.
Keratinocytes possess tyrosine kinase-like growth factor
receptors.
High tyrosine kinase activity has been associated with
high rates of cellular proliferation tCasneillie, Keri).
Inhibition of such kinase activity decreases the rate of
cellular proliferation. Several tyrosine kinase modulatory
peptides which are effective to inhibit tyrosine kinase
activity in vitro have been described in the literature,
and some are available commercially, for example from Pep-
tides International (Louisville, ~Y). Figure l shows
sequences of several exemplary tyrosine kinase modulatory
peptides which can be used to form anti-proliferative
compositions of the invention, such as STKS (SEQ ID NO: 1)
and STKSI ( SEQ ID ~0 . 2), seq~ences l and 2, respectively.
~lternatively, tyrosine kinase modulatory peptides can
be identified, bas_~ on peptide sequences surrounding
phosphorylation sites of endogenous protein kinase
substrates in highly proliferating cells. Preferably,
cellular sources us-~d for identification o~ such substrate
peptides are proliferating cells of the type to be
targeted; ho~ever, it is appreciated that tyrosine kinase
modulatory peptides derived from one cell source may
inhibit a tyrosine kinase derived from a different cell
type.
In order to isolate an endogenous protein kinase
substrate protein, proliferating cells, such as a lymphoma
cell line having a high level of tyrosine kinase activity,
are incubated with [32P]-phosphate, or a particulate
2150470
WO94/12~30 13 PCT~HU93/00065
fraction of the cells is incubated with radiolabelled
[gamma 32p] ATP. Cellular polypeptides are separated by
denaturing gel electrophoresis, and the phosphorylated
proteins are observed by autoradiography. Phosphoprotein-
containing bands are excised from the gel, and the
phosphoprotein is eluted from the gel band. The eluted
protein is subjected to partial hydrolysis, and the
identity of the phosphorylated amino acid determined,
according to methods known in the art (Casneillie). In
order to determine the peptide sequence in the vicinity of
the phosphotyrosine residue(s), phosphoproteins are
subjected to peptide fragmentation, such as by proteolysis
or chemical means, and phosphotyrosine-containing peptides
are sequenced, acco-ding to conventional methods (Casneil-
lie). Short peptides (approximately 6-20 amino acids)
corresponding to the peptide sequences in the vicinity of
the phosphorylation site can be synthesized and tested for
inhibition of tyrosine kinase activity.
Tyrosine kinase modulatory peptides can be prepared by
substituting for the tyrosine phosphorylation site residue
an amino acid residue that cannot be phosphorylated, such
as a phenylalanine residue. composition II in Figure 3
(D~-STKSI) shows 2n inhibitory fatty acyl-peptide
composition, the peptide portion of which has almost
complete identity with the peptide portion of composition
(D~-STKS), except that in composition II a phenylalanine
residue has been substituted for a tyrosine residue present
in composition I. ~oth peptides inhibit proliferation of
neoplastic cells. ~.ccordingly either peptide can be
conjugated to a fatty acyl group, to form an anti-tumor
fatty acyl-peptide composition of the invention, as
described in Section III, below.
2. Protein Kinase C (PKC) Pe~tide Substrates.
PKC is a component of the phospholipid metabolism/protein
kinase C signal transduction pathway which plays a critical
2 1 5 0 ~ PCTnlU93/00065 ~
role in normal cellular ~rowth control. Activation of PKC
is mediated by a family of G-protein-modulated receptors.
When activated, the cytosolic form of PKC binds to the
cytoplasmic face of the plasma membrane. One of the known
protein targets of PKC is the EGF receptor. Phosphoryla-
tion of EGF receptor by protein kinase C results in a
decrease in the affinity of the receptor for EGF and a
decrease in EGF receptor-associated tyrosine kinase
activity (Berridge). This illustrates the heterologous
interactions between signal transduction pathways,
specifically between the tyrosine kinase pathway and the
phospholipid metabolism/protein kinase C pathway.
Recent results demonstrate that protein kinase C
activity inhibition is directly related to the anti-
proliferative activity of certain gonadotropin-releasing
factor agonists. These agohists are effective in
inhibiting DNA synthesis in certain breast cancer cell
lines. Both follicin and buserelin, the two tested
gonadotropin-releasing factor agonists, inhibit protein
kinase C activit~y and tyrosine kinase activity lKeri)~ It
has also been demonstrated that somatostatin analogues can
stimulate a phosphatase activity in tumor cells thereby
inhibiting signal transduction and tumor growth (Schally).
The sequences of a number of peptide substrates for
PKC are known and are commercially available. Figure 1
lists the sequences of some of the ~nown peptide substrate
inhibitors for PKC, such as sequenc~ 3 (SEQ ID NO: 3) and
seqlence 17 (SEQ ID NO: 17). These peptides are suitable
PKC modulatory peptides for use in forming fatty acyl-pep-
tide compositions of the present invention. PKC peptide
inhibitors can also be identified from endogenous protein
substrates of protein kinase C, using the same general
procedures described in part 1, above, for tyrosine
kinases.
~ WO94/12530 2 1 5 0 4 7 0 PCT~HU93/00065
3. O~her Protein Kinases. Modulatory peptides
targeting protein kinases other than tyrosine kinase and
protein kinase C can also be used to form fatty acyl-pep-
tide anti-tumor compositions of the invention. One such
protein kinase target is a casein kinase II found in
epidermal keratinocytes. High proliferation rates are
related to the hyperphosphorylation of an oncogenic product
from a human papillomavirus which contains a casein kinase
II phosphorylation consensus sequence (Hashida). This
casein kinase II consensus phosphorylation sequence is
shown in as sequence 29 in Figure 1 (SEQ ID NO: 29).
