U.S. patent application number 09/956440 was filed with the patent office on 2002-01-31 for polymer stabilized neuropeptides.
This patent application is currently assigned to Shearwater Corporation. Invention is credited to Bentley, Michael David, Roberts, Michael James.
Application Number | 20020013266 09/956440 |
Document ID | / |
Family ID | 26854193 |
Filed Date | 2002-01-31 |
United States Patent
Application |
20020013266 |
Kind Code |
A1 |
Bentley, Michael David ; et
al. |
January 31, 2002 |
Polymer stabilized neuropeptides
Abstract
A substantially hydrophilic conjugate is provided having a
peptide that is capable of passing the blood-brain barrier
covalently linked to a water-soluble nonpeptidic polymer such as
polyethylene glycol. The conjugate exhibits improved solubility and
in vivo stability and is capable of passing the blood-brain barrier
of an animal.
Inventors: |
Bentley, Michael David;
(Huntsville, AL) ; Roberts, Michael James;
(Madison, AL) |
Correspondence
Address: |
ALSTON & BIRD LLP
BANK OF AMERICA PLAZA
101 SOUTH TRYON STREET, SUITE 4000
CHARLOTTE
NC
28280-4000
US
|
Assignee: |
Shearwater Corporation
|
Family ID: |
26854193 |
Appl. No.: |
09/956440 |
Filed: |
September 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09956440 |
Sep 19, 2001 |
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09678997 |
Oct 4, 2000 |
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60157503 |
Oct 4, 1999 |
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60166589 |
Nov 19, 1999 |
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Current U.S.
Class: |
514/1.2 ;
514/18.3; 514/18.5 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 25/04 20180101; A61K 47/60 20170801 |
Class at
Publication: |
514/2 |
International
Class: |
A61K 038/33 |
Claims
What is claimed is:
1. A substantially hydrophilic conjugate comprising a peptide
covalently linked to a water-soluble, nonpeptidic polymer, wherein
said peptide is stabilized in circulation and said conjugate can
transport across the blood-brain barrier of a mammal.
2. The conjugate of claim 1 wherein said peptide is an analgesic
peptide selected from the group consisting of dynorphins,
enkephalins, double enkephalins, endorphins, endomorphins, and
analogs and derivatives thereof.
3. The conjugate of claim 2, wherein said peptide is selected from
the group consisting of Met-enkephalin, Leu-enkephalin, endomorphin
I, endomorphin II, and analogs or dimeric forms thereof.
4. The conjugate of claim 2, wherein said peptide is dynorphin A or
fragments thereof.
5. The conjugate of claim 2, wherein said peptide is biphalin.
6. The conjugate of claim 2, wherein said peptide is DPDPE.
7. The conjugate of claim 1, wherein said water-soluble,
nonpeptidic polymer is polyethylene glycol or a copolymer of
polyethylene glycol and polypropylene glycol.
8. The conjugate of claim 1, wherein said water-soluble,
nonpeptidic polymer is polyethylene glycol.
9. The conjugate of claim 8, wherein said polyethylene glycol is
selected from the group consisting of monomethoxypolyethylene
glycol, branched polyethylene glycol, polyethylene glycol with
degradable linkages in the backbone, homobifunctional polyethylene
glycol, heterobifunctional polyethylene glycol, multi-arm
polyethylene glycol, pendant polyethylene glycol, and forked
polyethylene glycol.
10. The conjugate of claim 9, wherein said peptide is conjugated to
at least one polyethylene glycol molecule.
11. The conjugate of claim 3, wherein the dimeric form of said
peptide has two polyethylene glycol chains covalently attached.
12. The conjugate of claim 5, wherein said biphalin has two
polyethylene glycol chains covalently attached.
13. The conjugate of claim 8, wherein said polyethylene glycol has
a nominal average molecular weight of about 200 daltons to about
100,000 daltons.
14. The conjugate of claim 13, wherein said polyethylene glycol has
a nominal average molecular weight of about 1000 daltons to about
40,000 daltons.
15. The conjugate of claim 13, wherein said polyethylene glycol has
a nominal average molecular weight of 2000 daltons.
16. A composition comprising a conjugate according to claim 1 and a
pharmaceutically acceptable carrier for said conjugate.
17. A method for delivering a peptide into the brain of an animal
through the blood-brain barrier comprising: providing a conjugate
between a peptide and a water-soluble polymer, non-peptidic
polymer; administering said conjugate into the blood stream of an
animal; and transporting the conjugate across the blood-brain
barrier of said animal.
18. The method of claim 18, wherein said peptide is an analgesic
peptide.
19. The method of claim 18, wherein said polymer is selected from
the group consisting of copolymers of polyethylene glycol and
polypropylene glycol, monomethoxypolyethylene glycol, branched
polyethylene glycol, polyethylene glycol with degradable linkages
in the backbone, homobifunctional polyethylene glycol,
heterobifunctional polyethylene glycol, multi-arm polyethylene
glycol, pendant polyethylene glycol, and forked polyethylene
glycol.
20. The method of claim 19, wherein said polyethylene glycol has a
nominal average molecular weight from about 200 to about 100,000
daltons.
21. The method of claim 18, wherein said peptide is an analgesic
peptide selected from the group consisting of dynorphins,
enkephalins, endorphins, endomorphins, biphalin, and analogs and
derivatives thereof.
22. The method of claim 17, wherein said peptide is conjugated to
at least one polymer molecule.
23. The method of claim 17, wherein said peptide is conjugated to
at least two polymer molecules.
24. The method of claim 17, wherein said step of administering said
conjugate comprises parenterally injecting said conjugate into said
animal.
25. The method of claim 17, wherein said step of administering said
conjugate comprises of pulmonary and intranasal inhalation into
said animal.
26. The method of claim 17, wherein said step of administering said
conjugate is by oral, ocular, buccal, transdermal, or rectal
administration.
27. A method for delivering into the brain of an animal through the
blood-brain barrier an agent that is incapable of crossing the
blood-brain barrier comprising: providing a conjugate comprising a
water-soluble, nonpeptidic polymer, a peptide that is transportable
across the blood-brain barrier covalently linked to said polymer,
and a nontransportable agent also covalently linked to the polymer;
and administering the conjugate into the blood stream of said
animal and transporting the conjugate across the blood-brain
barrier of said animal.
28. The method of claim 27, wherein said non-transportable agent is
an imaging agent.
29. The method of claim 27, wherein said non-transportable agent is
doxorubicin.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to commonly owned copending
Provisional Applications Serial No. 60/157,503, filed Oct. 4, 1999,
and Serial No. 60/166,589, filed Nov. 19, 1999, and claims the
benefit of their earlier filing dates under 35 U.S.C. Section
119(e).
FIELD OF THE INVENTION
[0002] The invention relates to a conjugate between a peptide and
polyethylene glycol or a substantially substitutable polymer and a
method of use thereof.
