Note: Descriptions are shown in the official language in which they were submitted.
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ANALGESIC PEPTID . FROM VENOM OF
FIELD OF THE INVENTION
The present invention relates to peptides that induce analgesia in
mammals. More particularly, the present invention relates to analgesia-
inducing
peptides obtainable from venom of Grammostola spatulata, the Chilean pink
tarantula
spider.
BACKGROUND OF THE INVENTION
Pain is one of the basic clinical symptoms seen by every physician and is
usually categorized into three segments: mild, moderate and severe. The mild-
to-
moderate segment has multiple product entries including aspirin,
acetaminophen,
ibuprofen, and other non-steroidal, anti-inflammatory (NSAID) products.
Narcotic
analgesics remain the mainstay of currently marketed products for the
treatment of
moderate-to-severe pain.
Cancer and the post-operative surgical period are two conditions most
often associated with moderate-to-severe pain. Tumor infiltration of bone,
nerve, soft
tissue or viscera are the most common causes of cancer pain accounting for 65-
75% of
patients. Pain as a result of cancer treatment from surgery, chemotherapy or
radiation
accounts for 15-25% of patients, with the remaining ~-10% reporting pain
independent of their cancer or cancer therapy. Various factors influence the
prevalence of cancer pain including the primary tumor type, stage and site of
disease
and patient variables, especially psychological variables. Similarly, patient
response
to post surgical pain is dependent upon location and extent of intervention as
well as
personal attributes. However, post surgical pain is distinguished from cancer
pain by
length of treatment period.
The major concern with narcotics, which constitute the largest segment
of the U.S. market for treatment of moderate-to-severe pain, is the potential
for
addiction and loss of activity (i.e. tolerance) with continued use.
Consequently, there
is a need for other analgesics that can relieve pain, especially moderate-to-
severe pain
associated with caner. In order to improve analgesic responsiveness and reduce
side
SUBSTITUTE SHEET (RULE 26)
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2
effects, research efforts have focused on both drug delivery strategies and
novel drug
entities. Newer drug delivery strategies include transdermal narcotics, PCA,
intraspinal implantation of controlled release pumps and implantation of
encapsulated
living cells which release naturally-occurring endorphins or other analgesic
peptides.
New drug approaches reflect the varying pathways and causes of moderate-to
severe
pain. Classes of compounds in development for treating pain include
serotonergics,
noradrenergics, opioid partial agonists and kappa opioid agonists. Therapeutic
targets
with significant preclinical investigation include tachykinin/bradykinin
antagonists
and excitatory amino acid antagonists. Newer targets being exploited include
growth
factors, cytokines, nitride oxide regulators, etc. Natural sources including
folk
medicine remedies and frog venom extracts are also under investigation.
Investigations of spider venoms for identification of biological entities
with commercial potential has focused primarily on the agrochemical sector.
The
ultimate goal of these activities has been the search for chemical
constituents which
interact selectively with invertebrate species to induce paralysis and/or
death with
minimal mammalian toxicological properties. However, in recent years, spider
venoms have joined other predator-derived venoms being exploited for
identification
of compounds which identify mammalian targets and which assist the development
of
pharmaceuticals. The arachnid species Grammostola spatulata, commonly referred
to
as the Chilean pink tarantula spider, is a member of the Theraphosidae family
and the
Chelicerata order. Previous studies by Lampe et al. ( 1993) Molecular
Pharmacology
4:451-460 showed that venom of Grammostola spatulata contains a peptide which
interacts in a non-selective manner with voltage-sensitive calcium channels.
SUMMARY OF THE INVENTION
The present invention provides methods of treating pain comprising
administering to a mammal in need of such treatment an effective analgesic
amount of
a peptide having the amino acid sequence
Tyr-Cys-Gln-Lys-Trp-Leu-Trp-Thr-Cys-Asp-Ser-Glu-
Arg-Lys-Cys-Cys-Glu-Asp-Met-V al-Cys-Arg-Leu-Trp-
Cys-Lys-Lys-Arg-Leu-NH2 (referred to herein as GsAF I)
(SEQ ID NO:1)
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3
or
Tyr-Cys-Gln-Lys-Trp-Met-Trp-Thr-Cys-Asp-Glu-Glu-
Arg-Lys-Cys-Cys-Glu-Gly-Leu-Val-Cys-Arg-Leu-Trp-
Cys-Lys-Lys-Lys-Ile-Glu-Trp (referred to herein as GsAF II)
(SEQ ID N0:2).
The present invention thus provides for the use in the-manufacture of a
medicament for treatment of pain of the peptides of SEQ ID NO: 1 or SEQ ID NO:
2.
The present invention further provides for the use of the peptides of SEQ )D
NO: 1 or
SEQ ID NO: 2 in the treatment of pain.
An additional aspect of the invention provides a purified peptide having
the amino acid sequence
Tyr-Cys-Gln-Lys-Trp-Leu-Trp-Thr-Cys-Asp-Ser-Glu-
Arg-Lys-Cys-Cys-Glu-Asp-Met-Val-Cys-Arg-Leu-Trp-
Cys-Lys-Lys-Arg-Leu-NH2 (GsAF I) (SEQ ID NO:1 ).
Another aspect of the present invention provides pharmaceutical
compositions comprising a pharmaceutically acceptable carrier or diluent and a
peptide having the amino acid sequence
Tyr-Cys-Gln-Lys-Trp-Leu-Trp-Thr-Cys-Asp-Ser-Glu-
Arg-Lys-Cys-Cys-Glu-Asp-Met-Val-Cys-Arg-Leu-Trp-
Cys-Lys-Lys-Arg-Leu-NH2 (SEQ ID NO: 1 )..
Yet another aspect of the invention provides methods for identifying
compounds that mimic the analgesia-inducing activity of GsAF I and/or GsAF II.
The present invention additionally provides antibodies specific for GsAF I.
The antibodies can be monoclonal or polyclonal. Antibodies can be prepared
using
methods known in the art such as the methods in Harlow et al. eds.,
Antibodies: A
Laboratory Manual, New York, cold Spring Harbor Laboratory Press ( 1988).
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DETAILED DESCRIPTION OF THE INVENTION
Applicant has discovered that peptides from venom of the Chilean pink
tarantula spider, Grammostola spatulata, have analgesia-inducing properties
and are
thus useful as analgesics for treatment of pain in mammals, including humans,
and as
research tools for identification of compounds that mimic the analgesic
activity of the
peptides.
Thus, the present invention provides a method for treating pain comprising
administering to a mammal in need of such treatment an effective analgesic
amount of
a peptide having the-amino acid sequence
Tyr-Cys-Gln-Lys-Trp-Leu-Trp-Thr-Cys-Asp-Ser-Glu-
Arg-Lys-Cys-Cys-Glu-Asp-Met-Val-Cys-Arg-Leu-Trp-
Cys-Lys-Lys-Arg-Leu-NH2 (GSAF I) (SEQ ID NO:l) ; or
Tyr-Cys-Gln-Lys-Trp-Met-Trp-Thr-Cys-Asp-Glu-Glu-
Arg-Lys-Cys-Cys-Gl>i-Gly-Leu-Val-Cys-Arg-Leu-Trp-
Cys-Lys-Lys-Lys-Ile-Glu-Trp (GsAF II) (SEQ ID N0:2)
The peptides are useful for treating pain in mammals, including humans,
conventional laboratory animals such as rats, mice and guinea pigs, domestic
animals
such as cats, dogs and horses, and any other species of mammal. The peptides
can be
used to treat acute or chronic pain from any source or condition, such as
bums, cancer,
neuropathies, organ inflammation or surgical intervention. Preferably,
however, the
peptides are used to treat moderate-to-severe pain due to cancer or surgery.
The
peptides can be administered orally, parenterally, intrathecally, topically,
intraveneously, intramuscularly or intradermally/epineurally. A preferred
route of
administration is intrathecally.
The peptides thereof can be prepared for pharmaceutical use by
incorporating them with a pharnlaceutically acceptable carrier or diluent.