Another protein kinase, cyclic AMP-dependent protein
kinase (PKA), is a component of the cAMP-dependent protein
kinase signa] transduction pathway. PKA phosphorylates
threonine and serine residues of a protein substrate. The
activity of this enzyme is enhanced by cAMP. PKA may
regulate growth control. In some animal cells, an increase
in cAMP levels activates specific genes involved in growth
regulation. In neuroendocrine cells of the hypothalamus,
cyclic AMP turns on the gene that encodes the peptide hor-
mone release-inhibiting hormone, somatostatin.
Other target protein kinases, in conjunction with the
present invention, are components of signalling cascades
involved in cell proliferation. For example, when
2S epidermal growth factor (EGF) binds to the epidermal growth
factor receptor (EGFR), a tyrosine kinase, it stimulates
activation of myelin basic protein (MBP) kinase, through
phosphorylation of MBP kinase. MBP kinase, in turn,
activates S6 kinase by a phosphorylation event. The result
of activation of this signalling pathway is stimulation of
proliferation of EGFR-bearing cells, such as adipocyte
cells (Ahn). It can be appreciated that fatty acyl-peptide
compositions of the invention using as peptide components,
phosphorylation recognition sequence peptides for one or
. 3S more of EGFR kinase, MBP kinase and S6 kinase will serve to
inhibit the signalling cascade of which these kinases are a
W094/~530 16 PCTnlU93/00065
2150~7~ ~
part. Sequences of exemplary peptide inhibitors of these
kinases are shown in Figure 1 (e.g., sequence 11, SEQ ID
NO: 11, to sequëncê 31, SEQ ID NO:31).
Other examples of protein kinase activities involved
in cell proliferation whose activities can be modulated,
using fatty-acyl peptide compositions of the present
invention, include calmodulin-dependent kinases I and II,
cGMP-dependent protein kinase, ds-DNA-dependent protein
kinase, proline-dependent kinase, and AMP-activated ~inase.
Sequences of exemplary peptide modulatory peptides directed
to these kinases are listed in Figure 1 (SEQ ID NOs: 18-
31).
III. PreParation of FattY acYl-PePtide ComPositions
In accordance with the present invention, it has been
observed that conjugation of fatty acyl groups to the
above-described peptides enhances the biological activity
of the peptides. Enhancement of biological activity is
exemplified herein as enhancement of inhibition of cell
proliferation, such as neoplastic cell proliferation. In
experiments carried out in support of the present
invention, exemplary peptide hormones and protein kinase
modulatory peptides were selected, as described above, and
isolated from natural sources or synthesized, as described
below. Subsequently, the peptides were conjugated to fatty
acyl moieties, as described below and illustrated in Figure
2 and tested for the,~ activities in cell proliferation
assays, as described in Section III, below.
A. Solid Phase Pe~tide SYnthesis
Peptides shorter than about 30 amino acids in length
are conveniently prepared by methods commonly used in
solid-phase peptide synthesis, as detailed below (Stewart).
Briefly, N-alpha-protected amino acid anhydrides are
prepared in crystallized form and used for successive amino
acid addition to the peptide N-terminus. At each residue
2150~7~
094/12530 17 PCTnIU93/00065
addition, the growing peptide on a solid support is acid
treated to remove the protective group, and washed several
times to remove residual acid. The peptide is then reacted
with another N-protected amino acid. The amino acid
-5 addition reaction may be repeated two or three times to
increase the yield of growing peptide chains. After
completing the growing peptide chains, the protected pep-
tide resin is treated with liquid hydrofluoric acid to
deblock and release the peptides from the support.
B. Coniuaation of Fattv Acyl Moieties to Pe~tides
Preferred fatty acyl moieties for use in the present
invention are those with a high degree of unsaturation, and
include such fatty acids as cis 5,8,11,14,17-
eicosapentaenoic (EPA) and cis-4,7,10,13,16,19-
docosahexaenoic acid (DHA). Such polyunsaturated fatty
acids can be prepared synthetically according to standard
methods, isolated from the oils of marine fish, or obtained
from commercial sources.
Generally, fatty acids are linked to the peptides via
the terminal amine group or via internal amine groups, such
as the amine group of lysine, in an amide linkage. Figure
2 illustrates a scheme for coupling of sequence 1 to DHA by
the terminal amino group of the peptide through an amide
linkage to form the fatty acyl-peptide composition I in
Fig. 3. DHA is activated by reaction with N-
hydroxysuccinimide prior to reaction with an ami~o-group
containing polypeptide. In another embodiment, DHA is
coupled by the free amine group of a lysine residue in the
sequence to form the fatty acyl composition, such as
composition V in Fig. 3.
Examples 2-6 detail preparation of fatty acyl-peptide
compositions in which acylation of peptides is carried out
using activated ester or acyl chloride derivatives of DHA
or EPA. Activated esters useful in preparing compositions
of the current invention include DHA-O-benztriazole ester
WO94112530 18 PCTAHU93/00065
2150470 ~
(DHA-osT)~ DHA-O-pentafluorophenyl ester (DHA-opfp)t and
DHA-O-succinimidyl ester (DHA-O-N-Succ). The acylation is
carried out as detailed in Example 2. Crude products,
obtained after acylation, are purified by HPLC. The purity
of the final products is characterized by analytical HPLC
and TLC data, while the chemical characterization is
accomplished by mass spectrometry (MS).
C. FattY Acyl-Pe~tide Com~ositions
From the foregoing discussion, it can be appreciated
that peptides can be selected for use in anti-proliferative
fatty acyl-peptide compositions of the invention, based on
their abilities to inhibit cell proliferation or to inhibit
components, such as protein kinase components, of cell
proliferative stimulus pathways. According to an important
feature of the present invèntion, conjugating to such a
peptide, a polyunsaturated long chain carbon, such as a
polyunsaturated fatty acyl moiety described above, enhances
the biological activity of the peptide. Specifically,
conjugation of a polyunsaturated fatty acyl moiety to an
anti-proliferative peptide enhances its ability to inhibit
neoplastic cell proliferation. This aspect of the
invention will be better appreciated from the discussion
below.