BACKGROUND OF THE INVENTION
[0003] There has been significant progress in the discovery and
development of potential neuropharmaceuticals (small molecules,
peptides, proteins, and antisense) for treating pain and brain
disorders such as Alzheimer's and Parkinson's diseases over the
last decade. However, systemic delivery of many newly discovered
neuropharmaceuticals has been hampered by the lack of an effective
system for delivering them. Intravenous injection is usually
ineffective because of inadequate transport across the barrier
between the brain and the blood supply (the "blood-brain barrier"
or "BBB"). The blood-brain barrier is a continuous physical barrier
that separates the central nervous system, i.e., the brain tissue,
from the general circulation of an animal. The barrier is comprised
of microvascular endothelial cells that are joined together by
complex tight intracellular junctions. This barrier allows the
selective exchange of molecules between the brain and the blood,
and prevents many hydrophilic drugs and peptides from entering into
the brain. Many of the new potent neuroactive pharmaceuticals do
not cross the BBB because they have a molecular weight above 500
daltons and are hydrophilic. Compounds that are non-lipophilic and
have a molecular weight greater than 500 daltons generally do not
cross the BBB.
[0004] Several strategies for delivering high molecular weight,
non-lipophilic drugs to the brain have been developed including
intracerebroventricular infusion, transplantation of genetically
engineered cells that secrete the neuroactive compound, and
implantation of a polymer matrix containing the pharmaceutical. See
Pardridge, W. M., J. Controlled Rel., (1996) 39:281-286. However,
all of these involve invasive surgical procedures that can entail a
variety of complications.
[0005] Four nonsurgical transport mechanisms have been identified
for crossing the BBB, including: (i) transmembrane diffusion, (ii)
receptor-mediated transport, (iii) absorptive-mediated endocytosis,
and (iv) carrier-mediated transport. See Brownless et al., J.
Neurochemistry, (1993) 60(3):793-803. Vascular permeability can be
increased by opening the tight junctions with hyperosmotic
saccharide solutions and analogs of bradykinin. An inherent problem
in this method is that undesirable compounds in the general
circulation may enter the brain through the artificially enlarged
openings in the blood-brain barrier.
[0006] It has been discovered that capillary endothelial cells in
the blood-brain barrier have a high level of receptors to
transferrin, insulin, insulin-like growth factor I and II,
low-density lipoprotein and atrial natriuretic factor. See Friden,
P. M., J. Controlled Rel., (1996) 46:117-128. U.S. Pat. No.
5,833,988 to Friden describes a method for delivering a
neuropharmaceutical or diagnostic agent across the blood-brain
barrier employing an antibody against the transferrin receptor. A
nerve growth factor or a neurotrophic factor is conjugated to a
transferrin receptor-specific antibody. The resulting conjugate is
administered to an animal and is capable crossing the blood-brain
barrier into the brain of the animal.
[0007] U.S. Pat. No. 4,902,505 to Pardridge et al. describes the
use of chimeric peptides for neuropeptide delivery through the
blood-brain barrier. A receptor-specific peptide is used to carry a
neuroactive hydrophilic peptide through the BBB. The disclosed
carrier proteins, which are capable of crossing the BBB by
receptor-mediated transcytosis, include histone, insulin,
transferrin, insulin-like growth factor I (IGF-I), insulin-like
growth factor II (IGF-II), basic albumin, and prolactin. U.S. Pat.
No. 5,442,043 to Fukuta et al. discloses using an insulin fragment
as a carrier in a chimeric peptide for transporting a neuropeptide
across the blood-brain barrier.
[0008] Non-invasive approaches for delivering neuropharmaceutical
agents across the BBB are typically less effective than the
invasive methods in actually getting the agent into the brain. High
doses of the chimeric peptides are required to achieve the desired
therapeutic effect because they are prone to degradation. The
concentration of the chimeric peptides in the blood circulation can
be quickly reduced by proteolysis. An aqueous delivery system is
not generally effective for delivering hydrophobic drugs.
[0009] Another method for delivering hydrophilic compounds into the
brain by receptor-mediated transcytosis is described by Pardridge
et al. in Pharm. Res. (1998) 15(4):576-582. A monoclonal antibody
to the transferrin receptor (OX26 MAb) modified with streptavidin
is used to transport the cationic protein, brain-derived
neurotrophic factor (BDNF) through the BBB. BDNF is first modified
with PEG.sup.2000-biotin to form BDNF-PEG.sup.2000-biotin, which is
then bound to the streptavidin-modified antibody OX26 MAb. The
resulting conjugate was shown to be able to cross the BBB into the
brain.
[0010] Enhancing the duration of antinociceptive effects in animals
may result in less frequently administered analgesics, which can
improve patient compliance and reduce potential side effects. Maeda
et al. in Chem. Pharm. Bull. (1993) 41(11): 2053-2054, Biol. Pharm
Bull. (1994) 17(6):823-825, and Chem. Pharm. Bull. (1994)
42(9):1859-1863 demonstrate that by attaching polyethylene glycol
amine 4000 to the C-terminal leucine of Leu-enkephalin (distant
from the tyrosine residue needed for antinociception), they could
increase the potency and duration of Leu-enkaphalin when it was
directly administered to the brain by intracerebroventricular
injection.
[0011] There is a need in the art to deliver neuroactive agents
from systemic circulation across the blood-brain barrier and into
the brain that reduces or eliminates some of the drawbacks and
disadvantages associated with the prior art.
SUMMARY OF THE INVENTION
[0012] This invention provides a method for delivering a peptide
into the brain of a human or other animal through the blood-brain
barrier. The peptide to be delivered is bonded to a water soluble,
non-peptidic polymer to form a conjugate. The conjugate is then
administered to an animal into the blood circulation so that the
conjugate passes across the blood-brain barrier and into the brain.
The water-soluble nonpeptidic polymer can be selected from the
group consisting of polyethylene glycol and copolymers of
polyethylene glycol and polypropylene glycol activated for
conjugation by covalent attachment to the peptide.
[0013] In one embodiment of this invention, a substantially
hydrophilic conjugate is provided having a transportable analgesic
peptide, i.e., an analgesic peptide capable of passing the
blood-brain barrier, covalently linked to a water-soluble, and
nonpeptidic polymer such as polyethylene glycol. The conjugate is
capable of passing the blood-brain barrier of an animal.
[0014] Suitable transportable peptides for use in this embodiment
of the invention can include dynorphins, enkephalins, endorphins,
endomorphins, and biphalin. Typically, these small neuropeptides
are susceptible to degradation inside the body in blood circulation
and in the brain. In contrast, when conjugated to polyethylene
glycol or to a similar nonpeptidic, nonimmunogenic, water-soluble
polymer having similar properties, these peptides exhibit
significantly increased stability.
[0015] In another embodiment of this invention, a composition is
provided comprising a conjugate of this invention as described
above and a pharmaceutically acceptable carrier. The composition
can be directly administered into the general circulation of an
animal by any suitable means, e.g., parenteral injection, injection
of intracerebral vein, and intranasal, pulmonary, ocular, and
buccal administration.
[0016] In accordance with yet another embodiment of this invention,
a method is provided for delivering an analgesic peptide across the
blood-brain barrier into the brain of an animal. The method
comprises providing a conjugate of this invention as described
above, and administering the conjugate into the bloodstream of the
host animal.
