Note: Descriptions are shown in the official language in which they were submitted.
WO9G106626 PCT~9~/09~63
21 98348
ANALGESIC METHOD WITB DY..~ 1N ANALOGUES
TRUNCATED AT T~E N-TERMINUS
Field of the Invention:
The present invention generally relates to
dynorphin A analogues, and more particularly to
dynorphin A analogues truncated at the amino terminus
that are useful in analqesic methods. This invention
was made with government support under Grant 13Os. NIDA-
02643 and NIDA-06011 awarded by the ~lational Institutes
of Health. The Government has certain rights in this
invention.
Background of the Invention:
Opioids are a larqe class of drugs, used
clinically as painkillers, that include both plant-
derived and synthetic alkaloids and peptides found
endogenously in the mammalian brain. While the plant-
derived alkaloids have been known and used for thousands
of years, the endogenous opioid peptides were discovered
only in the mid-1970s. These are known to comprise
three distinct gene families: ~-endorphin and other
peptides derived from proopiomelanocortin; enkephalins,
derived from proenkephalin A; and the dynorphins,
derived from proenkephalin B.
opioid compounds interact with neuronal cells
~ and modulate physiological functions such as
nociception. Thus, one of the physioloqical effects
attributed to the opioid system is analgesia.
, . .. .... ..
wo g6,~6626 ~ 3 ~ ~ PCI/I~'S94109~.3
Endogenous opioids exist in multiple forms in
the central nervous system, and include the dynorphins,
which are a series of peptides derived from the
precursor prodynorphin (pro~nk~ph~l in 3~. The first of
the dynorphins to be isolated was the 17 amino acid
peptide having the structure shown (and designated SEQ
ID N0:1), sometimes also referred to as "dynorphin A~
17)":
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-
Leu-Lys-Trp-Asp-Asn-Gln ~S~Q ID N0:1]
Wlthin the last decade, various U.S. patents
have described and suggested uses of dynorphin.
U.S. Patent 4,396,606, îssued August 2, 1983,
inventor Goldstein, described isolation of a compound
~sometimes hereinafter called "dynorphin A-(1-13)") with
the structure:
Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-
Leu-Lys ( SEQ ID NO: 2 )
This fragment of the seventeen amino acid endogenous
peptide was ~ound to be substantially ~ore active than
the enkephaLins and r~-endorphin in a ~uinea pig ileum
test. Compositions cont~inin~ dynorphin were suggested
to be analgesic by virtue of their interaction with
opioid receptor sites, and administration in the same
manner as other opioid agonists ~e.g. morphine) was
disclosed.
U.S. Patent 4,462,941, issued July 31, 1984,
inventors Lee et al., described dynorphin amide analoqs
With ten amino acid residues. These dynorphin A-(1-10)
amide analogs do not have significant analgesic activity
in opioid tail flick tests (unless given in huge doses
where they tend to produce convulsions).
Enkephalin analogues that are conformationally
constrained~ by a cyclic structure ~such as with a
disulfide bridge) are described ~y U.S. Patent
~ ~ q~48
WV96106G26 PCT~1~9~l09~63
4,518,711, issued l~ay 21, 1985, inventors Hruby et al.
Subsequently, dynorphin analogues have become known that
have cysteine replacements at the amino acid residue 5
(usually leucine~ and at the ar~lino acid residue 11
(usually lysine). The amino acid residue 8 (usually an
isoleucine) and the amino acid residue 13 (usually a
lysine) have similarly been replaced by cysteines in a
bridged relationship. The bridges, or cyclic struc-
tures, appear to assist in stabili2ing the dynorphin
analogues against in vivo degradations.
In an international application published
under the Patent Cooperation Treaty on December 23,
1993, Lee et al. disclosed therapeutic uses of certain
truncated ~-terminal dynorphin A analogues in conjunc-
tion with narcotic analgesics in order to potentiateactivity of the narcotic analgesic and/or to block
withdrawal symptoms (International Publication ~o.
w093/25217). However, uses in conjunction with narcotic
analgesics for opioid effects require a presence of
opioid drugs.
Cpioid drugs are used clinically as
painkillers, but their usefulness is limited by the
tolerance and dependence that normally develops upon
chronic treatment. Tolerance may be defined as an
increase in the amount of drug needed to achieve a
certain level of analgesia, while dependence manifests
itself in the need to continue taking drug to prevent
withdrawal symptoms. Despite a great deal of research
on these phenomena, little is known about their
molecular basis. Opioid drugs, such as, for example,
morphine, are addictive and have central opioid side
effects such as drowsiness and impairment of mental
~ activity.
