U.S. patent application number 10/655756 was filed with the patent office on 2004-06-24 for compounds for intracellular delivery of therapeutic moieties to nerve cells.
Invention is credited to Hill, Craig, Kahl, Stephen B., McKee, Constance A., Webb, Robert R..
Application Number | 20040120891 10/655756 |
Document ID | / |
Family ID | 46299903 |
Filed Date | 2004-06-24 |
United States Patent
Application |
20040120891 |
Kind Code |
A1 |
Hill, Craig ; et
al. |
June 24, 2004 |
Compounds for intracellular delivery of therapeutic moieties to
nerve cells
Abstract
The invention features compounds of the general formula: B-L-M
where B is a binding agent capable of selectively binding to a
nerve cell surface receptor and mediating absorption of the
compound by the nerve cell; M is a moiety which performs a useful
non-cytotoxic function when absorbed by a nerve cell, and can be a
therapeutic moiety or an imaging moiety; and L is a linker coupling
B to M. The invention also features methods of use of the compounds
in, for example, treating conditions such as viral infections and
pain, as well as in labeling nerve cells.
Inventors: |
Hill, Craig; (Stockton,
CA) ; Kahl, Stephen B.; (Portola Valley, CA) ;
Webb, Robert R.; (Rancho Penasquitos, CA) ; McKee,
Constance A.; (Woodside, CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
200 MIDDLEFIELD RD
SUITE 200
MENLO PARK
CA
94025
US
|
Family ID: |
46299903 |
Appl. No.: |
10/655756 |
Filed: |
September 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10655756 |
Sep 5, 2003 |
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09707730 |
Nov 6, 2000 |
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09707730 |
Nov 6, 2000 |
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09217037 |
Dec 21, 1998 |
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6652864 |
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60409127 |
Sep 5, 2002 |
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Current U.S.
Class: |
424/1.41 ;
424/1.49; 424/178.1; 530/351; 530/391.1 |
Current CPC
Class: |
C07K 14/48 20130101;
A61P 25/00 20180101; A61K 47/6425 20170801; A61K 47/6807
20170801 |
Class at
Publication: |
424/001.41 ;
424/001.49; 424/178.1; 530/391.1; 530/351 |
International
Class: |
A61K 051/00; C12P
019/14; A61K 039/395; C07K 014/52; C07K 016/46 |
Claims
What is claimed is:
1. A conjugate comprising the formula: B-L-M where B is a binding
agent that selectively binds a nerve cell surface receptor and
mediating absorption of the compound by the nerve cell; M is a
therapeutic moiety or an imaging moiety which performs a useful
non-cytotoxic function when absorbed by a nerve cell, with the
proviso that M is other than a nerve growth factor or horse radish
peroxidase; and L is a linker covalently coupling B to M. wherein
said binding moiety selectively binds a trkA or trkB receptor of a
nerve cell and facilitates internalization of the conjugate into
the nerve cell.
2. The conjugate of claim 1, wherein the binding agent B is a
neurotrophin or a fragment or derivative thereof.
3. The conjugate of claim 1, wherein M is a therapeutic moiety
TM.
4. The conjugate of claim 3, wherein the therapeutic moiety TM is
chosen from an antiviral, an analgesic, an adrenergic agent, a
steroidal anti-inflammatory agent, a non-steroidal
anti-inflammatory agent, a steroid hormone, a Na+/K+/Ca++ channel
blocker, a mitochondrial modifier, an anti-oxidant, an
anti-bacterial agent, an anti-arrthymic agent, and an NMDA
antagonist,.
5. The conjugate of claim 3, wherein the therapeutic moiety TM
which is chosen from a nucleic acid, N-acetyl aspartic acid, and
WHI-P131.
6. The conjugate of claim 3, wherein the therapeutic moiety TM is
chosen from acyclovir, trifluridine, cortisone, prednisolone,
methylprednisolone, betamethasone, fluocinolone acetonide,
etodolac, tranylcypromine, ganciclovir, meclofenamate,
propoxycaine, dexamethasone, tauroursodeoxycholic acid,
flumethasone, N-acetyl-cysteine, procaine, piroxicam,
N-acetyl-aspartic acid, phentolamine, creatine, mexilitine,
minocycline, creatine phosphate, gabapentin, hyoscyamine,
ceftriaxone, baclofen, zidovudine (AZT), ketamine, lamivudine
(3TC), phenformin, enviroxime, pentamidine, adenosine, amantadine,
buprenorphine, butorphanol, celecoxib, dextromethorphan, DNA
oligomers, etorphine, levorphanol, morphine, naltrexone,
oxymorphone, testosterone, estradiol, progesterone and
WHI-P131;
7. The conjugate of claim 1, wherein the binding agent comprises an
amino acid sequence and the linker is covalently attached to the
binding agent at a lysine epsilon amino group.
8. The conjugate of claim 1, wherein the moiety M is an imaging
moiety IM.
9. The conjugate of claim 8, wherein the imaging moiety IM is
chosen from a fluorophore, a radioisotope, and a luminophores.