As an example, tyrosine kinase modulatory peptides
selected as described above are covalently linked to
polyunsaturated fatty acids to form compositions which are
effective to inhibit proliferation of neoplastic cells.
one such composition used in experiments carried out in
support of the current invention is DHA-STKS (DHA-SEQ. ID
NO. l) illustrated in Figure 3 (composition I).
Fatty acid-tyrosine kinase inhibitory peptide compo-
sitions can also be prepared by substituting for the tyro-
sine residue an amino acid residue that cannot be phos-
phorylated. In one such composition, DHA- STKSI (DHA-SEQ ID
NO: 2; composition II), the tyrosine residue of tyrosine
~150470
094/12530 19 PCTnIU93/00065
kinase substrate DHA-STKS (DHA-SEQ ID NO: li composition I)
has been substituted by a phenylalanine residue. Both
fatty acid-protein kinase peptide substrate and peptide
inhibitor compositions inhibit the proliferation of
- 5 neoplastic cells, as detailed in Example 8 and shown in
Table 1. Another such tyrosine ~inase inhibitor peptide is
DHA-EGFA (DHA-SEQ ID NO: 6, composition VI) also shown in
Table 1.
A PKC peptide substrate-fatty acid composition used in
the experiments described in Example 8 includes a peptide
fragment from myelin basic protein, and has the structure
shown in Figure 3 as composition III (DHA-SEQ. ID NO: 3).
Additionally, endogenous substrates of PKC may be
identified as described above for the tyrosine kinase
substrates. The polypeptide fragments that contain PKC
phosphorylation sites are then sequenced and used to form
the fatty acid peptide compositions of the invention.
IV. Anti-Proliferative Assavs
In accordance with the present invention, fatty acyl-
peptide derivatives are prepared as described in Section
II, above, and inhibition of neoplastic cell proliferation
by such compositions is measured, according to one or more
standard cell proliferation assays. In one exemplary
assay, described in Example 8, inhibition of cell
proliferation is measured directly, by measuring the number
of surviving cells after exposure of the cells to a test
composition, such as a fatty acyl-peptide composition
disclosed herein.
Alternatively, an activity which correlates to cell
proliferation can be measured to determine indirectly the
effect of a test compound on cell proliferation. Thus,
activities such as tyrosine kinase activity or release of a
growth-promoting hormone, which are known to correlate with
neoplastic cell proliferation, are used as indicators of
anti-proliferative activity. As exemplified herein,
WO94/12530 20 PCTnIU93/00065 ~
215~4~0
polyunsaturated fatty acyl-peptide anti-proliferative
compositions may exhibit one or more of the following in
vitro activities: (a) inhibition of cell proliferation,
(b) inhibition of tyrosine kinase activity, (c) stimulation
of release of a growth-1nhibiting hormone, such as lu-
teinizing hormone, and (d) inhibition of release of growth-
stimulating hormones, such as inhibition of growth-hormone
release. It is appreciated that other in vitro assays
which can be correlated with uncontrolled cell growth may
also serve as assays for selecting and determining the
activities of fatty acyl-peptide compositions of the
invention. More specifically, such assays can serve to
determine the relative potencies of compositions of the
invention as anti-proliferative agents.
A. Inhibition of Cel'l Proliferation
An assay for measuring the inhibitory effects of
polyunsaturated fatty acyl-peptide derivatives on cell
proliferation is described in detail in Example 8. In
experiments carried out in support of the invention, a
number of tumor cell lines, including a human prostatic
adenocarcinoma cell line, a human breast adenocarcinoma
cell line and a human colon adenocarcinoma cell line, were
used to test compounds of the invention for effects on cell
proliferation. Cells were exposed to the tyrosine kinase
synthetic peptide substrate fatty acid derivative DHA-STKS
(DHA-SEQ ID NO: 1), the protein kinase C peptide substrate
fatty acid derivative DHA-STKCS (DHA-SEQ ID NO:3), or the
gonadotropin release hormone fatty acid derivative DHA-D-
Lys6-GnRH (SEQ ID NO: 5), as described below.
Solutions of the peptide derivatives were added in
culture media to test cells for an incubation period of 2.5
hours. At the end of the test period, cells were
centrifuged, the pellet diluted with 1% BSA in saline and
viable tumor cell counts were determined by the trypan blue
exclusion method. Results of tests using peptide fatty
094/12530 21 2 1 5 0 4 7 o PCTnHU93/0006
acid derivatives DHA-STKS (DHA-SEQ. ID NO:1) and DHA-D-
lys6-GnRH (DHA-SEQ. ID NO: 5) in various transformed cell
lines are shown in Table 1. Cell proliferation was
inhibited by at least 50% at a peptide-fatty acid
~5 derivative concentration of 10 micrograms/ml of the TK
modulatory peptide substrate, the PKC modulatory peptide,
and the gonadotropin-releasing hormone analogue, as shown
in Table 1.
Table 1
Effect of Fatty Acid-Peptide Compositions on the
Inhibition of Cell Proliferation
(% Inhibition)
Concentr~tlon (ug/ml)
Compound
0.1 1 10 30
DHA-STKS (PC3)* - - qo
DHA-STKS (PC3) (1iposomal) - - '7
DHA-SP~CS (MCF 7) 5 25 ~3
nHA-D-Lys6-GnRH (MCF 7) - 33 52 61
DHA-D-Lys6-GnRH (HT 29) - 18 28 45
*Neoplastic cell types tested are indicated in
brackets, and are as follo~s:
PC 3, human prostatic adenocarcinoma cell line;
MCF 7, human breast adenocarcinoma cell line; and
HT 29: human colon adenocarcinoma cell line.