[0017] It has previously been considered that large hydrophilic
polymers such as polyethylene glycol, when attached to a peptide
that is capable of crossing the blood-brain barrier, would
interfere with the transport of the peptide across the blood-brain
barrier. In particular, it has been believed that direct
conjugation of large hydrophilic polymers to a peptide not only
would increase the hydrophilicity but would also impair the
interaction between the peptide and its receptor or other
structures in the BBB by steric interference from the large polymer
strands.
[0018] It has now been discovered that, although the conjugate is
substantially hydrophilic and contains a water-soluble and
nonpeptidic polymer, the conjugate is nevertheless capable of
passing the blood brain barrier of an animal. As compared to its
native state, peptides conjugated to a water-soluble and
non-peptidic polymer can exhibit reduced immunogenicity, enhanced
water solubility, and increased stability. In particular, peptides
conjugated to polyethylene glycol in accordance with this invention
have a longer circulation time, reduced susceptibility to metabolic
degradation and clearance, and once delivered into the brain
through the blood-brain barrier, exhibit extended lifetime in the
brain. Thus, this invention allows effective delivery of analgesic
peptides into human and other animal brains and can significantly
improve the efficacy of the peptides being delivered.
DETAILED DESCRIPTION OF THE INVENTION
[0019] As used herein, "passing the blood-brain barrier" or
"crossing the blood-brain barrier" means that, once administered
into the blood circulation of an animal at a physiologically
acceptable ordinary dosage, a conjugate or a peptide is capable of
passing the blood-brain barrier of the animal to such a degree that
a sufficient amount of the conjugate or peptide is delivered into
the brain of the animal to exert a therapeutic, antinociceptive, or
prophylactic effect on the brain, or to affect the biological
functioning of the brain to a detectable degree. "Passing the
blood-brain barrier" or "crossing the blood-brain barrier" can also
be used herein to mean that the conjugate or peptide is capable of
being taken up by an animal brain to a degree that is detectable by
a suitable method known in the art, e.g., in situ brain perfusion
as disclosed in Williams et al., J. Neurochem., 66 (3),
pp1289-1299, 1996, which is incorporated herein by reference.
[0020] The conjugate of this invention normally is substantially
hydrophilic. By the term "substantially hydrophilic," it is
intended to mean that the conjugate of this invention does not
contain a substantially lipophilic moiety such as fatty acids or
glycolipids. Fatty acids and glycolipids are used in the art to
increase the lipophilicity of a molecule in order to increase the
ability of the molecule to pass cell membranes.
[0021] The term "analgesic" as used herein means any chemical
substances that are desirable for delivery into the brain of humans
or other animals for purposes of alleviating, mitigating, or
preventing pain in humans or other animals, or otherwise enhancing
physical or mental well being of humans or animals. Analgesic
peptides can be introduced into the brain of an animal to exert a
therapeutic, antinociceptive, or prophylactic effect on the
biological functions of the animal brain, and can be used to treat
or prevent pain.
[0022] Agents not typically considered "analgesic" can be attached
to the peptide/polymer conjugate of the invention. For example,
diagnostic or imaging agents can be attached to the conjugate.
Fluoroscein, proteins, or other types of agents specifically
targeted to a particular type of cell or protein, such as
monoclonal antibodies, can all be used in the conjugate of this
invention for diagnostic or imaging purposes.
[0023] As described below, when an agent is incapable of passing
the blood-brain barrier, i.e., is non-transportable across the BBB,
then typically a peptide which is capable of passing the
blood-brain barrier, i.e., is transportable across the BBB, will be
used in a conjugate of this invention as a carrier.
[0024] In one embodiment of this invention, the peptide is a
transportable analgesic peptide. As used herein, the term
"transportable" means that the peptide is capable of crossing the
blood-brain barrier of an animal as defined above. Thus, a
conjugate is provided comprising a transportable peptide bonded to
a water-soluble, nonpeptidic, nonimmunogenic polymer, including
polyethylene glycol.
[0025] The term "peptide" means any polymerized .alpha.-amino acid
sequence consisting from 2 to about 40 amino acids having a peptide
bond (--CO--NH--) between each amino acid that can impact the
condition and biological function of the brain of an animal. An
analgesic peptide normally is an endogenous peptide naturally
occurring in an animal, or fragments or analogs thereof. However,
non-endogenous peptides that can impact the conditions and
biological functions of animal brain are also included.
[0026] Many peptides are generally known in the art that are
believed to be capable of passing the blood-brain barrier. Examples
of transportable peptides that are believed to be capable of
crossing the blood-brain barrier after PEGylation in accordance
with the invention include, but are not limited to, biphalin and
opioid peptides such as dynorphins, enkephalins, endorphins,
endomorphins etc. Many derivatives and analogues of these
transportable peptides can also be used in the practice of the
invention.
[0027] Opioid peptides are believed to be especially suitable for
practice of the invention. Opioid peptides exhibit a variety of
pharmacological activities, including among them pain relief and
analgesia.
[0028] Enkephalin is a pentapeptide having an amino acid sequence
of H-Tyr-Gly-Gly-Phe-Met-OH (methionine enkephalin) or
H-Tyr-Gly-Gly-Phe-Leu-OH (leucine enkephalin). Many enkephalin
analogs have been identified and synthesized which are specific to
different types of opiate receptors. See, e.g., Hruby and Gehrig,
(1989) Medicinal Research Reviews, 9(3):343-401. For example, U.S.
Pat. No. 4,518,711 discloses several enkephalin analogs including
DPDPE, [D-Pen.sup.2, D-Pen.sup.5] enkephalin, which is a cyclic
enkephalin analog made by substituting the second and fifth amino
acid residues of the natural pentapeptides with either cysteine or
with D- or L-penicillamine (beta, beta-dimethylcysteine) and
joining the two positions by a disulfide bond. DPDPE has been shown
to be able to pass the blood brain barrier into the brain. See,
e.g., Williams et al. (1996) Journal of Neurochemistry,
66(3):1289-1299. U.S. Pat. No. 5,326,751 discloses DPADPE prepared
by substituting the glycine residue at the third position of DPDPE
with an alanine residue. Both of the patents are incorporated
herein by reference.
[0029] Other enkephalin analogs include biphalin
(H-Tyr-D-Ala-Gly-Phe-NH-)- .sub.2, which is a synthetic analog of
enkephalin that is a dimerized tetramer produced by coupling two
units having the formula H-Tyr-D-Ala-Gly-Phe-OH at the C-terminus
with hydrazine. The dimeric form of enkephalin enhances affinity,
and specificity to the delta-opioid receptor. Dimeric enkephalin
analogs are disclosed in Rodbard et al. U.S. Pat. No. 4,468,383,
the contents of which are incorporated herein by reference.