10. The conjugate of claim 8, wherein the imaging moiety is Alexa
Fluor 488.RTM. or Alexa Fluor 647.RTM..
11. A method of delivering a therapeutic moiety to a nerve cell
having a trkA or a trkB receptor, the method comprising: contacting
a nerve cell having a trkA or a trkB receptor with a conjugate
according to claim 3.
12. The method of claim 11, wherein the therapeutic moiety TM is an
antiviral, and is administered in an amount effective to reduce or
retard advancement of symptoms caused by viral infection of a
trkA-positive or a trkB-positive neuron.
13. The method of claim 11, wherein said therapeutic moiety is a
steroid, and is administered in an amount effective to reduce
inflammation or pain.
14. A method of labeling a nerve cell, comprising: contacting a
nerve cell having a trkA or trkB receptor with a conjugate
according claim 8 in an amount effective to detectably label the
nerve cell or nerve cell components.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of provisional
application serial No. 60/409,127, filed Sep. 5, 2002; and this
application is a continuation-in-part U.S. application Ser. No.
09/707,730, filed Nov. 6, 2000, which is a continuation-in-part of
U.S. application Ser. No. 09/217,037, filed Dec. 21, 1998, each
which application is incorporated by reference in their
entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to compounds that can be used
to deliver moieties selectively to nerve cells, and methods of use
therefore. More specifically, the invention relates to compounds
that can be used to deliver moieties, including therapeutic
moieties and imaging moieties, selectively to sensory and motor
neurons, and methods of use therefore.
BACKGROUND
[0003] Although our understanding of the structure and function of
the nervous system has greatly advanced in recent years, a need
still exists for efficacious treatments of many neurological
disorders, including Alzheimer's disease, Parkinson's disease,
Huntington's disease, schizophrenia, severe pain, multiple
sclerosis, bipolar disease, and diseases of the nervous system
caused by infection by viruses and other microorganisms (herpes
simplex, HIV, cytomegalovirus, parasites, fungi, prions, etc.).
[0004] Many neuropharmaceutical agents have been developed to treat
diseases of the nervous system, but their usefulness has been
hampered by severe side effects partially due to nonspecific
interactions between these agents and cells or tissues other than
the targeted cells. For example, the corticosteroid hormone
cortisone (4-pregnen-17.alpha., 21-diol-3,11,20-trione) and its
derivatives are widely used to treat inflammation in the body
including the nervous system to reduce symptoms such as swelling,
tenderness and pain. However, the steroid dosage has to be kept at
the lowest effective level because of its severe side effects.
Steroid hormones like cortisone bind to their cognate nuclear
hormone receptors and induce a cascade of cellular effects,
including programmed cell death of the neurons in the brain (Kawata
et al., J. Steroid Biochem. Mol. Biol. 65: 273-280 (1998)). Since
steroid hormone receptors, such as the glucocorticoid receptor for
cortisone, are distributed in a wide variety of tissues and cells,
nonspecific interactions of the hormone with its cognate receptor
in different sites is unavoidable if the drug is circulated
systemically.
[0005] A need thus continues to exist for an effective system for
delivering therapeutic agents selectively to nerve cells and nerve
tissues. Various techniques have been developed to deliver drugs
selectively, but with only limited success.
[0006] For example, liposomes have been used as carrier molecules
to deliver a broad spectrum of agents including small molecules,
DNAs, RNAs, and proteins. Liposome mediated delivery of
pharmaceutical agents has major drawbacks because of its lack of
target specificity. Attempts have been made to overcome this
problem by covalently attaching whole site-specific antibody or Fab
fragments to liposomes containing a pharmaceutical agent (Martin et
al., Biochem. 20, 4229-4238, (1981)). However, an intrinsic problem
of particular importance in any liposome carrier system is that in
most cases the targeted liposome does not selectively reach its
target site in vivo. Whether or not liposomes are coated with
antibody molecules, liposomes are readily phagocytosed by
macrophages and removed from circulation before reaching their
target sites.
SUMMARY OF THE INVENTION
[0007] The invention features compounds of the general formula:
B-L-M
[0008] where B is a binding agent capable of selectively binding to
a nerve cell surface receptor and mediating absorption of the
compound by the nerve cell; M is a moiety which performs a useful
non-cytotoxic function when absorbed by a nerve cell, and can be a
therapeutic moiety or an imaging moiety; and L is a linker coupling
B to M. The invention also features methods of use of the compounds
in, for example, treating conditions such as viral infections and
pain, as well as in labeling nerve cells.
[0009] In certain embodiments, presently preferred, the binding
agent is further capable of being transported retrogradely to the
nerve cell body after internalization. In other particular
embodiments, M is a therapeutic moiety (TM)or an imaging moiety
(IM)
[0010] Thus, in one embodiment, the compounds have the general
formula:
B-L-TM
[0011] where:
[0012] B is a binding agent capable of selectively binding to a
nerve cell surface receptor and mediating absorption of the
compound by the nerve cell;
[0013] TM is a therapeutic moiety which has a non-cytotoxic
therapeutic effect when absorbed by a nerve cell; and
[0014] L is a linker coupling B to TM.