Thus, a
further aspect of the present invention provides pharmaceutical compositions
comprising a peptide from Grammostola spatulata as described herein and a
pharmaceutically acceptable carrier or diluent. The peptide can be prepared
for
pharmaceutical use by incorporating it in unit dosage form as tablets or
capsules for
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oral or parenteral administration either alone or in combination with suitable
carriers
such as calcium carbonate, starch, lactose, talc, magnesium stearate, and gum
acacia.
The peptide can be formulated for oral, parenteral or topical administration
in aqueous
solutions, aqueous alcohol, glycol or oil solutions or oil-water emulsions.
Buffered-
5 aqueous or carrier mediated aqueous/non-aqueous intrathecal and intraveneous
dosages can be formulated. These and other suitable forms for the
pharmaceutical
compositions of the invention can be found, for example, in Remin ton's
Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton,
Pennsylvania
(1980). The pharmaceutical compositions of the invention can comprise any
combination of one or both of the peptides.
The amount of the active component (i.e. peptide) in the pharmaceutical
compositions can be varied so that a suitable dose is obtained and an
effective
analgesic amount can be administered to the patient. The dosage administered
to a
particular patient will depend on a number of factors such as the route of
administration, the duration of treatment, the size and physical condition of
the
patient, the potency of the peptide and the patient's response thereto. An
effective
analgesic amount of the peptide when administered intrathecally is generally
in the
range of from about 5 nanograms per kilogram body weight of the patient to
about
500 micrograms per kilogram; preferably from about 50 nanograrns per kilogram
to
about 50 micrograms per kilogram; more preferably from about 500 nanograms per
kilogram to about 5 micrograms per kilogram. Effective amounts of the peptide
will
vary when administered by other routes. An effective analgesic amount can be
estimated by testing the peptide in one or more of the pain tests disclosed
herein to
arrive at a dose that can be varied according to one or more of the criteria
listed above
to provide a suitable amount of the peptide to the mammal.
The terms "inducing analgesia", "analgesia-inducing activity", "analgesia-
producing" and similar terms refer to the ability of the peptide to treat pain
in
mammals or attenuate pain as evidenced by favorable results in one or more
conventional laboratory models for testing pain or assessing analgesia such as
the tests
set forth herein.
Analgesic activity of the peptides is determined by testing in at least one,
and preferably more than one, of a series of tests which includes 1 ) tail
flick latency
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6
(Abbott, F.V. et al., Pharmacol. Biochem. Behav., 17, 1213-1219, 1982;
Cridland,
R.A. and Henry, J.L., Brain Res., 584:1-2, 163-168, 1992), 2) hot plate
threshold
(Woolfe,G and Macdonald, A.A., JPET, 80, 300, 1944; Ankier, S.L, European J.
Pharmacol., 27, 1-4, 1974), and 3) vonFrey filament threshold (Kim, S.H. et
al., Pain,
S5, 85-92, 1993).
The tail flick latency and hot plate threshold tests are measurements of
thermal nocicepdon. The von Frey filament threshold test evaluates mechanical
nociceptive activity. All three of these pain tests evaluate the analgesic
activity of
compounds against the phasic stimulation of either thermal- or mechanical-
nociceptors and reflect to a large degree the activation of A- and polymodal C-
fiber
afferents. Clinical analgesics with an opioid-based mechanism of activity are
efficacious in these tests, whereas those analgesics which either interact
preferentially
with peripheral targets or possess multiple sites of action are generally less
active.
These tests are good predictors of moderate to strong analgesic agents and
within the
opioid class of compounds the correlation with clinical effect is good. The
non-
steroidal anti-inflammatory (NSAID) class of analgesics, which clinically
target the
lower end of the pain scale, are not routinely detected under the parameters
normally
used for these tests.
Analgesic detection of NSAIDs is dependent upon the generation of a
nociceptive status of increased responsiveness (i.e. a lowering of threshold
to noxious
stimuli) in response to primary afferent tissue damage and inflammation.
Interaction
between the immune and nervous systems to induce this state represents the
target for
NSAID activity. Inhibition of this heightened activity of peripheral
nociceptors, and
of the corresponding central circuitry, is detected over longer time intervals
by either
monitoring spontaneous behavior or the response to subsequent noxious stimuli.
These more chronic measurements of the "hyperalgesic" status are considered to
mimic most clinical conditions of pain. They also broaden the detection
capability for
useful analgesic agents without exclusion of active agents detected in the
phasic pain
tests. Numerous pain tests have been developed to model this condition in
laboratory
animals. The noxious stimuli used to induce this condition are either chemical
irritants/caustic agents or inflammatory stimulators. Within these tests, the
major
defining variable is the time interval required for the development, and the
ethically
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justifiable duration, of the hyperalgesic/inflammatory state. Compounds can be
evaluated for their intrinsic activity to prevent the development of the
hyperalgesic
condition (i.e. compound administered prior to noxious stimulant) or to reduce
the
increased nociceptive response (i.e. compound administered post-noxious
stimulation)
or both. Primary end points in these tests are measurements of nociceptive and
inflammatory status.
The formalin test (Dubuisson, D. and Dennis, S. G., Pain, 4, 161-174,
1977) was used since it produces a well delineated bi-phasic response that is
considered to be indicative of tonic versus acute pain and can be performed
within a
reasonably short time period (i.e. <1 hr). The initial phase of this response
is triggered
by a substantial primary afferent barrage, similar in character to that
described for the
acute phasic tests except that chemical nociceptors are the mediators. The
second
phase is considered to be the hyperalgesic spontaneous activity that results
from the
initial tissue damage and reflects the lowering of the nociceptive threshold
plus the
priming or "wind up" of the corresponding spinal circuitry. Hence, both
peripheral
and central neuronal circuits and mediators are required to induce and sustain
this
painful tissue-injury condition.
The formalin model in rodents has been validated as a predictive test of
treating injury-induced pain in humans (Dennis, S. G. and Melzack, R., In:
Advances
in Pain Research and Therapy, vol. 3, 747-759, Eds. J.J. Bonica et al., Raven
Press:New York, 1979; Tjolsen, A. et al., Pain, 51: 5-17, 1992.). Evaluation
of
clinically used analgesics in this model has consistently demonstrated a
strong
correlation with human efficacy for opioid based compounds or drugs known to
interact with opioid systems (Wheeler-Aceto, H., "Characterization Of
Nocioception
And Edema After Formalin-Induced Tissue Injury In The Rat: Pharmacological
Analysis Of Opioid Activity", Doctoral Dissertation, Temple School of
Medicine,
Philadelphia, PA, 1994; Shibata, M. et al., 38, 347-352, 1989). Efficacy and
potency
profiles for milder analgesic drugs possessing primarily NSAID based
mechanisms of
action have produced equivocal results (Wheeler-Aceto, H., Doctoral
Dissertation,
Temple School of Medicine, Philadelphia, PA, 1994; Hunskaar, S. et al.,
Neurosci.
Meth., 14, 69-76, 1985; Shibata, M. et al., Pain, 38, 347-352, 1989; Malmberg,
A.B.
and Yaksh, T.L., J. Pharmacol. Exp. Ther., 263, 136-146, 1992). These
equivocal
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8
findings in the formalin model reflect experimental differences in how the
test is
conducted such as the parameters of the test (i.e. stimulus intensity
administered,
response measurement and response interval analyzed}, species and strain of
laboratory animal used and route/timing of administration of compounds (for
review,
S Wheeler-Aceto, H., Doctoral Dissertation, Temple School of Medicine,
Philadelphia,
PA, 1994}. However, consensus exists that high efficacy analgesics used to
treat
moderate to severe pain are detected in this test independent of these
experimental
differences. If these compounds have limited central nervous system
penetration, less
activity is detected.
