U.S. patent application number 13/934125 was filed with the patent office on 2013-10-24 for methods of use of epsilon inhibitor compounds for the attenuation of pain.
The applicant listed for this patent is Kai Pharmaceuticals, Inc.. Invention is credited to Derek Maclean, Qun Yin.
Application Number | 20130281380 13/934125 |
Document ID | / |
Family ID | 39636773 |
Filed Date | 2013-10-24 |
United States Patent
Application |
20130281380 |
Kind Code |
A1 |
Maclean; Derek ; et
al. |
October 24, 2013 |
METHODS OF USE OF EPSILON INHIBITOR COMPOUNDS FOR THE ATTENUATION
OF PAIN
Abstract
The disclosure herein relates to modified .epsilon.PKC
inhibitory peptides, methods of generating such peptides, and
method for using .epsilon.PKC inhibitory peptides for the treatment
of pain.
Inventors: |
Maclean; Derek; (Los Altos,
CA) ; Yin; Qun; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kai Pharmaceuticals, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
39636773 |
Appl. No.: |
13/934125 |
Filed: |
July 2, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13230724 |
Sep 12, 2011 |
8492346 |
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13934125 |
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12018111 |
Jan 22, 2008 |
8017583 |
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13230724 |
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60881396 |
Jan 19, 2007 |
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60903684 |
Feb 26, 2007 |
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60917876 |
May 14, 2007 |
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60977332 |
Oct 3, 2007 |
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Current U.S.
Class: |
514/18.3 ;
514/1.1 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 25/00 20180101; A61P 25/04 20180101; A61K 38/10 20130101; A61K
38/45 20130101; A61K 38/16 20130101; C07K 2319/10 20130101; A61P
43/00 20180101 |
Class at
Publication: |
514/18.3 ;
514/1.1 |
International
Class: |
A61K 38/16 20060101
A61K038/16 |
Claims
1. A method of treating a subject suffering from pain, comprising:
administering to the subject an effective amount of a hybrid
construct comprising an epsilon protein kinase C (.epsilon.PKC)
inhibitory peptide, a gamma PKC (.gamma.PKC) inhibitory peptide and
a carrier peptide, wherein the .epsilon.PKC inhibitory peptide is
covalently linked to the .gamma.PKC inhibitory peptide.
2. The method of claim 1, wherein the pain is selected from the
group consisting of acute pain, chronic pain, neuropathic pain, and
inflammatory pain.
3. The method of claim 1, wherein the pain is chronic pain.
4. The method of claim 1, wherein at least one of the carrier
peptide, the .epsilon.PKC inhibitory peptide and the .gamma.PKC
inhibitory peptide is modified at the C-terminus.
5. The method of claim 1, wherein the carrier peptide comprises the
amino acid sequence of SEQ ID NO:59.
6. The method of claim 1, wherein the carrier peptide comprises the
amino acid sequence of SEQ ID NO:4.
7. The method of claim 1, wherein the carrier peptide is modified
at its N-terminal end by an acyl, alkyl or sulfonyl group.
8. The method of claim 7, wherein the carrier peptide is modified
at its C-terminal end.
9. The method of claim 8, wherein the modification at the
N-terminal end of the carrier peptide is the formation of an
amide.
10. The method of claim 1, wherein the .epsilon.PKC inhibitory
peptide further comprises a terminal Cys.
11. The method of claim 10, wherein the terminal Cys is located at
the C-terminus of the .epsilon.PKC peptide.
12. The method of claim 10, wherein the terminal Cys is located at
the N-terminus of the .epsilon.PKC peptide.
13. The method of claim 1, wherein the .gamma.PKC inhibitory
peptide further comprises a terminal Cys.
14. The method of claim 13, wherein the terminal Cys is located at
the N-terminus of the .gamma.PKC inhibitory peptide.
15. The method of claim 13, wherein the terminal Cys is located at
the C-terminus of the .gamma.PKC inhibitory peptide.
16. The method of claim 10, wherein the carrier peptide further
comprises a terminal Cys.
17. The method of claim 16, wherein the carrier peptide is linked
to the .epsilon.PKC inhibitory peptide via a disulfide bond.
18. The method of claim 13, wherein the carrier peptide further
comprises a terminal Cys.
19. The method of claim 18, wherein the carrier peptide is linked
to the .gamma.PKC inhibitory peptide via a disulfide bond.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 13/230,724, filed Sep. 12, 2011, now allowed, which is a
divisional of U.S. application Ser. No. 12/018,111, filed Jan. 22,
2008, now U.S. Pat. No. 8,017,583, which claims the benefit of
priority of U.S. Provisional Application Nos. 60/881,396,
60/903,684, 60/917,876, and 60/977,332, all of which are
incorporated by reference in their entirety.
REFERENCE TO SEQUENCE LISTING
[0002] A Sequence Listing is being submitted electronically via EFS
in the form of a text file, created Jul. 2, 2013, and named
"0915088008US05seqlist.txt" (19,979 bytes), the contents of which
are incorporated herein by reference in their entirety.
TECHNICAL FIELD
[0003] This present disclosure relates to the development and use
of compound(s) that modulate different types of pain, and where
compounds that may have overlapping and/or non-overlapping
mechanisms of biochemical activity are incorporated into a single
compound entity (so-called "Hybrid" compounds or peptides) for the
treatment of pain and related conditions. Wherein the compounds
comprise one or more epsilon PKC (.epsilon.PKC) inhibitory peptides
coupled to at least one carrier moiety and where the inhibitory
peptides, the carrier moiety, or both have been modified from a
prototype sequence to increase the stability, potency, or both of
the resulting compound. The .epsilon.PKC inhibitory peptides may
also be coupled to one or more modulatory peptides with specific
activity against one or more of the other PKC isozymes, including
PKC alpha, beta, delta, gamma, theta or eta. The benefits of a
hybrid compound or peptide over an isozyme-specific PKC modulator
would be to provide a broader spectrum of activities to modulate
various types of pain and/or provide greater potency and/or greater
safety for a pain modulating compound and/or to provide dual
therapeutic activities to alleviate multiple aspects of a disease
condition (e.g., combining pain-reducing activity with an
anti-inflammatory activity).
BACKGROUND
[0004] Pain is an uncomfortable sensation caused by inflammation,
nerve injury, or overly-sensitive tissue reacting to mechanical,
thermal or chemical stimuli. It is a major health problem: every
year many work days are lost due to pain-related conditions. Among
a wide variety of pains, neuropathic pain is a disease arising from
nerve damage and affects >1 million Americans. This condition
arises from a variety of causes including diabetes, herpes zoster
infection (chicken pox/shingles), traumatic nerve injury, cancer,
or treatment of cancer with chemotherapeutic agents. Inflammatory
pain is another kind of pain that constitutes the single largest
category due to its multiple etiologies. The search for new
analgesic therapy is an area of great interest to the medical
community (Reichling & Levine, 1999) because it is associated
with other local or systemic diseases, such as auto-immune disorder
like rheumatoid arthritis and because it is chronic and thus causes
prolonged suffering.
[0005] Most of the current pain treatment use remedies of
systemically-administered drugs, such as non-steroidal
anti-inflammatory drugs (NSAIDS) or opioids. Many of these drugs
cause systemic sides effects ranging from the increase in heart
risk to addiction. There are only a few pain remedies that use
local routes of administration, such as capsaicin cream, which does
not work on all kinds of pain and causes local irritation (burning
sensation, skin pain, skin inflammation, etc).
[0006] Protein kinase C ("PKC") is a key enzyme in signal
transduction involved in a variety of cellular functions, including
cell growth, regulation of gene expression, and ion channel
activity. The PKC family of isozymes includes at least 11 different
protein kinases that can be divided into at least three subfamilies
based on their homology and sensitivity to activators. The families
are the classical, the novel, and the atypical subfamilies. Each
isozyme includes a number of homologous ("conserved" or "C")
domains interspersed with isozyme-unique ("variable" or "V")
domains. Epsilon PKC is a member of the "novel" subfamily, along
with .delta., .eta. and .theta.PKC. Members of this subfamily
typically lack the C2 homologous domain and do not require calcium
for activation. Individual isozymes of PKC have been implicated in
the mechanisms of various disease states. Epsilon PKC inhibitory
peptides derived from .epsilon.PKC have been generated and shown to
impact nociception. For example, see U.S. Pat. Nos. 6,376,467 and
6,686,334.
[0007] One problem with this approach is that the "naked" termini
of the excised fragments are different from their context in the
protein, revealing free amine and carboxyl groups at the points
where the fragment attaches to the remainder of the protein. These
extraneous moieties may render the peptide more susceptible to
proteases. As a result of these liabilities the potency of the
peptide may be less than desired and the in vivo half-life may be
significantly shortened.
[0008] A second area of the prior art makes use of a similar
strategy, wherein "carrier" peptides are designed as fragments of
HIV-Tat and other proteins. These peptide fragments mimic the
ability of the parent protein to cross cell membranes. Of
particular interest is the property that "cargo" peptides can be
attached to these carrier peptides such that both cargo and carrier
peptides are carried into the cell by these carrier peptide
fragments.
[0009] Recognizing that the carrier peptides are fragments, similar
deficiencies may apply as noted above for the cargo peptides. That
is, the exposed termini may confer undesirable properties including
protease susceptibility.
[0010] Prior art cargo/carrier peptide constructs have made use of
a Cys-Cys disulfide bond between cargo and carrier, which can be
cleaved by a number of agents, such as glutathione reduction when
the peptides enter cells. This property has been thought to be
important for biological activity, since the physical separation of
cargo and carrier allows the two moieties to exert their
independent effects within the cell. However, this hypothesis has
not been convincingly tested, and non-cleavable analogs may in fact
have good activity. Further, the disulfide bond is cumbersome to
assemble, and prone to chemical degradation.
[0011] The design of certain prior art cargo/carrier peptides is
based on a contiguous sequence of amino acids from the protein.
However, the optimal length of the peptide has not yet been well
defined, being based on sequence comparison analysis and
theoretical prediction of the desired sequence rather than on an
empirical basis of analog testing. Thus, increased potency may be
anticipated from analogs of the previously described cargo peptides
which contain additional residues corresponding to the .epsilon.PKC
domain from which the have been derived.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows a schematic representation of a modified
.epsilon.PKC inhibitory peptide (KP-1634) (SEQ ID
NOS:49,54,56).
[0013] FIG. 2 shows the chemical formula of an inhibitory
.epsilon.PKC inhibitory peptide (KP-1586).
[0014] FIG. 3 shows the impact of rat or human sera on the
stability of peptides KP-1586, KP-1630, and KP-1631, by plotting
the relative concentration of test peptides over time.
[0015] FIG. 4 shows the impact of rat or human sera on the
stability of peptides KP-1632, KP-1633, and KP-1634, by plotting
the relative concentration of test peptides over time.
[0016] FIG. 5 shows the impact of rat or human sera on the
stability of peptides KP-1635, KP-1636, and KP-1637, by plotting
the relative concentration of test peptides over time.
[0017] FIG. 6 shows the impact of time and temperature on the
chemical stability of peptides KP-1586, KP-1630, KP-1631, KP-1632,
KP-1633, KP-1634, KP-1635, KP-1636, KP-1637, and KP-1638, by
plotting the relative concentration of test peptides over time.
[0018] FIG. 7 shows the results of a formalin test to show the
attenuation of acute pain in a plot of flinches per minute versus
time in rats treated with control and two doses of KP-1586.
[0019] FIG. 8 shows the impact of .epsilon.PKC inhibitory peptide
on acute inflammatory pain in a bar graph plotting paw withdrawal
latency for peptide KP-1586 and control peptide KP-1587 in a
carrageenan model.
[0020] FIG. 9 shows a bar graph plotting paw withdrawal latency and
compares impact of inhibitory peptide KP-1586 or control peptide
KP-1587 in a chronic inflammatory pain model in rats where
carrageenan is administered followed 5 days later by PGE.sub.2 to
promote chronic hyperalgesia.
[0021] FIG. 10 shows a line plot indicating the effect of a
.epsilon.PKC inhibitory peptide on paw withdrawal threshold
measurements.
[0022] FIG. 11 shows bar graphs of the impact of subcutaneous
infusion of a .epsilon.PKC inhibitory peptide on thermal
hyperalgesia in rats following L5 nerve transection (a neuropathic
pain model).
[0023] FIG. 12 shows a line plot indicating the impact of a
subcutaneous bolus of a .epsilon.PKC inhibitory peptide on thermal
hyperalgesia in rats following L5 nerve transection measured using
paw withdrawal latency.
[0024] FIG. 13 shows a bar graph indicating the impact of
subcutaneous infusion of a .epsilon.PKC inhibitory peptide on
thermal hyperalgesia in rats following L5 nerve transection.
[0025] FIG. 14 Effects of local administered .epsilon.PKC
inhibitors on .lamda.-carrageenan-induced hyperalgesia in response
to mechanical stimulation. Increases in paw-withdrawal threshold
(PWT) upon injection of .epsilon.V.sub.1-2 via intraderminal (A)
and subcutaneous (B) routes. An increase in PWT indicates less
pain. An increase in PWT back to the levels before carrageenan
referred to anti-hyperalagesic effect. An increase in PWT above the
pre-carrageenan levels implicated a potential analgesic effect.
[0026] FIG. 15. Effects of unilateral de-afferentation on
anti-hyperalgesic effects of .epsilon.PKC inhibitors administered
to the limb on the same side of surgery, distal from the nerve
transaction. Intradermal (i.d.) administration of the compound on
the ipsilateral paw was still able to inhibit the
carageenan-induced pain in the de-afferenated animal.
[0027] FIG. 16. Effects of bilateral lumbar sympathectomy plus
bilateral suprarenal ganglionectomy on anti-hyperlagesic effects of
.epsilon.PKC inhibitors. Experiment was conducted 7-days after
surgery to allow degeneration of the SPGN (Sympathetic
post-ganglionic neurons) terminals. Surgical sympathectomy mimicked
the effects of the unilateral de-afferentation surgery.
Subcutaneous injection of an .epsilon.PKC inhibitor
(.epsilon.V.sub.1-2-TAT) no longer induced
anti-hyperalgesic/analgesic effects in the sympathectomized rats
but its effects on the sham surgery animal remained intact. The
reversal of hyperalgesic is very rapid within five minutes of
administering the .epsilon.PKC inhibitor.
[0028] FIG. 17. Effects of injection of phentolamine on
anti-hyperalgesic effects of .epsilon.PKC inhibitors. Injection of
phentolamine, which is a non-selective .alpha.-adrenoceptor
antagonist, abolished the pain-reducing effects of .epsilon.PKC
inhibitor (.epsilon.V.sub.1-2-TAT) injected to the distal aspect of
the limb injected previously with phentolamine. After phentolamine,
the anti-hyperalgesic effects of .epsilon.V.sub.1-2-TAT was
lost.
[0029] FIG. 18: Effects of Intradermal KAI-1678 on Mechanical
Hyperalgesia in the Carrageenan Inflammatory Pain Model in Rats.
Rats were treated with an intradermal bolus injection of KAI-1678
60 minutes after carrageenan injection into the plantar side of the
right hindpaw. KAI-1678 was dosed at 10 mcg/kg (triangles) or 100
mcg/kg (circles) to either the ipsilateral hindlimb (i.e. the same
limb that received carrageenan, filled symbols) or the
contralateral hindlimb (i.e. the limb that did not receive
carrageenan, open symbols). Data are presented as mean.+-.SEM
(standard error of the mean) for the (PWT) measurements for the
animals in each group (N=2-5 animals/group) at a given time point.
The dotted line at .about.90 g represents the pre-carrageenan
baseline PWT measurement; PWT indicative of the disease state is
.about.60 g. Measurements at or above the pre-carrageenan baseline
level indicate complete reversal of carrageenan-induced mechanical
hyperalgesia.
[0030] FIG. 19: Effects of Subcutaneous Bolus Administration of
KAI-1678 on Mechanical Hyperalgesia in the Carrageenan Inflammatory
Pain Model in Rats. A subcutaneous bolus injection of KAI-1678 or
KP-1723, the inactive analog of KAI-1678, was administered to rats
60 minutes after carrageenan injection into the plantar side of the
right hindpaw. KAI-1678 or KP-1723 was dosed at the indicated doses
to the contralateral hindlimb (i.e., the limb that did not receive
the carrageenan). Data are presented as mean.+-.SEM (standard error
of the mean) for the (PWT) measurements for the animals in each
group (N=2-5 animals/group) at a given time point. The dotted line
at .about.90 g represents the pre-carrageenan baseline PWT
measurement; PWT indicative of the disease state is .about.62 g.
Measurements at or above the pre-carrageenan baseline level
indicate complete reversal of carrageenan-induced mechanical
hyperalgesia.
[0031] FIG. 20: Effect of Dosing Location on Activity of
Subcutaneous Bolus Administration of KAI-1678 in the Rat
Carrageenan Inflammatory Pain Model. Rats injected with carrageenan
into the plantar surface of the right hindpaw were given two 10
mcg/kg subcutaneous bolus injections of KAI-1678 four hours apart
beginning 60 minutes after carrageenan injection. The two doses of
KAI-1678 were made to different sites on the rats as indicated.
Data are presented as mean.+-.SEM (standard error of the mean) for
the (PWT) measurements for the animals in each group (N=2
animals/group) at a given time point. The dotted line at .about.90
g represents the pre-carrageenan baseline PWT measurement; PWT
indicative of the disease state is .about.60 g. Measurements at or
above the pre-carrageenan baseline level indicate complete reversal
of carrageenan-induced mechanical hyperalgesia.
[0032] FIG. 21: Effects of Subcutaneous Infusion of KAI-1678 on
Mechanical Hyperalgesia in the Carrageenan Inflammatory Pain Model
in Rats. Rats were treated with a 6-hour subcutaneous infusion of
KAI-1678 or KP-1723, the inactive analog of KAI-1678, 60 minutes
after carrageenan injection into the plantar side of the right
hindpaw. KAI-1678 or KP-1723 was administered at the indicated dose
rates to the contralateral hindlimb (i.e. the limb that did not
receive the carrageenan). Data are presented as mean.+-.SEM
(standard error of the mean) for the (PWT) measurements for the
animals in each group (N=2-6 animals/group) at a given time point.