[0030] Dynorphins are another class of opioid peptides. Naturally
isolated dynorphin has 17 amino acids. Many dynorphin fragments and
analogs have been proposed in the art, including, e.g., dynorphin
(1-10), dynorphin (1-13), dynorphin (1-13) amide, [D-Pro.sup.10]
Dynorphin (1-11) (DPDYN), dynorphin amide analogs, etc. See, e.g.,
U.S. Pat. Nos. 4,684,624, 4,62,941, and 5,017,689, which are
incorporated herein by reference. Although such analgesic peptides
are capable of transporting across the blood-brain barrier, many of
them have a very short half-life due to their susceptibility to
biodegradation inside the body.
[0031] Even though polyethylene glycol normally has a large
molecular weight and is hydrophilic, conjugation to the
transportable peptides in the absence of a lipophilic moiety does
not interfere with transportability of the peptides. The conjugated
peptides remain capable of crossing the blood-brain barrier.
Typically, upon administration into the general circulation of an
animal, the conjugate of the invention, comprising a transportable
peptide bonded to polyethylene glycol or an equivalent polymer, is
taken up by the brain at a much greater percentage as compared to
an unconjugated form of the peptide. The peptides in the conjugates
of this invention have increased stability and exhibit extended
half-life inside the body.
[0032] In another embodiment of this invention, a conjugate is
provided comprising a first peptide, which is a transportable
peptide, and a second neuroactive agent linked to each other by
polyethylene glycol or an equivalent polymer. This second
neuroactive agent may or may not be capable of crossing the
blood-brain barrier by itself. The transportable peptide is used as
a carrier to transport a non-transportable neuroactive agent across
the blood-brain barrier into the brain of an animal. The linking
polymer serves not only as a linker but also increases solubility
and stability of the conjugate and reduces the immunogenicity of
both the neuropeptide and the other neuroactive agent to be
delivered.
[0033] In accordance with the invention, the transportable peptide
and, optionally, another neuroactive agent as described above, are
covalently linked to a water-soluble and nonpeptidic polymer to
form a conjugate of this invention. The water-soluble and
nonpeptidic polymers suitable for use in various aspects of this
invention include polyethylene glycol, other polyalkylene glycols,
and copolymers of polyethylene glycol and polypropylene glycol.
[0034] As used herein, the term polyethylene glycol ("PEG") is
inclusive and means any of a series of polymers having the general
formula:
HO--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--OH
[0035] wherein n ranges from about 10 to 2,000. PEG also refers to
the structural unit:
--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--
[0036] wherein n ranges from about 10 to about 2000. Thus, by PEG
is meant modified PEGs including methoxy-PEGs; PEGs having at least
one terminal moiety other than a hydroxyl group which is reactive
with another moiety; branched PEGs; pendent PEGs; forked PEGs; and
the like.
[0037] The polyethylene glycol useful in the practice of this
invention normally has an average molecular weight of from about
200 to 100,000 daltons. Molecular weights of from about 200 to
10,000 are somewhat more commonly used. Molecular weights of from
about 300 to 8,000, and in particular, from about 500 to about
5,000 daltons, are somewhat typical.
[0038] PEG is useful in biological applications because it has
properties that are highly desirable and is generally approved for
biological or biotechnical applications. PEG typically is clear,
colorless, odorless, soluble in water, stable to heat, inert to
many chemical agents, does not hydrolyze or deteriorate, and is
generally nontoxic. Poly(ethylene glycol) is considered to be
biocompatible, which is to say that PEG is capable of coexistence
with living tissues or organisms without causing harm. More
specifically, PEG, in itself, is normally considered
nonimmunogenic, which is to say that PEG does not tend to produce
an immune response in the body. Desirable terminal activating
groups by which PEG can be attached to various peptides should not
appreciably alter the nonimmunogenic character of the PEG, so as to
avoid immunogenic effects. Desirable PEG conjugates tend not to
produce a substantial immune response or cause clotting or other
undesirable effects.
[0039] PEG is a highly hydrated random coil polymer that can shield
proteins or peptides from enzymatic digestion, immune system
molecules and cells, and can increase the hydrodynamic volume to
slow reticuloendothelial system (RES) clearance. PEG is a useful
polymer having the properties of water solubility as well as
solubility in many organic solvents. The unique solubility
properties of PEG allow conjugation (PEGylation) to certain
compounds with low aqueous solubility, with the resulting conjugate
being water-soluble. However, PEGylation, which is conjugating a
PEG molecule to another molecule, is not without its difficulties.
The effects of a particular PEG derivative are not necessarily
predictable. The result depends on the specific interaction between
a particular compound and the functional non-peptidic PEG
polymer.
[0040] The polymer used in this invention normally can be linear or
branched. Branched polymer backbones are generally known in the
art. Typically, a branched polymer has a central core moiety and a
plurality of linear polymer chains linked to the central core. PEG
is commonly used in branched forms that can be prepared by addition
of ethylene oxide to various polyols, such as glycerol,
pentaerythritol and sorbitol. For example, the four-arm, branched
PEG prepared from pentaerythritol is shown below:
C(CH.sub.2--OH).sub.4+n
C.sub.2H.sub.4O.fwdarw.C[CH.sub.2O--(CH.sub.2CH.su-
b.2O).sub.n--CH.sub.2CH.sub.2--OH].sub.4
[0041] The central moiety can also be derived from several amino
acids. An example is lysine.
[0042] The branched polyethylene glycols can be represented in
general form as R(-PEG-OH).sub.n in which R represents the core
moiety, such as glycerol or pentaerythritol, and n represents the
number of arms. Suitable branched PEGs can be prepared in
accordance with U.S. Pat. No. 5,932,462, the contents of which are
incorporated herein in their entirety by reference. These branched
PEGs can then be used in accordance with the teachings herein.
[0043] Forked PEGs and related polymers should be useful in the
practice of the invention. The term "forked" is used to describe
those PEGs that are branched adjacent at least one terminus
thereof. The polymer has a branched moiety at one end of the
polymer chain and two free reactive groups, one on each end of the
branched moiety, for covalent attachment to another molecule. Each
reactive moiety can have a tethering group, including, for example,
an alkyl chain, linking a reactive group to the branched moiety.
Thus, the branched terminus allows the polymer to react with two
molecules to form conjugates. Forked PEGs and related forked
polymers are described in copending, commonly owned U.S. patent
application Ser. No. 09/265,989, which was filed Mar. 11, 1999 and
is entitled Poly(ethylene glycol) Derivatives with Proximal
Reactive Groups. This pending patent application is incorporated by
reference herein in its entirety. The forked PEGs can be either
linear or branched in the backbone attached to the branched
terminus.
[0044] Water-soluble, substantially nonimmunogenic, nonpeptidic
polymers other than PEG should also be suitable for practice of the
invention, although not necessarily with equivalent results. These
other polymers can be either in linear form or branched form, and
include, but are not limited to, other poly(alkylene oxides),
including copolymers of ethylene glycol and propylene glycol, and
the like. Exemplary polymers are listed in U.S. Pat. No. 5,990,237,
the contents of which are incorporated herein by reference in their
entirety. The polymers can be homopolymers or random or block
copolymers and terpolymers based on the monomers of the above
polymers, straight chain or branched.