In addition to their use as analgesics, the peptides are useful in biological
assays such as assays to detect compounds that mimic the analgesic activity of
the
peptides, assays to detect the anatomical site of action of the peptides, or
studies on
the mechanism of action of the peptides. Thus, another aspect of the invention
provides methods for detecting compounds that mimic the analgesic activity of
GsAF
I and/or GsAF II. Mimicking the activity of the peptides disclosed herein
refers to the
ability of test compounds to induce analgesia, bind to cellular receptors to
which the
peptides bind or otherwise act in the same or similar physiological manner as
the
peptides. Thus, the present invention provides methods for identifying
compounds
having analgesia-inducing activity or which otherwise mimic the activity of
GsAF I
and/or GsAF II comprising the steps of adding a test compound to a biological
assay
that determines activity of a peptide having the amino acid sequence of SEQ ID
NO: 1
or SEQ ID NO: 2; and detecting the activity of the test compound.
Biological assays to identify compounds that mimic the activity of GsAF I
and/or II can be in vivo assays, such as those described herein, or in vitro
assays, such
as the assays described below. For example, GsAF I and/or GsAF II can be used
in
competitive binding screening assays to identify compounds that mimic the
activity of
GsAF I and II according to the following method. A test compound and
detectably
labeled peptide are added to mammalian cells or tissue under conditions that
allow
binding to the cells or tissue. Binding of labeled peptide to the mammalian
cells or
tissue is then measured. Compounds that mimic the activity of the detectably
labeled
peptide will compete with the peptide for binding sites on the receptor.
Consequently,
a smaller amount of detectable label will be measured when the test compound
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9
mimics the activity of the peptide by binding to the receptor than when the
test
compound does not mimic the activity of the peptide and does not bind to the
receptor, or does so with much less affinity. In particular, GsAF I and/or II
could be
labeled with 1251 and used in the assay described in Stumpo et al, Eur. J.
Pharmacol.
206:155, 1991 and modified from Abe et al, Neurosci. Lett. 71:203, 1986.
Briefly,
individual test compounds are preincubated with brain or spinal cord membrane
tissue
prior to the addition of 125I_labeled GsAF I and/or II, followed by incubation
to
allow binding to occur. The reaction mixture is then filtered and the filters
containing
the brain or spinal cord membrane tissue are rinsed with buffer. Binding of
125I_
labeled peptide can be determined by scintillation counting. Compounds that
mimic
the action of GsAF I and II will compete with the labeled peptide and produce
lower
levels of labeled peptide binding to the receptor on the cells of the brain or
spinal cord
membrane tissue than compounds that do not mimic the activity of GsAF I or II.
Nonspecific binding will be defined as that remaining in the presence of
excess ( 100-
1,000X) unlabeled GsAF I or GsAF II.
For use as a reagent in biological assays, the peptides preferably
incorporate a detectable label. The detectable label can be any conventional
type of
label and is selected in accordance with the type of assay to be performed.
For
example, the detectable label can comprise a radiolabel such as 14C, 1251, or
3H, an
enzyme such as peroxidase, alkaline or acid phosphatase, a fluorescent label
such as
fluoroisothiocyanate (FITC) or rhodamine, an antibody, an antigen, a small
molecule
such as biotin, a paramagnetic ion, a latex particle, an electron dense
particle such as
ferritin or a light scattering particle such as colloidal gold. Suitable
method to detect
such labels include scintillation counting, autoradiography, fluorescence
measurement, calorimetric measurement or light emission measurement.
Detectable
labels, procedures for accomplishing such labeling and detection of the labels
are well
known in the art and can be found, for example, in An Introduction to
Radioimmunoassays and Related Techniques: Laboratory Technignes in
Biochemistry
and Molecular BioloQV, 4th Ed., T. Chard, Elsevier Science Publishers,
Amsterdam,
The Netherlands, 1990; Methods in Non-Radioactive Detection, Gary C. Howard,
Ed.,
Appleton and Lange, East Norwalk, Ct, 1993 or Radioisotopes in Biolo~y: A
Practical
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Approach, R.J. Slater, Ed., IRL Press at Oxford University Press, Oxford,
England,
1990.
Additionally, the peptides can be used in the assay of Keith et al, J. Auton.
Pharmacol., 9:243-252, 1989 and Mangano et al, Eur. J. Pharmacol. 192:9-17,
1991 to
5 identify compounds that mimic the activity of GsAF I or II. Briefly, this
assay
measures K+- evoked release of 3H-D-aspartate and 3H-norepinephrine from rat
brain
or spinal cord slices. Spinal cord or brain slices can be pre-equilibrated
with GsAF
I/GsAF II, test compound or vehicle for 15 min prior to K+ stimulation. Levels
of
K+-induced release of 3H-norepinephrine and 3H-D-aspartate are measured.
10 Inhibition of K+-induced release of 3H-norepinephrine and 3H-D-aspartate by
GsAF
I/GsAF or test compound versus inhibition due to the vehicle control is then
determined. Test compounds can be screened to determine both absolute
inhibitory
activity as well as activity relative to GsAF I/GsAF II.
The compounds that mimic the analgesic activity of GsAF I and/or II will
themselves have analgesic activity and can be used as analgesics or for other
purposes
such as determining the anatomical site of action, determining the mechanism
of
action of the peptides and in screening assays to identify other compounds
that mimic
the analgesic activity of the peptides. Preferably test compounds used in the
screening
assay are small organic molecules but analgesic activity of any type or size
of
compound such as proteins and peptides can also be tested with the methods of
the
invention.
GsAF I and/or II can be used in assays to identify its site of action and for
further physiological characterization of its activity. For example, the
peptides can be
used to study inhibition of binding/interaction of labelled ligand to
mammalian
tissues, isolated cells or subcellular components derived therefrom.
Similarly, the
peptides can be used to study inhibition of binding/interaction of labelled
ligand to
specific recombinantly expressed proteins generated following either cDNA or
genomic transformations/transfections of eukaryotic or prokaryotic host
systems. The
peptides can be used to study analogous biochemical interaction with mammalian
tissue function to include receptor mediated activation/inhibition of
specified
transduction pathways, movement of ionic species across biological membranes
and
alteration of transcriptional/translational profile of specific pain-induced
gene activity.
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Specifically, methods to measure the alteration of potassium, sodium, calcium,
chloride or hydrogen ionic distribution across mammalian cell derived membrane
barriers as measured by either radioisotopic or fluorescent detection of
specified ionic
species can be utilized. The effects of these ionic movements upon the
regulation of
specific immediate early genes can be studied as well.
The peptides can additionally be used for electrophysiological
measurements of potassium, sodium, calcium and chloride distribution across
mammalian cell membranes to include macroscopic analysis of synaptic
transmission
as well as microscopic analysis of specified ionic currents. Specifically,
inhibition of
noxious-mediated neuronal firing and synaptic transmission within spinal
dorsal hom
neurons can be analyzed as well as inhibition of isolated specific ionic
currents within
individual dorsal root ganglion or spinal dorsal horn neurons.
The peptides can further be used in studies of inhibition of physiological
response to nociofensive/noxious stimuli administered to mammalian species.
Specifically, motor parameters (i.e. limb withdrawal thresholds or response
time
latencies/durations) can be quantitated in response to either thermal,
mechanical or
chemical noxious stimuli administered to either naive animals or animals in
which a
painful condition has been experimentally induced.
GsAF I and II can be prepared by isolation from Grammostola spatulata
venom, chemical synthesis or recombinant DNA methods. Grammostola spatulata
venom is commercially available from Spider Pharm, Feasterville, Pennsylvania,
USA. The peptides are preferably isolated from spider venom by sequential
fractionation using reverse phase-high pressure liquid chromatography on C-8
and C-
18 silica supports with trifluoroacetic acid/acetonitrile buffer. A preferred
C-8 silica
support is Zorbax~ Rx C-8 (Mac-Mod Analytical, Inc., West Chester,
Pennsylvania)
which is comprised of 5 micron diameter silica particles having 300 ~ pore
size and
covalently modified to contain diisopropyloctyl side chains. The C-18 silica
support
is preferably comprised of 5 micron diameter silica particles having 300 pore
size
and covalently modified to contain an octadecyl side chain. Other types of C-8
and C-
18 silica supports are also suitable for use in isolating the peptides. A
preferred buffer
is 0.1 % trifluoroacetic acid in acetonitrile. In a preferred method, crude
venom is
initially fractionated on a C-8 semi-preparative column using a broad 20-50%
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12
gradient of 0.1 % trifluoroacetic acid in acetonitrile buffer. The peptides
are further
purified using a C-8 column and shallower gradients of the same buffer,
followed by
additional fractionation using a C-8 column and the broad buffer gradient.