The dotted line at .about.90 g represents the pre-carrageenan
baseline PWT measurement; PWT indicative of the disease state is
.about.55 g. Measurements at or above the pre-carrageenan baseline
level indicate complete reversal of carrageenan-induced mechanical
hyperalgesia.
[0033] FIG. 22: Effects of Intravenous Infusion of KAI-1678 on
Mechanical Hyperalgesia in the Carrageenan Inflammatory Pain Model
in Rats. Rats were treated with an intravenous infusion of KAI-1678
starting 60 minutes after carrageenan injection into the plantar
side of the right hindpaw. KAI-1678 was infused via the jugular
vein at the indicated dose rates for either 2 hours (triangles) or
5 hours (circles). After the end of the 5-hour infusion, 10 mg/kg
indomethacin was administered by oral gavage to test the
responsiveness of the model. Data are presented as mean.+-.SEM
(standard error of the mean) for the (PWT) measurements for the
animals in each group (N=3 or 4 animals/group) at a given time
point. The dotted line at .about.90 g represents the
pre-carrageenan baseline PWT measurement; PWT indicative of the
disease state is .about.55 g. Measurements at or above the
pre-carrageenan baseline level indicate complete reversal of
carrageenan-induced mechanical hyperalgesia.
[0034] FIG. 23: Effect of Cutting the Sciatic and Saphenous Nerves
on the Activity of KAI-1678 in the Carrageenan Inflammatory Pain
Model in Rats. A 1-cm section of the sciatic and saphenous nerves
was surgically removed from the left (contralateral) hindleg of
rats. The next day, carrageenan was injected into the plantar side
of the right hindpaw of the surgically-treated rats. Sixty minutes
after carrageenan injection, a 4-hour subcutaneous infusion of
KAI-1678 at 25 mcg/kg/hr was initiated on the contralateral limb at
a site proximal (open circles) or distal (closed circles) to the
site of the nerve transection. Data are presented as mean.+-.SEM
(standard error of the mean) for the (PWT) measurements for the
animals in each group (N=4 animals/group) at a given time point.
The dotted line at .about.90 g represents the pre-carrageenan
baseline PWT measurement; PWT indicative of the disease state is
.about.60 g. Measurements at or above the pre-carrageenan baseline
level indicate complete reversal of carrageenan-induced mechanical
hyperalgesia.
[0035] FIG. 24: Effect of Surgical Sympathectomy on the Activity of
Subcutaneously Administered KAI-1678 on Mechanical Hyperalgesia in
the Carrageenan Inflammatory Pain Model in Rats. One week following
bilateral lumbar sympathectomy and suprarenal ganglionectomy,
carrageenan was injected into the plantar side of the right hindpaw
of surgically treated rats (Sympathectomized animals--closed
circles) or rats in which the lumbar sympathetic chain and
suprarenal ganglia were exposed but not removed (Sham surgery
animals--open circles). Sixty minutes after carrageenan injection,
a 4-hour subcutaneous infusion of KAI-1678 at 25 mcg/kg/hr was
initiated into the hindlimb of each animal, contralateral to the
site of carrageenan administration. Data are presented as
mean.+-.SEM (standard error of the mean) for the (PWT) measurements
for the animals in each group (N=2-4 animals/group) at a given time
point. The dotted line at .about.90 g represents the
pre-carrageenan baseline PWT measurement; PWT indicative of the
disease state is .about.55 g. Measurements at or above the
pre-carrageenan baseline level indicate complete reversal of
carrageenan-induced mechanical hyperalgesia.
[0036] FIG. 25: Effects of Subcutaneous Bolus Administration of
KAI-1678 on Mechanical Allodynia in the L5 Transection Neuropathic
Pain Model in Rats. Seven days after transection of the L5 spinal
nerve, rats were given a subcutaneous bolus of KAI-1678 at the
indicated doses (time zero). At the indicated times after compound
administration, animals were tested with von Frey filaments to
determine (left) the number of paw withdrawals out of 30 tests (5
tests at each filament) or (right) the paw withdrawal threshold
determined as the lowest von Frey filament that produced at least 3
withdrawals out of 5 tests. Data are presented as mean.+-.SEM
(standard error of the mean) for the animals in each group (N=6
animals/group) at a given time point.
[0037] FIG. 26: Effects of Subcutaneous Infusion of KAI-1678 on
Allodynia in the L5 Transection Neuropathic Pain Model in Rats. The
day after transection of the L5 spinal nerve, osmotic minipumps
containing KAI-1678 were implanted subcutaneously to deliver the
indicated daily doses of compound. At the indicated times, animals
were tested (left) with von Frey filaments to determine the number
of paw withdrawals out of 30 tests (5 tests at each filament) or
(right) the paw withdrawal latency in response to exposure to a
radiant heat source. Data are presented as mean.+-.SEM (standard
error of the mean) for the animals in each group (N=6
animals/group) at a given time point.
[0038] FIG. 27: KAI-1678 Plasma Concentrations in Rats After IV
Bolus. Plasma concentrations of KAI-1678 after intravenous bolus
administration at 300 and 3,000 mcg/kg. Averaged data from three
rats (300 mcg/kg) and two rats (3,000 mcg/kg) are shown. A
preliminary estimation of terminal half-life from these data is
.about.38 and .about.66 minutes (300 mcg/kg and 3,000 mcg/kg,
respectively).
[0039] FIG. 28: KAI-1678 Plasma Concentrations in Rats Dosed by IV
Infusion. Plasma concentrations of KAI-1678 administered as an
intravenous infusion at 50 mcg/kg/hr. Averaged data from three rats
are shown.
[0040] FIG. 29: KAI-1678 Plasma Concentrations in Rats after
Subcutaneous Bolus Injection. Plasma concentration of KAI-1678
after subcutaneous bolus administration at approximately 80 and 800
mcg/kg. Averaged data from three rats (80 mcg/kg) and four rats
(800 mcg/kg) are shown. A preliminary estimation of terminal
half-life from these data is .about.35 minutes.
[0041] FIG. 30: KAI-1678 Plasma Concentrations in Rats Dosed by
Subcutaneous Infusion. Plasma concentrations of KAI-1678 in rats
dosed by subcutaneous infusion for two hours. Averaged data from
two rats at each dose level are shown.
[0042] FIG. 31: KAI-1678 Plasma Concentrations in Dogs Dosed by
Subcutaneous Infusion over 5 Days. Plasma concentrations of
KAI-1678 in dogs dosed by subcutaneous infusion at 3, 8 and 25
mg/kg/day. Note that samples were recovered on Day 1 for the first
four hours and on Day 6 at the end of infusion (EOI), with no
sampling in the period between.
DISCLOSURE OF THE INVENTION
[0043] The disclosure herein relates to modified .epsilon.PKC
inhibitory peptides, methods of generating such peptides, and
method for using .epsilon.PKC inhibitory peptides for the treatment
of pain. The disclosed invention also relates to the role of
locally-administered protein kinase C epsilon (.epsilon.PKC)
inhibitors play in suppressing pain perception. Methods to suppress
pain systemically with an .epsilon.PKC inhibitor, particularly
through mechanisms that require affecting primary afferent function
and modulation of the sympathetic nervous system. Hybrid peptides
comprising an .epsilon.PKC-specific inhibitor and another PKC
modulatory peptide are also contemplated as falling within the
scope of the presently disclosed invention. Any PKC modulatory
peptide can be used to prepare the hybrid construct so that the
activity of more than one isozyme-specific PKC modulator is
combined into a single hybrid compound/peptide. Other aspects and
embodiments will be apparent to those skilled in the art form the
following detailed description.
DESCRIPTION OF THE INVENTION
[0044] The presently described invention relates to modified
peptides which inhibit the epsilon protein kinase C (.epsilon.PKC)
isozyme and are coupled to another isozyme-specific PKC modulator.
Typically, the .epsilon.PKC inhibitory peptides discussed herein
are coupled to a carrier moiety to facilitate transport of the
inhibitory peptide to a target cell. The cargo inhibitory peptide,
the carrier peptide, or both can be modified relative to a
prototype control to increase the stability of the resulting
cargo/carrier peptide constructs. The disclosed modified
.epsilon.PKC peptides are useful in preventing, reversing and
otherwise treating various types of pain, such as acute pain,
chronic pain, neuropathic pain and inflammatory pain. The
.epsilon.PKC inhibitory proteins can also be used to construct a
hybrid peptide construct, which comprises one or more
isozyme-specific PKC modulatory peptides with activity against
other PKC isozymes.
DEFINITIONS
[0045] As used in the present specification, the following words
and phrases are generally intended to have the meanings as set
forth below, except to the extent that the context in which they
are used indicates otherwise.
[0046] A "PKC modulatory compound" is any compound, including small
molecules and peptides, which is capable of modulating the
enzymatic activity of a PKC isozyme. The term "modulation" refers
to increasing or decreasing the enzymatic activity and other
functional activities of a PKC isozyme. A specific PKC modulator
("isozyme-specific PKC modulator") is any compound which measurably
modulates, either positively or negative, one PKC isozyme over
another.
[0047] A "PKC activator" is any compound, including small molecules
and peptides, which is capable of activating the enzymatic activity
of a PKC isozyme. A specific PKC activator is any compound which
measurably activates one PKC isozyme over another.
[0048] A "PKC inhibitor" is any compound, including small molecules
and peptides, which is capable of inhibiting the enzymatic activity
and other functional activities of a PKC isozyme. A specific PKC
inhibitor is any compound which measurably inhibits one PKC isozyme
over another.
[0049] An ".epsilon.PKC activator peptide" refers to a peptide that
can activate an .epsilon.PKC enzyme.
[0050] An ".epsilon.PKC inhibitory peptide" refers to a peptide
that can inhibit or inactivate an .epsilon.PKC enzyme.
[0051] An ".gamma.PKC activator peptide" refers to a peptide that
can activate an .gamma.PKC enzyme.
[0052] An ".gamma.PKC inhibitory peptide" refers to a peptide that
can inhibit or inactivate an .gamma.PKC enzyme.
[0053] The term "KAI-1586" refers to an peptide derived from the
first variable region of .epsilon.PKC conjugated via a Cys-Cys
disulfide linkage to a "capped" HIV Tat-derived transporter
peptide, and can be represented as follows:
##STR00001##
[0054] The term "KAI-1634" refers to two modified .epsilon.PKC
peptides derived from the first variable region of .epsilon.PKC,
which are covalently linked and a capped HIV Tat-derived
transporter peptide. The construct is depicted in FIG. 1.
[0055] The term "capped" refers to a peptide that has been
chemically modified to alter the amino terminus, carboxy terminus,
or both. A capped carrier peptide disulfide bonded to an unmodified
cargo peptide is shown in FIG. 2.
[0056] The term "carrier" refers to a moiety that facilitates
cellular uptake, such as cationic polymers, peptides and antibody
sequences, including polylysine, polyarginine, Antennapedia-derived
peptides, HIV Tat-derived peptides and the like, as described, for
example, in US Patents and Publications Nos., U.S. Pat. Nos.
4,847,240, 5,888,762, 5,747,641, 6,593,292, US2003/0104622,
US2003/0199677 and US2003/0206900. An example of a carrier moiety
is a "carrier peptide," which is a peptide which facilitates
cellular uptake of an .epsilon.PKC inhibitory peptide which is
chemically associated or bonded to the transporter peptide.
[0057] The term "prophylaxis" is intended as an element of
"treatment" to encompass both "preventing" and "suppressing" as
defined herein. It will be understood by those skilled in the art
that in human medicine it is not always possible to distinguish
between "preventing" and "suppressing" since the ultimate inductive
event or events may be unknown, latent, or the patient is not
ascertained until well after the occurrence of the event or
events.
[0058] The term "stability" refers generally to modifications that
improve shelf-life times, for example, retarding shelf life-based
cys-cys exchange, by retarding proteolytic degradation, or both.
The term "potency" relates to the amount of a particular peptide
composition required to achieve a particular result. One peptide
composition is more potent than another when dosages of the
composition can be reduced to achieve a desired end point. Certain
modifications of a given peptide composition can be made with
improve potency of that composition.
[0059] Epsilon Protein Kinase C (.epsilon.PKC) Inhibitors
[0060] There are many known inhibitors of .epsilon.PKC that can be
used in the instant invention. Small molecule inhibitors of PKC are
described in U.S. Pat. Nos. 5,141,957, 5,204,370, 5,216,014,
5,270,310, 5,292,737, 5,344,841, 5,360,818, 5,432,198, 5,380,746,
and 5,489,608, (European Patent 0,434,057), all of which are hereby
incorporated by reference in their entirety. These molecules belong
to the following classes:
N,N'-Bis-(sulfonamido)-2-amino-4-iminonaphthalen-1-ones;
N,N'-Bis-(amido)-2-amino-4-iminonaphthalen-1-ones;
vicinal-substituted carbocyclics; 1,3-dioxane derivatives;
1,4-Bis-(amino-hydroxyalkylamino)-anthraquinones;
furo-coumarinsulfonamides; Bis-(hydroxyalkylamino)-anthraquinones;
and N-aminoalkyl amides,
2-[1-(3-Aminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide,
2-[1-[2-(1-Methylpyrrolidino)ethyl]-1H-indol-3-yl]-3-(1H-indol-3-yl)malei-
mide, Go 7874. Other known small molecule inhibitors of PKC are
described in the following publications (Fabre, S., et al. 1993.
Bioorg. Med. Chem. 1, 193, Toullec, D., et al. 1991. J. Biol. Chem.
266, 15771, Gschwendt, M., et al. 1996. FEBS Lett. 392, 77,
Merritt, J. E., et al. 1997. Cell Signal 9, 53, Birchall, A. M., et
al. 1994. J. Pharmacol. Exp. Ther. 268, 922. Wilkinson, S. E., et
al. 1993. Biochem. J. 294, 335, Davis, P. D., et al. 1992. J. Med.
Chem. 35, 994), and belong to the following classes:
2,3-bis(1H-Indo)-3-yl)maleimide (Bisindolylmaleimide IV);
2-[1-(3-Dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)
maleimide (Go 6983);
2-{8-[(Dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl}-3-
-(1-methyl-1H-indol-3-yl)maleimide (Ro-32-0432);
2-[8-(Aminomethyl)-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl]-3-(1-methyl-
-1H-indol-3-yl)maleimide (Ro-31-8425); and
3-[1-[3-(Amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl)male-
imide Bisindolylmaleimide IX, Methanesulfonate (Ro-31-8220) all of
which are also hereby incorporated by reference in their
entirety.
[0061] Epsilon Protein Kinase C (.epsilon.PKC) Inhibitory
Peptides
[0062] Various .epsilon.PKC inhibitors are described herein and can
be used with the presently disclosed methods. The inhibitory
peptide can be derived from any domain, whether variable or
constant. Thus, inhibitory peptides can be derived from V1, V2, V3,
V4, or V5. Inhibitory peptides can also be derived from the
constant regions C1 (C1a, C1 b), C3, C4, or C5. Peptides
overlapping one or more of these regions are also contemplated.
Another source of prototype peptides can be found in U.S. patent
application Ser. No. 11/011,557, entitled, "Isozyme-specific
antagonists of protein kinase C," which is hereby incorporated by
reference in its entirety.
[0063] In one embodiment, the cargo peptide is an .epsilon.PKC
inhibitory peptide derivative of .epsilon.V1-2 comprising the amino
acid sequence of E-A-V-S-L-K-P-T (SEQ ID NO:58), a cysteine residue
located at the amino or carboxy terminal ends of the peptide, or
internally, and a carrier peptide linked to the cargo peptide. The
cargo peptide described above can further comprise one or more
additional cargo peptides, attached to one another and ultimately
to the carrier peptide.
[0064] Modifications to both the carrier and cargo have been made
with the goals of improving potency, stability in biological
fluids/tissues, and chemical stability. These changes provide an
.epsilon.PKC inhibitor with enhanced properties for use in a
variety of clinical indications.
[0065] Some of the modifications which have been applied include:
[0066] 1. Capping the cargo and/or carrier peptides to hinder
proteolysis in vivo, and thereby to increase potency and/or
duration of efficacy; [0067] 2. Generating overlap peptides
incorporating additional contiguous regions of the parent protein
to improve potency; [0068] 3. Making linear peptides which have
cargo and carrier in a single peptide chain to improve the chemical
stability and shelf-life of drug product; [0069] 4. Making multimer
peptides which have two or more copies of the active peptide to
improve protease resistance and potency; [0070] 5. Making
retro-inverso analogs of peptides to hinder proteolysis; and [0071]
6. Introducing disulfide analogs to provide improved chemical
stability.
[0072] The modifications described herein improve the potency,
plasma stability, and chemical stability of the modified
.epsilon.PKC inhibitory peptides. Effective modifications to
.epsilon.PKC inhibitory peptides are identified by selecting a
prototype .epsilon.PKC inhibitory peptide and modifying these
peptides to serve as cargo peptides for the treatment of pain. The
prototype peptide can be a presently known peptide or one as of yet
unidentified as an .epsilon.PKC inhibitory peptide. A preferred
prototype sequence is E-A-V-S-L-K-P-T (SEQ ID NO:58), where the
peptide is unmodified and conjugated to a carrier via Cys residues
located at the amino termini of the cargo and carrier peptides,
although any inhibitory .epsilon.PKC peptide can be used as the
starting cargo sequence. A variety of modified or analog peptides
are contemplated. Some such analogs comprise amino acid sequences
that overlap and extend beyond the prototype sequence. Other analog
peptides are truncated relative to the prototype. Additionally,
analogs of the prototype sequence may have one or more amino acid
substitutions relative to the prototype sequence, wherein the amino
acid substituted is an alanine residue or an aspartic acid residue.