[0015] And thus, in another embodiment, the compounds have the
general formula:
B-L-IM
[0016] where:
[0017] B is a binding agent capable of selectively binding to a
nerve cell surface receptor and mediating absorption of the
compound by the nerve cell;
[0018] IM is a non-cytotoxic imaging moiety which can be used to
image a nerve cell or an intracellular component of the nerve cell;
and
[0019] L is a linker coupling B to IM.
[0020] In regard to each of the above embodiments, particular
classes of binding agents B which may be used include, but are not
limited to, nucleic acid sequences, peptides, peptidomimetics,
peptoids, antibodies and antibody fragments. As used herein, the
term "peptides" includes polypeptides and oligopeptides of any
length, and is generic to antibodies and antibody fragments.
[0021] Examples of nucleic acids that can serve as the binding
agent B include, but are not limited to, DNA, RNA, and other
nucleomimetic ligands that function as antagonists of nerve growth
factors or inhibit binding of other growth factors to nerve cell
surface receptors, such as aptamers that function as antagonists or
nerve growth factors or inhibit binding of growth factors to nerve
cell surface receptors.
[0022] Examples of peptides that can serve as the binding agent B
include, but are not limited to, members of the nerve growth factor
(neurotrophin) family such as NGF, BDNF, NT-3, NT-4, NT-6;
derivatives (e.g., biochemically or chemically modified proteins
having, for example, different glycosylation or other modification
relative to a native protein), analogues (e.g., proteins that
differ in amino acid sequence relative to an amino acid sequence of
a native protein), and fragments of such nerve growth factors
(e.g., a recombinant, naturally-occurring, or synthetic protein
fragment or peptide or peptidomimetic that selectively binds a
nerve growth factor receptor, e.g., recombinant molecules of NGF
and BDNF); and synthetic peptides; where B selectively binds to a
nerve cell surface receptor, and which may have agonist or
antagonist activities of a nerve growth factor.
[0023] Antibodies, derivatives of antibodies and antibody fragments
can also serve as the binding agent B. Examples of this type of
binding agent B include, but are not limited to, anti-human trkA
monoclonal antibody 5C3 and anti-human p75 monoclonal antibody MC
192.
[0024] Where M is a therapeutic moiety, the therapeutic moiety TM
is selected to perform a non-cytotoxic therapeutic function within
nerve cells. Examples of non-cytotoxic functions which the
therapeutic moiety TM may perform include, but are not limited to,
the functions performed by adrenergic agents (agonist or
antagonist) (e.g., epinephrine, norepinephrine, dopamine,
atenolol), analgesics (e.g., opioids, morphine, codeine,
oxycodone), anti-inflammatories (steroidal-e.g., cortisone,
prednisolone, methylprednisolone, betamethasone, dexamethasone, and
nonsteroidal-eg., piroxicam, meclofenamate, etodolac), anti-trauma
agents (e.g., adenosine, epinephrine, dopamine, epinephrine, and
other agents used in the treatment of shock), anti-viral agents
(e.g., acyclovir, ganciclovir, AZT, ddI, ddC, trifluridine, etc.),
antibacterial and anti-infective agents, anti-arrthymic agents
(e.g., adenosine), gene therapy agents (e.g., DNAs, RNAs, or other
nucleomimetics which introduce a gene or replace a mutated gene),
hormones (e.g., peptide hormones (e.g., growth factors) and steroid
hormones ((e.g., cortisone, testosterone, progesterone, estrogen)),
anti-oxidants, NMDA antagonists or modifiers, and immune system
modulators, particularly those with antiviral activity such as
interferons, etc.). In general, the therapeutic moiety is not a
nerve growth factor.
[0025] Examples of classes of therapeutic moieties include, but are
not limited to, adrenergic agents (e.g., epinephrine,
norepinephrine, dopamine, atenolol), analgesics (e.g., opioids,
morphine, codeine, oxycodone), anti-inflammatories (steroidal-e.g.,
prednisolone, methylprednisolone, betamethasone, dexamethasone and
nonsteroidal--e.g., piroxicam, meclofenamate, etodolac) anti-trauma
agents, anti-viral agents (e.g., acyclovir, ganciclovir, AZT, ddI,
ddC, trifluridine, etc.), gene therapy agents (e.g., DNAs, RNAs, or
nucleomimetics which introduce a gene or replace a mutated gene),
steroids (e.g., pregnanes, estranes, and androstanes, such as
corticosteroids, including cortisone, progestins, such as
progesterone, and estranes, such as estradiol), and nonsteroidal
hormones (e.g., growth factors); and immune system modulators, such
as interferons, etc.). Exemplary therapeutic moieties of interest
are described in more detail below.
[0026] Where M is an imaging moiety (IM), IM is a non-cytotoxic
agent that can be used to determine whether a nerve cell or an
internal component of the nerve cell is specifically associated
with (e.g.,. has absorbed) the imaging moiety, and optionally
locate and, further optionally, visualize, such nerve cells or
nerve cell internal components. For example, fluorescent dyes may
be used as an imaging moiety IM. In another example, radioactive
agents that are non-cytotoxic may also be an imaging moiety IM. In
some embodiments, the IM is a moiety other than horse radish
peroxidase (HRP). In other embodiments, the imaging moiety provides
a detectable signal that does not require the addition of a
substrate for detection.