Additional methods are available for monitoring the
viability of cells, including assays based on the
differential dye uptake by viable cells in comparison to
that taken up by non-viable cells. For e~ample, viable
cells take up diacetyl fluorescein and hydrolyze it to
fluorescein, to which the cell membrane of live cells is
impermeable. Live cells fluoresce green. Nonviable cells
may be counter-stained with ethidium bromide and will
fluoresce red. These methods may be used in flow
cytometric assays, in accordance with protocols known in
the art.
B. Stimulation of Luteinizinq Hormone (LH) Release
WO94/12530 22 PCTnlU93/00065 ~
21~047~ --
Peptide hormone-releasing hormones increase the
extracellular levels of peptide-hormones that interact with
specific cellular receptors. Compounds, such as peptide
hormone-releasing hormones, which stimulate production of
LH have also been shown to inhibit cellular proliferation.
In accordance with the invention, fatty acyl derivatives of
such compounds are effective to inhibit neoplastic cell
proliferation at concentrations which are several-fold
lower than the concentration of peptide alone required to
inhibit such cell proliferation.
Specifically, as shown below, an analog of gonado-
tropin releasing hormone analog conjugated to the poly-
unsaturated fatty acid, DHA, (D-Lys6(DHA)-GnRH; DHA-SEQ ID
NO:5) was tested in an assay of LH release by anterior
pituitary cell suspensions as detailed in Example 9. In
this assay, luteinizing hormone levels were quantitated by
a double antibody radioimmunoassay procedure. As shown in
Table 2, the polyunsaturated fatty acid-gonadotropin
release hormone analogue (D-Lys6(DHA)-GnRH) was about 5-
fold more effective in eliciting the release of luteinizing
hormone when compared to equivalent concentrations of GnRH
or to the D-Lys6-GnRH analogue lacking the fatty acid
moiety. The polyunsaturated fatty acid DHA alone had no
effect on luteinizing hormone release at the concentration
tested (Table 2). These results further demonstrate the
effectiveness of compositions of the invention in enhancing
biological activity of their peptide components.
215~470
094112530 23 PCTAHU93100065
Table 2
Effect of the Fatty Acid-GnRH Analogue
Composition on LH Release (% Changea)
Concentration (M)
Compound
10-10 10-9 1o-8
D-Lys6(DHA)- lO 550 900
GnRH
DHA - O
GnRH 23 lO0 220
Lys6-GnRH 20 95
aCompared to the amount of LH released by lO-9M
GnRH
C. Inhibition of Growth Hormone Release
Peptide hormone-release inhibiting hormones decrease
the extracellular levels of peptide hormones which interact
with specific cellular receptors. Generally, binding of
peptide hormone to the receptor triggers a cascade of
biochemical events mediated through second messengers. In
some cases, an end-result of such hormonal activity is
cellular proliferation. Peptide hormone-release inhibiting
hormones which act to inhibit such hormones are useful in
forming fatty acid-peptide compositions described by the
present invention. Analogues of such peptide hormone-
release inhibiting hormones are also useful in forming such
compositions.
Somatostatin is an exemplary peptide hormone-release
inhibiting hormone analogue which inhibits hormonally
activated cell proliferation. Somatostatin is a growth
hormone release inhibitory hormone which inhibits release
of growth hormone. Growth hormone (GH) binds to specific
cell-surface receptors distributed widely throughout the
body. Binding of GH agonists to GH receptors results in
increased cellular division, and hence, cell proliferation.
In experiments carried out in support of the present
invention, a somatostatin-fatty acyl analogue (SEQ ID NO:
WO94/12530 24 ~CT~HU93/0006~ _
215047~ ~ ~
4) was synthesized~as detailed in Example 5. Inhibition of
growth hormone release by the DHA-acylated somatostatin
analogue was measured, as described in Example 10. GH
levels were determined by a double-antibody radio-
immunoassay for the hormone. In this assay, the DHA-
somatostatin analogue was 1000 times more effective in
stimulating release of growth hormone than was either
unacetylated somatostatin analogue or somatostatin.
D. Inhibition of TYrosine Kinase ActivitY
As described above, proliferative activity of certain
cells has been correlated to tyrosine kinase activity
present in the cells. As described herein (Example 11),
tyrosine kinase activity can be measured in cells from the
human breast adenocarcinoma line MDA-MB-231. Briefly, these
cells are incubated with a ~est compound, such as the
polyunsaturated fatty acid-tyrosine kinase peptide
inhibitor DHA-STKSI (DHA-SEQ ID NO: 2), then harvested and
homogenized. Tyrosine kinase activity is measured by
incorporation of radiolabeled phosphate into an endogenous
protein substrate in the cells. In this case the
endogenous protein substrate is the EGF receptor, and the
phosphorylation event is an autophosphorylation event.
The effect of DHA-STKSI on EGF receptor autophos-
phorylation is shown in Table 3. At a concentration of 20
micrograms/ml, the underivatized peptide inhibitor, STKSI
(SEQ ID NO:2), did not inhibit autophosphorylation of EG~.
In contrast, D~A-STKSI (DHA-SEQ ID NO:2) inhibited
phosphorylation by 50% at 1 microgram/ml and by 70% at 20
micrograms/ml. DHA present by itself at 20 micrograms/ml
decreased the extent of phosphorylation by only 5%. It can
be appreciated, in accordance with the present invention,
that similar cellular as well as in vitro tyrosine kinase
assays can be used to monitor activity and to test
compounds for their abilities to inhibit such activity.