The systematic generation of such alanine or aspartic acid
containing peptides is known as "scanning." The generation of
linear peptides comprising the analogs and modified carrier
peptides is further contemplated.
[0073] Additional modifications to prototype sequences are directed
at modifying specific degradation sites within the cargo peptide or
peptides, the carrier peptide or peptides, or both, and introducing
amino acid substitutions or other chemical modifications which
blocks these sites from degradation.
[0074] Table 1 lists a number of exemplary epsilon PKC inhibitory
peptides for use with the present invention as prototype
sequences.
TABLE-US-00001 TABLE 1 Peptides derived from epsilon PKC Peptide
SEQ ID NO. Sequence Position V1-1 SEQ ID NO: 15 N-G-L-L-K-I-K
PKC(5-11) V1-2 SEQ ID NO: 58 E-A-V-S-L-K-P-T PKC(14-21) V1-3 SEQ ID
NO: 16 L-A-V-F-H-D-A-P- PKC(81-91) I-G-Y V1-4 SEQ ID NO: 17
D-D-F-V-A-N-C-T- PKC(92-100) I V1-5 SEQ ID NO: 18 W-I-D-L-E-P-E-G-
PKC(116-125) R-V V1-6 SEQ ID NO: 19 H-A-V-G-P-R-P-Q- PKC(27-36) T-F
V1-7 SEQ ID NO: 20 N-G-S-R-H-F-E-D PKC(108-115) V1-7.1 SEQ ID NO:
21 H-D-A-P-I-G-D-Y -- V1-7.2 SEQ ID NO: 22 H-D-A-P-I-G -- V1-7.3
SEQ ID NO: 26 H-D-A-A-I-G-Y-D -- V1-7.4 SEQ ID NO: 27
H-D-A-P-I-P-Y-D -- V1-7.5 SEQ ID NO: 28 H-N-A-P-I-G-Y-D -- V1-7.6
SEQ ID NO: 29 H-A-A-P-I-G-Y-D -- V1-7.7 SEQ ID NO: 30
A-D-A-P-I-G-Y-D -- V1-7.8 SEQ ID NO: 31 H-D-A-P-A-G-Y-D -- V1-7.9
SEQ ID NO: 32 H-D-A-P-I-G-A-D -- V1-7.10 SEQ ID NO: 33
H-D-A-P-I-A-Y-D -- V1-7.11 SEQ ID NO: 34 H-D-A-P-I-G-Y-A -- V3-1
SEQ ID NO: 35 S-S-P-S-E-E-D-R- PKC(336-344) S V3-2 SEQ ID NO: 36
P-C-D-Q-E-I-K-E PKC(351-358) V3-3 SEQ ID NO: 37 E-N-N-I-R-K-A-L-
PKC(360-368) S V3-4 SEQ ID NO: 38 G-E-V-R-Q-G-Q-A PKC(393-400) V5-1
SEQ ID NO: 39 E-A-I-V-K-Q PKC(714-719) V5-2 SEQ ID NO: 40
I-K-T-K-R-D-V PKC(689-695) V5-2.1 SEQ ID NO: 41 I-K-T-K-R-L-I --
V5-3 SEQ ID NO: 42 C-E-A-I-V-K-Q PKC(714-719) V5-4 SEQ ID NO: 43
T-K-R-D-V-N-N-F- PKC(791-800) D-Q
[0075] As discussed more fully below, it is preferable that the
.epsilon.PKC inhibitory peptide be chemically associated with a
carrier moiety, such as a carrier peptide. In one embodiment, the
inhibitory peptide and the carrier peptide are linked via a
disulfide bond. Electrostatic and hydrophobic interactions can also
be exploited to associate chemically the carrier moiety with the
.epsilon.PKC inhibitory peptide. In the case of the forming a
disulfide bond, it may be advantageous to add a Cys residue to the
PKC inhibitory peptide sequence or to the carrier peptide sequence.
The Cys residue can be added to the amino or carboxy termini, or
both. The Cys residue can also be located within the amino acid
sequence of the cargo or carrier peptides. Such endogenous Cys
residues have been shown to stabilize a disulfide bond linkage
between the carrier and cargo peptides. Another linking system
involves linearizing peptides of interest using glycine residue
linkers. One preferred embodiment is KP-1678, which has the
sequence of .epsilon.V1-2 and a TAT carrier peptide, wherein the
amino terminus is acetylated and the carboxy terminus is modified
with an amino group (Ac--EAVSLKPTGGYGRKKRRQRRR-NH.sub.2) (SEQ ID
NO:57).
[0076] Hybrid Peptide Constructs
[0077] As discussed above a variety of modifications to PKC
modulatory peptides are contemplated. One example of such a
modification includes the construction of linear peptides
comprising, for example a cargo PKC modulatory peptide and a
carrier peptide. Another example is a multimer peptide construct
comprising a plurality of PKC modulatory peptides and a carrier
peptide. Either peptide design model can be modified such that
multiple modulatory PKC peptides are included, and those modulatory
peptides can be selected such that different PKC isozymes can be
modulated with the same construct.
[0078] The hybrid peptide approach has various advantages over a
single functionality peptide construct. For example, using multiple
PKC modulatory peptides in the same construct permits one to
modulate simultaneously two or more different PKC isozymes using
otherwise isozyme specific modulatory peptides. Joint modulation in
this manner, while less specific than the use of isozyme specific
peptides alone, is still more specific than using isozyme
non-specific peptide modulators and other small molecule type
kinase inhibitors. The use of isozyme specific modulatory peptides
is merely exemplary. Hybrid peptide constructs can also comprise
modulatory peptides which are either specific or non-specific for
any PKC isozyme.
[0079] Peptide modulators of any of the PKC isozymes are
contemplated for use to construct hybrid peptide constructs. The
PKC family of isozymes includes at least 11 different protein
kinases that can be divided into at least three subfamilies based
on their homology and sensitivity to activators. Each isozyme
includes a number of homologous ("conserved" or "C") domains
interspersed with isozyme-unique ("variable" or "V") domains.
Members of the "classical" or ".epsilon.PKC" subfamily, .alpha.,
.beta..sub.I, .beta..sub.II, and .gamma.PKC, contain four
homologous domains (C1, C2, C3 and C4) and require calcium,
phosphatidylserine, and diacylglycerol or phorbol esters for
activation. In members of the "novel" or "nPKC" subfamily, .delta.,
.epsilon., .eta. and .theta.PKC, a C2-like domain precedes the C1
domain. However, that C2 domain does not bind calcium and therefore
the nPKC subfamily does not require calcium for activation.
Finally, members of the "atypical" or "aPKC" subfamily, .zeta. and
.lamda.PKC, lack both the C2 and one-half of the C1 homologous
domains and are insensitive to diacylglycerol, phorbol esters and
calcium. Modulatory peptides with activity against one or more of
the PKC isozymes can be used to prepare hybrid peptide
constructs.
[0080] In a preferred embodiment, hybrid peptide constructs
comprising one or more .epsilon.PKC modulatory peptides and one or
more .gamma.PKC modulatory peptides. One a preferred embodiment,
one or more .epsilon.PKC inhibitory peptides are used with one or
more .gamma.PKC inhibitory peptides to construct an
.epsilon.PKC-.gamma.PKC hybrid inhibitory peptide.
[0081] Various .epsilon.PKC inhibitory peptides are discussed
above, and these peptides are examples of some of the peptides that
can be used to prepare hybrid peptide constructs. Various
.gamma.PKC inhibitors are described herein and can be used with the
presently disclosed methods. The inhibitory peptide can be derived
from any domain, whether variable or constant. Thus, inhibitory
peptides can be derived from V1, V2, V3, V4, or V5. Inhibitory
peptides can also be derived from the constant regions C1 (C1a,
C1b), C3, C4, or C5. Peptides overlapping one or more of these
regions are also contemplated. The cargo peptides derived from the
various domains and range in length from 5 to 30 amino acids in
length. More particularly, the peptides derived from the PKC domain
are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, or 30 residues in length. Another
source of prototype peptides can be found in U.S. patent
application Ser. No. 11/011,557, entitled, "Isozyme-specific
antagonists of protein kinase C," which takes activator peptides
and converts them to inhibitor peptides, and which is hereby
incorporated by reference in its entirety. A preferred .gamma.PKC
inhibitory peptide prototype sequence is R-L-V-L-A-S (SEQ ID NO:1).
All of the modifications described above in relation to the PKC
peptides are equally applicable to the .gamma.PKC inhibitory
peptides contemplated for use in the hybrid constructs.
[0082] The following tables list a number of exemplary gamma PKC
inhibitory peptides for use with the present invention as prototype
sequences.
TABLE-US-00002 TABLE 2 BASIC SET CARGO CARRIER SEQ SEQ ID LINKER ID
N-term Cargo NO: C-term Linker N-term Carrier NO: C-term Amine
CRLVLAS 2 Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Acetyl
CRLVLAS 2 Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Acetyl
CRLVLAS 2 Amide Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Amine
CRLVLAS 2 Amide Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Amine
CRLVLAS 2 Carboxyl Disulfide Acetyl CYGRKKRRQRRR 4 Carboxyl Amine
CRLVLAS 2 Carboxyl Disulfide Acetyl CYGRKKRRQRRR 4 Amide Amine
CRLVLAS 2 Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Amide Acetyl
CRLVLAS 2 Amide Disulfide Acetyl CYGRKKRRQRRR 4 Amide Amine RLVLASC
3 Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Acetyl RLVLASC 3
Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Acetyl RLVLASC 3
Amide Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Amine RLVLASC 3 Amide
Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Amine RLVLASC 3 Carboxyl
Disulfide Acetyl CYGRKKRRQRRR 4 Carboxyl Amine RLVLASC 3 Carboxyl
Disulfide Acetyl CYGRKKRRQRRR 4 Amide Amine RLVLASC 3 Carboxyl
Disulfide Amine CYGRKKRRQRRR 4 Amide Acetyl RLVLASC 3 Amide
Disulfide Acetyl CYGRKKRRQRRR 4 Amide Amine RLVLASC 3 Carboxyl
Disulfide Amine YGRKKRRQRRRC 5 Carboxyl Acetyl RLVLASC 3 Carboxyl
Disulfide Amine YGRKKRRQRRRC 5 Carboxyl Acetyl RLVLASC 3 Amide
Disulfide Amine YGRKKRRQRRRC 5 Carboxyl Amine RLVLASC 3 Amide
Disulfide Amine YGRKKRRQRRRC 5 Carboxyl Amine RLVLASC 3 Carboxyl
Disulfide Acetyl YGRKKRRQRRRC 5 Carboxyl Amine RLVLASC 3 Carboxyl
Disulfide Acetyl YGRKKRRQRRRC 5 Amide Amine RLVLASC 3 Carboxyl
Disulfide Amine YGRKKRRQRRRC 5 Amide Acetyl RLVLASC 3 Amide
Disulfide Acetyl YGRKKRRQRRRC 5 Amide Amine CRLVLAS 2 Carboxyl
Disulfide Amine YGRKKRRQRRRC 5 Carboxyl Acetyl CRLVLAS 2 Carboxyl
Disulfide Amine YGRKKRRQRRRC 5 Carboxyl Acetyl CRLVLAS 2 Amide
Disulfide Amine YGRKKRRQRRRC 5 Carboxyl Amine CRLVLAS 3 Amide
Disulfide Amine YGRKKRRQRRRC 5 Carboxyl Amine CRLVLAS 2 Carboxyl
Disulfide Acetyl YGRKKRRQRRRC 5 Carboxyl Amine CRLVLAS 2 Carboxyl
Disulfide Acetyl YGRKKRRQRRRC 5 Amide Amine CRLVLAS 2 Carboxyl
Disulfide Amine YGRKKRRQRRRC 5 Amide Acetyl CRLVLAS 2 Amide
Disulfide Acetyl YGRKKRRQRRRC 5 Amide
TABLE-US-00003 TABLE 3 HOMOCYSTEINE (homoC) CARGO CARRIER SEQ SEQ
ID LINKER ID N-term Cargo NO: C-term Linker N-term Carrier NO:
C-term Amine homoC-RLVLAS 6 Carboxyl Disulfide Amine
homoC-YGRKKRRQRRR 8 Carboxyl Acetyl homoC-RLVLAS 6 Carboxyl
Disulfide Amine homoC-YGRKKRRQRRR 8 Carboxyl Acetyl homoC-RLVLAS 6
Amide Disulfide Amine homoC-YGRKKRRQRRR 8 Carboxyl Amine
homoC-RLVLAS 6 Amide Disulfide Amine homoC-YGRKKRRQRRR 8 Carboxyl
Amine homoC-RLVLAS 6 Carboxyl Disulfide Acetyl homoC-YGRKKRRQRRR 8
Carboxyl Amine homoC-RLVLAS 6 Carboxyl Disulfide Acetyl
homoC-YGRKKRRQRRR 8 Amide Amine homoC-RLVLAS 6 Carboxyl Disulfide
Amine homoC-YGRKKRRQRRR 8 Amide Acetyl homoC-RLVLAS 6 Amide
Disulfide Acetyl homoC-YGRKKRRQRRR 8 Amide Amine RLVLAS-homoC 7
Carboxyl Disulfide Amine homoC-YGRKKRRQRRR 8 Carboxyl Acetyl
RLVLAS-homoC 7 Carboxyl Disulfide Amine homoC-YGRKKRRQRRR 8
Carboxyl Acetyl RLVLAS-homoC 7 Amide Disulfide Amine
homoC-YGRKKRRQRRR 8 Carboxyl Amine RLVLAS-homoC 7 Amide Disulfide
Amine homoC-YGRKKRRQRRR 8 Carboxyl Amine RLVLAS-homoC 7 Carboxyl
Disulfide Acetyl homoC-YGRKKRRQRRR 8 Carboxyl Amine RLVLAS-homoC 7
Carboxyl Disulfide Acetyl homoC-YGRKKRRQRRR 8 Amide Amine
RLVLAS-homoC 7 Carboxyl Disulfide Amine homoC-YGRKKRRQRRR 8 Amide
Acetyl RLVLAS-homoC 7 Amide Disulfide Acetyl homoC-YGRKKRRQRRR 8
Amide Amine RLVLAS-homoC 7 Carboxyl Disulfide Amine
YGRKKRRQRRR-homoC 9 Carboxyl Acetyl RLVLAS-homoC 7 Carboxyl
Disulfide Amine YGRKKRRQRRR-homoC 9 Carboxyl Acetyl RLVLAS-homoC 7
Amide Disulfide Amine YGRKKRRQRRR-homoC 9 Carboxyl Amine
RLVLAS-homoC 7 Amide Disulfide Amine YGRKKRRQRRR-homoC 9 Carboxyl
Amine RLVLAS-homoC 7 Carboxyl Disulfide Acetyl YGRKKRRQRRR-homoC 9
Carboxyl Amine RLVLAS-homoC 7 Carboxyl Disulfide Acetyl
YGRKKRRQRRR-homoC 9 Amide Amine RLVLAS-homoC 7 Carboxyl Disulfide
Amine YGRKKRRQRRR-homoC 9 Amide Acetyl RLVLAS-homoC 7 Amide
Disulfide Acetyl YGRKKRRQRRR-homoC 9 Amide Amine homoC-RLVLAS 6
Carboxyl Disulfide Amine YGRKKRRQRRR-homoC 9 Carboxyl Acetyl
homoC-RLVLAS 6 Carboxyl Disulfide Amine YGRKKRRQRRR-homoC 9
Carboxyl Acetyl homoC-RLVLAS 6 Amide Disulfide Amine
YGRKKRRQRRR-homoC 9 Carboxyl Amine homoC-RLVLAS 6 Amide Disulfide
Amine YGRKKRRQRRR-homoC 9 Carboxyl Amine homoC-RLVLAS 6 Carboxyl
Disulfide Acetyl YGRKKRRQRRR-homoC 9 Carboxyl Amine homoC-RLVLAS 6
Carboxyl Disulfide Acetyl YGRKKRRQRRR-homoC 9 Amide Amine