[0027] In one embodiment, the IM is a charged moiety. Cells have
difficulty transporting charged molecules across cell membranes.
According to this embodiment, the binding agent B serves to
facilitate transport of a charged imaging moiety IM into a cell.
Within the cell, the compound (i.e. the conjugate formed between B
and IM) is metabolized to form a metabolite product that comprises
the charged imaging moiety IM. The metabolite product is less prone
to being transported across the cell membrane out of the cell
relative to the conjugate because of the metabolism of the
conjugate resulting in the separation of the imaging moiety IM from
the binding agent B. The metabolite product is also less prone to
being transported across the cell membrane out of the cell relative
to a non-charged version of the imaging moiety due to the charge
that the imaging moiety carries.
[0028] According to this embodiment, compounds are provided which
comprise a charged imaging moiety, the charged derivative being
conjugated to a binding agent (also interchangeably referred to
herein as a "binding moiety") B that facilitates transport of the
IM across a cell membrane into a cell, the cell metabolizing at
least a portion of the imaging agent to form a charged metabolite
product that provides for a detectable signal, the charged
metabolite product being less prone to being transported across the
cell membrane out of the cell relative to the conjugate and less
prone to being transported across the cell membrane out of the cell
relative to an uncharged imaging agent.
[0029] In one particular embodiment, the charged imaging moiety IM
is Alexa Fluor 488.RTM., Molecular Probes, a fused heterocyclic 3
ring aromatic system with a pendant phenyl ring with an amino, a
quaternary amine, two sulfonic acid lithium salts, a carboxylic
acid, and one carboxylic acid N-hydroxy-succinnimidyl ester groups
attached. It is this last group that forms an amide crosslink to
the epsilon amino group of lysine. This compound is a highly
modified derivative of the imaging moiety fluorescein with very
similar absorption and emission spectra but with a much higher
extinction coefficient. In another embodiment, the charged imaging
moiety IM is a similarly modified derivative of Texas Red.RTM. but
of proprietary makeup, Alexa Fluor 647.RTM., Molecular Probes.
[0030] In general, the linker may be any moiety that can be used to
link the binding agent B to the moiety M. In one particular
embodiment, the linker is a cleavable linker. The use of a
cleavable linker enables the moiety M linked to the binding agent B
to be released from the compound once absorbed by the nerve cell
and transported to the cell body. The cleavable linker may be
cleavable by a chemical agent, by an enzyme, due to a pH change, or
by being exposed to energy. Examples of forms of energy that may be
used include light, microwave, ultrasound, and radiofrequency.
[0031] In certain applications, it is desirable to release the
moiety M, particularly where M is a therapeutic moiety TM, once the
compound has entered the nerve cell, resulting in a release of the
moiety M. Accordingly, in one variation, the linker L is a
cleavable linker. This enables the moiety M to be released from the
compound once absorbed by the nerve cell. This may be desirable
when, for example, M is a therapeutic moiety TM which has a greater
therapeutic effect when separated from the binding agent. For
example, the therapeutic moiety TM may have a better ability to be
absorbed by an intracellular component of the nerve cell when
separated from the binding agent. Accordingly, it may be necessary
or desirable to separate the therapeutic moiety TM from the
compound so that the therapeutic moiety TM can enter the
intracellular compartment.
[0032] The present invention also relates to a method for
selectively delivering a moiety into nerve cells comprising the
steps of:
[0033] delivering to a patient a compound having the general
formula:
B-L-M
[0034] where:
[0035] B is a binding agent capable of selectively binding to a
nerve cell surface receptor and mediating absorption of the
compound by the nerve cell;
[0036] M is a moiety which performs a useful non-cytotoxic function
when absorbed by a nerve cell; and
[0037] L is a linker coupling B to M,
[0038] having the compound selectively bind to a nerve cell surface
receptor via the binding agent B; and
[0039] having the compound be absorbed by the nerve cell mediated
by the binding of the binding agent B to the nerve cell surface
receptor.
[0040] In one embodiment, moiety M is a therapeutic moiety TM as
described herein and in another embodiment is an imaging moiety
IM.
[0041] The above method can be used to deliver therapeutic moieties
for treating a variety of neurological disorders when the
therapeutic moiety TM is a moiety useful for treating such
neurological disorders.
[0042] The above method can be used to deliver therapeutic moieties
for treating pain when a therapeutic moiety TM for treating pain,
such as an analgesic or anti-inflammatory, is included as the
therapeutic moiety TM in the compound.
[0043] The above method can also be used to deliver steroid
hormones for treating nerve damage when a therapeutic moiety TM for
treating nerve damage, such as a steroid hormone, is included as
the therapeutic moiety TM in the compound.
[0044] The above method can also be used to stimulate nerve growth
when a therapeutic moiety TM for inducing the production of a nerve
growth factor is included as the therapeutic moiety TM in the
compound.