[0045] Specific examples of suitable additional polymers include,
but are not limited to, poly(acryloylmorpholine) ("PAcM") and
poly(vinylpyrrolidone)("PVP"), and poly(oxazoline). PVP and
poly(oxazoline) are well known polymers in the art and their
preparation should be readily apparent to the skilled artisan. PAcM
and its synthesis and use are described in U.S. Pat. Nos. 5,629,384
and 5,631,322, the contents of which are incorporated herein by
reference in their entirety.
[0046] To couple PEG to a peptide, e.g., a transportable peptide,
to form a conjugate of this invention, it is often necessary to
"activate" the PEG to prepare a derivative of the PEG having a
reactive group at the terminus for reaction with certain moieties
on the peptide. Many activated derivatives of PEG have been
described in the art and can all be used in this invention,
although not necessarily with equivalent results. An example of
such an activated derivative is the succinimidyl succinate "active
ester":
CH.sub.3O-PEG-O.sub.2C--CH.sub.2CH.sub.2--CO.sub.2--NS
[0047] where NS= 1
[0048] The succinimidyl active ester is a useful compound because
it reacts rapidly with amino groups on proteins and other molecules
to form an amide linkage (--CO--NH--). For example, U.S. Pat. No.
4,179,337 to Davis et al. describes coupling of this derivative to
proteins (represented as PRO-NH.sub.2):
mPEG-O.sub.2CCH.sub.2CH.sub.2CO.sub.2NS+PRO-NH.sub.2.fwdarw.mPEG-O.sub.2C--
-CH.sub.2CH.sub.2--CONH-PRO
[0049] Other activated PEG molecules known in the art include PEGs
having a reactive cyanuric chloride moiety, succinimidyl carbonates
of PEG, phenylcarbonates of PEG, imidazolyl formate derivatives of
PEG, PEG-carboxymethyl azide, PEG-imidoesters, PEG-vinyl sulfone,
active ethyl sulfone derivatives of PEG, tresylates of PEG,
PEG-phenylglyoxal, PEGs activated with an aldehyde group,
PEG-maleimides, PEGs with a terminal amino moiety, and others.
These polyethylene glycol derivatives and methods for conjugating
such derivatives to an agent are generally known in the art and are
described in Zalipsky et al., Use of Functionalized Poly(Ethylene
Glycol)s for Modification of Polypeptides, in Use of Polyethylene
Glycol Chemistry: Biotechnical and Biomedical Applications, J. M.
Harris, Ed., Plenum Press, New York (1992), and in Zalipsky,
Advanced Drug Reviews (1995) 16:157-182, all of which are
incorporated herein by reference.
[0050] Typically, conjugation of a water-soluble, nonimmunogenic
polymer to a peptide in accordance with this invention results in
the formation of a linkage between the polymer and the peptide. The
term "linkage" is used herein to refer to groups or bonds normally
formed as a result of a chemical reaction.
[0051] Covalent linkages formed in the practice of this invention
can be hydrolytically stable. The linkage can be substantially
stable in water and does not react with water at a useful pH, under
physiological conditions, for an extended period of time,
preferably indefinitely. Alternatively, the covalent linkage can
also be hydrolytically degradable under physiological conditions so
that the neuroactive agent can be released from the PEG in the body
of an animal, preferably after it is delivered into the brain of
the animal.
[0052] The approach in which drugs to be delivered are released by
degradation of more complex agents under physiological conditions
is a powerful component of drug delivery. See R. B. Greenwald, Exp.
Opin. Ther. Patents, 7(6):601-609 (1997). For example, conjugates
of the invention can be formed by attaching PEG to transportable
peptides and/or neuroactive agents using linkages that are
degradable under physiological conditions. The half-life of a
PEG-neuroactive agent conjugate in vivo depends upon the type of
reactive group of the PEG molecule that links the PEG to the
neuroactive agent. Typically, ester linkages, formed by reaction of
PEG carboxylic acids or activated PEG carboxylic acids with alcohol
groups on neuroactive agents, hydrolyze under physiological
conditions to release the neuroactive agent. See, e.g., S.
Zalipsky, Advanced Drug Delivery Reviews, 16:157-182 (1995). For
example, in PCT Publication No. WO 96/23794, it is disclosed that
paclitaxel can be linked to PEG using ester linkages and the linked
paclitaxel can be released in serum by hydrolysis. Antimalarial
activity of dihydroartemisinin bonded to PEG through a hydrolyzable
ester linkage has also been demonstrated. See Bentley et al.,
Polymer Preprints, 38(1):584 (1997). Other examples of suitable
hydrolytically unstable linkages include carboxylate esters,
phosphate esters, disulfides, acetals, imines, orthoesters,
peptides and oligonucleotides.
[0053] Typically, the degradation rate of the conjugate should be
controlled such that substantial degradation does not occur until
the conjugate passes into the brain of an animal. Many peptides in
their native state are subject to substantial degradation in blood
circulation and in organs such as liver and kidney. The
hydrolytically degradable linkages can be formed such that the
half-life of the conjugate is longer than the time required for the
circulation of the conjugate in the bloodstream to reach the
blood-brain barrier. Some minor degree of experimentation may be
required for determining the suitable hydrolytically unstable
linkage between specific neuroactive agents and PEG derivatives,
this being well within the capability of one skilled in the art
once apprised of the present disclosure.
[0054] The covalent linkage between a peptide and a polymer can be
formed by reacting a polymer derivative such as an activated PEG
with an active moiety on the peptide. One or more PEG molecules can
be linked to one peptide.
[0055] Conversely, multiple peptides, including transportable
peptides and/or other types of neuroactive agents, can be linked to
one PEG molecule. Typically, such a PEG molecule has multiple
reactive moieties for reaction with the peptide and neuroactive
agents. For this purpose, bifunctional PEGs, pendant PEGs, and
dendritic PEGs can all be used. Reactive PEGs have also been
synthesized in which several active functional groups are placed
along the backbone of the polymer. For example, lysine-PEG
conjugates have been prepared in the art in which a number of
activated groups are placed along the backbone of the polymer.
Zalipsky et al., Bioconjugate Chemistry, (1993) 4:54-62.
[0056] In one embodiment of this invention, a conjugate having a
dumbbell structure is provided wherein a transportable peptide or
other transportable neuroactive agent capable of passing the
blood-brain barrier of an animal is covalently linked to one end of
a polyethylene glycol molecule, and another neuroactive agent to be
delivered into the brain of an animal is linked to the other end of
the PEG molecule. This other neuroactive agent can be a
transportable peptide, or any other neuroactive agent. Typically,
it is not transportable and cannot in itself pass the blood-brain
barrier. Therefore, the transportable peptide or other agent at one
end of the PEG molecule acts as a carrier for delivering the
non-transportable neuroactive agent into the brain. For this
purpose, bifunctional PEGs, either homobifunctional or
heterobifunctional PEGs, can be used. As used herein "bifunctional
PEG" means a PEG derivative having two active moieties each being
capable of reacting with an active moiety in another molecule. The
two active moieties can be at two ends of a PEG chain, or proximate
to each other at a forked end of a PEG chain molecule, allowing for
steric hindrance, if any. Suitable transportable peptides for use
in this invention are described above including, but not limited
to, dynorphins, enkephalins, biphalin, endorphins, endomorphins,
and derivatives and analogues thereof.