094/12530 21~ O ~ 7 o PCTnHU93/00065
Table ~
Effect of DHA-STKSI on the
Autophosphorylation of EGF Receptor
Compound Concentr~tlon Inhibition
(ug/ml) (%)
STKSI 20 0
DHA-STKSI 1 50
DHA 20 5
IV. UtilitY
Fatty acyl-peptide compositions of the invention are
useful in inhibiting uncontrolled proliferation of cells,
such as benign and malignant tumor cells, virally-infected
cells, psoriatic cells, and the like. The use of the
composition of the invention for inhibition of such cell
proliferation is illustrated by experiments summarized in
Table 1. Generally, peptides are selected for use in
forming compositions of the invention, based on their known
or experimentally determined activities in inhibiting cell
proliferation. Selected peptides are then used to form
polyunsaturated fatty acyl-peptide compositions, according
to the general methods described in Section II, above and
detailed in Examples 1-7. As illustrated herein,
polyunsaturated fatty acyl-peptide compositions of the
present invention are effective to produce a several-fold
enhancement of anti-proliferative activity, in comparison
to peptides alone.
2S More specifically, fatty acyl-GnRH and -GnRH analogues
will find use in treating androgen-dependent prostate
adenocarcinomas. Test GnRH compounds can be screened in an
LH release assay, as described in Example 9, then tested in
an experimental animal model, such as a rat bearing the
Dunning R-3327-H prostate adenocarcinoma. Additionally,
GnRH derivatives of the invention are expected to find
usefulness in treatment of estrogen-dependent mammary
tumors, and their efficacy can be measured in rats (such as
WO94/12~30 26 PCT~HU93100065
21~ O ~ 7 Wistar Furth rats) carrying a mammary tumor such as the
MT/W9A mammary tumor, according to methods known in the
art. Likewise, it is anticipated that such LHRH analog
compositions will inhibIt growth of certain pituitary
tumors, chondrosarcomas, and osteosarcomas (Schally).
An anti-neoplastic or anti-tumor treatment method, as
described herein, includes exposing target neoplastic cells
to a concentration of fatty acyl-peptide compound effective
to inhibit neoplastic cell proliferation at least about
50~, and preferably about 90%. Such effective
concentrations can be determined in an in vi tro assay, as
described in Section IV, above.
Further, it can be appreciated from the foregoing
discussion that the method of the invention has general
utility in enhancing biological activity of a biologically
active peptide, by attaching to the peptide a
polyunsaturated fatty acyl moiety.
The following examples illustrate, but in no way are
intended to limit, the present invention.
Exam~le l
Svnthesis of PePtides
Solid-phase synthesis was carried out according to
standard methods. In one exemplary method, the synthesis
is performed using a Beckman model 900 peptide synthesizer.
Each BOC protected amino acid (2.4 mmol) is dissolved in 5
ml dichloromethane (DCM) and cooled to 0 degrees. The
volume of dichloromethane used for soC-leucine is 12 ml,
and the solution is not cooled. 2 ml of 0.6 M N,N-
dicyclohexylcarbodiimide (DCCD) in DCM is added and the
mixture is stirred at 0 degrees for 15 minutes. Coupling
reactions are monitored at each step using the ninhydrin
assay. Coupling reactions that are incomplete are repeated
using the appropriate symmetric anhydride.
After the coupling reaction any remaining free amino
acids are acetylated by using acetylimidazole.
~094/12~30 27 21~ 0 4 7 ~ PCTnHU93/00065
Precipitation of N, N-dicyclohexylurea is completed by
storage at -20 degrees for 1.5 hours, after which the
precipitate is filtered and washed with ethyl ether (5 ml).
The filtrate is evaporated to remove solvents, and the
- 5 product is crystallized by precipitation (Meyers).
Side chain protecting groups include for cysteine, 4-
Met-Benzyl; for lysine, 2-chlorobenzyloxycarbonyl; for
serine, benzyl; for arginine, tosyli for threonine, benzyl;
for aspartate, benzyl; and for tyrosine, 2-bromobenzyl-
oxycarbonyl. After peptide synthesis products are
deprotected in liquid hydrofluoric acid. For example, a
mixture of protected peptide resin (1.32 g), 2-
mercaptopyridine (0.5 g), p-cresol (2.6 g), and liquid
hydrogen fluoride (HF) (25 ml) is stirred at 0 degrees with
a rapid stream of nitrogen gas, first below 0 degrees, then
at 24 degrees. The mixture is stirred in ethyl acetate (25
ml) until a finely divided solid is obtained. The solid is
filtered, washed with ethyl acetate, and air dried. The
solid is stirred in 50~ aqueous acetic acid (10 ml) to
dissolve the peptide, filtered and washed with 20 ml water.
The filtrate is freeze dried.
Exam~le 2
PreDaration of DHA-STKS (DHA-SEO ID NO: 1)
A. PreDaration of DHA-O-N-Succ Ester
0.340 mM DHA (112.5 mg) (Aldrich Chemical Co., catalog
No.:27,155-1) and 0.341 mM TBTU (113.5 mg) were stirred in
4 ml of dry dimethylformamide (DMF) in the presence of 0.1
mM (15 mg) of diisopropylethylamine (DIEA). Activation was
carried out under He atmosphere for under an hour. 0.341
mM (39.6 mg) of N-hydroxysuccinnimide (HO-N-Succ) and 0.341
mM of DIEA were added to the reaction mixture. The mixture
was stirred overnight under He atmosphere for the
completion of the transesterification reaction. This
reaction mixture can be used directly for the N-terminal
acylation of the required peptide or worked up for the
WO94/12530 28 PCTnlU93/00065 _
2~0~7~
preparation of pure DHA-O-N-Succ. The reaction mixture was
diluted to 30 ml with water and extracted with 3 x 10 ml of
peroxide-free ether (treated and stored over alumina). The
combined ether phase was back-extracted with 3 x 5 ml of
water. The ether solution was dried over anhydrous sodium
sulphate, evaporated to dryness and taken up in 4 ml of
acetone.