[0045] The above method can also be used to treat infected nerve
cells infected with viruses or immunize nerve cells from viruses
when the therapeutic moiety TM in the compound is an antiviral
agent.
[0046] The above method can also be used to perform gene therapy
when the therapeutic moiety TM is a gene therapy agent.
[0047] The present invention also relates to a method for improving
intracellular administration of a therapeutic agent, by
administration of a B-L-TM compound of the invention.
[0048] In one embodiment, this method is used in conjunction with
the conjugates of the present invention and hence is used in
conjunction with the methods of the present invention for
selectively delivering a moiety into nerve cells.
DETAILED DESCRIPTION OF THE INVENTION
[0049] The present invention relates to compounds which include a
binding agent that binds to a nerve cell surface receptor and
facilitates absorption of the compound by the nerve cell; and a
moiety. Different moieties may be included in the compounds of the
present invention including therapeutic moieties that are
non-cytotoxic to the nerve cells and imaging moieties that can be
used to image nerve cells that absorb these compounds.
[0050] In one embodiment, compounds of the present invention have
the general formula:
B-L-M
[0051] where:
[0052] B is a binding agent capable of selectively binding to a
nerve cell surface receptor and mediating absorption of the
compound by the nerve cell;
[0053] M is a moiety, which can be a therapeutic moiety TM which
has a non-cytotoxic therapeutic effect when absorbed by a nerve
cell, or an imaging moiety IM which provides for detectable
labeling of a nerve cell; and
[0054] L is a linker coupling B to TM.
[0055] According to this embodiment, the binding agent B serves as
a homing agent for nerve cells by selectively binding to nerve cell
surface receptors. The binding agent B also serves to facilitate
absorption of the compound by the nerve cell. In certain
embodiments, presently preferred, binding agent B is retrogradely
transported to the cell body.
[0056] The binding agent B can be any molecule that can perform the
first two, and preferably the third, of these functions. Particular
classes of binding agents which may be used include, but are not
limited to, nucleic acid sequences, peptides, peptidomimetics,
antibodies and antibody fragments. Further exemplary binding agents
are described in detail below.
[0057] The linker L serves to link the binding agent B to the
moiety M. A wide variety of linkers are known in the art for
linking two molecules together, particularly, for linking a moiety
to a peptide or nucleic acid, all of which are included within the
scope of the present invention.
[0058] Examples of classes of linkers that may be used to link the
binding agent B to the moiety M include amide, alkylamine,
carbamate, phosphoramide, ester, ether, thioether, alkyl,
cycloalkyl, aryl, and heteroaryl linkages such as those described
in Hermanson, GT., Bioconjugate Techniques (1996), Academic Press,
San Diego, Calif.
[0059] In certain applications, it is desirable to release the
moiety M, particularly where M is a therapeutic moiety TM, once the
compound has entered the nerve cell, resulting in a release of the
moiety M. Accordingly, in one variation, the linker L is a
cleavable linker. This enables the moiety M to be released from the
compound once absorbed by the nerve cell. This may be desirable
when, for example, M is a therapeutic moiety TM which has a greater
therapeutic effect when separated from the binding agent. For
example, the therapeutic moiety TM may have a better ability to be
absorbed by an intracellular component of the nerve cell when
separated from the binding agent. Accordingly, it may be necessary
or desirable to separate the therapeutic moiety TM from the
compound so that the therapeutic moiety TM can enter the
intracellular compartment.
[0060] Cleavage of the linker releasing the therapeutic moiety may
be as a result of a change in conditions within the nerve cells as
compared to outside the nerve cells, for example, due to a change
in pH within the nerve cell. Cleavage of the linker may occur due
to the presence of an enzyme within the nerve cell that cleaves the
linker once the compound enters the nerve cell. Alternatively,
cleavage of the linker may occur in response to energy or a
chemical being applied to the nerve cell. Examples of types of
energies that may be used to effect cleavage of the linker include,
but are not limited to light, ultrasound, microwave and
radiofrequency energy.
[0061] In one embodiment, compounds of the present invention have
the general formula:
B-L-TM
[0062] where:
[0063] B is a binding agent capable of selectively binding to a
nerve cell surface receptor and mediating absorption of the
compound by the nerve cell;
[0064] TM is a therapeutic moiety which has a non-cytotoxic
therapeutic effect when absorbed by a nerve cell; and
[0065] L is a linker coupling B to TM.
[0066] In another embodiment, compounds of the present invention
have the general formula:
B-L-IM
[0067] where:
[0068] B is a binding agent capable of selectively binding to a
nerve cell surface receptor and mediating absorption of the
compound by the nerve cell;
[0069] IM is a non-cytotoxic imaging moiety which can be used to
image the nerve cell or an intracellular component of the nerve
cell; and
[0070] L is a linker coupling B to IM.
[0071] According to this embodiment, the binding agent B and linker
L may be varied as described above with regard to compounds having
the general formula B-L-TM. Further according to this embodiment,
the imaging moiety IM may be a non-cytotoxic moiety which can be
used to image nerve cells. Examples of imaging moieties that may be
used include fluorescent dyes, radioisotopes and other detectable
moieties such as luminophores which are non-cytotoxic.