GsAF I and II can be prepared by recombinant DNA techniques. A DNA
sequence coding for one of the peptides is prepared, inserted into an
expression vector
and expressed in an appropriate host cell. The peptide thus produced is then
purified
from the host cells and/or cell culture medium. Methods for preparing DNA
coding
for the peptides and expression of the DNA are well-known and can be found,
for
example, in Sambrook et al. ( 1989) Molecular Cloning: A Laboratory! Manual,
Cold
Spring Harbor, New York: Cold Spring Harbor Laboratory Press, Guide to
Molecular Cloning Techniques: Methods in Enzymoloay, vol.152, S.L. Berger and
A.R. Kimmel, Ed., Academic Press (San Diego, CA), 1987 and Gene Transfer and
Expression Protocols: Methods in Molecular Biolo~v. vol.7, E.J. Murray, Ed.,
Humana Press (Clifton, NJ), 1991.
The peptides can also be prepared by chemical synthesis using either
automated or manual solid phase synthetic technologies. These techniques are
well
known in the art and are differentiated on the basis of features such as
selection of
synthetic resin backbone, selection of amino, carboxyl and side chain
protecting
groups and selection of deprotection strategies. Methods for synthesizing
peptides can
be found in standard texts such as E. Atherton and R.C. Sheppard, Solid Phase
Peptide Synthesis: A Practical Approach, IRL Press/Oxford University Press,
Oxford,
UK, 1989 and M. Bodanszky, Peptide Chemistry: A Practical Textbook, Springer-
Verlag, New York, USA, 1988.
In a preferred synthetic method, synthesis of GsAF I and GsAF II can be
done using Fmoc chemistry on an automated synthesizer. Dependent upon
quantitative yields, production of the linear reduced peptide can be performed
in either
a single process or in two different processes followed by a condensation
reaction to
join the fragments. A variety of protecting groups can be incorporated into
the
synthesis of linear peptide to facilitate isolation, purification, and/or
yield of the
desired peptide. Protection of cysteine residues found in the peptide can be
accomplished using, for example, a triphenylmethyl, acetamidomethyl andlor 4-
methoxybenzyl group in any combination. Such a strategy may offer advantages
for
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13
subsequent oxidation studies to yield folded peptide. Differential proteolytic
digestion
of native GsAF I and GsAF II coupled to mass spectrometric analysis of the
resultant
fragments can be utilized for assignment of intramolecular disulfide bonds.
This data
can be subsequently incorporated into synthetic peptide strategies to increase
yields.
Oxidative strategies include random air oxidation, iodine assisted oxidation,
and
dimethylsulfoxide assisted oxidation, as well as the use of small quantities
of thiol
reagents in an oxidation reaction to attain the desired folded peptide. Crude,
linear,
reduced peptides, as well as homogeneous, oxidized peptides, can be purified
using
reverse-phase high pressure liquid chromatography-HPLC (RP-HPLC) or other
standard techniques.
A further aspect of the invention provides a novel peptide which has the
amino acid sequence
Tyr-Cys-Gln-Lys-Trp-Leu-Trp-Thr-Cys-Asp-Ser-Glu-
Arg-Lys-Cys-Cys-Glu-Asp-Met-Val-Cys-Arg-Leu-Trp-
1 S Cys-Lys-Lys-Arg-Leu-NH2 (GsAF I) (SEQ ID NO: 1 )
The leucine at the carboxy terminus of the peptide is amidated, i.e., the free
end of the
terminal leucine residue ends with -CO-NH2 instead of -COOH. Both amidated and
non-amidated peptides are within the scope of the present invention.
As used herein, a purified or isolated peptide refers to a peptide that is
substantially free of contaminating cellular components, other venom
constituents or
other material such as reagents used in chemical synthesis of the peptide:
Preferably,
the peptide is present in a mixture containing the peptide in an amount
greater than
about 50% of the total mixture, more preferably in an amount greater than
about 80%,
most preferably in an amount greater than about 90%.
EXAMPLES
Example 1- Isolation and Characterization of Peptide GsAF I from Venom of
Grammostola spatulata
A. Isolation of Peptide
Crude Grammostola spatulata venom was supplied as frozen aliquots by
Spider Pharm, Inc. (Feasterville, Pennsylvania, USA 19053). Reverse phase-high
pressure liquid chromatography (RP-HPLC) of the venom was performed using C-8
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semi-preparative (25 cm x 9.4 mm) and analytical (25 cm x 4.6 mm) columns
(Zorbax~ RX-C8, Mac-Mod Analytical, Inc. West Chester, PA, which is comprised
of 5 micron silica microsphere particles having a 300 pore size and covalently
modified with diisopropyl octyl side chains); and a C-18 analytical (25 cm. x
4.6 mm)
column (Vydac, Hesperia, CA, which is comprised of 5 micron silica microsphere
particles having a 300 pore size and covalently modified with octadecyl side
chains).
Semi-preparative scale RP-HPLC was done using a five milliliter per minute
flow rate
whereas a one milliliter per minute flow rate was used for the analytical
analyses.
Detection of eluting entities was monitored via ultraviolet spectroscopy at
215 nm and fractions were either collected at 1 minute intervals or manually
based
upon ultraviolet intensity. Initial injection volumes of 30-50 microliter
crude venom
were made. Consequently, multiple fractionations were carried out at each
stage of
the purification with pooling of individually identical fractions. All
fractions were
lyophilized prior to resuspension in HPLC grade H20 for subsequent
purification or in
vivo analgesic testing. Resuspension volumes were based upon original crude
venom
volumes. Analgesic evaluation was done on samples deemed to be greater than
90%
homogeneous by RP-HPLC. Samples were stored at 4 C following resuspension. No
detectable loss of activity was witnessed with storage or with adherence to
either
plastic or glass.
Initial fractionation of crude Grammostola spatulata venom on the
Zorbax~ RX-C8 semipreparative column was done with a 20-50% gradient of
TFA/CH3CN Buffer (0.1 % trifluoroacetic acid in acetonitrile) over 30 minutes
with a
3 minute delay. (TFA/CH3CN Buffer was prepared by adding 4 ml of
trifluoroacetic
acid to 4 liters of acetonitrile.) Column flow rate was 5 milliliters per
minute and
fractions collected at one minute intervals. Fraction 18 was highly enriched
for GsAF
I. Fraction 17 also contained GsAF I peptide but in smaller quantities than
fraction
18 with most purifications. Following lyophilization and resuspension of
fraction 18,
and optionally fraction 17, further separations were performed with shallower
gradients of TFA/CH3CN Buffer.
Fraction 18 (and optionally 17) were applied to a Zorbax C~ RX-C8 semi-
preparative column and fractionated using a 24-30% gradient of TFA/CH3CN
Buffer
over 24 minutes, with 3 minute delay. The major UV absorbing peak was manually
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collected with removal of peak tails. After this step, sample purity was
usually found
to be at least 85%.
The major UV absorbing peak collected in the previous step was further
purified using a 20 - 50% gradient of TFA/CH3CN Buffer on a Zorbax ~ RX-C8
5 semi-preparative column (flow rate 5 ml/min) over 30 min with a 3 minute
delay. The
primary peak which elutes at 22 minutes was collected manually with removal of
peak
tails. GsAF I sample purity was found to be about 98% pure.
On occasion, exposure of the GsAF I sample to a very shallow gradient of
48-51 % TFA/CH30H buffer on a Zorbax ~ RX-C8 semi-preparative column over 21
10 minutes, followed by lyophilization, resulted in the appearance of two RP-
HPLC
resolvable peptides that differ in mass by 16 Daltons. From internal studies
done with
another peptide sample, this mass differential does not translate into a
differential
primary amino acid sequence but most likely reflects a side-chain adduct.