B. Pre~aration of the N-DHA-derivative of STKS
0.03 mM STKS (37.0 mg) was first suspended in a
water-acetone mixture (1:1), before DIEA (0.15 mM, 18.0 mg)
was added. 0.05 mM of DHA-O-N-Succ ester in 1 ml of ace-
tone was added to the suspension and the ratio of
water-acetone was adjusted to 1:1. The reaction mix~ture
was stirred overnight under a He atmosphere, and then eva-
porated to dryness. The dried residue was triturated with
3 x 5 ml of the following solvents: petroleum ether,
diethyl ether, and HPLC grade water.
Using this procedure, a yield of 80% and at least a
90.0% product purity can be achieved, according to HPLC
analysis. A Hewlett-Packard HP-1089 liquid chromatograph
equipped with diode array detector was used with a Zorbax
SB-300-C18 column with the dimensions 4.6 x 150 mm. The
product~s absorbance at 210 nm was monitored. The solvent
composition was used with a gradie~t from 0 to 100% B, where
buffer A consisted of 0.05% TFA in water (v/v); and buffer
B consisted of 0.05% TFA in acetonitrile (v/v). The
product eluted at about 64% buffer s. The chemical
characterization of the compound was carried out by mass
spectrometry (MS) .
MH+ theoretical:1533.3,
MH+ measured:1533.2
Exam~le 3
Pre~aration of DHA-STKSI (DHA-SEO ID NO. 2)
~ 094/12530 21~ 0 4 7 0 PCTnHU93/0006~
A procedure similar tv that described in Example 2 was
used to generate the activated ester group of DHA (DHA-O-N-
succinimide). Then, 0.03 mM of STKSI (36.2 mg) was
acylated with 0.05 mM of DHA-O-N-Succ ester in the presence
5of 0.15 mM of DIEA according to Example 2. The product was
eluted from the same HPLC gradient as described in Example
2, the product eluted at 64.5% B. The final yield was 82%.
The chemical characterization of the compound was carried
out by mass spectrometry. MH+theoretical:1517.4,
10MH+measured:1515.5.
ExamPle g
Pre~aration of DHA-SPKCS (DHA-SEO ID NO: 3)
SPKC-(Lys/epsilon-TFA/)2,g (54.8 mg, 0.04 mM, final
15concentration) was suspended in 3 ml of water-acetone (1:1)
containing 0.08 r~ DIEA. ~Crude DHA-O-N- Succ activated
ester in DMF solution (0.04mM), as described in Example 2,
was added, and the suspension was stirred overnight under a
He atmosphere. The reaction mixture was evaporated to
20dryness and the product was TFA-deprotected in the
following fashion. TFA-deprotection and purification of
the end product was performed in the following fashion. The
dry residue was dissolved in 3 ml of 1 M aqueous piperidine
and stirred at room temperature. Deprotection was complete
25within 135 min. The reaction mixture was neutrali7ed
with acetic acid, evaporated to dryness and triturated with
3 x 5 ml of petroleurn ether. The rest of the solvent was
evaporated, the residue was taken up in 4 ml of
water-glacial acetic acid (1:1) and purified by preparative
30HPLC. Preparative HPLC conditions were as follows. An
Aquapore RP-300 column (Source) 2 x 22 cm was used with a
buffer gradient of 0-100% buffer B. Buffer A was 0.05~ TFA
in wateri buffer B was 0.05% TFA in acetonitrile. The flow
rate was 10 ml/min and the eluent program used was the
35following:
t~min) A% B%
WO94/12530 ~ 30 PCTnEU93/00065 ~
21~0 ~7 ~ o lOO o
0 100
s .
,, ~
The product waS eluted at 11.5 minutes, the yield was
65%, the purity of the product was 97%. The product was
analyzed by mass spectrometry, with the following results:
MH+theoretical:1700.8; MX+measured:1700.8
Exam~le 5
PreParation of DHA-Somatostatin Analoque
(DHA-SEO ID NO: 4)
360 ul each of 0.5 mM solutions of DHA, N,N' -diiso-
propyl carbodiimide (DIC), and pentafluorophenol in
dimethylformamide (DMF) were mixed and kept at 25 oC. After
15 min 54 mg (45 umol) of H-D-Phe-Cys-Tyr-D-Trp-
-Lys(Tfa)-Val-Cys-Thr-NH2 x HCl (dissolved in 1 ml of DMF)
were added to this solution and-the pH of the reaction
mixture was adjusted to 8 with triethylamine and kept at
room temperature overnight. The DMF was evaporated in
vacuo, and the oily residue was dissolved in 1 ml of
ethanol. To the solution was added 2 ml of 2 M hydrazine
hydrate in ethanol. and the reaction mixture was stirred at
45'C for 48 hours.
The solvent was removed in vacuo and the oily residue
was TFA-deprotected by dissolving the residue in 3 ml of 1
M aqueous piperidine and stirring at room temperature, and
dissolved in 28 ml of a 2-propanol:acetic acid:water
(30:35:35) solvent mixture and purified by HPLC under the
following conditions: A reversed-phase chromatography
column was used (Prepex C-18, 25-40 um, 42 x 1.4 cm); the
buffer was a 2-propanol:acetic acid:water mixture
(30:35:35) and separation was followed by TLC and HPLC.
THe purest fractions were pooled and repurified by MPLC.
The elution procedure was the following:
~ WO94/12530 321 5 0 4 7 0 PCTnIU93/00065
Step 1: Isocratic elution with 50 ml of 2-propa-
nol-acetic acid-water (20:40:40).
Step 2: Gradient elution with 400 ml of elution
mixture applied in step 1 and 400 ml of 2-propanolacetic
~ 5 acid-water (35:32.5:32.5).