[0057] The conjugate of this invention can be administered to an
animal for purposes of treating, mitigating, or alleviating pain.
Examples of animal hosts include, but are not limited to, mammals
such as humans, and domestic animals including cats, dogs, cows,
horses, mice, and rats.
[0058] The conjugate of this invention can be administered in any
suitable manner to an animal. For example, the conjugate can be
administered parenterally by intravenous injection, intramuscular
injection, or subcutaneous injection. Alternatively, the conjugate
of this invention can also be introduced into the body by
intranasal and pulmonary inhalation or by oral and buccal
administration. Preferably, intravenous injection is utilized such
that substantially all of the conjugate in an injection dose is
delivered into the bloodstream of the animal, through which the
conjugate circulates to the blood-brain barrier of the animal.
[0059] The conjugate can be injected in the form of any suitable
type of formulation. For example, an injectable composition can be
prepared by any known methods in the art containing the conjugate
of this invention in a solvent such as water or solution, including
saline, Ringer's solution. One or more pharmaceutically acceptable
carriers that are compatible with the other ingredients in the
formulation may also be added to the formulation. Excipients,
including mannitol, sodium alginate, and carboxymethyl cellulose,
can also be included. Other pharmaceutically acceptable components,
including antiseptics such as phenylethylalcohol; stabilizers such
as polyethylene glycol and albumin; isotonizing agents such as
glycerol, sorbitol, and glucose; dissolution aids; stabilizing
buffers such as sodium citrate, sodium acetate and sodium
phosphate; preservatives such as benzyl alcohol; thickeners such as
dextrose, and other commonly used additives can also be included in
the formulations. The injectable formulation can also be prepared
in a solid form such as lyophilized form.
[0060] The PEGylated transportable peptides of the invention can be
administered in a variety of formulations, including, for example,
intranasal, buccal, and oral administration. The dosage of the
conjugate administered to a human or other animal will vary
depending on the animal host, the types of transportable peptides
and/or neuroactive agents used, the means of administration, and
the symptoms suffered by the animal. However, the suitable dosage
ranges in a specific situation should be readily determinable by a
skilled artisan without undue experimentation.
[0061] The invention is further illustrated by the following
examples, which are intended only for illustration purposes and
should not be considered in anyway to limit the invention.
EXAMPLE 1 Modification and Purification PEG-Dynorphin A
[0062] Dynorphin A (1-11)
(H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-N- H.sub.2) (1.47
mg) was dissolved in 0.25 ml deionized water and 0.25 ml of 25 mM
NaP, pH 5.8 buffer in a 1.5 ml microcentrifuge tube. The reagent,
NHS-PEG.sub.2K-Fluoroscein (1.0 mg), was added to the peptide
solution in approximately 2-fold mole excess. After 30 minutes of
reaction time, 0.1 ml of 25 mM sodium phosphate buffer, pH 7.4 was
added and the reaction was allowed to proceed at room temperature
for 3 hours.
[0063] Conjugation of NHS-PEG.sub.2K-Fluoroscein was monitored by
capillary electrophoresis (CE) and mass spectrometry (MALDI).
Purification of the PEG-Dynorphin A conjugate was performed on a
HiTrap SP cation exchange column from Amersham/Pharmacia using a
gradient elution from 5 mM sodium phosphate buffer, pH 4.0 to 50 mM
sodium phosphate, 1.5M NaCl buffer, pH 7.5 in 53 minutes. Fractions
were collected and the contents were analyzed by MALDI. These
fractions were pooled and stored frozen prior to in vivo assay.
EXAMPLE 2
Modification and Purification PEG-Endomorphin II
[0064] Endomorphin II (H-Tyr-Pro-Phe-Phe-NH.sub.2, 2.3 mg) was
dissolved in 1.15 ml of 5 mM sodium phosphate buffer, pH 8.0.
Modification of Endomorphin II was performed in 1.5 hours at room
temperature by adding mPEG.sub.2000-SPA (38 mg) in a 5 mole excess.
The reaction mixture was analyzed by mass spectrometry (MALDI) to
determine the extent of modification. MALDI was used to verify that
the reaction between mPEG.sub.2000-SPA and Endomorphin II went to
completion. The sample was dialyzed against water using a 2000 MWCO
membrane and lyophilized prior to in vivo assay.
EXAMPLE 3
In situ Perfusion, Capillary Depletion, Brain Extraction and
Protein Binding Studies of PEG-Dynorphin A and PEG-Endomorphin
II
[0065] The protocol for the rat brain perfusion experiments was
approved by the Institutional Animal Care and Use Committee at the
University of Arizona. The in situ perfusion, capillary depletion,
brain extract and protein binding studies were carried out as
previously reported (Williams et al., J. Neurochem., 66 (3),
pp1289-1299, 1996). PEG-dynorphin A (PdynA) had a very high in situ
uptake R.sub.Br value of 0.343.+-.1.84. In contrast in situ
perfusion with I.sup.125 Dynorphin, gave a very high R.sub.Br of
approximately 0.96. The entire radioactivity was recovered in the
solvent front of the subsequent HPLC, showing that labeled
dynorphin A (1-11) rapidly degrades, probably to I.sup.125Tyr.
[0066] Capillary depletion studies of the PdynA were carried out,
and revealed that approximately 88% of the radioactivity associated
with the capillary fraction rather than the brain parenchyma.
[0067] In situ uptake of PEG-endomorphin II (Pend) gave an R.sub.Br
value of 0.057.+-.0.008, similar to those previously reported for
peptides. Subsequent capillary depletion showed that of the
radioactivity entering the brain, 32% was associated with the
capillary fraction with 67% in the brain parenchyma.
[0068] The protein binding of Pend was studied using the centrifree
filter system. It was found that 30% of 25,000 dpm Pend was bound
to a 1% BSA solution.
[0069] The major contribution is that PEGylation improved brain and
blood enzymatic stability dramatically. Endomorphin and dynorphin
are very unstable in either brain or blood with half-lives on the
order of minutes. After PEGylation, those half-lives increased to
hours for endomorphin II. In the case of endomorphin II, the
half-life in blood plasma was 3.2 minutes, and brain tissue was 13
minutes. After PEGylation, those half-lives increased to greater
than two hours.
EXAMPLE 4
Conjugation of PEG-Doxorubicin to Endomorphin I
[0070] Endomorphin I (H-Tyr-Pro-Trp-Phe-NH.sub.2, 3.0 mg, 4.9 E-6
moles) was dissolved in 1 ml of 50 mM sodium phosphate, pH 8.2
buffer containing 150 mM NaCl and 50 mM DTT. A four fold molar
excess of Traut's reagent (2.7 mg) was added and was allowed to
react at room temperature for 2 hours. The thiol-modified
endomorphin was purified from DTT and Traut's reagent using a
Superdex 30 size exclusion column (Pharmacia). The modified
endomorphin fractions were collected and lyophilized.