Some non-opiate compositions have been
suggested for relieving chronic pain, such as
, . . ~
~ q~
wos6/o6c26 PCT~iS9~l95
~ 4
experienced~as burning or hyperesth;esia pain. Thus,
U.5. Patent 5,0a6,510, issued April 9, l~gl, lnventor
Ellis, describes somatostatin analogue compositions for
topical administration i.n the treatment of pain where
opiates do not significantly change the experience of
the patient's pain.
Nevertheless, a variety of painful conditions
exist that are relatively resistant to analgesic relief
by narcotic analgesics such as morphine.
Summary of the Invention:
In one aspect of the present invention,
peptides are used to reli.eve pain. The peptides have at
least six, preferably bave at least seven, amino acid
residues, and are ana..,gues of dynorphin A that are
truncated lwith respect to endogenous dynorphin) at the
N-terminus, such as being des-(Tyr), des-(Tyr-Gly), or
des-~Tyr-Gly-Gly) with respect to the endogenous
dynorp~in A~ These peptides may be iormulated in a
pharmaceutically accepta~le solution or with a pharma-
ceutically acceptable carrier, and are usefully admini-
stered to a patient experiencing pain.
Administration of dynorphin~ A analogues in
practicing the invention is preferably syste~ic, such as
intravenous, and includes tr~nsnS~1, transrectal,
intrathecal, intramuscular, transdermal electrotran-
sport, or subcutaneous procedures in dose ranges of
between about 50 to about. 2,000 yg/kg ~or by continuous
infusion). Administration may also be topical ~e.g. to
wounds or mucus membranes) and oral administration may
also be feasible.
Practlce of the invention is useful generally
for analgesia to counter pain, and is particularly
useful for painful conditions, often having unknown
etioloqy, such as "neuropathic pain," "neuroqenic pain,l~
~1 983~3
W096/OC626 PCTI~S9~109~63
"hyperesthesla" (a morbid response to normal sensory
stimuli perceived as extremely painful)l "allodynia,"
"causalgia" (meaning a pathological response to nerve
injury that is disabling), persistent lower back pain,
visceral pain, bone pain (such as is experienced by some
cancer patients), post-operative pain, and wounds such
as from burns. Because practice of the invention
provides non-opiate analgesia, the central nervous
system side effects of a drug such as morphine ~e.g.
drowsiness, impaired mental functioning and the like)
are avoided.
Detailed Description of the Preferred Embodiments:
The present invention concerns the interaction
of dynorphin A analogues with non-opioid receptors in an
analgesic manner. In particular, preferred practice of
the invention pertains to use of dynorphin A analogues
that are des-~Tyr)~ des-(Tyr-Gly), or des-(Tyr-Gly-Gly)
with respect to dynorphin A.
It is known that dynorphin A-(2-17) does not
bind to ~, ~, or ~ opioid receptors. However, we have
discovered that the non-opioid binding dynorphin A
analogues show substantial antinociceptive (that is,
analgesic~ activity, particularly when administered
systemically (such as by i.v.). This potency is
retained even in morphine-tolerant animals. That is,
there is a lack of cross-tolerance.
At this point, we do not know the identity of
the non-opioid receptors with which dynorphin A
analogues are interacting. However, data obtained from
3U i~ vivo assays used for demonstrating non-opioid
analgesic activity shows that practice of the present
invention is useful to relieve pain where the analgesic
effect is not mediated by opioid receptors.
q ~ 4 ~ --
W096/06626 L ~ PCT~59~tO9~h3
For example, many patients suffer from pain
associated with an injury or a disease that interrupts
some or all of the axons in a nerve (e.g. painful
neuropathies3. Following nerve in~ury, the regenerating
tips of damaged primary afferent axons acquire abnormal
properties, such as firing spontaneously and developing
abnormal sensitivity to certain types of stimulation.
It is believed that such axons with abnormal properties
are present in other conditions such as dia~etic
disorders. Among the syndromes of painful peripheral
neuropathies are those usually classified on the basis
of an associated disease, such as phantom pain when the
nerve has been amputated. These types of pain are often
not relieved by or are relatively resistant to opioid
analgesics.
Pe~tides used in practicing the invention have
at least six, and preferably have seven, amino acids.