homoC-RLVLAS 6 Carboxyl Disulfide Amine YGRKKRRQRRR-homoC 9 Amide
Acetyl homoC-RLVLAS 6 Amide Disulfide Acetyl YGRKKRRQRRR-homoC 9
Amide
TABLE-US-00004 TABLE 4 HOMOCYSTEINE (homoC)-Cargo only CARGO
CARRIER SEQ SEQ ID LINKER ID N-term Cargo NO: C-term Linker N-term
Carrier NO: C-term Amine homoC-RLVLAS 6 Carboxyl Disulfide Amine
CYGRKKRRQRRR 4 Carboxyl Acetyl homoC-RLVLAS 6 Carboxyl Disulfide
Amine CYGRKKRRQRRR 4 Carboxyl Acetyl homoC-RLVLAS 6 Amide Disulfide
Amine CYGRKKRRQRRR 4 Carboxyl Amine homoC-RLVLAS 6 Amide Disulfide
Amine CYGRKKRRQRRR 4 Carboxyl Amine homoC-RLVLAS 6 Carboxyl
Disulfide Acetyl CYGRKKRRQRRR 4 Carboxyl Amine homoC-RLVLAS 6
Carboxyl Disulfide Acetyl CYGRKKRRQRRR 4 Amide Amine homoC-RLVLAS 6
Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Amide Acetyl homoC-RLVLAS 6
Amide Disulfide Acetyl CYGRKKRRQRRR 4 Amide Amine RLVLAS-homoC 7
Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Acetyl
RLVLAS-homoC 7 Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Carboxyl
Acetyl RLVLAS-homoC 7 Amide Disulfide Amine CYGRKKRRQRRR 4 Carboxyl
Amine RLVLAS-homoC 7 Amide Disulfide Amine CYGRKKRRQRRR 4 Carboxyl
Amine RLVLAS-homoC 7 Carboxyl Disulfide Acetyl CYGRKKRRQRRR 4
Carboxyl Amine RLVLAS-homoC 7 Carboxyl Disulfide Acetyl
CYGRKKRRQRRR 4 Amide Amine RLVLAS-homoC 7 Carboxyl Disulfide Amine
CYGRKKRRQRRR 4 Amide Acetyl RLVLAS-homoC 7 Amide Disulfide Acetyl
CYGRKKRRQRRR 4 Amide Amine RLVLAS-homoC 7 Carboxyl Disulfide Amine
YGRKKRRQRRRC 5 Carboxyl Acetyl RLVLAS-homoC 7 Carboxyl Disulfide
Amine YGRKKRRQRRRC 5 Carboxyl Acetyl RLVLAS-homoC 7 Amide Disulfide
Amine YGRKKRRQRRRC 5 Carboxyl Amine RLVLAS-homoC 7 Amide Disulfide
Amine YGRKKRRQRRRC 5 Carboxyl Amine RLVLAS-homoC 7 Carboxyl
Disulfide Acetyl YGRKKRRQRRRC 5 Carboxyl Amine RLVLAS-homoC 7
Carboxyl Disulfide Acetyl YGRKKRRQRRRC 5 Amide Amine RLVLAS-homoC 7
Carboxyl Disulfide Amine YGRKKRRQRRRC 5 Amide Acetyl RLVLAS-homoC 7
Amide Disulfide Acetyl YGRKKRRQRRRC 5 Amide Amine homoC-RLVLAS 6
Carboxyl Disulfide Amine YGRKKRRQRRRC 5 Carboxyl Acetyl
homoC-RLVLAS 6 Carboxyl Disulfide Amine YGRKKRRQRRRC 5 Carboxyl
Acetyl homoC-RLVLAS 6 Amide Disulfide Amine YGRKKRRQRRRC 5 Carboxyl
Amine homoC-RLVLAS 6 Amide Disulfide Amine YGRKKRRQRRRC 5 Carboxyl
Amine homoC-RLVLAS 6 Carboxyl Disulfide Acetyl YGRKKRRQRRRC 5
Carboxyl Amine homoC-RLVLAS 6 Carboxyl Disulfide Acetyl
YGRKKRRQRRRC 5 Amide Amine homoC-RLVLAS 6 Carboxyl Disulfide Amine
YGRKKRRQRRRC 5 Amide Acetyl homoC-RLVLAS 6 Amide Disulfide Acetyl
YGRKKRRQRRRC 5 Amide
TABLE-US-00005 TABLE 5 HOMOCYSTEINE (homoC)-Carrier only CARGO
CARRIER SEQ SEQ ID LINKER ID N-term Cargo NO: C-term Linker N-term
Carrier NO: C-term Amine CRLVLAS 2 Carboxyl Disulfide Amine
homoC-YGRKKRRQRRR 8 Carboxyl Acetyl CRLVLAS 2 Carboxyl Disulfide
Amine homoC-YGRKKRRQRRR 8 Carboxyl Acetyl CRLVLAS 2 Amide Disulfide
Amine homoC-YGRKKRRQRRR 8 Carboxyl Amine CRLVLAS 2 Amide Disulfide
Amine homoC-YGRKKRRQRRR 8 Carboxyl Amine CRLVLAS 2 Carboxyl
Disulfide Acetyl homoC-YGRKKRRQRRR 8 Carboxyl Amine CRLVLAS 2
Carboxyl Disulfide Acetyl homoC-YGRKKRRQRRR 8 Amide Amine CRLVLAS 2
Carboxyl Disulfide Amine homoC-YGRKKRRQRRR 8 Amide Acetyl CRLVLAS 2
Amide Disulfide Acetyl homoC-YGRKKRRQRRR 8 Amide Amine RLVLASC 3
Carboxyl Disulfide Amine homoC-YGRKKRRQRRR 8 Carboxyl Acetyl
RLVLASC 3 Carboxyl Disulfide Amine homoC-YGRKKRRQRRR 8 Carboxyl
Acetyl RLVLASC 3 Amide Disulfide Amine homoC-YGRKKRRQRRR 8 Carboxyl
Amine RLVLASC 3 Amide Disulfide Amine homoC-YGRKKRRQRRR 8 Carboxyl
Amine RLVLASC 3 Carboxyl Disulfide Acetyl homoC-YGRKKRRQRRR 8
Carboxyl Amine RLVLASC 3 Carboxyl Disulfide Acetyl
homoC-YGRKKRRQRRR 8 Amide Amine RLVLASC 3 Carboxyl Disulfide Amine
homoC-YGRKKRRQRRR 8 Amide Acetyl RLVLASC 3 Amide Disulfide Acetyl
homoC-YGRKKRRQRRR 8 Amide Amine RLVLASC 3 Carboxyl Disulfide Amine
YGRKKRRQRRR-homoC 9 Carboxyl Acetyl RLVLASC 3 Carboxyl Disulfide
Amine YGRKKRRQRRR-homoC 9 Carboxyl Acetyl RLVLASC 3 Amide Disulfide
Amine YGRKKRRQRRR-homoC 9 Carboxyl Amine RLVLASC 3 Amide Disulfide
Amine YGRKKRRQRRR-homoC 9 Carboxyl Amine RLVLASC 3 Carboxyl
Disulfide Acetyl YGRKKRRQRRR-homoC 9 Carboxyl Amine RLVLASC 3
Carboxyl Disulfide Acetyl YGRKKRRQRRR-homoC 9 Amide Amine RLVLASC 3
Carboxyl Disulfide Amine YGRKKRRQRRR-homoC 9 Amide Acetyl RLVLASC 3
Amide Disulfide Acetyl YGRKKRRQRRR-homoC 9 Amide Amine CRLVLAS 2
Carboxyl Disulfide Amine YGRKKRRQRRR-homoC 9 Carboxyl Acetyl
CRLVLAS 2 Carboxyl Disulfide Amine YGRKKRRQRRR-homoC 9 Carboxyl
Acetyl CRLVLAS 2 Amide Disulfide Amine YGRKKRRQRRR-homoC 9 Carboxyl
Amine CRLVLAS 2 Amide Disulfide Amine YGRKKRRQRRR-homoC 9 Carboxyl
Amine CRLVLAS 2 Carboxyl Disulfide Acetyl YGRKKRRQRRR-homoC 9
Carboxyl Amine CRLVLAS 2 Carboxyl Disulfide Acetyl
YGRKKRRQRRR-homoC 9 Amide Amine CRLVLAS 2 Carboxyl Disulfide Amine
YGRKKRRQRRR-homoC 9 Amide Acetyl CRLVLAS 2 Amide Disulfide Acetyl
YGRKKRRQRRR-homoC 9 Amide
TABLE-US-00006 TABLE 6 MERCAPTOPROPIONIC ACID (MerPC) CARGO CARRIER
SEQ SEQ ID LINKER ID N-term Cargo NO: C-term Linker N-term Carrier
NO: C-term Amine MerPC-RLVLAS 10 Carboxyl Disulfide Amine
MerPC-YGRKKRRQRRR 11 Carboxyl Acetyl MerPC-RLVLAS 10 Carboxyl
Disulfide Amine MerPC-YGRKKRRQRRR 11 Carboxyl Acetyl MerPC-RLVLAS
10 Amide Disulfide Amine MerPC-YGRKKRRQRRR 11 Carboxyl Amine
MerPC-RLVLAS 10 Amide Disulfide Amine MerPC-YGRKKRRQRRR 11 Carboxyl
Amine MerPC-RLVLAS 10 Carboxyl Disulfide Acetyl MerPC-YGRKKRRQRRR
11 Carboxyl Amine MerPC-RLVLAS 10 Carboxyl Disulfide Acetyl
MerPC-YGRKKRRQRRR 11 Amide Amine MerPC-RLVLAS 10 Carboxyl Disulfide
Amine MerPC-YGRKKRRQRRR 11 Amide Acetyl MerPC-RLVLAS 10 Amide
Disulfide Acetyl MerPC-YGRKKRRQRRR 11 Amide Amine MerPC-RLVLAS 10
Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Acetyl
MerPC-RLVLAS 10 Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Carboxyl
Acetyl MerPC-RLVLAS 10 Amide Disulfide Amine CYGRKKRRQRRR 4
Carboxyl Amine MerPC-RLVLAS 10 Amide Disulfide Amine CYGRKKRRQRRR 4
Carboxyl Amine MerPC-RLVLAS 10 Carboxyl Disulfide Acetyl
CYGRKKRRQRRR 4 Carboxyl Amine MerPC-RLVLAS 10 Carboxyl Disulfide
Acetyl CYGRKKRRQRRR 4 Amide Amine MerPC-RLVLAS 10 Carboxyl
Disulfide Amine CYGRKKRRQRRR 4 Amide Acetyl MerPC-RLVLAS 10 Amide
Disulfide Acetyl CYGRKKRRQRRR 4 Amide Amine CRLVLAS 2 Carboxyl
Disulfide Amine MerPC-YGRKKRRQRRR 11 Carboxyl Acetyl CRLVLAS 2
Carboxyl Disulfide Amine MerPC-YGRKKRRQRRR 11 Carboxyl Acetyl
CRLVLAS 2 Amide Disulfide Amine MerPC-YGRKKRRQRRR 11 Carboxyl Amine
CRLVLAS 2 Amide Disulfide Amine MerPC-YGRKKRRQRRR 11 Carboxyl Amine
CRLVLAS 2 Carboxyl Disulfide Acetyl MerPC-YGRKKRRQRRR 11 Carboxyl
Amine CRLVLAS 2 Carboxyl Disulfide Acetyl MerPC-YGRKKRRQRRR 11
Amide Amine CRLVLAS 2 Carboxyl Disulfide Amine MerPC-YGRKKRRQRRR 11
Amide Acetyl CRLVLAS 2 Amide Disulfide Acetyl MerPC-YGRKKRRQRRR 11
Amide
TABLE-US-00007 TABLE 7 MERCAPTOACETIC ACID (MerAC) CARGO CARRIER
SEQ SEQ ID LINKER ID N-term Cargo NO: C-term Linker N-term Carrier
NO: C-term Amine MerAC-RLVLAS 24 Carboxyl Disulfide Amine MerAC- 25
Carboxyl YGRKKRRQRRR Acetyl MerAC-RLVLAS 24 Carboxyl Disulfide
Amine MerAC- 25 Carboxyl YGRKKRRQRRR Acetyl MerAC-RLVLAS 24 Amide
Disulfide Amine MerAC- 25 Carboxyl YGRKKRRQRRR Amine MerAC-RLVLAS
24 Amide Disulfide Amine MerAC- 25 Carboxyl YGRKKRRQRRR Amine
MerAC-RLVLAS 24 Carboxyl Disulfide Acetyl MerAC- 25 Carboxyl
YGRKKRRQRRR Amine MerAC-RLVLAS 24 Carboxyl Disulfide Acetyl MerAC-
25 Amide YGRKKRRQRRR Amine MerAC-RLVLAS 24 Carboxyl Disulfide Amine
MerAC- 25 Amide YGRKKRRQRRR Acetyl MerAC-RLVLAS 24 Amide Disulfide
Acetyl MerAC- 25 Amide YGRKKRRQRRR Amine MerAC-RLVLAS 24 Carboxyl
Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Acetyl MerAC-RLVLAS 24
Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Acetyl
MerAC-RLVLAS 24 Amide Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Amine
MerAC-RLVLAS 24 Amide Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Amine
MerAC-RLVLAS 24 Carboxyl Disulfide Acetyl CYGRKKRRQRRR 4 Carboxyl
Amine MerAC-RLVLAS 24 Carboxyl Disulfide Acetyl CYGRKKRRQRRR 4
Amide Amine MerAC-RLVLAS 24 Carboxyl Disulfide Amine CYGRKKRRQRRR 4
Amide Acetyl MerAC-RLVLAS 24 Amide Disulfide Acetyl CYGRKKRRQRRR 4
Amide Amine CRLVLAS 2 Carboxyl Disulfide Amine MerAC- 25 Carboxyl
YGRKKRRQRRR Acetyl CRLVLAS 2 Carboxyl Disulfide Amine MerAC- 25
Carboxyl YGRKKRRQRRR Acetyl CRLVLAS 2 Amide Disulfide Amine MerAC-
25 Carboxyl YGRKKRRQRRR Amine CRLVLAS 2 Amide Disulfide Amine
MerAC- 25 Carboxyl YGRKKRRQRRR Amine CRLVLAS 2 Carboxyl Disulfide
Acetyl MerAC- 25 Carboxyl YGRKKRRQRRR Amine CRLVLAS 2 Carboxyl
Disulfide Acetyl MerAC- 25 Amide YGRKKRRQRRR Amine CRLVLAS 2
Carboxyl Disulfide Amine MerAC- 25 Amide YGRKKRRQRRR Acetyl CRLVLAS
2 Amide Disulfide Acetyl MerAC- 25 Amide YGRKKRRQRRR
TABLE-US-00008 TABLE 8 MERCAPTOBUTYRIC ACID (MerBC) CARGO CARRIER
SEQ SEQ ID LINKER ID N-term Cargo NO: C-term Linker N-term Carrier
NO: C-term Amine MerBC-RLVLAS 23 Carboxyl Disulfide Amine MerBC- 12
Carboxyl YGRKKRRQRRR Acetyl MerBC-RLVLAS 23 Carboxyl Disulfide
Amine MerBC- 12 Carboxyl YGRKKRRQRRR Acetyl MerBC-RLVLAS 23 Amide
Disulfide Amine MerBC- 12 Carboxyl YGRKKRRQRRR Amine MerBC-RLVLAS
23 Amide Disulfide Amine MerBC- 12 Carboxyl YGRKKRRQRRR Amine
MerBC-RLVLAS 23 Carboxyl Disulfide Acetyl MerBC- 12 Carboxyl
YGRKKRRQRRR Amine MerBC-RLVLAS 23 Carboxyl Disulfide Acetyl MerBC-
12 Amide YGRKKRRQRRR Amine MerBC-RLVLAS 23 Carboxyl Disulfide Amine
MerBC- 12 Amide YGRKKRRQRRR Acetyl MerBC-RLVLAS 23 Amide Disulfide
Acetyl MerBC- 12 Amide YGRKKRRQRRR Amine MerBC-RLVLAS 23 Carboxyl
Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Acetyl MerBC-RLVLAS 23
Carboxyl Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Acetyl
MerBC-RLVLAS 23 Amide Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Amine
MerBC-RLVLAS 23 Amide Disulfide Amine CYGRKKRRQRRR 4 Carboxyl Amine
MerBC-RLVLAS 23 Carboxyl Disulfide Acetyl CYGRKKRRQRRR 4 Carboxyl
Amine MerBC-RLVLAS 23 Carboxyl Disulfide Acetyl CYGRKKRRQRRR 4
Amide Amine MerBC-RLVLAS 23 Carboxyl Disulfide Amine CYGRKKRRQRRR 4
Amide Acetyl MerBC-RLVLAS 23 Amide Disulfide Acetyl CYGRKKRRQRRR 4
Amide Amine CRLVLAS 2 Carboxyl Disulfide Amine MerBC- 12 Carboxyl
YGRKKRRQRRR Acetyl CRLVLAS 2 Carboxyl Disulfide Amine MerBC- 12
Carboxyl YGRKKRRQRRR Acetyl CRLVLAS 2 Amide Disulfide Amine MerBC-
12 Carboxyl YGRKKRRQRRR Amine CRLVLAS 2 Amide Disulfide Amine
MerBC- 12 Carboxyl YGRKKRRQRRR Amine CRLVLAS 2 Carboxyl Disulfide
Acetyl MerBC- 12 Carboxyl YGRKKRRQRRR Amine CRLVLAS 2 Carboxyl
Disulfide Acetyl MerBC- 12 Amide YGRKKRRQRRR Amine CRLVLAS 2
Carboxyl Disulfide Amine MerBC- 12 Amide YGRKKRRQRRR Acetyl CRLVLAS
2 Amide Disulfide Acetyl MerBC- 12 Amide YGRKKRRQRRR
TABLE-US-00009 TABLE 9 Ala-Cys CARGO CARRIER SEQ SEQ ID LINKER ID
N-term Cargo NO: C-term Linker N-term Carrier NO: C-term Amine
ACRLVLAS 13 Carboxyl Disulfide Amine ACYGRKKRRQRRR 14 Carboxyl
Acetyl ACRLVLAS 13 Carboxyl Disulfide Amine ACYGRKKRRQRRR 14
Carboxyl Acetyl ACRLVLAS 13 Amide Disulfide Amine ACYGRKKRRQRRR 14
Carboxyl Amine ACRLVLAS 13 Amide Disulfide Amine ACYGRKKRRQRRR 14
Carboxyl Amine ACRLVLAS 13 Carboxyl Disulfide Acetyl ACYGRKKRRQRRR
14 Carboxyl Amine ACRLVLAS 13 Carboxyl Disulfide Acetyl
ACYGRKKRRQRRR 14 Amide Amine ACRLVLAS 13 Carboxyl Disulfide Amine
ACYGRKKRRQRRR 14 Amide Acetyl ACRLVLAS 13 Amide Disulfide Acetyl
ACYGRKKRRQRRR 14 Amide Amine ACRLVLAS 13 Carboxyl Disulfide Amine
CYGRKKRRQRRR 4 Carboxyl Acetyl ACRLVLAS 13 Carboxyl Disulfide Amine
CYGRKKRRQRRR 4 Carboxyl Acetyl ACRLVLAS 13 Amide Disulfide Amine
CYGRKKRRQRRR 4 Carboxyl Amine ACRLVLAS 13 Amide Disulfide Amine
CYGRKKRRQRRR 4 Carboxyl Amine ACRLVLAS 13 Carboxyl Disulfide Acetyl
CYGRKKRRQRRR 4 Carboxyl Amine ACRLVLAS 13 Carboxyl Disulfide Acetyl
CYGRKKRRQRRR 4 Amide Amine ACRLVLAS 13 Carboxyl Disulfide Amine
CYGRKKRRQRRR 4 Amide Acetyl ACRLVLAS 13 Amide Disulfide Acetyl
CYGRKKRRQRRR 4 Amide Amine CRLVLAS 2 Carboxyl Disulfide Amine
ACYGRKKRRQRRR 14 Carboxyl Acetyl CRLVLAS 2 Carboxyl Disulfide Amine
ACYGRKKRRQRRR 14 Carboxyl Acetyl CRLVLAS 2 Amide Disulfide Amine
ACYGRKKRRQRRR 14 Carboxyl Amine CRLVLAS 2 Amide Disulfide Amine
ACYGRKKRRQRRR 14 Carboxyl Amine CRLVLAS 2 Carboxyl Disulfide Acetyl
ACYGRKKRRQRRR 14 Carboxyl Amine CRLVLAS 2 Carboxyl Disulfide Acetyl
ACYGRKKRRQRRR 14 Amide Amine CRLVLAS 2 Carboxyl Disulfide Amine
ACYGRKKRRQRRR 14 Amide Acetyl CRLVLAS 2 Amide Disulfide Acetyl
ACYGRKKRRQRRR 14 Amide
[0083] All of the PKC peptides discussed in U.S. Provisional No.