B. Characterization of Peptide
15 1. Electrospray Mass Spectrometry (ES-MS) Analysis of Molecular
Weight and Disulfide Bridge Assignment:
Electrospray spectra were acquired for the peptide using a mass
spectrometer (VG/Fisons QUATTRO, Fisons Instruments, Inc. Manchester, UK) in
the continuum acquisition mode. The (M+3H)3+, (M+4H~ and (M+SH)5+ charge
states were observed for each sample and mathematically transformed to yield a
zero
charge state spectrum. Analyses were performed on both the native/oxidized and
the
reduced state of the peptide. Lyophilized GsAF I was reduced in O.SM dithio-
threitol
(DTT) O.1M N-ethylmorpholine, pH 8.5, at 38C for 10 min. Flow injections
containing approximately 200-400 picomoles of peptide were measured. The
average
molecular weight of GsAF I was determined to be 3707.5 Daltons (Da). After
thiol
reduction, the average molecular weight was measured at 3713.5 Daltons. Since
each
reduction of a disulfide bond increases the mass of a peptide by 2 Da, the
peptides
contain three disulfide linkages based upon the 6 Da mass shift.
The native oxidized peptide was digested with a combination of modified
trypsin (Boehringer Mannheim) and endoproteinase Asp-N proteases. The
resulting
mixture of proteolysis products was subjected to liquid chromatography-
electrospray
mass spectral analysis to assign disulfide linkage. Multiple peptides
containing a
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disulfide bridge linking amino acids 9 and 21 of the GsAF I peptide were
observed.
Scrambling of disulfide bonds may occur when proteolysis is performed at pH 8;
however, randomization of proteolysis products was not observed, and disulfide
bond
scrambling is thought to be unlikely in this case. Links for the remaining
disulfide
bridges were not established. Subsequent analysis of the crude proteolysis
mixture by
matrix-assisted laser desorption ionization - time-of flight (MALDI-TOF) mass
spectrometry (VG Analytical/Fisons TOFSpec-SE, Fisons Instruments, Inc.
Manchester, UK) provided confirmation of the electrospray mass spectral
measurements.
2. N-terminal Sequence Analysis of Reduced, Pyridylethylated Native
Peptides and Proteolytically Digested Fragments:
N-terminal sequencing was performed on a gas phase sequencer (Applied
Biosystems 475, Foster City, CA). SDS-Page was performed using a I6.5% high
cross linked Tris-Tricine gel (Schagger,H. and G. von Jagow, Anal. Biochem.
166:368-379, 1987) and electroblotted to ProBlot (Applied Biosystems, Foster
City,
CA)) as described by Matsuidara, P., J. Biol. Chem. 262:10035-10038.
Electroblotted
bands were pyridylethylated in the gas phase according to the method described
in
Andrews, P.C. and J.E. Dixon, Anal. Biochem. 161:524-528, 1987. Covalent
attachment of peptides via activation of carboxyl groups and reaction with
arylamine
derivatized polyvinylidene difluoride using sequalon membranes (Millipore,
Inc.,
Milford, MA) was performed according to the manufacturer's instructions. V8
proteolytic digestion of reduced ( 100 x dithiothreitol vs. Cys on mole basis)
GsAF I
peptide was done in 50 mM Na phosphate buffer, pH 7.8, for 18 hr. using an
enzymeaubstrate ratio of 1:44. Fragments were isolated using RP-HPLC and their
mass analyzed using laser desorption/ionization mass spectrometry prior to
sequence
analysis. Samples were applied to the sequencer either as direct solutions
onto a
coated disc or as covalent coupled entities to ascertain carboxyl terminal
acidification/amidation. Shown below is the sequence obtained for peptide GsAF
I.
Amidation of the GsAF I peptide is supported by the ES-MS data for the intact,
native
peptide and for the respective V8 (or tryptic as well) carboxyl terminal
fragment.
The sequence obtained for peptide GsAF I is
Tyr-Cys-Gln-Lys-Trp-Leu-Trp-Thr-Cys-Asp-
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Ser-Glu-Arg-Lys-Cys-Cys-Glu-Asp-Met-Val-
Cys-Arg-Leu-Trp-Cys-Lys-Lys-Arg-Leu-NH2
(SEQ ID NO: l )
Leu-NH2 denotes that the terminal leucine residue is amidated, i.e., the free
end of the
terminal leucine residue ends with -C(=O)-NH2 instead of -COON. The amino acid
sequence of the peptide is presented starting with the amino terminus.
3. UV Spectroscopy:
A complete spectrum was obtained for the peptides using a 8452A diode
array spectrophotometer (Hewlett Paclcard, Avondale, Pennsylvania, USA).
Concentrations of the final peptides were deduced from the Abs~"",. Based upon
the
differential contributions from 3 Trp, 1 Tyr and 6 Cys, the calculated molar
extinction
coefficient of GsAF I at 280 nm is 18710. In cases where sufficient peptide
was
isolated for accurate mass weighing (and assuming appropriate peptide content
as a
result of the TFA salt), the respective concentration values were in good
agreement.
Using either method of quantitadon, and multiple preparations of native GsAF
I, the
venom concentration of GsAF I is estimated to be approximately 500-750 E.tM.
Example 2 - Isolation and Characterization of Peptide GsAF II
A. Isolation of Peptide
Crude Grammostola spatulata venom was supplied, as frozen aliquots, by
the commercial vendor Spider Pharm, Inc. (Feasterville, Pennsylvania 19053,
USA).
Reverse phase-high pressure liquid chromatography (RP-HPLC) of the venom was
performed using Zorbax~ Rx-CS semi-preparative (25 cm x 9.4 mm) and analytical
(25 cm x 4.6 mm) columns (Mac-Mod Analytical, Inc. West Chester, PA; Zorbax~
Rx-C8 is comprised of 5 micron silica microsphere particles having a 300th
pore size
and covalently modified with diisopropyl octyl side chains) and a C-18
analytical (25
cm x 4.6 mm) column (Vydac, Hesperia, CA; the C-18 support is comprised of 5
micron silica microsphere particles having a 300A pore size and covalently
modified
with octadecyl side chains). Semi-preparative scale RP-HPLC was done using a S
milliliter/minute flow rate whereas a 1 milliliter per minute flow rate was
used for the
analytical analyses.
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Detection of eluting entities were monitored via ultraviolet (UV)
spectroscopy at 215 nm and fractions were either collected at 1 minute
intervals or
manually based upon UV intensity. Initial injection volumes of 30-50
microliter (lCl)
crude venom were made. Consequently, multiple fractionations were carried out
at
each stage of the purification with pooling of individually identical
fractions. All
fractions were lyophilized prior to resuspension in HPLC grade H20 for
subsequent
purification or in vitro testing. Resuspension volumes were based upon
original crude
venom volumes. Evaluation was done on samples deemed to be greater than 90%
homogeneous by RP-HPLC. Samples were stored at 4 C following resuspension. No
detectable loss of activity was witnessed with storage or with adherence to
either
plastic or glass.
Initial fractionation of crude Grammostola spatulata venom on the Zorbax
~ RX-C8 semi-preparative column was done with a 20-50% gradient of TFA/CH3CN
Buffer (0.1 % trifluoroacetic acid in acetonitrile) over 30 min with a 3
minute delay.
(TFA/CH3CN Buffer was prepared by adding 4 ml of trifluoroacetic acid to 4
liters of
acetonitrile). Column flow rate was 5 milliliters per minute and fractions
collected at
one minute intervals. Fraction 19 was highly enriched for GsAF II. Following
lyophilization and resuspension of fraction 19, further separations of this
fraction were
performed with shallower gradients of TFA/CH3CN Buffer.