[0071] Doxorubicin hydrochloride (3.0 mg, 5.2 E-6 moles) was
dissolved in 1.0 ml of 50 mM sodium phosphate, pH 7.2 buffer
containing 150 mM NaCl. The pH of the solution was titrated to 8.0
with 0.1N sodium hydroxide. A ten molar excess of
heterobifunctional PEG (NHS-PEG.sub.2K-OPSS) was added to the
doxorubicin solution. The reaction was allowed to proceed at room
temperature for 2 hours. OPSS-PEG.sub.2K-doxorubicin was purified
from unreacted PEG and free doxorubicin using a Superdex 30 size
exclusion column. The OPSS-PEG.sub.2K-doxorubicin fractions were
collected and lyophilized.
[0072] The lyophilized powders of modified endomorphin I and
OPSS-PEG.sub.2K-doxorubicin were reconstituted in 50 mM sodium
phosphate buffer, pH 6.0. An equimolar amount of each solution was
mixed together and the two were reacted at room temperature for 6
hours. The doxorubicin-PEG.sub.2K-endomorphin conjugate was
purified on a Superdex 30 size exclusion column.
EXAMPLE 5
Conjugation of PEG to DPDPE
[0073] 3.0 mg of DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen) was dissolved in 5
ml of anhydrous acetonitrile. A 20% molar excess of PEG reagent
(either mPEG-SPA 5K [27.9 mg] or mPEG-SPA 2K [11.1 mg]) and
triethylamine (0.8 .mu.l) was added to the DPDPE. The reaction was
allowed to proceed at room temperature under an argon atmosphere
for 2 days. The sample was diluted to 15 ml with deionized water
and lyophilized. The PEG-DPDPE powder was reconstituted in 5 ml of
deionized water and purified on a Superdex 30 size exclusion
column. The pertinent fractions were pooled together, dialyzed
against water and frozen until in situ perfusion experiments.
[0074] Both PEG.sub.2k-DPDPE and PEG.sub.5k-DPDPE were iodinated
and tested in in situ perfusion, capillary depletion, brain
extraction and protein binding studies as in Example 3. A
significant increase in brain uptake was observed for both
PEG.sub.2k-DPDPE and PEG.sub.5k-DPDPE. It was determined that for
both of these compounds, the increase in uptake was due to peptide
entering the brain rather than being trapped in the
capillaries.
EXAMPLE 6
Conjugation of PEG to Biphalin
[0075] a. (mPEG.sub.2K).sub.2-Biphalin
[0076] Biphalin (21.1 mg, 0.046 mmol) was dissolved into 15 ml of
anhydrous acetonitrile and treated with 16 .mu.l of triethylamine
(0.115 mmol, 2.5 fold molar excess). At the same time,
mPEG.sub.2K-SPA (110 mg, 0.055 mmol, 1.2 fold molar excess) was
dissolved into 5 ml of acetonitrile. The dissolved mPEG.sub.2K-SPA
was slowly added into the above biphalin solution and the reaction
mixture was stirred 66 hours at room temperature under nitrogen
atmosphere.
[0077] Di-pegylated [(mPEG.sub.2K).sub.2-biphalin] and
monopegylated biphalin [mPEG.sub.2K-biphalin] were separated from
unreacted PEG and free biphalin on a Vydac C18 reverse-phase column
at 1 ml/min and 215 nm UV detector using a gradient elution of 30%
to 60% solvent B. Solvent A is 0.1% TFA in water and solvent B is
0.1% TFA in acetonitrile.
[0078] b. (mPEG.sub.5K).sub.2-Biphalin
[0079] Dissolve 118.7 mg mthoxy-PEG.sub.5K-SPA
(2.374.times.10.sup.-5 moles, 1.5 fold molar excess) in 3.0 mL
anhydrous acetonitrile. Under a slow Argon flow, add 10.0 mg
Biphalin (1.583.times.10.sup.-5 moles of --NH.sub.2 group) followed
by pipette 4.4 .mu.L triethylamine (3.166.times.10.sup.-5 moles,
2.0 fold molar excess) into the solution. Stir at ambient solution
for overnight.
[0080] Evaporate solvent on rotary evaporator at 40.degree. C. till
near dryness, then further dry on high vacuum for 5 minutes (Use a
liquid nitrogen trap when apply vacuum). Dissolve the remaining in
10 mL deionized water. The solution pH is 4.5. Load the solution by
injection into a prehydrated Slide-A-Lyzer dialysis cassette with
3500 MWCO (from PIERCE) and dialysis against 2.times.900 mL
deionized water over three days.
[0081] Load the solution onto 2 mL DEAE Sepharose column. Collect
the eluent. Elute the column with additional 125 mL deionized
water, and collect the eluent (pH7.6). Combine the two fractions,
freeze the solution by liquid nitrogen, and then lyophilize on a
freeze dryer.
[0082] c. (mPEG.sub.2K).sub.2-Biphalin
[0083] Dissolve 141.4 mg methoxy-PEG.sub.2K-SPA
(1.187.times.10.sup.-5 moles, 1.5 fold molar excess) in 2.0 mL
anhydrous acetonitrile. Under a slow Argon flow, add 5.0 mg
Biphalin.multidot.2TFA (7.915.times.10.sup.-6 moles of --NH.sub.2
group) followed by pipette 2.2 .mu.L triethylamine
(1.583.times.10.sup.-5 moles, 2.0 fold molar excess) into the
solution. Stir at ambient solution for overnight.
[0084] Evaporate solvent on high vacuum at room temperature till
dryness (Use a liquid nitrogen trap when apply vacuum). Dissolve
the remaining in 10 mL deionized water. Load the solution by
injection into a prehydrated Dialysis Cassette with 10000 MWCO
(from PIERCE) and dialysis against 2.times.800 mL deionized water
over three days.
[0085] Dilute the solution to 18 mL by deionized water. Load the
solution onto 10 mL DEAE Sepharose column. Collect the eluent.
Elute the column with additional 90 mL deionized water. Combine the
fractions, frozen by liquid nitrogen, and then lyophilize on a
freeze dryer.
[0086] d. (mPEG.sub.20K).sub.2-Biphalin
[0087] Dissolve 255.2 mg Methoxy-PEG.sub.20K-SPA
(1.187.times.10.sup.-5 moles, 1.5 fold molar excess) in 3.0 mL
anhydrous acetonitrile. Under a slow Argon flow, add 5.0 mg
Biphalin.multidot.2TFA (7.915.times.10.sup.-6 moles of --NH.sub.2
group) followed by pipette 2.2 .mu.L triethylamine
(1.583.times.10.sup.-5 moles, 2.0 fold molar excess) into the
solution. Stir at ambient solution for overnight.
[0088] Evaporate solvent on high vacuum at room temperature until
dryness (Use a liquid nitrogen trap when apply vacuum). Dissolve
the remaining in 10 mL deionized water. Load the solution by
injection into a prehydrated Dialysis Cassette with 10000 MWCO
(from PIERCE) and dialysis against 2.times.800 mL deionized water
over three days.