60/910,588, filed Apr. 6, 2007 are hereby incorporated by
reference.
[0084] Carrier Moiety
[0085] A wide variety of molecules (particularly macromolecules
such as peptides) intended for cellular uptake have been found to
be poorly transported across cell membranes. Among the solutions
proposed to facilitate cellular uptake have been the use of carrier
moieties such as cationic (i.e., positively charged) polymers,
peptides and antibody sequences, including polylysine,
polyarginine, Antennapedia-derived peptides, HIV Tat-derived
peptides and the like. (See, for example, U.S. Patents and
Publications Nos., U.S. Pat. Nos. 4,847,240, 5,888,762, 5,747,641,
6,593,292, US2003/0104622, US2003/0199677 and US2003/0206900.)
[0086] A particular example of a cargo/carrier conjugate is KP-1634
(SEQ ID NO:49), which is made up of two .epsilon.PKC-derived
peptides with amino terminal caps and a HIV Tat-derived carrier
peptide which has been capped at both its amino and carboxy
terminal ends.
[0087] Additional Inhibitors
[0088] Additional inhibitors of .epsilon.PKC can be identified
using assays that measure the activation, intracellular
translocation, binding to intracellular receptors (e.g. RACKs) or
catalytic activity of .epsilon.PKC. Traditionally, the kinase
activity of PKC family members has been assayed using at least
partially purified PKC in a reconstituted phospholipid environment
with radioactive ATP as the phosphate donor and a histone protein
or a short peptide as the substrate (T. Kitano, M. Go, U. Kikkawa,
Y. Nishizuka, Meth. Enzymol. 124, 349-352 (1986); R. O. Messing, P.
J. Peterson, C. J. Henrich, J. Biol. Chem. 266, 23428-23432
(1991)). Recent improvements include a rapid, highly sensitive
chemiluminescent assay that measures protein kinase activity at
physiological concentrations and can be automated and/or used in
high-throughput screening (C. Lehel, S. Daniel-Issakani, M.
Brasseur, B. Strulovici, Anal. Biochem. 244, 340-346 (1997)) and an
assay using PKC in isolated membranes and a selective peptide
substrate that is derived from the MARCKS protein (B. R.
Chakravarthy, A Bussey, J. F. Whitfield, M. Sikorska, R. E.
Williams, J. P. Durkin, Anal. Biochem. 196, 144-150 (1991)).
Inhibitors that affect the intracellular translocation of
.epsilon.PKC can be identified by assays in which the intracellular
localization of .epsilon.PKC is determined by fractionation (R. O.
Messing, P. J. Peterson, C. J. Henrich, J. Biol. Chem. 266,
23428-23432 (1991)) or immunohistochemistry (U.S. Pat. No.
5,783,405; U.S. patent application Ser. No. 08/686,796 now U.S.
Pat. No. 6,255,057, now U.S. Pat. No. 6,255,057). To identify an
inhibitor of .epsilon.PKC, the assays should be performed with
.epsilon.PKC. The selectivity of such .epsilon.PKC inhibitors can
be determined by comparing the effect of the inhibitor on
.epsilon.PKC with its effect on other PKC isozymes. The relevant
portions of foregoing patents and publications are hereby
incorporated by reference.
[0089] Additional assays for identifying EPKC inhibitors can be
found in U.S. Pat. Nos. 5,783,405, 6,156,977, and 6,423,684, all of
which are hereby incorporated by reference in their entirety.
[0090] Mechanisms
[0091] Experimental data reported here shows that (1)
locally-delivered .epsilon.PKC inhibitory peptides to the hindlimbs
of test animals produced anti-hyperalgesic/analgesic effects in
neuropathetic and inflammatory pain models; (2) such effects of the
.epsilon.PKC inhibitory peptides are though to be mediated by
nerves traveling inside the sciatic and saphenous afferents, which
project to the hindlimbs and thus could be part of a neuronal
reflex subject to be inhibited by the .epsilon.PKC inhibitory
peptides; (3) the effects of the .epsilon.PKC inhibitory peptides
appears to be sympathoadrenal-dependent, which coincides with the
reports that .epsilon.PKC not only exists in sympathetic ganglia
and functions to the neurotransmitter-releasing process (e.g.,
Scholze et al.: J. Neurosci., 22: 5823-32, 2002) but also modulates
sensitization of the C-fibers (a pain-sensing nerve) (Khasar, et
al., Neuron, 24: 253-260, 1999); and (4) the
anti-hyperalgesic/analgesic effects of the .epsilon.PKC inhibitory
peptides is mediated by adrenoceptors. Of note, epinephrine (EPI),
one of the neurotransmitters released from the adrenal medulla and
sympathetic post-ganglionic neuron (SPGN) terminals, is known to
sensitize the C-fibers (Chen & Levine, J. Pain, 6: 439-446,
2005; Khasar, et al., Neuron, 24: 253-260, 1999). It was reported
that .epsilon.PKC modulates Ca.sup.++ influx (Boehm et al., J.
Neurosci., 16: 4516-603, 1996) by phosphorylating the N-type
calcium channels (Zhu and Ikeda, J. Neurophysiol., 74: 1546-60,
1994) and thus regulates the release of catecholamines from the
sympathetic neurons (Scholze et al.: J. Neurosci., 22: 5823-32,
2002).
[0092] Identification of the anti-hyperalgesic effects of
.epsilon.PKC inhibitory peptides differs from that discussed by
Messing & Levine (U.S. Pat. No. 6,686,334 B2, U.S. Pat. No.
6,376,467 B1 and 2002/0151465 A1), in which exogenous EPI or
isopropranolol (ISO, a synthetic .beta.-adrenoceptor agonist) acts
on the C-fibers via stimulating .beta.-adrenoceptors. In that work,
the pro-hyperalgesic effects of exogenous EPI or ISO are
.epsilon.PKC-dependent and do not act on the autoregulatory
.alpha..sub.2-adrneoceptors on the SPGN terminals. U.S. Pat. Nos.
6,686,334 and 6,376,467 and Pub. No. 2002/0151465 A1 focused on the
anti-hyperalgesic effects of .epsilon.V.sub.1-2 on the downstream,
that is, the effects caused by exogenous catecholamines or, in
theory by endogenous catecholamines after they are released from
the sympathoadrenal system. The presently disclosed data does not
contradict Messing & Levine, but rather focuses and extend the
understanding the role .epsilon.PKC plays in pain
neurotransmission. Furthermore, the findings presented here
demonstrate that the anti-hyperalgesic effects of the .epsilon.PKC
inhibitory peptides are also mediated via "the upstream"
neurotransmission in the SPGN, and more specifically highlights the
effects of the .epsilon.PKC inhibitory peptides have on the
sympathoadrenal system.
[0093] This invention shows that administration of a .epsilon.PKC
inhibitor locally at a remote site can inhibit pain by a mechanism
that is mediated via the SPGN, and is not limited by nor does it
require systemic distribution of the inhibitor throughout the body.
The remote action, rapid onset and low doses of the .epsilon.PKC
inhibitor needed to inhibit the pain response require intact nerve
since transection of the sciatic and saphenous nerves
(de-afferentation) on the contralateral limb blocked the ability of
S.C administered .epsilon.PKC to inhibit pain from a remote site.
Furthermore, bilateral lumbar sympathectomy plus bilateral
suprarenal ganglionectomy as well as unilateral injection of
phentolamine (which is a non-selective .alpha.-adrenoceptor
antagonist) abolished the pain-reducing effects of .epsilon.PKC
inhibitors.
[0094] The very low doses, rapid onset and remote action observed
with .epsilon.PKC inhibitor support the conclusion that perhaps
even a much less selective inhibitor of .epsilon.PKC (including
inhibitors that might otherwise be toxic if systemically
administered) could be used to suppress a pain response.
Furthermore, since we have shown that very low doses of an
.epsilon.PKC administered locally can have this effect, it is
conceivable that a similar very low dose of an otherwise
non-selective and systemically toxic .epsilon.PKC inhibitor could
be administered locally at very low doses (i.e., much below the
dose-limiting systemic toxic levels of that drug) to achieve
inhibition of a pain response without producing any or only very
limited systemic toxic side effects.
Methods of Use and Formulations
[0095] The modified peptides described herein are useful for the
prevention and treatment of pain. For the purposes of this
discussion, pain, and the treatment thereof, is categorized into
different classes: treatment of acute, chronic, neuropathic, and
inflammatory pain. The modified .epsilon.PKC inhibitory peptides
described herein are useful for the treatment of acute, chronic,
neuropathic, and inflammatory pain.
[0096] Interestingly, the compounds disclosed herein are also
useful in attenuated or preventing the development of neuropathic
pain caused by a plurality of stimuli. For example, as discussed in
Example 7 below, chronic inflammatory pain can be induced by the
administration of carrageenan followed by the administration of
prostaglandin E2. This phenomenon serves as a model for a variety
of systems where a subject receiving a plurality of pain stimuli or
pain sensitizing agents results in chronic inflammatory or
neuropathic pain. It has been noted that chemotherapy patients
receiving TAXOL develop neuropathic pain, which typically resolves
after the initial dose or doses of the drug. However, chemotherapy
patients receive a full course of TAXOL treatments are left with
on-going neuropathic pain. The present disclosure contemplates that
the administration of the .epsilon.PKC inhibitory peptides
described herein, either prophylactically, with the
chemotherapeutic agent, or subsequent to the chemotherapy will be
effective to attenuate or prevent the development of the chronic
inflammatory or neuropathic pain condition.
[0097] Once a cargo/carrier peptide construct has been assembled
and tested for increased stability, potency, or both as compared to
a prototype, the construct is placed into a pharmaceutically
acceptable formulation for administration to a subject prior to,
during, or continuously through a pain inducing event.
[0098] A "pharmaceutically acceptable formulation" comprises one
that is suitable for administering the modified .epsilon.PKC
inhibitor in a manner that gives the desired results and does not
also produce adverse side effects sufficient to convince a
physician that the potential harm to a patient is greater than the
potential benefit to that patient. The components of a suitable
pharmaceutically acceptable formulation for use with a modified
.epsilon.PKC inhibitors are determined in part by the route and
method of administration. The formulations generally comprise one
or more modified .epsilon.PKC inhibitory peptides incorporated into
a pharmaceutically acceptable carrier typically comprising simple
chemicals such as sugars, amino acids or electrolytes. Exemplary
solutions are typically prepared with saline or buffer. The
pharmaceutically acceptable carrier may contain excipients which
are well known in the art, and may be used in a variety of
formulations. See, e.g., Remington's Pharmaceutical Sciences, 18th
Edition, A. R. Gennaro, Editor, Mack Publishing Company (1990);
Remington: The Science and Practice of Pharmacy, 20th Edition, A.
R. Gennaro, Editor, Lippincott Williams & Wilkins (2000);
Handbook of Pharmaceutical Excipients, 3rd Edition, A. H. Kibbe,
Editor, American Pharmaceutical Association, and Pharmaceutical
Press (2000); and Handbook of Pharmaceutical Additives, compiled by
Michael and Irene Ash, Gower (1995).
[0099] Inhibitor dosage in the formulation will vary according to a
variety of parameters influenced by the stability and potency of
the cargo/carrier construct, the route of administration, and
desired dosing regime. Daily dosages in the range of 1 .mu.g/kg-100
mg/kg of body weight, preferably 1 .mu.g/kg-1 mg/kg and most
preferably 10 .mu.g/kg-1 mg/kg are contemplated.
[0100] Modified .epsilon.PKC inhibitors can be administered locally
or systemically. Local administration can be achieved by topical
administration, transdermal, intradermal administration,
intrathecal administration, intraperitoneal administration, or
subcutaneous injection. Systemic administration of a modified
.epsilon.PKC inhibitor is preferably parenteral, although oral,
buccal, and intranasal administration is also contemplated.
Parenteral administration is generally characterized by injection,
either subcutaneously, intramuscularly, intraperitoneal, and
intravenously. Injectable forms of the modified inhibitory peptides
can be prepared in conventional forms, either as liquid solutions
or suspensions, solid (e.g., dried or lyophilized) forms suitable
for reconstitution into solution or suspension in liquid prior to
injection, or as emulsions. Generally, suitable excipients include,
for example, water, saline, dextrose, glycerol, ethanol or the
like. In addition, minor amounts of non-toxic auxiliary substances
can be employed, such as wetting or emulsifying agents, pH
buffering agents, solubility enhancers, tonicifiers and the like
including, for example, sodium acetate, sorbitan monolaurate,
triethanolamine oleate, cyclodextrins, etc.
[0101] vThe modified .epsilon.PKC inhibitory peptides can be
administered to treat pain as necessary. For prophylaxis, the
modified .epsilon.PKC compound may be administered prior to a
pain-inducing event. For example, the peptide can be administered
5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, one hour,
several hours, one day, several days, one week, or weeks prior
ahead of an anticipated pain-inducing event. Even longer periods of
prophylactic administration can be achieved using modified peptides
that are particularly stable in vivo, or by using a sustained
release formulation of the peptide, e.g. delivery by intrathecal
pump.
EXAMPLES
[0102] The following examples serve to describe more fully the
manner of using the above-described invention, as well as to set
forth the best modes contemplated for carrying out various aspects
of the invention. It is understood that these examples in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes. All references cited herein
are incorporated by reference in their entirety.
Example 1
Analogs of .epsilon.PKC Inhibitor Optimization
[0103] A prior art .epsilon.PKC inhibitory sequence (KP-1636) was
used as the prototype to study the impacts of various chemical
modifications on potency and stability. The prototype sequence,
KP-1586 was used as the template in the work described below.
Various cargo and carrier peptides were modified for this work, and
these are provided in Table 10.
TABLE-US-00010 TABLE 10 Compound Description Cargo Peptide
Sequences Carrier Peptide Description KP-1586 C-E-A-V-S-L-K-P-T Cap
Tat KP-1630 Ac-C-E-A-V-S-L-K-P-T-NH.sub.2 Cap Tat KP-1631
Ac-C-L-K-P-T-A-W-S-L-R-NH.sub.2 Cap Tat KP-1632
Ac-C-E-A-V-S-L-K-P-T-A-W-S-L-R Cap Tat KP-1633
E-A-V-S-L-K-P-T-G-G-TaT-NH.sub.2 Linear KP-1634
[Ac-E-A-V-S-L-K-P-T-G-G]-K-C Cap Tat KP-1635 t-p-k-l-s-v-a-e-c Cap
Tat KP-1636 C-E-A-V-S-L-K-P-T Tat KP-1637
Ac-C-E-A-V-S-L-K-P-T-NH.sub.2 Ac-hCys-Tat KP-1638
Ac-hC-E-A-V-S-L-K-P-T-NH.sub.2 Ac-hCys-Tat KP-1678
Ac-E-A-V-S-L-K-P-T-G-G-Tat-NH.sub.2 Linear
[0104] "Tat" in Table 10 refers to the fragment 47-57 of HIV Tat,
and "Cap Tat refers to an N-acetyl or C-amide analog of the same
peptide. The term "hC" or "hCys" refers to a homocysteine amino
acid.
Example 2
Stability of .epsilon.PKC Inhibitor Analogs
[0105] The plasma and chemical stability of various cargo/carrier
peptide constructs described in Example 1 was tested. Plasma
stability of the compounds was tested using both human and rat sera
and the amount of starting material was determined after 30 minutes
of treatment. Chemical stability was evaluated by determining the
amount of starting material remaining after 9 days of
treatment.
TABLE-US-00011 TABLE 11 Plasma Stability % Remaining at 30 min.
Chemical Stability Compound Human Rat % Remaining at 9 days 1586
Lead 85 77 57 1630 Cap cargo/Tat 76 77 67 1631 Overlap 1 49 67 76
1632 Overlap 2 54 61 89 1633 Linear 83 77 99 1634 Dimer/cTat 87 106
82 1635 retro-inverso 63 72 95 1636 no caps 79 79 27 1637
half-HomoCys 60 78 100 1638 full HomoCys nd nd 100
Example 3
Time Course Plasma Stability of .epsilon.PKC Inhibitor Analogs
[0106] The plasma stability of various cargo/carrier peptide
constructs described in Example 1 was tested using both human and
rat sera and the amount of starting material was determined over
time to 30 minutes of treatment. The time course data for
cargo/carrier peptides KP-1586, KP-1630, and KP-1631 is shown in
FIG. 3, the data for peptides KP-1632, KP-1633, and KP-1634 is
shown in FIG. 4, and the data for peptides KP-1635, KP-1636, and
KP-1637 is shown in FIG. 5. The dimer peptide and the analog
containing an uncapped carrier peptide were more stable that of the
prototype material. Interestingly, capping of the cargo peptide had
little impact on plasma stability as seen when comparing the
stability of KP-1586 and KP-1630.