In one aspect of this invention, suitable peptides can
~e viewed as having amino acid residues analogous to
20 endogenous dynorphin A ~SEQ ID NO:l~, but the peptides
used prefera~ly are des-(Tyr~, as shown by the amino
acid residue sequences oE SEQ ID N05:4-l2:
Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-
Trp-Asp-Asn (SR0 ID N0:4~;
Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-
~rp-Asp (SEQ ID N0:5);
Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-
Trp (SEQ ID N0:6);
Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Ly s
(SEQ ID N0:7);
Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-1eu (SEQ
ID N0~
Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys (SRQ ID
~0 : 9 ) ;
2 1 9~3~'B
W096l06C26 PCT~S9~109~63
Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro (SEQ ID
N0:10~;
Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arq ~SEQ ID NO:11);
and,
Gly-Gly-Phe-Leu-Arg-Arg-Ile ~SEQ ID NO:12).
We sometimes refer to the SEQ ID NOS:4-12
peptides as dynorphin A-(2-16) through dynorphin A-(2-
8), respectively. Further, the variations known to the
art (e.g. some of which are discussed in U.S. Patent
4,462,941~ are meant to be within the scope of these
suitable peptides. Yet further, in any of the SEQ ID
NOS:4-12, any one or two of the residues may be replaced
with the same or a different amino acid residue in the
D-configuration (to increase in vivo stability), such as
where the N-terminal Gly is replaced by D-Ala, or a
modification for conformational stability or rigidity
may be made, such as where a plurality of the specified
amino acid residues are replaced by moieties capable of
forming a cyclic structure, or bridge le.g., the
disulfide bridge). An illustrative bridged such
dynorphin A analogue is where the normal leucine at the
5 position and the lysine at the 11 position (these
positions referred to as though in endogenous
dynorphin), are replaced by cysteines, whose disulfide
bridge provides conformational stability. Any of the
peptides used in practicing this invention may have the
C-terminal and/or the N-terminal including a blocking
group, for example, where the N-terminus has been
acetylated. All the peptides can also be used in their
free acid or amide form.
Des-(Tyr) peptides for this invention can also
lack the next adjacent one or two glycine residues of
the endogenous dynorphin A and thus be des-(Tyr-Gly) or
des-(Tyr-Gly-Gly), as shown by SEQ ID NOS:13-21 and SEQ
ID NOS:22-29, respectively, which can similarly be
~Og6~u~626 ~q~3~ PCT~IS94/~l~S63
modified to increase conformational stability or
rigidity as already described for SEQ ID ~OS:4-12 and
where the des-(Tyr-Gly) peptides can also have the N-
terminal Gly replaced by D-Ala, the C-terminus and/or
the N-terminus can include a blocking group as are well
known to the art Isuch as to retard degradation of the
peptide within the body), and the peptides can be in
free acid or amide form:
Des-(T~r-&lY)
Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-
Asp-Asn-Gln ~SLO ID NO:13);
Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-
Asp-Asn ~SEQ ID NO:14);
Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-
Asp (SEQ ID NO:15);
Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Tr p
(SEQ ID NO:16);
Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys (SEQ
NO: 17 );
~0 Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu (SEQ ID
NO:18);
Gly-Phe-Lell-Arg-Arg-Ile-Arg-Pro-Lys (SEQ ID
NO:19);
Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro (SEQ ID ~0:20);
2 5 and
Gly-Phe-Leu-Arg-Arg-Ile-Arg (SEQ ID NO:21).
Ne sometimes refer to the SEQ ID NOS:13-21
peptides as :dynorphin A-~3-17) through dynorphin A-(3-
9), respectively.
Des-(Tyr-Gly-Gly~
Phe-Leu-Arg-Arg-Ile-Arg-Pro-1ys-Leu-Lys-Trp-Asp-
Asn-Gln (SEQ ID NO:22);
~096/06626 ~ 1 9 ~ 3 i~ ~ Pc~ JS~4109~;63
Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn
t ~SEQ ID NO:23);
Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-As p
~SEQ ID NO:24);
Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp ~SEQ ID
~0:25);
Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys (SEQ ID
~0:26);
Phe-Leu-Arg-Arq-Ile-Arg-Pro-Lys-Leu (SEQ ID 130:27j;
Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys (SEQ ID NO:28); and
Phe-Leu-Arg-Arg-Ile-Arg-Pro (SEQ ID NO:29).
We sometimes refer to the SEQ ID 110S:22-29
peptides as dynorphin A-(4-17) through dynorphin A-(4-
10), respectively.
The des-(Tyr) dynorphin A-(2-17) is likewise
useful in practicing this invention (SEQ ID NO:30) with
the variations and modifications known to the art and as
earlier noted.