The purity of the fractions was checked by HPLC, the
purest fractions were pooled, evaporated, lyophilized and
yielded 57% product. The purity of the product was 92%.
The composition of the product was analyzed by mass
spectrometry.
MH+theoretical:1356.5.
MH+measured:1356.7
Exam~le 6
Pre~aration of DH.~-D-LYs6-GnRH Analoaue (SEO ID NO: 5)
360 ul each of 0.2 mM 'solutions of DHA, DIC, and
pentafluorophenol in DMF were mixed and kept at room
temperature. After 15 minutes 56 mg (0.05 mM) of
Glp-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-NH2 x HCl
(D-Lys6-GnRH) (dissolved in 1 ml of DMF) were added to this
so]ution and the pH of the reaction mixture was adjusted to
8 with triethylamine and kept at~ room temperature
overnight. The product was TFA-deprotected as described in
Example 4. The solvent was removed in vacuo and the oily
residue was dissolved in 25 ml of 2-propanol-acetic
acid-water (30:35:35) solvent mixture and purified by MPLC
as described in Example 5. The final product yield was
47%. The sample identity was confirmed by mass
spectrometry.
MH+theoretical:1564.9,
MH+measured:1565.0
Exam~le 7
Synthesis of DHA-T Cell E~ito~e Pe~tide
(DHA-SEO ID NO: 12)
WO94/12~30 32 PCT~IU93/00065 _
2i~0~7~ _
T-cell epitope peptide (TCEP) (132 mg, 0.075 mmole),
with TFA and formyl protecting groups, was dissolved in
DMSO (2 ml) containing DIEA (40 ~l, 0.30 mmole) and DHA
succinyl ester (0.075 mmole). The reaction mixture was
stirred for 3 hours at room temperature at which time
acetonitrile/water (1:1, v/vj-, 0.5 ml) and TFA (0.5 ml)
were added. The reactio~ mixture was then filtered through
a 0.2 ~m filter and this~solution was applied directly to a
preparative reversed-phase HPLC column for purification.
DHA-TCEP was collected and identified by mass spectroscopy.
To remove the TFA and formyl protecting groups the purified
peptide was treated in 1 M aqueous piperidine for 8 hours
at room temperature at which time the product was isolated
by lyophilization. The product was identified by mass
spectrometry (MWcalc = 1886.7, MWfoUnd = 1888 5)
Exam~le 8
AssaY of the Inhibition of Cell ProliferatiQn
The growth inhibitory effect of peptide hormone
analogues, such as DHA-STKS, DHA-STKCS and D-Lys6(DHA)-
GnRH, was evaluated on the basis of changes in the total
cell number. Cells were cultivated in RPMI 1640 medium
(GIsCo BRL, Eggenstein, Germany, Cat. No.: 074-01800)
supplemented with 10% fetal calf serum. Tumor cells were
washed with phosphate-buffered saline, then 0.25% trypsin
was added to detach them. The cells were suspended in RPMI
1640 medium and after resuspending the cells were stained
with trypan blue and counted in a Burker chamber. 0.1 ml
cell suspension and 0.1 ml 0.1% trYpan blue were mixed.
The viability of tumor cells was estimated on the basis of
trypan blue (0.4%) exclusion using a hematocytometer.
Exam~le 9
Luteinizina Hormone (LH) Release AssaY
Anterior pituitaries were obtained from adult (male
or female) Wistar strain rats according to the method
~ 094/12530 33 2 1 5 0 ~ 7 PCTnHug3looo6~
previously described (Shaw). The pituitaries were cut into
small pieces, incubated with collagenase then dispersed
mechanically to single cells. The cell-suspension was
mixed with Sephadex G-10 beads as support material and
transferred into a superfusion chamber. The cells were
continuously perfused with oxygenated medium or with a
medium containing the peptide to be tested, such as D-
Lys6(DHA)-GnRH. The LH content from each 1 ml fraction of
the superfusate was measured by radioimmunoassay (RIA)
using rat an LH RIA kit or as described in Example 9 using
radiolabeled LH and anti-luteinizing hormone antibodies.
The biological potency of a given analogue was determined
based on the pituitary hormone responses (peaks) to the
peptide stimulation over the baseline secretion.
Exam~le 10
Assav for Growth Hormone (GH)
The release of GH was measured during various DHA-
peptide hormone or peptide analogue treatments, such as
somatotsatin analogue treatments, on rat hypophysis by
using the superfusion method (Myers). Following is a
description of the method. The hypophyses are cut into
small pieces, incubated with collagenase, and dispersed to
single cells. The cells in a superfusion chamber are
continuously perfused with oxygenated medium or with medium
containing the peptide to be tested.
The levels of GH are determined by a double-antibody
radioimmunoassay for the hormone. The following
methodology can be used. Several commercial sources now
supply reagent grade hGH of sufficient purity to be
reliably used in the assay. Antibody is produced by
dissolving an appropriate amount of growth hormone (2-3 mg)
in 200 microliters 0.01 N sodium hydroxide. The solution
is diluted to 1 mg/ml with protein free Pss and mixed 1:1
with Freund's adjuvant. 0.5-1 mg growth hormone is in-
jected into three subcutaneous sites of a young guinea pig,
WO 94/12~30 34 PCT~U93/00065
2~5047~ ~
and repeated at two- to three-week intervals. The
anti-hGH serum is harvested from the guinea pigs and tested
for its immunopotency. Antisera which will allow for a
sensitive assay usually will precipitate 50% of the
labelled hGH at a final dilution of 1:50,000 using test
radiolabelled hGH. Second antibodies (goat anti-guinea pig
antibodies) are prepared in a;similar fashion. The
following radioiodination procedure for hGH is used. One
ampoule containing hGH in 20 microliters is thawed and
placed on ice. I-125 is usually obtained from New England
Nuclear (Boston, ~A). High specific activity exceeding 200
mCi/ml is necessary for the highest degree of sensitivity
in the assay. 0.5 M phosphate buffer (pH 7.6) is added to
the ampoule containing hGH. About 1 mCi of the radioactive
iodine is added to the ampoule.