Example 4
Time Course Chemical Stability of .epsilon.PKC Inhibitor
Analogs
[0107] The chemical stability of various cargo/carrier peptide
constructs described in Example 1 was tested by examining the
relative concentration of the peptides over a time period of more
than 200 hours at 37.degree. C. The time course data for the
cargo/carrier peptides is shown in FIG. 6. The data from this study
showed that the prototype sequence was only moderately stable as
compared to the analogs. Both the linear and
homocysteine-containing constructs showed improved stability
relative to the prototype sequence. For example, KP-1637 showed
marked stability.
Example 5
Attenuation of Acute Pain with a Modified .epsilon.PKC Inhibitory
Peptide
[0108] A formalin-induced pain test was used to study the ability
of a modified .epsilon.PKC peptide to attenuate acute pain.
Formalin was administered by an intraplantar route to all rats used
in the present study. Test subjects received a formulation
containing modified .epsilon.PKC peptide KP-1586 by intrathecal
administration 15 minutes prior to the pain-causing agent
(prophylactic mode). Two different concentrations of the modified
peptide were used in the test subjects. The data from this
experiment is shown in FIG. 7. The results of this study indicate
that the prophylactic administration of the modified .epsilon.PKC
inhibitory peptide was effective to reduce flinches per minute in
the test animals. Thus, the administration of modified .epsilon.PKC
peptides is effective to attenuate an acute pain stimulus.
Example 6
Attenuation of Chronic Pain with a Modified .epsilon.PKC Inhibitory
Peptide
[0109] The Chung (L5 nerve transection) is a well-known model for
chronic (neuropathic) pain. A representative modified .epsilon.PKC
peptide KP-1586 provided in the disclosure has been efficacious in
reducing pain in this model when delivered systemically. The
results from this work are shown in FIG. 8.
[0110] The test peptide KP-1586, but not the control peptide,
suppressed thermal hyperalgesia in a modified Chung's model when
delivered for many days by subcutaneous osmotic pump. Such
inhibitory effects were dose-dependent, with an initial dose of
10-50 pmol/day. Anti-hyperalgesic effects became detectable as
early as the next day after implantation and continued throughout
at least one week with continuous compound delivery.
[0111] In the same model, chronic sub-cutaneous delivery of
KP-1586, but not the control peptide KP-1587, suppressed mechanical
allodynia. This inhibition was dose-dependent in some observation
windows, particularly in the tests of the 7th day following
establishment of the model. Anti-allodynic effects became
detectable as early as the day after implantation.
[0112] KP-1586 was further able to modify pain responses in the
Chung model following intrathecal administration. The efficacy of
the drug in this mode lasted at least 90 minutes following single
bolus administration.
Example 7
Attenuation of Chronic Inflammatory Pain Using a Modified
.epsilon.PKC Inhibitory Peptide
[0113] The intraplantar administration of carrageenan followed 5
days later with prostaglandin E2 (PGE2) to the rat paw causes both
acute and chronic pain through an inflammatory mechanism. As shown
in FIG. 9, local delivery of compounds described herein is able to
attenuate the development of the pain response.
[0114] Representative compound was KP-1586 was able to reverse the
painful effects of carrageenan when delivered by intradermal
administration while the control peptide KP-1587 did not.
Example 8
Subcutaneous .epsilon.PKC Inhibitor Reverses Inflammatory Pain
[0115] The intraplantar administration of carrageenan followed 1
hour later with a subcutaneous bolus administration of KP-1634 was
tested in a mechanical pain model to demonstrate the effect of
.epsilon.PKC inhibition on paw withdrawal. As shown in FIG. 10, paw
withdrawal thresholds increased markedly after the administration
of the inhibitor while the control peptide KP-1587 did not induce
the same effect.
Example 9
Prevention of Neuropathic Pain with Subcutaneous Administration of
a .epsilon.PKC Inhibitory Peptide
[0116] The .epsilon.PKC peptide KP-1586 was used in the Chung model
to test the ability of .epsilon.PKC inhibitory peptides
administered subcutaneously to prevent neuropathic pain. The test
peptides were administered at 1, 10, 50, and 100 pmol/day. The
results from this work are shown in FIG. 11.
[0117] The test peptide KP-1586, but not the control peptide 1587,
suppressed thermal hyperalgesia in a modified Chung's model when
delivered by subcutaneous osmotic pump and tested as 1, 3 and 5
days post-surgery.
Example 10
Reversal of Neuropathic Pain with Subcutaneous Administration of a
.epsilon.PKC Inhibitory Peptide
[0118] The .epsilon.PKC peptide KP-1586 was used in the Chung model
to test the ability of .epsilon.PKC inhibitory peptides
administered subcutaneously to reverse neuropathic pain. The test
peptides were administered at 0.1, 1, 10, 50, 1000 pmol/day. The
results from this work are shown in FIG. 12. FIG. 13 shows the
effect of an inhibitor administered at 10 pmole per day by
subcutaneous infusion with the pump being implanted 7 days after
the transection event.
Example 11
The Role of Sympathetic Terminals in Suppressing
Carrageenan-Induced Mechanical Hyperalgesia by Inhibitors of PKC
Epsilon in Rats
[0119] .lamda.-carrageenan (Carr)-induced inflammatory pain
involves activity of the sympathetic nervous system. Since the
sympathetic neurons, like the sensory neurons, are rich in the
epsilon isozyme of protein kinase C (.epsilon.PKC), and since
.epsilon.PKC inhibitors reduce Carr-induced mechanical
hyperalgesia, it is hypothesized that the mechanism of action for
.epsilon.PKC inhibitors requires the sympathetic post-ganglionic
nerve (SPGN) terminals.
[0120] The nociceptive flexior reflex was quantified on lightly
restrained rats by using a Basile Analgesymeter (Randall-Selitto
test). Inflammatory hyperlagesia was induced by injection of Carr
(1%, 5 uL, i.d.), which was injected unilaterally to the dorsum of
the hindpaw one-hour prior to compound administration. The
.epsilon.PKC inhibitor peptide (.epsilon.V1-2) was injected via
systemic routes.
[0121] Subcutaneous injection of .epsilon.V1-2 dose-dependently
suppressed Carr-induced mechanical hyperalgesia, independent of the
site of administration (ipsilateral or contralateral hindlimb or
the dorsal trunk).
[0122] In contrast, when .epsilon.V1-2 was administered
subcutaneously distal to a transection of the sciatic and saphenous
nerves, there was no longer suppression of Carr-induced mechanical
hyperalgesia of the contralateral paw. This result suggested that
tonic neuronal signaling in these nerves may be the site of action
for .epsilon.V1-2. Furthermore, surgical removal of bilateral
lumbar sympathetic chain (L2-L4) and bilateral suprarenal ganglia
7-day prior to the experiment completely abolished
anti-hyperalgesic effects of .epsilon.V1-2. The effect of the
surgical sympathectomy was mimicked by acute treatment with
adrenergic antagonists such as phentolamine (10 ug, s.c. injection
to the contralateral limb, just before Carr injection).
[0123] Results from the current study suggest a mechanism of action
for .epsilon.PKC and demonstrate anti-hyperalgesic effects of
systemically-delivered .epsilon.V1-2. These results support the
potential development of .epsilon.PKC inhibitors as novel therapies
for inflammatory pain.
Example 12
Modulation of Pain Responses Using Peptides for Selective
Modulation of Protein Kinase C
[0124] It is known that .lamda.-carrageenan (Carr)-induced
inflammatory pain involves activity of the sympathetic nervous
system. Since the sympathetic neurons, like the sensory neurons,
are rich in the c isozyme of protein kinase C (.epsilon.PKC), and
since .epsilon.PKC inhibitors reduce Carr-induced mechanical
hyperalgesia, it is hypothesized that the mechanism of action for
.epsilon.PKC inhibitors requires the sympathetic post-ganglionic
nerve (SPGN) terminals.
[0125] The nociceptive flexior reflex was quantified on lightly
restrained rats by using a Basile Analgesymeter (Randall-Selitto
test). Inflammatory hyperlagesia was induced by injection of Carr
(1%, 5 .mu.L, i.d.), which was injected unilaterally to the dorsum
of the hindpaw one-hr prior to compound administration. The
.epsilon.PKC inhibitor peptide (.epsilon.V.sub.1-2) was injected
via systemic routes.
[0126] Subcutaneous injection of .epsilon.V.sub.1-2
dose-dependently suppressed Carr-induced mechanical hyperalgesia,
independent of the site of administration (ipsilateral or
contralateral hindlimb or the dorsal trunk).
[0127] In contrast, when .epsilon.V.sub.1-2 was administered
subcutaneously distal to a transaction of the sciatic and saphenous
nerves, there was no longer suppression of Carr-induced mechanical
hyperalgesia of the contralateral paw. This result suggested that
tonic neuronal signaling in these nerves may be the site of action
for .epsilon.V.sub.1-2. Furthermore, surgical removal of bilateral
lumbar sympathetic chain (L.sub.2-L.sub.4) and bilateral suprarenal
ganglia 7-day prior to the experiment completely abolished
anti-hyperalgesic effects of .epsilon.V.sub.1-2. The effect of the
surgical sympathectomy was mimicked by acute treatment with
adrenergic antagonists such as phentolamine (10 ug, s.c. injection
to the contralateral limb, just before Carr injection). See FIGS.
14-17.
Example 12
Nonclinical Pharmacology of KAI-1678
[0128] The ability of KAI-1678 to reduce allodynia, the heightened
response to normally innocuous stimuli, and hyperalgesia, the
heightened response to painful stimuli, has been evaluated in rat
models of inflammatory pain (carrageenan-induced pain) and
neuropathic pain (L5 spinal nerve transection). In the
carrageenan-induced inflammatory pain model, local intradermal
administration of KAI-1678 has been shown to be effective at
reducing mechanical hyperalgesia. However, intradermal
administration of KAI-1678 to a remote site was equally effective;
suggesting that local administration of KAI-1678 could provide
system-wide pain relief. This conclusion is supported by the
observation that KAI-1678 is effective with subcutaneous
administration, either as a bolus or as a prolonged infusion, to
any site on the animal, including those distant from the site of
carrageenan injection. Although KAI-1678 appears to have
system-wide activity, intravenous infusion of the compound, even at
dose rates sufficient to achieve plasma steady-state levels 5- to
10-fold greater than those measured at the end of subcutaneous
infusions that achieve maximal efficacy, does not inhibit
carrageenan-induced mechanical hyperalgesia. Elucidation of the
site-of action for KAI-1678 is ongoing, but two observations help
to define the target of the compound. Surgical disruption of the
sciatic and saphenous nerves on the opposite hindlimb from the site
of carrageenan injection abrogates the activity of compound
administered distal, but not proximal, to the site of nerve
disruption, suggesting a requirement for functional innervations of
the injection site for the compound to achieve efficacy. Further
studies indicate that an intact sympathetic nervous system and a
adrenergic receptor signaling is required for KAI-1678 to be active
when administered distant from the site of injury. One hypothesis
arising from these studies is that KAI-1678 acts on local nerves at
the site of injection, perhaps within the dermis or epidermis, and,
by an unidentified mechanism, elicits a descending pain-suppressing
signal that is dependent on a adrenergic receptor signaling.
[0129] Results similar to those obtained in the carrageenan-induced
inflammatory pain model were obtained in he L5 spinal nerve
transection mononeuropathic pain model used to investigate the
efficacy of KAI-1678 in neuropathic pain. In the L5 spinal nerve
transection mononeuropathic pain model subcutaneous administration
of KAI-1678 caused a reduction in the level of injury-induced
allodynia. However, although KAI-1678 was active in both
neuropathic pain models, the maximum response and the dose required
to achieve maximal response varied substantially in the two models.
In these studies, the injury induced by L5 nerve transection
appeared to be much more susceptible to treatment with KAI-1678 in
that complete reversal of injury-induced allodynia was achieved
with low total doses of compound administered either as a
subcutaneous bolus or infusion.
[0130] Taken together, the nonclinical pharmacology studies support
the activity of KAI-1678 in inflammatory and neuropathic pain.
These studies also suggest that the maximum effect and the dose
required to achieve maximum effect may vary depending on the type
and source of injury causing the pain. Efforts are ongoing to
identify the site of action and the mechanism of action in each of
these models.
[0131] Activity of KAI-1678 in the Carrageenan Inflammatory Pain
Model
[0132] The rat carrageenan model has been extensively used to
evaluate the response to modulators of inflammatory pain. In the
model used for the studies described below, a single 5 microL
injection of a 1% solution of carrageenan into the plantar surface
of the right hindpaw was used to elicit an inflammatory response
resulting in local edema and mechanical hyperalgesia. The
mechanical hyperalgesia was quantitated as previously described
using measurements of the nociceptive flexion reflex
(Randall-Selitto paw-withdrawal test) in response to mechanical
painful stimulation at the site of carrageenan injection. Under the
conditions used in these studies, the paw withdrawal threshold
(PWT) in the Randall-Selitto test typically decreased from
.about.90 g prior to carrageenan injection to .about.55 g measured
1 hour after carrageenan injection for the untreated animals. In
the absence of further treatment, the PWT remained stable at
.about.55 g for several hours, making it possible to use this model
to evaluate the time-course of the response to treatment over the
course of at least six hours after the establishment of the disease
state.
[0133] Intradermal Administration of KAI-1678
[0134] Previous publications have reported that local intradermal
administration of analogs of KAI-1678 is efficacious in the
carrageenan-induced inflammatory pain model. To determine whether
intradermal administration of KAI-1678 was active in this model,
rats injected with carrageenan into the right hindpaw were treated
one hour later with an intradermal injection of 10 or 100 mcg/kg of
KAI-1678 either on the same (ipsilateral) hindpaw injected with the
carrageenan or the other (contralateral) hindpaw. As shown in FIG.
18, intradermal administration of 10 mcg/kg KAI-1678 to the
ipsilateral site was able to completely reverse the mechanical
hyperalgesia as indicated by the fact that the PWT returned to
pre-carrageenan levels. Administration of 100 mcg/kg KAI-1678 to
the ipsilateral site was more effective than the 10 mcg/kg dose,
increasing the PWT beyond the pre-carrageenan levels and
maintaining the PWT at or above pre-carrageenan levels for a longer
period. Strikingly, the effect of KAI-1678 demonstrated a rapid
onset of action with the mechanical hyperalgesia substantially
reversed five minutes after administration of the 10 mcg/kg dose
and completely reversed by the 5-min time point following
administration of the 100 mcg/kg dose.
[0135] Intradermal administration of KAI-1678 to the contralateral
hindpaw was also able to completely reverse the mechanical
hyperalgesia, with the maximum extent of reversal at each dose
similar to that seen for the same dose administered to the
ipsilateral hindpaw (FIG. 18). Of note, as in the case in which
KAI-1678 was administered close to the site of carrageenan
injection, intradermal administration of KAI-1678 to the
contralateral paw resulted in a rapid onset of action with near or
complete reversal of mechanical hyperalgesia at the 5-min time
point for the lower and higher doses, respectively. There was,
however, a difference in the duration of the effect with the effect
being more prolonged at both dose levels when KAI-1678 was
administered to the ipsilateral hindpaw as compared to the
contralateral hindpaw.
[0136] While the efficacy of local (ipsilateral) administration of
selective peptidic .epsilon.PKC inhibitors structurally related to
KAI-1678 has been reported previously, the demonstration that
intradermal administration of KAI-1678 to the contralateral hindpaw
was similarly efficacious was not anticipated. This result suggests
that local administration of KAI-1678 at a remote site may elicit
system-wide pain suppression based in its ability to reduce
mechanical hyperalgesia in an inflammatory pain model.
Subcutaneous Administration of KAI-1678
[0137] KAI-1678 was administered subcutaneously to test whether
administration of the compound by this route could suppress
mechanical hyperalgesia in the carrageenan inflammatory pain model.
As shown in FIG. 19, subcutaneous bolus administration of KAI-1678
on the thigh of the contralateral hindlimb caused a dose-dependent
suppression in the pain response, with a dose as low as 0.1 mcg/kg
completely reversing mechanical hyperalgesia for a short period of
time. At doses up to 100 mcg/kg the duration of the response
increased. However, at doses greater than 100 mcg/kg the duration
of the response decreased as the dose increased, suggesting a
parabolic dose-response for duration.
[0138] A group of animals was also dosed with 100 mcg/kg of
KP-1723, the inactive control peptide (see above). As shown in FIG.
19, KP-1723 had no effect on mechanical hyperalgesia, indicating
that the effects seen with KAI-1678 are not a consequence of
non-specific effects of the carrier moiety.
[0139] To determine whether the location at which KAI-1678 is
administered as a subcutaneous bolus affects its activity in this
model, carrageenan-injected rats were given a 10 mcg/kg
subcutaneous bolus of KAI-1678 either on the ipsilateral hindlimb
close to the site of carrageenan injection, on the contralateral
hindlimb, or on the central trunk above the contralateral hindlimb
one hour after carrageenan injection. Four hours later, each animal
was given a second 10 mcg/kg subcutaneous administration of
compound to a different location as a control for possible
animal-to-animal variability. The results shown in FIG. 20 indicate
that the efficacy of the first dose of compound was similar
regardless of the site of compound administration, and that
duration of the response with the second dose was reduced relative
to the first administration. This latter observation suggests that
repeated subcutaneous bolus administrations may result in
tachyphylaxis. Consistent with this observation, repeat
administration of an PKC inhibitor analog of KAI-1678 has shown a
reduction in both the magnitude and duration of the response that
is inversely correlated with the length of time between
administrations (data not shown). The basis for the apparent
tachyphylaxis is under investigation.