All the peptides suitable for practicing this
invention may be readily prepared synthetically, such as
by solid phase peptide synthesis techniques. When a
chloromethylated resin or a hydroxymethyl resin has been
used as a resin support, then the peptide cleaved from
the resin support will be in the form of the carboxyl
terminal benzyl ester, which may then be readily
converted by methods well known in the art to provide
the carboxyl terminal amide form of the peptide.
Table 1 summarizes peptides useful for
analgesic, but non-opioid, properties in practicing the
present invention as set out by SEQ ID NOS:22-29, and
wh.ich are believed to be novel peptides.
~: ?~
WO ~filO6626 ~ ' PCTIU~94~9563
T~BLE 1
Novel Peptides
With Analgesic,
Noll-Opioid Propert~ SE0 ID N0.
~yn A-~4-171 22
Dyn A-(4-16I 23
Dyn A-(4-15I 24
~Dyn A-(4-14I 25
Dyn A-I4-13I 26
Dyn A-I4-12l 27
byn A-~4-11I 2
Dyn A-(4-1OI 29
Researchers in the field seeking to clone cDI~
encoding an opioid receptor had recently noted that a
des-(Tyr) dynorphin did not compete at all in assays
with various ligands ~Xie et al., Proc. Natl. Acad. Sci.
USA, ~9, pp. 4124-41~8 (1992)). However, the des-(Tyr;
dynorphin peptides useful in practicing this invention
20 not only lack the N-terminal tyrosine, but indeed the
des-(Tyr-~ly-~ly) dynorphin peptides summarized in Table
1 lack the first three N-tPrm;n~ amino acid residues.
Nevertheless, these novel peptides have substantial
analgesic pr~operties that are useful in treating pain,
such as pain that is otherwise relatively resistant to
opiates. Thus, the present invention i5 a new method of
providing effective anal~esia with completely non-opioid
compounds. This means practice of the invention will
avoid the addiction, tolerance, dependence, and central
opioid side effects that are serious disadvantages in
the use of cpioid drugs.
Pe~ptides of the invention are preferably
formulated in a pharmaceutically acceptable solution or
with a pharmaceutically acceptable carrier, and then are
administered in such a solution or carrier. Depending
upon the mode of administration, the peptides may thus
W0~)6/06626 ~ 9 1~ 3 4 ~ PCTI~S9~/0~563
be formulated with a wide variety of physiologically
acceptable carriers, such as aqueous saline and
phosphate buffered saline, and may include physio-
~ logically acceptable excipients, such as glucose,
mannitol, or the like
Administration may be by transdermalelectrotransport ~sometimes also referred to as using an
iontophoretic current). For example, such a means of
drug administration is described by U.S. Patent
5,312,326, issued May 17, 1994, inventors IMyers et al.
Other assemblies that may be suitable for selective drug
release are described by U.S. Patent 5,290,240, issued
March 1, 1934, inventor Horres, and by U.S. Patent
5,2~8,209, issued February 22, 1994, inventors Haak et
al.
EXPERIMENTAL
Randomly bred male ICR mice (Sasco, Omaha,
Nebraska) weighing 20 to 25 g were used in all
experiments. All animals were supplied food and water
20 ad libitu~ and were housed in a temperature (22+1~C)-
and humidity (40-50%)- controlled animal room for at
least one day before experimentation. Each mouse was
used only once.
The abdominal stretching ~writhing) assay as
described by Hayashi and Takemori ~1971) was used as an
antinociceptive assay. Mice were injected intraperi-
toneally with 10 ml~kg of 0.6% acetic acid, and the
number of writhing responses per animal was counted for
a six-minute period commencing five minutes after acetic
acid injection. A writhe was defined as a wave of
contraction of the abdominal musculature followed by a
stretching of hind limbs. ~umber of writhes per animal
was expressed as mean ~ S.E. Antinociceptive activlty
. _ . . . _ _ _ .
W096/06~26 PCT~Sg~4~63
2l 983~8 12
was expressed as percentage decrease in the mean number
of writhes observed in the drug-treated animals compared
with the mean number of writhes in the saline control
group. Administration of drugs was timed so that the
peak action coincided with the center of the observation
period. A minimum of ten mice were used at each of
three dose levels to determine the dose response curve
and EDs~ Of the drug.
The parallel line assay of Finney (1964) was
1~ used to estimate ~Dsl. values, 95% confidence intervals
and potency ratios.
The data of the following Tables 2-4 use the
terminology "ADsO" to indicate analgesic doses. The
analgesic doses represent three data points taken from
at least 30 animals (and thus are the summation from
dose response curves) and represent the dose at which
50~ of the animals showed an analgesic effect.