[0072] The present invention also relates to a method for
selectively delivering a non-cytotoxic therapeutic moiety into
nerve cells comprising the steps of:
[0073] delivering to a patient a therapeutic amount of a compound
having the general formula:
B-L-TM
[0074] where:
[0075] B is a binding agent capable of selectively binding to a
nerve cell surface receptor and mediating absorption of the
compound by the nerve cell,
[0076] TM is a therapeutic moiety which has a non-cytotoxic
therapeutic effect when absorbed by a nerve cell, and
[0077] L is a linker coupling B to TM;
[0078] having the compound selectively bind to a nerve cell surface
receptor via the binding agent B; and
[0079] having the compound be absorbed by the nerve cell mediated
by the binding of the binding agent B to the nerve cell surface
receptor.
[0080] The method of the present invention offers the advantage of
specifically targeting a non-cytotoxic therapeutic moiety to nerve
cells where the therapeutic moiety is absorbed by the nerve cells.
The method utilizes the fact that internalization of the conjugated
agent is mediated by the binding of the binding agent B to nerve
cell surface receptors. Once internalized, the therapeutic moiety
can accumulate within the nerve cells where it has a therapeutic
effect. In certain embodiments, the compound of the present
invention is transported retrogradely to the cell body after
internalization; in such cases, the therapeutic moiety can
accumulate within the nerve cell body.
[0081] The ability to deliver the compound selectively to nerve
cells reduces the overall amount of therapeutic moiety that needs
to be administered. Selective delivery of the therapeutic moiety to
the nerve cell reduces the amount of side effects observed due to
non-specific administration of the therapeutic moiety. In addition,
the therapeutic moiety is less likely to be separated from the
binding agent and non-specifically administered as compared to
delivery methods involving the use of a binding agent and a
therapeutic moiety in combination. Various and exemplary
applications of the conjugates of the invention having a
therapeutic moiety are described below.
[0082] The method of the present invention can be used to deliver
therapeutic moieties for treating a variety of neurological
disorders including, but not limited to, Alzheimer's disease,
Parkinson's disease, multiple sclerosis, neurodegenerative disease,
epilepsy, seizure, migraine, trauma and pain. Examples of
neuropharmaceuticals that may be used include proteins,
antibiotics, adrenergic agents, anticonvulsants, analgesics,
anti-inflammatories, anti-viral agents, gene therapy agents,
hormones (growth factors), and immune system modulators,
particularly those with antiviral activity such as interferons,
nucleotide analogues, anti-trauma agents, peptides and other
classes of agents used to treat or prevent a neurological
disorders. For example, analgesics such as opioids, morphine,
codeine, and oxycodone can be conjugated to the binding agent B and
specifically delivered to the nerve cells. Since the same level of
pain relief can be achieved using a smaller dosage of analgesics,
side effects such as respiratory depression or potential drug
addiction can be avoided or at least ameliorated.
[0083] Steroid hormones such as corticosteroids can also be
conjugated with nerve cell-specific binding agents and used to
treat inflammation of the nerves, which may reduce the side effects
associated with high doses of steroids, such as weight gain,
redistribution of fat, increase in susceptibility to infection, and
avascular necrosis of bone. Corticosteroids include, inter alia,
cortisol (4-pregnen-11,17,21-triol-3- ,20-dione), cortisone
(4-pregnen-17,21-diol-3,11,20-trione), deoxycorticosterone
(4-pregnen-21-hydroxy-3,20-dione), prednisone
(1,4-pregnadien-17.alpha.,21-diol--3,11,20-trione), prednisolone
(1,4-pregnadiene-11.beta.,17.alpha.,21-triol-3,20-dione),
methylprednisolone (1,4-pregnadiene-6.alpha.-methyl-11.beta.,
17.alpha.,21-triol-3,20-dione), beclomethasone
(1,4-pregnadiene-9-chloro--
11.beta.,17,21-triol-16.beta.-methyl-3,20-dione-17,21-dipropionate),
triamcinolone (1,4-pregnadiene-9-fluoro-11.beta., 16.alpha.,
17,21-tetrahydroxy-3,20-dione) and its derivative triamcinolone
acetonide, (1,4-pregnadiene-9-fluoro-11.beta., 16.alpha.,
17,21-tetrahydroxy-3,20-dione cyclic 16,17-acetal with acetone),
desonide (1,4-pregnadiene-3,20-dione,
11.beta.,21-dihydroxy-16.alpha., 17-[(1-methylethylidene)bis(oxy)],
alclometasone (typically as the dipropionate:
1,4-pregnadiene-7a-chloro-11.beta., 17, 21-trihydroxy,
16.alpha.-methyl, 3,20-dione, 17,21-dipropionate), flurandrenolide
(4-pregnene-3,20-dione, 6.alpha.-fluoro-11.beta.,
21-dihydroxy-16.alpha.,- 17-[(1-methylethylidene) bis (oxy)]),
dexamethasone (1,4-pregnadiene-9-fluoro-11.beta.,
17,21-trihydroxy-16.alpha.-methyl, 3,20-dione), desoximetasone
(1,4-pregnadiene-3,20-dione,9-fluoro-11.beta.-
,21-dihydroxy-16.alpha.-methyl), flumethasone
(1,4-pregnadiene-3,20-dione--
9.alpha.-fluoro-16.alpha.-methyl-11.beta., 17,21-trihydroxy), and
betamethasone (1,4-pregnadiene,
9-Fluoro-1,16,17,21-trihydroxy-16.beta.-m- ethyl-3,20-dione) and
its derivatives (such as the dipropionate: 1,4-pregnadiene,
9-Fluoro-1,16,17,21-trihydroxy-16.beta.-methyl-3,20-dion- e
17,21-dipropionate).