[0140] A comparison of the results shown in FIG. 17 (intradermal
dosing) with those of FIG. 18 (subcutaneous dosing) indicates that
both routes of administration cause a rapid response, with complete
reversal of mechanical carrageenan-induced hyperalgesia occurring
as early as five minutes after dosing at the higher doses. However,
the duration of the effect following subcutaneous bolus
administration was shorter than that seen with intradermal
administration adjacent to the carrageenan injection site. In an
attempt to extend the duration of the anti-hyperalgesic effect,
KAI-1678 was administered as a subcutaneous infusion to rats
starting one hour after intradermal injection of carrageenan. As
shown in FIG. 21, a subcutaneous infusion of KAI-1678 extended the
duration of the response in a dose-dependent fashion. However, the
response to the subcutaneous infusion occurred in two phases. The
first phase, which lasted approximately one hour, consisted of a
rapid and complete reversal of the mechanical hyperalgesia at all
doses tested. The second phase, which developed typically between
two and three hours after the start of the infusion, was dose
dependent with only the higher doses (.gtoreq.25 mcg/kg/hr) of
KAI-1678 completely reversing mechanical hyperalgesia as indicated
by a return of the paw withdrawal threshold to pre-carrageenan
baseline levels. At 250 mcg/kg/hr the reversal of pain was rapid
and sustained without any recurrence of hyperalgesia; although
during the infusion a small decline in paw withdrawal threshold was
observed at .about.60 minutes after the start of infusion. Of note,
a 25,000 mcg/kg/hr dose rate of KAI-1678 was tested in a subsequent
subcutaneous infusion experiment. This dose rate, which is 100-fold
higher than the 250 mcg/kg/hr dose rate tested in the experiment
shown in FIG. 21, produced an effect comparable to that seen with
the 250 mcg/kg/hr infusion (data not shown). Therefore,
subcutaneous infusion of KAI-1678 does not appear to display the
parabolic dose-response observed with bolus subcutaneous
administration of the compound in this model (see FIG. 19).
[0141] At subcutaneous infusion rates .gtoreq.25 mcg/kg/hour the
efficacy achieved during the second phase was maintained for the
duration of the infusion but was rapidly reversed following the end
of infusion (FIG. 21). As discussed below, plasma levels of
KAI-1678 declined at the end of a subcutaneous infusion but with a
terminal half-life of .about.30 minutes. Thus the decline in
efficacy at the end of a subcutaneous infusion appears to be more
rapid than the drop in plasma concentrations, implying that plasma
concentrations are not the primary factor in determining
efficacy.
Intravenous Administration of KAI-1678
[0142] The intradermal and subcutaneous studies described above
indicate that KAI-1678 is efficacious when administered at a site
remote from the site of injury, suggesting that the compound may
have a system-wide effect. We therefore investigated whether
systemic administration of KAI-1678 via the intravenous (IV) route
was efficacious in the carrageenan inflammatory pain model. The IV
infusion dose rates used in these studies were selected to achieve
plasma levels of KAI-1678 that matched or exceeded those associated
with fully efficacious subcutaneous infusions of KAI-1678 (e.g.,
.gtoreq.25 mcg/kg/hr) based on the results of pharmacokinetic
experiments (see below).
[0143] As shown in FIG. 22, IV infusions of KAI-1678 at rates as
high as 1000 mcg/kg/hr for as long as 5 hours had no effect on
carrageenan-induced mechanical hyperalgesia. Although plasma
samples were not taken as part of these efficacy studies,
pharmacokinetic studies suggest that plasma levels of KAI-1678 at
the 100 mcg/kg/hr IV dose rate would be comparable to or greater
than those achieved with the 25 mcg/kg/hr subcutaneous infusion
(FIG. 20), and that plasma levels of KAI-1678 at the 1,000
mcg/kg/hr IV dose rate would be 5- to 10-fold higher than those
achieved with the 250 mcg/kg/hr subcutaneous infusion. The
inability of IV KAI-1678 to affect carrageenan-induced hyperalgesia
does not appear to be due to any technical issues, since oral
indomethacin, a known analgesic that has been shown to reduce
carrageenan-induced hyperalgesia, completely reversed the
mechanical hyperalgesia when administered after the end of the 5-hr
IV infusion of KAI-1678. Moreover, the lack of IV activity does not
appear to be unique to KAI-1678, as structurally related
.epsilon.PKC inhibitors that were efficacious in this model
following intradermal or subcutaneous administration were not
active when administered as an IV infusion (data not shown).
Site of Action Studies with KAI-1678 in the Rat Carraqeenan
Model
[0144] The demonstration that KAI-1678 appears to elicit a
system-wide response, but only when administered intradermally or
subcutaneously suggested that the primary site of action for the
compound might be in the periphery.
[0145] This hypothesis, combined with the observation that the
response to KAI-1678 administration was very rapid, even when the
compound was administered to the contralateral limb, suggested that
the afferent nerves in the skin at the site of compound
administration might be essential for the rapid onset of action.
Since signals propagated via the peripheral afferent nerves in the
skin on the contralateral hindlimb would have to travel via the
sciatic and saphenous nerves to affect mechanical hyperalgesia at
the site of carrageenan injection on the ipsilateral hindpaw, the
need for intact sciatic and saphenous nerves on the side of
compound administration was investigated.
[0146] In the experiment shown in FIG. 23, a 1-cm section of the
sciatic and saphenous nerves was removed from the left hindleg of
rats. The next day, after the rats had recovered from surgery,
carrageenan was injected into the plantar surface of the right
hindpaw of the rats to elicit an inflammatory response. Sixty
minutes after injection of carrageenan into the right hindpaw,
KAI-1678 was administered as a subcutaneous infusion to the left
hindlimb at a site either proximal or distal to the site where the
1-cm section of the sciatic and saphenous nerves had been removed.
As shown in FIG. 23, a 25 mcg/kg/hr subcutaneous infusion of
KAI-1678 was effective at eliminating carrageenan-induced
mechanical hyperalgesia when the site of compound administration
was proximal, but not distal, to the site of nerve transection.
Comparing with historical data (FIG. 21), the response to KAI-1678
administered proximal to site of the transection of the sciatic and
saphenous nerves was similar to the response observed in surgically
naive rats (see FIG. 21), although the onset of the second phase of
activity appears to be more rapid in the rats administered KAI-1678
as a subcutaneous infusion proximal to the site of nerve
transection. Although it is possible that other secondary effects
of the surgical procedure may have impacted the response to
subcutaneous infusion distal to the site of nerve transection, the
results of this experiment suggest that innervation at the site of
administration is required for KAI-1678 efficacy. As the sciatic
and saphenous nerves contain motor neurons, primary sensory
afferent neurons, and sympathetic neurons, these data do not allow
us to definitively assign the site of action to one of these
subsets of neurons.
[0147] This finding suggests that 1678 may be acting on `normal`
neurons to transmit a signal centrally resulting in system-wide
pain suppression. This modulation could be occurring in the dorsal
horn; however, efficacy is observed when KAI-1678 is administered
to sites outside of the dermatome affected by the pain stimulus,
suggesting that system-wide pain suppression is occurring above the
level of the spinal cord (see below).
[0148] Earlier studies have reported that catecholamines from the
sympathoadrenal system are involved in carrageenan-induced
inflammatory pain and that .epsilon.PKC is expressed in the
sympathetic system. We therefore investigated whether an intact
sympathoadrenal system was required to maintain the efficacy of
distally administered KAI-1678. As shown in FIG. 24, bilateral
lumbar sympathectomy plus suprarenal ganglionectomy did not alter
the pain responses to carrageenan. However, the ability of
KAI-1678, administered as a 25 mcg/kg/hr subcutaneous infusion to
the contralateral hindlimb, to reverse the carrageenan-induced
mechanical hyperalgesia was abrogated in these animals. As shown in
FIG. 24, both the early and the later phases of the bi-phasic
response to subcutaneous KAI-1678 infusion are eliminated by the
sympathectomy and suprarenal ganglionectomy. In previous
experiments with V1-2-containing analogs of KAI-1678, we have also
demonstrated that surgical sympathectomy, as described above,
eliminates the effect of .epsilon.PKC inhibitors administered as a
bolus injection at a remote subcutaneous site, as well as at an
intradermal site adjacent to the injury.
[0149] The sympathectomy experiments described above require
surgical intervention and recovery of animals for a week prior to
intervention with the carrageenan. As shown in FIG. 24, sham
surgery, without sympathectomy, may have partially reduced the
efficacy of KAI-1678. Consequently we sought to complement this
study by investigating the effect of pharmacological blockade of
the sympathetic nervous system function, using the alpha-adrenergic
antagonist phentolamine, thus eliminating the need for surgery.
Administration of this agent at the time of carrageenan injection
did not influence the establishment of pain, although the effect of
remote site subcutaneous bolus administration of an V1-2-containing
analog of KAI-1678 was eliminated, as was the effect of an
intradermal administration local to the site of injury (data not
shown). Furthermore, the efficacy of an V1-2-containing analog of
KAI-1678 following subcutaneous bolus administration in a second,
neuropathic, pain model was also eliminated by prior administration
of phentolamine, although, again, the phentolamine did not prevent
the establishment of pain in this model (data not shown).
[0150] Thus, surgical ablation of the sympathetic nervous system
and pharmacologic blockade of alpha adrenergic receptors block the
anti-hyperalgesic activity of .epsilon.PKC inhibitors. It is not
clear if these effects reflect a dependence on the sympathetic
nervous system or on adrenergic receptor signaling in the CNS.
Summary of Carrageenan Inflammatory Pain Model Studies
[0151] The data above demonstrate that KAI-1678 can reverse
inflammatory pain when administered intradermally at the site of
injury and intradermally or subcutaneously at a remote site.
Efficacy has not been observed with intravenous administration,
even at KAI-1678 plasma levels equivalent to those obtained during
an efficacious subcutaneous infusion. We have also demonstrated
that the remote site subcutaneous efficacy of KAI-1678 is dependent
on innervation at the site of administration, which suggests that
the remote-site efficacy of KAI-1678 is mediated by an effect on
subcutaneous neurons that are exposed to KAI-1678 at the site of
administration. One hypothesis to explain these data is that, by
acting on skin nociceptors, KAI-1678 elicits an ascending signal in
the primary afferent neurons that initiates a spinal or supraspinal
reflex, ultimately resulting in descending pain suppression of the
carrageenan-induced hyperalgesia. Alternatively KAI-1678 might be
acting by reducing tonic signaling in neurons at the injection site
and this inhibition might lead to the descending pain suppression.
Descending modulation might involve alpha adrenergic signaling,
which would be consistent with the effect of phentolamine on
KAI-1678 efficacy. Thus, in the spinal cord, nor epinephrine
released from descending pathways suppresses pain by inhibitory
action on alpha-2A-adrenoceptors on central terminals of primary
afferent nociceptors (presynaptic inhibition), by direct
alpha-2-adrenergic action on pain-relay neurons (postsynaptic
inhibition), and by alpha-1-adrenoceptor-mediated activation of
inhibitory interneurons. This hypothesis contrasts with other
models of .epsilon.PKC action, in which a role has been proposed
for .epsilon.PKC in primary afferent nerve terminals modulating
membrane channels and membrane depolarization in response to
inflammatory stimuli.
Activity of KAI-1678 in Neuropathic Pain Models
[0152] The activity of KAI-1678 has been evaluated in the L5
transection model or modified Chung model, is a mononeuropathic
pain model in which the L5 spinal nerve is surgically transected,
resulting in rapid development of sustained pain. The activity of
KAI-1678 in this model is described below.
Activity of KAI-1678 in the L5 Spinal Nerve Transection
Mononeuropathic Pain Model in Rats
[0153] The rat L5 spinal nerve transection model has been used to
evaluate the response to modulators of neuropathic pain. In the
studies described below surgical transaction of the L5 spinal nerve
was used to elicit mechanical allodynia, mechanical hyperalgesia
and thermal hyperalgesia that characterize neuropathic pain. In
this model, the neuropathic effects develop within a day and are
reported to last for 1-2 months.
[0154] Mechanical allodynia and mechanical hyperalgesia are
measured using calibrated von Frey filaments to elicit a flexion
withdrawal response (paw withdrawal response) by pressing them
against the plantar surface of the hindpaw on the same side as the
nerve transection. The 2 g, 6 g and 10 g von Frey filaments are
used to assess allodynia since these stimuli are normally innocuous
to naive animals. The 15 g, 26 g and 60 g filaments are used to
assess algesia (response to noxious stimulus) since these stimuli
elicit paw withdrawals in naive animals and are considered to
represent painful stimuli.
[0155] The existence of the disease state following nerve
transection is confirmed based on tests performed with the 2 g and
the 10 g filaments. When tested with a total of 3 sets of 10 tests
(30 tests total), the number of paw withdrawals in response to the
2 g von Frey filament typically increases from 1-2 prior to nerve
injury to 12-15 paw withdrawals following surgery. Similarly, the
number of paw wthdrawals in response to the 10 g filament typically
increases from 2-3 prior to nerve injury to 20-23 paw withdrawals
following surgery. In the absence of further treatment, the
mechanical allodynia remains stable for at least 3 weeks, making it
possible to use this model to evaluate the time course of the
response to treatment either as a bolus administration or an
extended infusion, with sequential measurements typically over the
course of 1-2 weeks after establishment of the disease state.
[0156] Once the disease state is confirmed, evaluation of the
extent of mechanical allodynia or hyperalgesia is determined by
observing the number of paw withdrawals in responsse to 5 tests
with each of the von Frey filaments. Results are typically
expressed either as the total number of paw withdrawals for the
allodynic filaments (2 g, 6 g and 10 g for a total of 15 tests) or
the algesic filaments (15 g, 26 g and 60 g for a total of 15
tests), or as the lowest filament to which the animals in a group
have an average positive response defined as withdrawals out of 5
tests with a particular filament.
[0157] Along with measurements of the effects of compound treatment
on mechanical allodynia and hyperalgesia, changes in the level of
thermal algesia in response to L5 spinal nerve transection have
also been determined in this model using, the Hargreaves test. In
these studies, rats are placed on a glass surface above a radiant
heat source focused on the lateral plantar surface of the affected
hindpaw. When the heat source is turned on, the glass surface heats
up over time. The time until the animal lifts its hindpaw (paw
withdrawal latency, measured in seconds) is an indicator of thermal
algesia. Under the conditions used in this study, paw withdrawal
latency decreases from 10-12 seconds prior to nerve injury to 6-8
seconds after L5 nerve transection, indicating the development of
thermal hyperalgesia. In the absence of further treatment, the
thermal hyperalgesia remains stable for at least three weeks.
Subcutaneous Administration of KAI-1678
[0158] Rats given an L5 spinal nerve transection were treated with
a bolus subcutaneous dose of KAI-1678 ranging from 0.00025-0.25
mcg/rat (.about.0.001-1 mcg/kg) on day 7 post-transection. The
degree of mechanical allodynia and hyperalgesia was evaluated based
on the number of paw withdrawals (out of 30) in response to 6
different von Frey filaments before and up to 3 hours after
administration of the compound. For comparison, some rats were
dosed with 0.250 mcg/rat (-1 mcg/kg) KP-1723, the inactive analog
of KAI-1678.
[0159] As shown in FIG. 25, KAI-1678 treatment resulted in a
dose-dependent decrease in mechanical allodynia and mechanical
hyperalgesia at doses 0.01 mcg/kg. Based on composite measurements
with the 15 g, 26 g and 60 g von Frey filaments (FIG. 25, left),
KAI-1678 at 0.1 and 1 mcg/kg completely reverses mechanical
hyperalgesia, as indicated by the return to pre-surgery responses.
Complete reversal of the hyperalgesia at these two doses was
observed between 30 and 60 minutes after compound administration,
and an anti-hyperalgesic effect was observed for at least 2 hours.
In contrast to the results obtained with KAI-1678, KP-1723
treatment at 1 mcg/kg had no effect on mechanical hyperalgesia.
Comparable results to those described above were obtained when the
2 g, 6 g and 10 g von Frey filaments were used in order to measure
KAI-1678 effects on L5-transection-induced mechanical allodynia
(data not shown).
[0160] The efficacy of KAI-1678 in this model was also apparent
when paw withdrawal threshold was measured by determining the
minimum von Frey filament to elicit 3 paw withdrawals out of 5
stimulations (FIG. 25, right).
[0161] To determine whether the efficacy of KAI-1678 in this model
could be prolonged with continuous delivery, subcutaneous exposure,
osmotic minipumps delivering 0.00025-0.25 mcg/day (.about.0.001-1
mcg/kg/day) of KAI-1678 or 0.25 mcg/day (.about.1 mcg/kg/day)
KAI-1723 were implanted subcutaneously between the scapula on day 2
post-transection. Mechanical allodynia was examined on Days 3, 5,
7, 9, 11 post-transection (Days 1, 3, 5, 7, 9 post-pump
implantation) using the 2 g and the 10 g filaments. As shown in
FIG. 26 (left), results from the tests with the 10 g filament
indicate that KAI-1678 dose rates .gtoreq.0.0025 mcg/day
(.about.0.01 mcg/kg/day) are able to substantially reduce the level
of mechanical allodynia, with the 0.025 and 0.25 mcg/day
(.about.0.1 and 1 mcg/kg/hr) dose rates producing nearly complete
reversal of mechanical allodynia that lasted until Day 7 post-pump
implantation. At the highest dose rate, evidence of reduction in
mechanical allodynia was observed as late as Day 11
post-transection. Similar results were obtained using the 2 g
filament to evaluate mechanical allodynia (data not shown) and
using the Hargreaves test to thermal hyperalgesia (FIG. 26, right).
After about 5 days of complete reversal of hyperalgesia in the
presence of pumps containing KAI-1678, we noted a loss of efficacy.
This was not due to a tolerance to the drug, as a subcutaneous
bolus administration of the drug at this time elicited complete
reversal of pain (data not shown) and it may be that the loss of
effect was due to a failure of the pumps to maintain delivery.