To start the iodinatioh reacti~n, 35 micrograms of a
freshly prepared solution of chloramine-T in 25 microliters
0.05 M phosphate buffer (pH 7.6) is added. The iodine, hor-
mone, and oxidant are gently agitated for 15 seconds. To
stop the reaction, 125 micrograms of a freshly prepared
solution of sodium metabisulfate in 100 microliters of 0.05
M phosphate (pH 7.6) is added. The entire contents of the
ampoule are then placed on Sephadex G-50 column and the
labeled hormone separated from the iodine. The hormone is
diluted in 1% BSA-PBS and stored in small aliquots at
-20 C.
The hGH radioimmunoassay is performed in the following
manner. The standard~s concentrations in hGH assays are
the following: 5, 2.5, 1.25, 0.625, 0.313, 0.156, and 0.078
ng/ml. 50 ml of the unknown growth hormone concentration
is added to tubes not containing the standard. The next
step is to add the guinea pig anti-hGH serum (first anti-
body). The radiolabeled hGH is added to every tube. The
assay tubes are shaken gently, left at room temperature for
two hours, and then placed in a refrigerator at 4 C for at
least three days. After the three-day incubation, the
2150470
094/~530 35 PCT~HU93/00065
tubes are removed from the refrigerator and the appropriate
dilution of the goat anti-guinea pig serum added (second
antibody). The second antibody is diluted in 1% BSA PBS
buffer to obtain approximately 50% precipitation of
~5 antibody-bound labeled hGH. Aliquots of 100 microliters of
the second antibody are added to every tube. The tubes are
gently agitated, left at room temperature for about two
hours, and then placed at 4 C. for at least eight hours.
Following incubation with the second antibody, the
tubes are centrifuged at 2000 g for 30 minutes at 4 degrees
in a refrigerated IEC-PR6 centrifuge. The supernatants are
aspirated by vacuum suction, with care taken not to disturb
the precipitates packed in the bottom of tubes. The pellet
is resuspended in 2 ml cold Pss and recentrifuged as
described above. Washing the precipitates with buffer
increases the reproducibility.
Exam~le 11
Measurement of Auto~hos~horvlation of the EGF Rece~tor
Autophosphorylation of the EGF receptor was measured
according to Bellot et al. (Bellot). Cells were washed
once with binding buffer. The plates were then placed in a
water bath at 37 degrees and different concentrations of
the analogues were added to the cells for specific periods
of time. The buffer was removed and cells were scraped off
the plates with 0.5 ml of Laemmli's sample buffer, boiled
for 5 minutes, and sonicated for 10 seconds. Aliquots of
each sample were run on two different SDS-polyacrylamide
gels (7%) and each gel was transferred to nitrocellulose
paper. One was immunoblotted with RK2, an anti-EGF-R
antibody, and the other with an antiphosphotyrosine-
specific polyclonal antibody. Blots were then labeled with
radioiodinated Protein A (New England Nuclear) and
autoradiograms of the nitrocellulose papers were made on
Kodak X-Omat paper. The activity of the analogues was
characterized on the basis of their inhibitory effect on
W094/~530 36 PCTnlU93/00065 _
21~47~ _
the phosphotyrosine content in comparison to that of
untreated cells used as control. Alternatively, the
tyrosine kinas~e ~ct~ivity was determined according to the
following method (Keri). The cells were incubated with the
substrate analogues, such as DHA-SPRCS or DHA-STRS for 24
hours, then harvested and homogenized. The reaction volume
of 100 microliters, and homogenized in a Dounce homogenizer
30 times in 5 vol buffer (50 mM Tris-HCl, pH 7.8, 50 mM
MgC12, 10 micromolar sodiumvanadate, 1 mM EDTA, and 50
microgram/ml aprotinin. The reaction volume of 100 ml
contained 50 mM Tris-HC1, pH 7.8, 50 mM MgC12, 10
micromolar sodium vanadate, 0.1% nonidet P-40, 5 micromole
gamma 32p-ATP, lmM substrate and 60 microliter homogenate.
The assay was initiated by addition of the ATP.
After incubation the reaction was stopped by addition
of trichloroacetic acid, and the supernatant was spotted on
a 2 x 2 cm phosphocellulose paper (Whatman P-81). The
paper sa;uares were washed with phosphoric acid and acetone,
and the dried papers were counted for radioactivity in
scintillation fluid. For each sample an appropriate
reaction mixture containing no peptide was run a control.
The activity of the analogues was characterized on the
basis of their inhibitory effect in comparison to the
incorporation of 32P isotope to untreated cells used as
control.
Exam~le 12
PreQaration of DHA-EGFA (SEO. ID. NO: 6)
O.035 mM of EGFA was suspended in 2.5 ml of water-
acetone (1:1), then 0.035 mM DIEA (6.5 ul) and 0.035 mM of
DHA-O-N-Succ ester (40.2 mg) in 2.Oml DMF were added to the
solution. The acylation was carried out by stirring the
reaction mixture overnight under He atmosphere. The
solvents were evaporated and the dry residue was triturated
with 3 x 5 ml of petroleum ether. The solvent residue was
~ 094/12530 21~ 0 4 7 0 PCTnHU93/00065
removed and the crude product was purified by preparative
HPLC under the following circumstances:
Eluent programme:
t(min) A% B%
, 5 o 100 0
~5 5
0 100
All the other parameters were identical to those in
Example 3.
While the invention has been described with reference
to specific methods and embodiments, it will be appreciated
that various modifications and changes may be made without
departing from the invention.