Fraction 19 was applied to a Zorbax ~ RX-C8 semi-preparative column
and fractionated using either a 29-33% or a 30-34% gradient of TFA/CH3CN
Buffer
over 24 minutes with a 3 minute delay. The major UV absorbing peak was
manually
collected with removal of peak tails. After this step, sample purity was
usually found
to be at least 85%. The major UV absorbing peak was further purified using a
20-
50% gradient of TFA/CH3CN Buffer over 30 min with a 3 minute delay. The
primary
peak which elutes at 23.5 minutes was collected manually with removal of peak
tails.
GsAF II sample purity was found to be about 98% pure.
B. Characterization of Peptide
The peptide GsAF II was characterized using the methods described for
- peptide GsAF I in Example 1. The average molecular weight of GsAF II was
determined to be 3979.9 Daltons (Da). After thiol reduction, the average
molecular
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19
weight was 3985.9 Da. Since each reduction of a disulfide bond increases the
mass of
a peptide by 2 Da, the peptide contains three disulfide linkages based upon
the 6 Da
mass shift.
Amino acid composition analyses were performed using an amino acid
analyzer (Applied Biosystems 420H, Foster City, CA). Data normalization was
done
with respect to leucine. No discrepancies (excluding those residues which are
either
partially or totally destroyed during hydrolysis) in residue/mol values were
recorded
with respect to the Edman N-terminal sequencing analysis.
Amino acid composition analysis yielded the data presented in the table
below. Since tryptophan is completely destroyed and cysteine is partially
destroyed in
this analysis, their presence was inferred from UV spectroscopy and
electrospray mass
spectral analysis, respectively. Residue/mol values were calculated on the
basis of
using Leu as the standard.
Residue Total Amount (pmole)Residue/mol
Asp/Asn 701.2 1.2
Glu/Gln 2767.6 4.7
Ser 108.9 0.2
Gly 618.1 1.0
His 0 -
Arg 1050.0 1.8
Thr 518.9 0.9
Ala 35.5 0.1
Pro 36.7 0.1
Tyr 547.5 0.9
Val 523.9 0.9
Met 875.7 1.5
Cys 2124.1 3.6
Ile 545.3 0.9
Leu 1186.1 2.0
Phe 48.5 0.1
Lys 2639.7 4.5
Shown below is the amino acid sequence for the peptide GsAF II:
Tyr-Cys-Gln-Lys-Trp-Met-Trp-Thr-Cys-Asp-
Glu-Glu-Arg-Lys-Cys-Cys-Glu-GIy-Leu-Val-Cys-
Arg-Leu-Trp-Cys-Lys-Lys-Lys-lle-Glu-Trp (SEQ ID N0:2)
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Deduction of Trp at position 31 of GsAF II is based upon amino acid
compositional data and ES-MS analysis. Specifically, the unaccounted mass
difference between the calculated mass value for the Edman deduced sequence
and the
mass spectral analysis for the native peptide is 186 Da assuming a free acid
carboxyl
5 terminus or 187 Da if the carboxyl terminus is amidated. This mass
difference (+ or -
1 Da) could be accounted for by multiple amino acid combinations. However,
none
of those combinations are in good agreement with the amino acid composition
data.
Since the mass of an internal Trp is 186 Da, and Trp is destroyed under the
hydrolysis
conditions, assignment of Trp to position 31 as a free-acid was tentatively
made. This
10 assignment was subsequently supported by analysis of the carboxyl fragment
of GsAF
II isolated following cryptic digestion. Both high resolution mass spectral
analysis and
MS-MS sequencing analysis of the fragment demonstrate the presence of a free
acid
Trp at the carboxyl terminus. These data were further corroborated when
identical
analyses were obtained for a synthetically prepared Lys-Ile-Glu-Trp peptide.
15 Additionally, the RP-HPLC retention profile of both the native fragment and
the
synthetic fragment were identical.
Based upon the presence of 4 Trp, 1 Tyr and slight contribution from 6 Cys
residues, a molar extinction coefficient of 24310 at 280 nm was deduced for
GsAF II.
Using this value , UV spectroscopy analyses of native GsAF II preparations
indicate
20 that the venom concentration of this peptide is approximately 3-5 mM.
Example 3 - Analgesic Evaluation - Tail FIick Latency
This test measures the time interval required for a rat to withdraw its tail,
via a spinally mediated reflex mechanism, from a high intensity light source
(ITTC
Inc./ Life Sciences Instruments, Woodland Hills, CA 91367) focally applied to
the
dorsal surface of the appendage. The intensity of the light beam has been
experimentally defined such that naive animals will withdraw their tails
within 2 to 4
seconds. A maximum cut off time for the light source has been set at ten
seconds to
reduce the amount of secondary tissue damage.
Data is expressed either as absolute time or a percentage of the maximal
possible effect (MPE) which is described by the following equation where 10
seconds
is the maximum:
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% MPE = !,post-treatment latency - pretreatment latency )
- pretreatment latency
(Latency refers to the amount of time before the animal removed its appendage
from
the light source.)
5 GsAF I Administration:
With minimal restraint, peptide GsAF I was injected intrathecally (i.th.)
into young (75-150 gram) male Sprague-Dawley rats (Charles Rivers
Laboratories,
Wilmington, MA 01887). Lth. injections were made into the spinal subarachnoid
space between lumbar spinous processes L4 and LS using 10 microliter Hamilton
10 syringes equipped with 3/8 inch by 28G needles. Dosing levels were based
upon
concentrations deduced from ultraviolet absorbance values at 280 nm using an
extinction coefficient of 18710. Injection volume was 10 microliters. The
injection
vehicle was saline or 0.1 % bovine serum albumin(BSA)/saline. The rats were
pretreated with GsAF I 30 minutes prior to exposure to the light source.
Complete inhibition of the tail flick response (i.e., latency value greater
than 10 seconds) was recorded in most rats following administration of 180
picomoles
(666 nanograms) of GsAF I. A 95% MPE was attained for this dose and
confounding
side effects such as motor disturbances, limb impairment/paralysis, righting
reflex,
sedation, etc.) were either minimal or not present. Logarithmic decreases in
the dose
resulted in rapid loss of effect. 18 picomoles ( 66 nanograms) of GsAF I
produced
29% MPE and 1.8 picomoles (6.6 nanograms) was inactive. Maximal activity was
detected with a 30 minute pretreatment time.
GsAF II Administration:
With minimal restraint, peptide GsAF II was injected intrathecally (i.th.)
into young (75-150 gram) male Sprague-Dawley rats (Charles Rivers
Laboratories,
Wilmington, MA)) into the spinal subarachnoid space between lumbar spinous
processes L4 and LS using 10 microliter Hamilton syringes equipped with 3/8
inch by
28G needles. Dosing levels were based upon concentrations deduced from
ultraviolet
absorbance values at 280 nm using the deduced molar extinction coefficient of
24310.
Injection volume was 10 microliters. The injection vehicle was saline or 0.1 %
bovine
serum albumin(BSA)/saline. The rats were pretreated with GsAF II 15
minutes.prior
to exposure to the light source.
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Complete inhibition of the tail flick response (i.e. latency greater than ten
seconds) was recorded with all animals (n = 8) dosed with 2.33 nanomoles (9.27
micrograms) of GsAF II. When animals were dosed with 583 picomoles (2.33
micrograms) of GsAF II, 100% MPE was recorded for five of six animals, with
the
average MPE at this dose of 92%. Similar to GsAF I, no confounding side
effects
were detected at these doses.
Example 4 - Analgesic Evaluation - Hot Plate Threshold
This test measures the temperature at which point a rat voluntarily removes
one of its hindlimbs from the heated surface and either shakes or lick the
affected
appendage. The temperature of the heated surface is pre-set to the
experimentally
deduced value of 38 C and a maximum cutoff value of either 53 C or 54 C has
been
used. Data is expressed either as absolute temperature in degrees C or as
percentage
maximal possible effect as described by the formula
% MPE = (postlatency value - prelatency value)
53 - prelatency value
(54 is substituted for 53 in the above formula if it is the maximal value.)