[0084] The method according to the present invention can also be
used to deliver agents that induce the production of nerve growth
factor in the target nerve cells, especially under conditions of
pathogenic under-expression of NGFs (See Riaz, S. S. and Tomlinson,
D. R. Prog. Neurobiol. 49: 125-143 (1996)). NGF induction has been
demonstrated in a wide variety of cell types, such as fibroblasts
(Furukawa, Y. et al., FEBS Lett. 247: 463-467(1989)), astrocytes
(Furukawa, Y. et al., FEBS Lett. 208: 258-262 (1986)), Schwann
cells (Ohi, T. et al., Biochem. Int. 20:739-746 (1990)) with a
variety of agents including cytokines, steroids, vitamins,
hormones, and unidentified components of serum. Specific examples
of agents known to induce NGF include 4-methylcatechol,
clenbuterol, isoprenaline, L-tryptophan, 1,25-dihydroxyvitamin D3,
forskolin, fellutamide A, gangliosides and quinone derivatives
(Riaz, S. S. and Tomlinson, D. R. Prog. Neurobiol. 49: 125-143
(1996)).
[0085] The method according to the present invention can also be
used to deliver antiviral drugs into nerve cells in order to treat
diseases caused by viral infection, to eliminate viruses spread to
the nerves, and to inhibit infection by such viruses. Examples of
viruses that infect the nervous system include but are not limited
to rabies viruses, herpes viruses, polioviruses, arboviruses,
reoviruses, pseudorabies, corona viruses, and Borna disease
viruses. For example, antiviral drugs such as acyclovir,
ganciclovir, cidofovir, and trifluridine can be conjugated to the
binding agent and used to inhibit active or latent herpes simplex
viruses in the peripheral and central nervous system. Specific
delivery of the conjugate containing these antiviral drugs to the
nervous system can reduce the side effects associated with high
doses or long-term administration of these drugs, such as
headaches, rash and paresthesia. The method according to the
present invention can also be used to deliver marker compounds to
image intracellular components of the nerve cells. Such marker
compounds include but are not limited to fluorescent dyes,
radioactive compounds, and other luminophores.
[0086] The method according to the present invention can also be
used to perform gene therapy wherein nucleic acids (DNA, RNA or
other nucleomimetics) are delivered to the nerve cells. These
nucleic acids may serve to replace genes that are either defective,
absent or otherwise not properly expressed by the patient's nerve
cell genome.
[0087] The above and other features and advantages of the present
invention will become more apparent in the following description of
the preferred embodiments in greater detail.
[0088] 1. Binding Agent (B)
[0089] According to the present invention, a compound with a
binding agent B is used to selectively deliver the conjugated M,
which can be a therapeutic moiety TM or an imaging moiety IM, to
nerve cells. At the surface of the nerve cell, the binding agent B
interacts with a receptor expressed on the nerve cell and is
absorbed by the nerve cell mediated by this interaction. Any
molecules possessing these two physical properties are intended to
fall within the scope of a binding agent B as it is used in the
present invention. In particular, peptides or proteins with these
features can serve as a binding agent B, examples including but not
limited to nerve growth factors (neurotrophins), antibodies against
nerve cell-specific surface proteins, mutants and synthetic
peptides derived from these peptides or proteins.
[0090] In one embodiment, neurotrophins are preferably used as the
binding agent B. Neurotrophins are a family of small, basic
polypeptides that are required for the growth, development and
survival of neurons. A particular "survival" factor is taken up by
the neuron via binding to one or more of a related family of
transmembrane receptors. Table I lists several members of the
neurotrophin family and their cognate receptors. The neurotrophin
from which the binding agent is derived may be of any suitable
origin, e.g., human, mouse. Where the conjugate is to be delivered
to a human, the neurotrophin is preferably a human
neurotrophin.
[0091] As listed in Table 1, nerve growth factor (NGF) is the first
identified and probably the best characterized member of the
neurotrophin family. It has prominent effects on developing sensory
and sympathetic neurons of the peripheral nervous system.
Brain-derived neurotrophic factor (BDNF) has neurotrophic
activities similar to NGF, and is expressed mainly in the CNS and
has been detected in the heart, lung, skeletal muscle and sciatic
nerve in the periphery (Leibrock, J. et al., Nature, 341:149-152
(1989)). Neurotrophin-3 (NT-3) is the third member of the NGF
family and is expressed predominantly in a subset of pyramidal and
granular neurons of the hippocampus, and has been detected in the
cerebellum, cerebral cortex and peripheral tissues such as liver
and skeletal muscles (Ernfors, P. et al., Neuron 1: 983-996
(1990)). Neurotrophin-4 (also called NT-4/5) is the most variable
member of the neurotrophin family. Neurotrophin-6 (NT-6) was found
in teleost fish and binds to p75 receptor.