Summary of Neuropathic Pain Model Studies
[0162] The data above demonstrate that KAI-1678 is extremely
effective in the L5 spinal nerve transection mononeuropathic pain
model. The potency of KAI-1678 administered as a subcutaneous bolus
is comparable in reversing mechanical and thermal allodynia in the
L5 spinal transaction model. When administered by subcutaneous
infusion, KAI-1678 is about 1000-fold more potent than in the
carrageenan-induced inflammatory pain model. The reason for the
high sensitivity of the L5-transection model to KAI-1678 treatment
is not known, but it likely reflects the responsiveness of this
model to selective .epsilon.PKC inhibitors, since this model is
exhibits a similar high sensitivity to structurally-related analogs
of KAI-1678. The demonstration that KAI-1678 has activity in two
neuropathic pain models suggests that this compound may have
clinical utility for the management of neuropathic pain in
humans.
CONCLUSION FROM RAT PHARMACOLOGY STUDIES
[0163] The activity of KAI-1678 has been evaluated in rat
inflammatory and neuropathic pain models. Subcutaneous
administration of the compound is efficacious in the models shown.
The potency of KAI-1678 administered by subcutaneous bolus is
similar across the models, as is the rapid onset and duration of
effect tested, although the overall effect size does vary between
models. The response to different infusion dose rates is also
different between models. In particular, the L5 transection
mononeuropathic pain model appears to be particularly sensitive to
KAI-1678 with doses approximately one thousand fold lower being
active in this model than in the carrageenan model.
[0164] While experiments with these models are continuing,
additional work is being done in other pain models. In particular,
preliminary results in the Brennan incision model and the single
spared nerve model suggest that KAI-1678 is also active in these
models at doses similar to those that are active in the carrageenan
model.
[0165] Taken together, the available animal efficacy data suggest
that KAI-1678 may be active in a variety of pain models and support
the continued development of the compound.
[0166] Pharmacokinetics in Animals
[0167] The pharmacokinetics of KAI-1678 are being studied in rats
administered the compound as an intravenous bolus and infusion or
as a subcutaneous bolus and infusion. The toxicokinetics of
KAI-1678 are being studied in dogs administered the compound as a
subcutaneous infusion as part of the toxicity studies. In these
studies, plasma levels of KAI-1678 have been determined using a
sandwich-based ELISA assay developed at KAI Pharmaceuticals. The
lower limit of quantitation of this assay is approximately 0.3
ng/mL. A similar assay has been developed to measure tissue levels
of KAI-1678.
[0168] In rats KAI-1678 is rapidly cleared from systemic
circulation following intravenous bolus administration. When
administered as a 2-hr intravenous infusion at 50 mcg/kg/hr, steady
state plasma concentrations of approximately 10-20 ng/mL appeared
to be achieved by 60 minutes, and dropped off rapidly after the end
of infusion. The terminal half-lives for the bolus and infusion
administrations of KAI-1678 via the intravenous route are
approximately 50 and 25 minutes, respectively.
[0169] Subcutaneous administration of KAI-1678 to rats as either a
bolus (approximately 80 or 800 mcg/kg) or as an infusion (19 and
190 mcg/kg/hr) resulted in dose-proportional increases in exposures
over the doses tested. Plasma concentrations of KAI-1678 reached
maxima at approximately 10 minutes and 1 hour for the subcutaneous
bolus and infusions, respectively, and then decreased with a
half-life of approximately 35 and 45 minutes, respectively, once
the peptide was no longer being administered. The bioavailability
of KAI-1678 administered by the subcutaneous route is approximately
10% in rats.
[0170] Although the terminal half-life was not determined, KAI-1678
was also rapidly cleared from the systemic circulation in dogs
administered KAI-1678 following an extended subcutaneous infusion
over 5 days. In this study, KAI-1678 was detected in tissue samples
from a variety of organs removed at the end of infusion (most
notably the kidney, liver and lung), while levels of KAI-1678 in
brain and spinal chord were consistently low.
[0171] Rat Pharmacokinetics
[0172] Intravenous Administration
[0173] To initially characterize the pharmacokinetics of KAI-1678
in rats, male rats were administered KAI-1678 at doses of 100 and
1,000 mcg/rat (-300 and .about.3,000 mcg/kg) as an IV bolus
injection via the tail vein. Blood samples were withdrawn
periodically for up to two hours following the injection for plasma
analysis of KAI-1678 levels. There were three and two animals in
the 100 and 1,000 mcg dose groups, respectively, and there was
consistency in KAI-1678 plasma concentrations between animals in
each dose group. As shown in FIG. 27, C.sub.max plasma levels of
KAI-1678 increased in a dose-proportional manner over the doses
tested. Preliminary analysis of these data indicates a terminal
half-life of approximately 38 and 66 minutes for the .about.300
mcg/kg and the .about.3,000 mcg/kg dose levels, respectively.
[0174] Subcutaneous Administration
[0175] The majority of the animal efficacy studies have been
performed with KAI-1678 administered to rats as a subcutaneous
bolus or infusion. Therefore, the pharmacokinetics of KAI-1678
administered by the subcutaneous route has been evaluated based on
measurements of plasma concentrations of KAI-1678.
[0176] In the experiment shown in FIG. 29, male rats were
administered KAI-1678 as a subcutaneous bolus injection (200 microL
into the left hindleg) at doses of 25 or 250 mcg/animal
(approximately 80 and 800 mcg/kg, respectively). Blood samples were
taken at various time points over two hours after administration of
the compound to determine plasma concentrations of KAI-1678. There
were three and four animals in the 25 and 250 mcg/animal dose
groups, respectively, and the KAI-1678 plasma concentration
measurements were consistent between animals in each dose group.
C.sub.max occurred within 10 minutes after injection and increased
in a dose-proportional manner over the two doses tested. A
preliminary estimation of terminal half-life from these data is 35
minutes for both dose levels. A comparison of the areas under the
plasma concentration versus time curves for KAI-1678 administered
as an intravenous bolus and as a subcutaneous bolus indicates that
the bioavailability of KAI-1678 administered via the subcutaneous
route is approximately 10% in rats at this site of
administration.
[0177] In the experiment shown in FIG. 30, male rats were
administered KAI-1678 as a continuous subcutaneous infusion for 2
hours at doses of 19 and 190 mcg/kg/hr. Blood samples were taken at
various time points throughout the infusion and for 1 hour after
the end of infusion to determine plasma concentrations of KAI-1678.
There were two animals per dose group and the plasma KAI-1678
concentration measurements were consistent between animals in each
dose group. At the lower dose rate (19 mcg/kg/hr), plasma
concentrations reached steady state (-3 ng/mL) by 60 minutes and
remained relatively constant throughout the rest of the infusion.
At the higher dose rate (190 mcg/kg/hr), plasma concentrations of
KAI-1678 rose throughout the infusion and did not appear to reach a
plateau, or steady state, within the two hours of infusion
(C.sub.max.about.60 ng/mL). At both dose rates, plasma
concentrations of KAI-1678 dropped quickly after the end of
infusion although compound was still detectable at both dose levels
one hour after the end of infusion.
[0178] A comparison of the data in FIG. 28 and FIG. 30 indicates
that a 50 mcg/kg/hr intravenous infusion of KAI-1678 will produce
plasma concentrations of compound (10-20 ng/mL) that are higher
than those achieved with a subcutaneous infusion at 19 mcg/kg/hr
(.about.3 ng/mL) and nearly at the level achieved with a
subcutaneous infusion at 190 mcg/kg/hr (.about.60 ng/mL).
Interestingly, there does not seem to be a correlation with plasma
concentration and efficacy, as efficacy is not observed with iv
administration of KAI-1678 even at dose rates higher than those
required to achieve maximal efficacy via subcutaneous infusion of
the compound.
[0179] Dog Pharmacokinetics
[0180] As part of a range-finding toxicology study, 6 beagle dogs
(one per sex per group) were administered continuous subcutaneous
infusions of KAI-1678 for 5 days at dose levels of 3, 8 and 25
mg/kg/day. Blood samples were collected at various times on Day 1
(start of infusion) and Day 6 (after the end of infusion). Plasma
concentrations of KAI-1678 increased over the course of the first
four hours of infusion, and in most cases increased another two- to
three-fold by the end of infusion on the fifth day, suggesting that
steady state was not reached within 4 hours of subcutaneous
infusion at the dose levels tested (FIG. 31). However, at all dose
levels, plasma levels declined quickly upon cessation of infusion
although terminal half-life was not calculated because of the small
number of data points available.
[0181] As part of this study, tissues were harvested from a subset
of the animals at the end of dosing. Tissue extracts were prepared
and analyzed for the presence of KAI-1678, which was detected in a
subset of tissues from the two highest dose groups: liver, lung,
kidney, brain (cerebrum), spinal cord, injection site (consisting
of skin and underlying skeletal muscle), a peripheral nerve in the
left fore-limb, and muscle in the leg (not proximal to infusion
site). In general, tissue levels of KAI-1678 in the major organs
(kidney, liver and lung) increased with increasing dose and
reflected differences that were seen in plasma levels. As expected,
KAI-1678 concentrations were highest in the infusion site skin
(i.e., for 25,000 mcg/kg/day animals), although the underlying
muscle had relatively low levels. KAI-1678 levels in nervous system
(spinal cord and ulnar nerve) and peripheral tissues (muscle and
skin) were less consistent between animals and dose groups. The
levels in the brain and spinal cord were, for the most part,
consistently low, in many cases just above the limit of
quantitation, indicating relatively low exposure of these tissues
to subcutaneously infused KAI-1678.
TABLE-US-00012 TABLE 12 Tissue Levels of KAI-1678 in Dogs
Administered KAI-1678 as a Subcutaneous Infusion at 25 mg/kg/day
for Five Days Animal 4001 Animal 4501 (25 mg/kg/day (25 mg/kg/day
male) female) Mean (ng KAI- Std. Mean (ng KAI- Std. 1678/g tissue)
Dev. 1678/g tissue) Dev. Brain 6* 1.2 7 1.3 Spinal Cord 11 1.3 140
13 Peripheral Nerve 429 56 155 14 Lung 254 22 1364 150 Liver 224 15
1505 176 Kidney 689 84 3054 522 Skeletal Muscle 133 20 33 4 Dose
Site Muscle 111 14 1003 155 Dose Site Skin 1139 88 6165 998
*includes at least one data point that was below the specified
limits of quantitation.
Sequence CWU 1
1
6216PRTArtificial SequenceSynthetic construct 1Arg Leu Val Leu Ala
Ser1 5 27PRTArtificial SequenceSynthetic construct 2Cys Arg Leu Val
Leu Ala Ser1 5 37PRTArtificial SequenceSynthetic construct 3Arg Leu
Val Leu Ala Ser Cys1 5 412PRTArtificial SequenceSynthetic construct
4Cys Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5 10
512PRTArtificial SequenceSynthetic construct 5Tyr Gly Arg Lys Lys
Arg Arg Gln Arg Arg Arg Cys1 5 10 67PRTArtificial SequenceSynthetic
construct 6Xaa Arg Leu Val Leu Ala Ser1 5 77PRTArtificial
SequenceSynthetic construct 7Arg Leu Val Leu Ala Ser Xaa1 5
812PRTArtificial SequenceSynthetic construct 8Xaa Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg1 5 10 912PRTArtificial
SequenceSynthetic construct 9Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Xaa1 5 10 107PRTArtificial SequenceSynthetic construct
10Xaa Arg Leu Val Leu Ala Ser1 5 1112PRTArtificial
SequenceSynthetic construct 11Xaa Tyr Gly Arg Lys Lys Arg Arg Gln
Arg Arg Arg1 5 10 1212PRTArtificial SequenceSynthetic construct
12Xaa Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5 10
138PRTArtificial SequenceSynthetic construct 13Ala Cys Arg Leu Val
Leu Ala Ser1 5 1413PRTArtificial SequenceSynthetic construct 14Ala
Cys Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5 10
157PRTArtificial SequenceSynthetic construct 15Asn Gly Leu Leu Lys
Ile Lys1 5 1611PRTArtificial SequenceSynthetic construct 16Leu Ala
Val Phe His Asp Ala Pro Ile Gly Tyr1 5 10 179PRTArtificial
SequenceSynthetic construct 17Asp Asp Phe Val Ala Asn Cys Thr Ile1
5 1810PRTArtificial SequenceSynthetic construct 18Trp Ile Asp Leu
Glu Pro Glu Gly Arg Val1 5 10 1910PRTArtificial SequenceSynthetic
construct 19His Ala Val Gly Pro Arg Pro Gln Thr Phe1 5 10
208PRTArtificial SequenceSynthetic construct 20Asn Gly Ser Arg His
Phe Glu Asp1 5 218PRTArtificial SequenceSynthetic construct 21His
Asp Ala Pro Ile Gly Asp Tyr1 5 226PRTArtificial SequenceSynthetic
construct 22His Asp Ala Pro Ile Gly1 5 237PRTArtificial
SequenceSynthetic construct 23Xaa Arg Leu Val Leu Ala Ser1 5
247PRTArtificial SequenceSynthetic construct 24Xaa Arg Leu Val Leu
Ala Ser1 5 2512PRTArtificial SequenceSynthetic construct 25Xaa Tyr
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5 10 268PRTArtificial
SequenceSynthetic construct 26His Asp Ala Ala Ile Gly Tyr Asp1 5
278PRTArtificial SequenceSynthetic construct 27His Asp Ala Pro Ile
Pro Tyr Asp1 5 288PRTArtificial SequenceSynthetic construct 28His
Asn Ala Pro Ile Gly Tyr Asp1 5 298PRTArtificial SequenceSynthetic
construct 29His Ala Ala Pro Ile Gly Tyr Asp1 5 308PRTArtificial
SequenceSynthetic construct 30Ala Asp Ala Pro Ile Gly Tyr Asp1 5
318PRTArtificial SequenceSynthetic construct 31His Asp Ala Pro Ala
Gly Tyr Asp1 5 328PRTArtificial SequenceSynthetic construct 32His
Asp Ala Pro Ile Gly Ala Asp1 5 338PRTArtificial SequenceSynthetic
construct 33His Asp Ala Pro Ile Ala Tyr Asp1 5 348PRTArtificial
SequenceSynthetic construct 34His Asp Ala Pro Ile Gly Tyr Ala1 5
359PRTArtificial SequenceSynthetic construct 35Ser Ser Pro Ser Glu
Glu Asp Arg Ser1 5 368PRTArtificial SequenceSynthetic construct
36Pro Cys Asp Gln Glu Ile Lys Glu1 5 379PRTArtificial
SequenceSynthetic construct 37Glu Asn Asn Ile Arg Lys Ala Leu Ser1
5 388PRTArtificial SequenceSynthetic construct 38Gly Glu Val Arg
Gln Gly Gln Ala1 5 396PRTArtificial SequenceSynthetic construct
39Glu Ala Ile Val Lys Gln1 5 407PRTArtificial SequenceSynthetic
construct 40Ile Lys Thr Lys Arg Asp Val1 5 417PRTArtificial
SequenceSynthetic construct 41Ile Lys Thr Lys Arg Leu Ile1 5
427PRTArtificial SequenceSynthetic construct 42Cys Glu Ala Ile Val
Lys Gln1 5 4310PRTArtificial SequenceSynthetic construct 43Thr Lys
Arg Asp Val Asn Asn Phe Asp Gln1 5 10 449PRTArtificial
SequenceSynthetic construct 44Cys Glu Ala Val Ser Leu Lys Pro Thr1
5 459PRTArtificial SequenceSynthetic construct 45Xaa Glu Ala Val
Ser Leu Lys Pro Xaa1 5 4610PRTArtificial SequenceSynthetic
construct 46Xaa Leu Lys Pro Thr Ala Trp Ser Leu Xaa1 5 10
4714PRTArtificial SequenceSynthetic construct 47Xaa Glu Ala Val Ser
Leu Lys Pro Thr Ala Trp Ser Leu Arg1 5 10 4821PRTArtificial
SequenceSynthetic construct 48Glu Ala Val Ser Leu Lys Pro Thr Gly
Gly Tyr Gly Arg Lys Lys Arg1 5 10 15 Arg Gln Arg Arg Xaa 20
4912PRTArtificial SequenceSynthetic construct 49Xaa Ala Val Ser Leu
Lys Pro Thr Gly Gly Lys Cys1 5 10 509PRTArtificial
SequenceSynthetic construct 50Thr Pro Lys Leu Ser Val Ala Glu Cys1
5 519PRTArtificial SequenceSynthetic construct 51Cys Glu Ala Val
Ser Leu Lys Pro Thr1 5 529PRTArtificial SequenceSynthetic construct
52Xaa Glu Ala Val Ser Leu Lys Pro Xaa1 5 539PRTArtificial
SequenceSynthetic construct 53Xaa Glu Ala Val Ser Leu Lys Pro Xaa1
5 5421PRTArtificial SequenceSynthetic construct 54Xaa Ala Val Ser
Leu Lys Pro Thr Gly Gly Tyr Gly Arg Lys Lys Arg1 5 10 15 Arg Gln
Arg Arg Xaa 20 5510PRTArtificial SequenceSynthetic construct 55His
Cys Glu Ala Val Ser Leu Lys Pro Thr1 5 10 5612PRTArtificial
SequenceSynthetic construct 56Xaa Tyr Gly Arg Lys Lys Arg Arg Gln
Arg Arg Arg1 5 10 5721PRTArtificial SequenceSynthetic construct
57Xaa Ala Val Ser Leu Lys Pro Thr Gly Gly Tyr Gly Arg Lys Lys Arg1
5 10 15 Arg Gln Arg Arg Xaa 20 588PRTArtificial SequenceSynthetic
construct 58Glu Ala Val Ser Leu Lys Pro Thr1 5 5911PRTArtificial
SequenceSynthetic construct 59Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg1 5 10 609PRTArtificial SequenceSynthetic construct 60Glu
Ala Val Ser Leu Lys Pro Thr Cys1 5 6112PRTArtificial
SequenceSynthetic construct 61Xaa Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Xaa1 5 10 6221PRTArtificial SequenceSynthetic construct
62Glu Ala Val Ser Leu Lys Pro Thr Gly Gly Tyr Gly Arg Lys Lys Arg1
5 10 15 Arg Gln Arg Arg Xaa 20
* * * * *