GsAF I Administration:
With minimal restraint, peptide GsAF I was injected intrathecally (i.th.)
into young (75-150 gram) male Sprague-Dawley rats (Charles Rivers
Laboratories,
Wilmington, MA) into the spinal subarachnoid space between lumbar spinous
processes L4 and LS using 10 microliter Hamilton syringes equipped with 3/8
inch by
28G needles. Dosing levels were based upon concentrations deduced from
ultraviolet
absorbance values at 280 nm using an extinction coefficient of 18710.
Injection
volume was 10 microliters. The injection vehicle was saline or 0.1 %bovine
serum
albumin(BSA)/saline. The rats were pretreated with GsAF I 30 minutes prior to
exposure to heat.
With a 30 minute pretreatment interval, 180 picomoles of GsAF I (666
nanograms) produced a 70% MPE. Lowering the dose ten-fold (to 18 picomoles)
resulted in the loss of significant activity. Greater efficacy at multiple
doses could be
obtained with one hour pretreatment. With one hour pretreatment, a 180
picomole (66
nanograms) dose of GsAF I produced a 91% MPE and 18 picomoles {6.6 nanograms)
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produced a 24% MPE. No adverse motor effects were evident at these doses. This
is
corroborated by the observation that the animals' front paws were responsive
at sub
threshold temperatures and were quickly lifted off the hot surface at the
elevated
temperatures.
GsAF II Administration:
With minimal restraint, peptide GsAF II was injected intrathecally (i.th.)
into young (75-150 gram) male Sprague-Dawley rats (Charles Revers
Laboratories,
Wilmington, MA) into the spinal subarachnoid space between lumbar spinous
processes L4 and L5 using -10 microliter Hamilton syringes equipped with 3/8
inch by
28G needles. Dosing levels were based upon concentrations deduced from
ultraviolet
absorbance values at 280 nm using an extinction coefficient of 18710.
Injection
volume was 10 microliters. The injection vehicle was saline or 0.1% bovine
serum
albumin(BSA)/saline. The rats were pretreated with GsAF II 15 minutes prior to
exposure to heat.
IS A I00 % MPE was recorded for all animals receiving a 2.33 naiiomole
(9.27 micrograms) dose of GsAF II. When the dose was lowered to 583 picomoles
(2.32 micrograms) , an average MPE of 59% was recorded. No motor coordination
problems were witnessed at these doses.
Example 5 - Analgesic Evaluation - von Frey Threshold
In this test filaments of increasing thickness are applied to the dorsal
surface of a hindlimb until the rat either voluntarily removes the appendage
with a
forcible escape movement or vocalizes. The thickness of the filaments are
arbitrarily
labelled with a value which can be transformed into a grams force reading
according
to the following equation:
grams force = 10(von Frey score - 1 )
1000
Data are expressed either as absolute gram force values or a percentage of the
maximum possible effect (MPE) as described in the following equation:
MPE = (postlatency value - prelatency value)
446.? - prelatency value
GsAF I Administration:
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When tested 30 minutes post injection, doses of 180 picomoles (666
nanograms) and 18 picomoles (66 nanograms) of GsAF I produced 93% and 24%
MPE's, respectively. Extending the pretreatment period to one hour resulted in
a
100% MPE for the 180 picomole dose and a 22% MPE for the 18 picomole (6.6
nanograms) dose. No confounding side effects were present.
GsAF II Administration:
When tested 30 minutes post injection, a 2.33 nanomole (9.27 micrograms}
dose of GsAF II produced an MPE of 83%. A 583 picomole (2.32 micrograms) dose
of GsAF II produced an MPE of 48%. At the higher dose, 75% of the rats tested
demonstrated maximal analgesic activity (i.e. 100% MPE). Based upon the GsAF I
data, it is assumed that greater analgesic efficacy with GsAF II would be
obtained if
longer pretreatment were used.
Example 6 - Analgesic Evaluation - Formalin Pain Test
"15 The noxious stimulus for this test is the sub-cutaneous injection of a 5%
solution of formalin into the dorsal surface of one of the hindlimbs of the
animal.
Motor activity indices used in this test are 1 ) the total time spent licking
that
appendage and 2) the total number of flinching/shaking responses of the
affected
appendage. Data collection is initiated immediately upon injection of the
formalin
solution into the limb. The acute phase response is defined by the time
interval of 0-5
minutes post formalin injection. The tonic phase response is defined by the
interval of
20-35 minutes post formalin injection Data collection is done in a
computerized
format. Expression of the data is done using either absolute values or as
percent
control which is defined by the level of response following injection of
saline vehicle.
GsAF I Administration:
With minimal restraint, peptide GsAF I was injected intrathecally (i.th.)
into young (75-150 gram) male Sprague-Dawley rats (Charles Rivers
Laboratories,
Wilmington, MA) into the spinal subarachnoid space between lumbar spinous
processes L4 and LS using 10 microliter Hamilton syringes equipped with 3/8
inch by
28G needles. Dosing levels were based upon concentrations deduced from
ultraviolet
absorbance values at 280 nm using an extinction coefficient of 18710.
Injection
volume was 10 microliters. The injection vehicle was saline or 0.1 % bovine
serum
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albumin(BSA)/saline. The rats were pretreated with GsAF I 30 minutes prior to
injection with the formalin solution..
The following results were obtaiped 30 minutes post injection of 180
picomoles (666 nanograms) 18 picomoles (66 nanograms) and 1.8 picomoles (6.6
5 nanograms) of GsAF I. Values are presented as % control with absolute levels
in
parentheses.
Acute Flinches Tonic Flinches Tonic Licking
Control (42.5) ( 139) ( 181.8 sec)
10 180 pmoles 8.9% (3.8) 3.0% (4.3) 0% (0 sec.}
18 pmoles 20% (8.5) 21% (29.5) 8.4% (15.3
sec)
1.8 pmoles 28% (12) 32% (44.3) 46% (84.3
sec)
GsAF II Administration:
With minimal restraint, peptide GsAF II was injected intrathecally (i.th.)
into young (75-150 gram) male Sprague-Dawley rats (Charles Rivers
Laboratories,
Wilmington, MA) into the spinal subarachnoid space between lumbar spinous
processes L4 and LS using 10 microliter Hamilton syringes equipped with 3/8
inch by
28G needles. Dosing levels were based upon concentrations deduced from
ultraviolet
absorbance values at 280 nm as stated previously. Based upon a mass of 3980
Da, 583
pmoles corresponds to 2.3 micrograms of GsAF II, and 2.33 nmoles corresponds
to
9.3 micrograms of GsAF II. Injection volume was 10 microliters. The injection
vehicle was saline or 0.1% bovine serum albumin(BSA)/saline. The rats were
pretreated with GsAF II 15 minutes prior to injection with formalin.
Acute Flinches Tonic Flinches Tonic Licking
Vehicle Control ( 16.8) (86) (95.0 sec)
2.33 nmoles 19% (3.25) 1.4% ( 1.2) 0% (0 sec)
583 pmoles 41% (6.8) 9.5% (8.2) 8.2% (7.8 sec)
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In addition to the above analyses, 180 pmoles (666 nanograms) of GsAF I
was administered 5 minutes after injection of 5% formalin and the tonic phase
responses were recorded as described previously. The highly efficacious
activity of
GsAF I was retained. Tonic flinch response was inhibited 86% (i.e. 14% of
control)
and tonic lick duration was reduced 91% (i.e. 9% of control). This property
has only
been reported for strong analgesic compounds that interact with p-opioid
receptors. It
also demonstrates that the analgesic activity of GsAF I is not dependent upon
the
interruption of the initial rapid firing of sensory fibers (primarily c-
fibers) or occlusion
of wind-up within dorsal horn neurons.
Example 7 - Analgesic Evaluation - Opioid Receptor Testing
In order to determine if the anti-nociceptive effects of GsAF I and II are
mediated by opioid receptors, young (75-150 gram) male Sprague-Dawley rats
(Charles Rivers Laboratories, Wilmington, MA) were pretreated with opioid
antagonists at doses that reversed the anti-nociceptive activity of morphine.