W0~6/06626 2 ~ 3 ~ i~ PCT~ss4io~63
TABLE 2
Antinociceptive Response
in Writbing Assav (~aive Mice~
Embodiments of the Invention AD~ (umole/kq)
Dyn A-(2-17~, i.v., 5 min. 1.1 10.99-1.201
Dyn A-(2-17~, i.v., 30 min. 1.1 (0.99-1.20)
Dyn A-(2-17), i.v., 60 min. 5.9 (5.2-7.8)
Dyn A-(2-17), i.v., 2 hrs. 16.1 (13.4-20.5)
Dyn A-(2-17), i.v., 5 min.
+ ~al, s.c., 50 umole~kg 1.8 (1.7-1.9)
Dyn A-(2-17), i.t., 5 min. 5.1 (4.0-6.4~
nmole/mouse
Dyn A-(2-17;, i.c.v., 5 min. 7.3 (6.9-7.8~
nmole/mouse
Dyn A-(2-17), i.p., 5 min. 3.1 (2-5)
Dyn A-(2-12~, i.v., 5 min. 3.8 (2.5-5.4)
Dyn A-(3-17), i.v., 5 min. 2.4 (1.6-3.7)
Dyn A-(3-13), i.v., 5 min. 7.5 (4.8-11.5)
Dyn A-(4-17), i.v., 5 min. 5.7 (3.6-8.80)
ZO Comparati~e
Dyn A-(7-17), i.v., 5 min. >100
As seen from the data of TabLe 2, dynorphin A-
(2-17), when administered i.v. at 1.1 umole/kg of
animal weiqht, was an analgesic dose for 50% of the
animals tested despite the animals being subjected to an
injection five minutes later with acetic acid and then
judged by the writhing assay. Calculations show that
the amounts administered of dynorphin A analogues (which
are basic compounds) are insufficient to neutralize even
1% of the acidity of the administered acetic acid, and
thus the effects shown from the writhing assay are not
~Og~lQ6626 ~ 4 ~ PcT~ss~Qg.~3
due to a simple chemical neutralization. As further
seen by the data of Table 2, the duratlon of action was
substantially the same when administered 30 minutes
before the acetic acid, although at 60 minutes one sees
S the analgesic property begins to decline in duration of
action.
Stlll with reference to the data of Table 2,
the non-opioid effect of the analgesia being measured is
indicated by the fact that when naloxone (50 ymole/kg)
is administered in conjunction with dynorphin A-(2-17),
substantially equivalent analgesic properties are still
obtained. Naloxone, of course, is ~n antagonist o~
opioid receptors, and thus this data shows the analgesic
effect is not being mediated through opioid receptors.
The amount of naloxone administered was more than
sufficient for antagonism of opioid analge&ia, both with
morphine and V5Q,488H which act primarily at ~ and K
opioid receptors, respectively, as shown by Table 3.
Still with re~erence to the data o~ Table 2, one sees
that the peptide dynorphin A-(4-17), even though it ifi
missing the fLrst three amino acid residues of dynorphin
A~ 17), continued to provide qood analgesia. However,
by contrast,~ dynorphin A-~7-17~, did not.
ABLE 3
25 Comparative ADc~ ole/kq)
U50,488H, i v., 30 min. 2.0 (1.9-2.2)
U50,488H, + Nal., 10 ~mole/kg 17.2 (14.9-19.9)
Morphine, s.c., 30 min. 2.6 (2.1-3.2)
Morphine + Nal., 10 ~mole/kg 23.~ (15-35)
The data of Table 4 were collected from
morphine tolerant mice (where the mice were made
tolerant to mDrphine by implantation of a 75 mg pellet
W096/06626 ~ 3 ~ ~ PCT~Ss~ 63
according to standard procedures), and these data show
that the morphine-tolerant animals are not cross-
tolerant to the des-(Tyr) dynorphin A analogue.
.
TABLE 4
Antinoclceptive Response
in ~rithinq AssaY (Morphine-Tolerant Mice)
ADs,~ ( umole/k
Dyn A-(2-17), i.v., 5 min. 2.2 (1.5-3.1)
(one morphine pellet
for 3 days)
Dyn A-(2-17), i.v., 5 min. l.9 (1.1-3.2
(one morphine pellet
for 5 days)
In conclusion, practice of the invention
provides a non-opioid analgesia, where the anti-
nociception is likely not an effect on the central
nervous system.
It is to be understood that while the
invention has been described above in conjunction with
preferred specific embodiments, the description and
examples are intended to illustrate and not limlt the
scope of the invention, which is defined by the scope of
the appended claims.