[0089] Dilute the solution to 25 mL by deionized water. Load the
solution onto 15 mL DEAE Sepharose column. Collect the eluent.
Elute the column with additional 150 mL deionized water. Combine
the fractions, frozen by liquid nitrogen, and then lyophilize on a
freeze dryer.
[0090] Purity of each sample was determined by reverse-phase HPLC
and by mass spectrometry (MALDI).
EXAMPLE 7
Analgesia Assay
[0091] Animals
[0092] Male ICR mice (20-25 g) or male Sprague-Dawley rats (250-300
g) (Harlan Sprague-Dawley Inc., Indianapolis, Ind.) were used for
these experiments. Animals were housed four per cage in an animal
care facility maintained at 22.+-.0.5.degree. C. with an
alternating 12 hr light-dark cycle. Food and water were available
ad libitum. Animals were used only once.
[0093] Protocol
[0094] All drugs were dissolved in sterile saline and were prepared
so that the proper dose would be delivered in 5 .mu.l (i.c.v.), 100
.mu.l (i.v.), 100 .mu.l (s.c.) and 100 .mu.l (i.m.) of the vehicle.
All rodents were recorded for baseline latency before injection of
the drug. A morphine control was used with the i.c.v. and i.v.
injection procedures to compare the analgesic efficacies of test
compounds.
[0095] I.C.V. Inection
[0096] Rodents were placed into a jar containing gauze soaked with
ethyl ether until they went into a light sleep. The rodents were
immediately removed from the jar and a 1/2" incision was made with
a scalpel to expose the top of the skull. The right lateral
ventricle was located by measuring 2 mm lateral of the midline and
2 mm caudal to Bregma. At this point, a Hamilton syringe (22 G,
1/2") was placed through the skull 2 mm and a 5 .mu.l injection of
the compound was delivered. The rodents were then placed back into
their cages until the specified testing time. Methylene blue was
placed into the injection site to insure proper delivery of the
compound into the lateral ventricle.
[0097] L V. Injection
[0098] Rodents were placed into a restraint holder and their tails
were placed into a beaker of warm water and then swabbed with
ethanol to maximize vasodilation in the tail veins. A vein was
selected and the restraint was braced to prevent excessive
movement. A 30 G needle was selected as the proper size for
delivery of the compounds. The needle was carefully inserted into
the vein of each mouse and a 100 .mu.l bolus was slowly delivered.
Blanching of the vein up towards the body was indicative of proper
delivery.
[0099] S.C. Injection
[0100] Rats were restrained by hand to prevent excessive movement.
A 30 G needles was selected as the proper size for delivery of the
compounds. The needle was carefully inserted into the scruff of the
neck of each rat and a 100 .mu.l bolus was slowly delivered.
[0101] I.M. Injection
[0102] Rats were restrained by hand to prevent excessive movement.
A 30 G needles was selected as the proper size for delivery of the
compounds. The needle was carefully inserted into the right hind
leg muscle of each rat and a 100 .mu.l bolus was slowly
delivered.
[0103] Analgesia Testing
[0104] The rodents were placed into restraint holders and their
tails were properly placed under the radiant heat beam. The beam
was turned on and the time until the animal flicked their tail from
under the beam was recorded at each time point. In instances where
the animals moved their tails without a flick, the animals were
retested only if the elapsed time under the radiant beam was less
than 5 seconds.
[0105] Assessment of Analgesic Data
[0106] The raw data (recorded times) was converted to a percentage
of the maximum possible effect (% M.P.E.) which was determined as
15 seconds. % M.P.E. was determined by the following equation:
% M.P.E.=(Recorded time-Baseline)/(15-Baseline).times.100
[0107] These percentages then allow the compound to be plotted
according to % M.P.E. vs. Time. The curve can then be analyzed to
determine the area under the curve (AUC).
[0108] The results of the i.c.v. administration of the PEG-DPDPE
clearly indicates that PEGylation does not interfere with DPDPE's
ability to produce an analgesic effect. (FIG. 1) Furthermore, the
study showed a trend toward a prolongation of analgesic effect of
the PEGylated compound when compared to the parent compound.
[0109] Intraveneous injection of PEG-DPDPE showed that the
PEGylated compound is able to cross the blood brain barrier, in
sufficient amounts, as to maintain its analgesic properties. (FIG.
2) This study also helped confirm that PEGylation for DPDPE
significantly prolongs the duration of the analgesic effect.
[0110] All PEGylated biphalin and biphalin samples exhibited a
potent analgesic response in mice with a maximum response of 80-90%
reached between 30-45 minutes. The (mPEG.sub.2K).sub.2-biphalin
continued to prolong the analgesic effect with a 50% M.P.E. being
seen at the 400 minute mark of the study as compared to the 90
minute mark for native biphalin. The data also shows an inverse
relationship between the molecular weight of PEG and the % M.P.E.
(FIG. 3)
[0111] When comparing the analgesic effect of monopegylated
biphalin (mPEG.sub.2K-biphalin) to that of the dipegylated biphalin
[(mPEG.sub.2K).sub.2-biphalin] at the same concentration in mice,
the duration of analgesic effect for mPEG.sub.2K-biphalin is nearly
half of that for (mPEG.sub.2K).sub.2-biphalin at 50% M.P.E. (FIG.
4) In fact there is nearly equivalent analgesic effect of
mPEG.sub.2K-biphalin at half the dose of
(mPEG.sub.2K).sub.2-biphalin.
[0112] Intravenous administration of (mPEG.sub.2K).sub.2-biphalin
gives a longer lasting analgesic effect in rats than native
biphalin at the various doses tested. (FIG. 5) Rats given
(mPEG.sub.2K).sub.2-biphalin by subcutaneous or intramuscular
administration show elevated and sustained levels of analgesic
activity as compared to native biphalin at the same concentration.
(FIG. 6)
EXAMPLE 8
In situ Perfusion Studies of PEG-DPDPE and PEG-Biphalin
[0113] The protocol for the rat brain perfusion experiments was
approved by the Institutional Animal Care and Use Committee at the
University of Arizona. The in situ perfusion studies were carried
out as previously reported (Williams et al., J. Neurochem., 66(3),
pp1289-1299, 1996). PEG.sub.2K-DPDPE had a very high in situ uptake
R.sub.Br value of 3.41.+-.0.15. The in situ perfusion of I.sup.125
DPDPE is comparable to that of the monopegylated DPDPE,
R.sub.Br=3.54.+-.0.30. I.sup.125 labeled Biphalin has an in situ
perfusion uptake of 7.26.+-.0.11, while the in situ uptake of
(mPEG.sub.2K).sub.2-Biphalin was dramatically lower, R.sub.Br value
of 2.70.+-.0.27.
[0114] Many modifications and other embodiments of the invention
will come to mind to one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing descriptions and the associated drawings. Therefore, it
is to be understood that the invention is not to be limited to the
specific embodiments disclosed and that modifications and other
embodiments are intended to be included within the scope of the
appended claims. Although specific terms are employed herein, they
are used in a generic and descriptive sense only and not for
purposes of limitation.
* * * * *