Animals
were then tested in the tail flick test (Example 3) and von Frey tests
(Example 5).
Subcutaneous administration of lOmg/kg naloxone 5 minutes prior to the
intrathecal
administration of 180 picomoles (666 nanograms) GsAF I failed to inhibit the
analgesic activity of GsAF I (180 pmoles i.th.) measured 10 minutes later
(i.e. 10
minutes post i.th. administration of GsAF I and 15 minutes post subcutaneous
adminitration of naloxone), but completely antagonized the analgesic effect of
morphine (3pg i.th.). Additionally, intrathecal administration of naloxone (50
~tg)
immediately prior to intrathecal dosing of 180 pmoles GsAF I failed to inhibit
analgesic response measured 10 minutes later whereas reversal of intrathecal
morphine ( 3 pg) was determined.
Similarly, pretreatment with the irreversible opioid antagonist B-
funaltrexamine (5 micrograms administered intrathecally 18 hours prior to the
injection of GsAF I) failed to inhibit the analgesic activity of GsAF I in
either the hot
plate test (Example 4) or formalin test (Example 6). The analgesic profile for
GsAF I,
and presumably GsAF II, indicates high efficacy mediated by a non-opioid
receptor
related mechanism.
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Example 8 - Analgesic Evaluation for Cross Tolerance of GsAF I with Morphine
Repeated administration of morphine to both humans and rodents results in a
decreased analgesic response for any individual dose or a leftward shift in
the dose
response curve. This phenomenon, termed tolerance, leads to an escalation of
morphine dose for maintenance of equivalent pain relief over time. Clinically,
the
difference in tolerance development for analgesia versus negative side effects
(i.e.
sedation, constipation, respiratory depression, etc) can limit the utility of
morphine in
a chronic treatment regime. Although the physiological basis of tolerance is
not
completely understood, putative analgesic compounds can be evaluated to
determine
if their efficacy is altered as a result of morphine administration (i.e.
morphine cross
tolerance). Young (75-150 gram) male Sprague Dawley rats (Charles Rivers
Laboratory, Wilmington, Massachusetts) were subcutaneously dosed twice daily
for 6
days with escalating quantities (i.e. 2.Smg/kg/dose - day 1; Smg/kg/dose - day
2;
lOmg/kg/dose - day 3; 20mg/kg/dose - days 4 and S; 25mg/kg/dose - day 6) of
morphine or with saline as a vehicle control. On the seventh day, the
analgesic
activity of morphine and GsAF I was tested using the tonic flinch response of
the
formalin pain test (as described in Example 6). Dose response determinations
for
morphine and GsAF I were done using intrathecal administration into young (75-
150
gram) male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA).
With minimal restraint, either morphine or GsAF I was injected into the spinal
subarachnoid space between lumbar spinous processes L4 and LS using 10
microliter
Hamilton syringes equipped with 3/8 inch by 28G needles. Dosing levels of GsAF
I
were based upon concentrations deduced from ultraviolet absorbance values at
280
nm using an extinction coefficient of 18710. Injection volumes were 10 ul for
both
morphine and GsAFI. Vehicle controls for morphine and GsAFI were saline and
0.1 % BSA/saline, respectively.
Animals treated with morphine for 6 days and then evaluated for analgesia
induction as a result of morphine administration on the seventh day exhibited
a
significant leftward shift (300x) in the dose response curve versus animals
receiving
the saline vehicle (EDSO = 0.1 ug i.th. for saline control group; EDsa >30ug
i.th. for
morphine treatment group). EDso is the dose required to give 50% of the
maximal
analgesic effect. In contrast, the dose response properties of GsAF I were not
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28
significantly different for the two treatment arms of the study (EDso - 26.7
pmoles
i.th. for saline control group; EDso - 20 pmoles i.th. for morphine treatment
group)
indicating that prior exposure to morphine does not alter the analgesic
properties of
GsAF I or produce cross tolerance.
Example 9 - Analgesia Evaluation - Acute Inflammatory Pain Testing
Tissue injury results in inflammation and hyperalgesia (i.e. increased
magnitude or duration of pain response to supra threshold noxious stimuli) at
both the
site of injury and at adjacent tissue sites. To assess the anti-hyperalgesic
activity of
GsAF II in inflammatory pain conditions, paw withdrawal latencies were
determined
in adult 350-400 gram male Sprague Dawley rats following the unilateral
injection of
carrageenan, a seaweed extract, into the hindpaw in accordance with the method
of
Hargreaves et al, Pain, 32:77-88, 1988. Briefly, withdrawal latencies are
measured by
placing the rat on a glass plate and focusing radiant heat from the underside
of the
plate toward the hindpaw surface. Latencies values are recorded in seconds to
withdrawal of the hindpaw from the surface of the plate. Basal measurements
are
made prior to injection of the carrageenan (4 mg/hindpaw), followed by a
measurement at 150 min post carrageenan injection to obtain the level of
hyperalgesic
response. Subsequent to the second measurement, GsAF II or vehicle (i.eØ1 %
BSA/saline) is administered through an indwelling intrathecal cannula
positioned
within the lumbar enlargement of the spinal cord. Anti-hyperalgesic activity
is
determined by measuring paw withdrawal latencies at various time intervals
following
compound administration. Concurrent with the paw withdrawal latencies,
physical
measurements of paw volume and paw temperature are recorded to detect anti-
inflammatory and anti-pyretic activities.
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29
Mean Latency Time In Seconds t SEM
Vehicle Control 3 nmol GsAFII
(n = 8) (n = 4)
Base latency prior to
carrageenan injection 10.12 ~ 0.05 8.3 ~ 1.2
150 minutes post carrageenan
injection 2.14 t 0.24 3.0 t 0.81
30 minutes post treatment 2.73 ~ 0.08 17.82 t 3.78
60 minutes post treatment 3.61 t 0.40 20.8 ~ 0.00
S Development of hyperalgesia was detected in all animals tested. As shown in
the table above, paw withdrawal times were significantly reduced at 150 min
post
injection of carrageenan. Administration of 3 nmoles of GsAF II completely
reversed
this hyperalgesic, thermal-induced response by increasing paw withdrawal
latencies to
near maximal values ( 17.82 ~ 3.78 sec) at 30 min and to a predetermined cut-
off value
(20.8 sec) at 60 min. No significant reduction in either paw volume or paw
temperature was detected and there was no indication of confounding motor
deficits
or presence of overt negative side effects. GsAF II is an effective
analgesic/anti-
hyperalgesic compound for acute peripheral inflammatory pain. GsAF II does not
appear to possess anti-inflammatory properties since the induction of
analgesia was
not associated with an acute reduction of edema or of body temperature.
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1
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: ZENECA Limited
(ii) TITLE OF INVENTION: Analgesic Peptides from Venom of
Grammostola Spatulata and Use Thereof
(iii) NUMBER OF SEQUENCES: 2
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: ZENECA Pharmaceuticals
(B) STREET: Mereside, Alderley Park
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(D) SOFTWARE: PatentIn Release #1.0, Version
#1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
3O (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: BILL, Kevin
(B) REFERENCE/DOCKET NUMBER: PHM.70122
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: +44-1625-512461
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
CA 02319038 2000-07-25
WO 99/42480 PCT/GB98/00534
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
$ (A) NAME/KEY: Peptide
(B) LOCATION: 29
(D) OTHER INFORMATION: /note= "Xaa is amidated leucine"
IO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Tyr Cys Gln Lys Trp Leu Trp Thr Cys Asp Ser Glu Arg Lys Cys Cys
1 5 10 15
1$ Glu Asp Met Val Cys Arg Leu Trp Cys Lys Lys Arg Xaa
20 25
(2) INFORMATION FOR SEQ ID N0:2:
ZO (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
2$ (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
30 Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Glu Glu Arg Lys Cys Cys
1 5 10 15
Glu Gly Leu Val Cys Arg Leu Trp Cys Lys Lys Lys Ile Glu Trp
20 25 30
3$