[0092] As listed in Table 1 at least two classes of transmembrane
glycoproteins (trk and p75) have been identified which serve as
endogenous receptors for neurotrophins. The trk receptors (tyrosine
kinase-containing receptor) bind to neurotrophins with high
affinity, whereas the p75 receptors possess lower affinity for
neurotrophins. For example, nerve growth factor (NGF) binds to a
relatively small number of trkA receptors with high affinity
(K.sub.D=10.sup.-11) and to more abundant p75 with lower affinity
(K.sub.D=10.sup.-9). The receptor-bound NGF is internalized with
membrane-bound vesicles and retrogradely transported to the
neuronal cell body. Thus, native neurotrophins, or fragments or
other modified form of neurotrophins, can serve as the binding
agent B in the compound according the present invention to deliver
the conjugated moiety M to the neuronal cell body whether it be a
therapeutic moiety, TM, or an imaging moiety, IM.
1TABLE 1 The Neurotrophin Family and Its Receptors. Responsive
Receptor neurons Factor Kinase isoforms Nonkinase forms (examples)
NGF TrkA p75 Cholinergic forebrain neurons Sympathetic ganglia DRG
nociceptive BDNF TrkB p75.sup.LNTR Many CNS trkB.sub.T1 populations
trkB.sub.T2 Vestibular ganglia Nodose ganglia DRG mechanoreceptors
NT-3 TrkC p75.sup.LNTR Many CNS trkB and trkA trkC.sub.TK-113
populations Nonpreferred trkC.sub.TK-108 Cochlear ganglia DRG
proprioceptive NT-4 TrkB p75 Many CNS trkB.sub.T1 populations
trkB.sub.T2 Nodose ganglia Petrosalganglia NT-6 TrkA p75
[0093] Thus specific examples of peptides that can serve as the
binding agent B include, but are not limited to, members of the
nerve growth factors (neurotrophin) family such as nerve growth
factor (NGF), brain-derived neurotrophic factor (BDNF),
neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), neurotrophin-6
(NT-6), etc. (see reviews: Frade, J. M., et al., Bioessays 20:
137-145 (1998); Shieh, P. B., Curr. Biol. 7: R627-R630 (1997);
Dechant, G., et al., Curr. Opin. Neurobiol. 7: 413-418 (1997);
Chao, M. V. and Hempstead, B. L., Trends Neurobiol. 18: 321-326
(1995)); and derivatives, analogues, and fragments of nerve growth
factors such as recombinant molecules of NGF and BDNF (Ibanez et
al., EMBO J. 10: 2105-2110; Ibanez et al., EMBO J. 12: 2281-2293),
synthetic peptides that bind to nerve cell surface receptors and
have agonist or antagonist activities of nerve growth factors
(Longo, F. M., et al., Cell Regulation 1: 189-195 (1990);
LeSauteur, L. et al., J. Biol. Chem. 270: 6564-6569 (1995); Longo
F. M., et al., J. Neurosci. Res. 48: 1-17; Longo, et al., Nature
Biotech. 14: 1120-1122 (1997)).
[0094] In addition to the neurotrophins described above, analogues
and derivatives of neurotrophins may also serve as the binding
agent B. The structure of mouse NGF has been solved by X-ray
crystallography at 2.3 A resolution (McDonald et al., Nature, 345:
411-414, (1991)). Murine NGF is a dimeric molecule, with 118 amino
acids per protomer. The structure of the protomer consists of three
antiparallel pairs of beta strands that form a flat surface, four
loop regions containing many of the variable residues between
different NGF-related molecules, which may determine the different
receptor specificities, and a cluster of positively charged side
chains, which may provide a complementary interaction with the
acidic low-affinity NGF receptor. Murine NGF has a tertiary
structure based on a cluster of three cysteine disulfides and two
extended, but distorted beta-hairpins. One of these .beta.-hairpin
loops was formed by the NGF 29-35 region.
[0095] Structure/function relationship studies of NGF and
NGF-related recombinant molecules demonstrated that mutations in
NGF region 25-36, along with other .beta.-hairpin loop and non-loop
regions, significantly influenced NGF/NGF-receptor interactions
(Ibanez et al., EMBO J., 10, 2105-2110, (1991)). Small peptides
derived from this region have been demonstrated to mimic NGF in
binding to trkA receptor and affecting biological responses
(LeSauteur et al. J. Biol. Chem. 270, 6564-6569, 1995). Dimers of
cyclized peptides corresponding to .beta.-loop regions of NGF were
found to act as partial NGF agonists in that they had both
survival-promoting and NGF-inhibiting activity while monomer and
linear peptides were inactive (Longo et al., J. Neurosci. Res., 48,
1-17, 1997). Cyclic peptides have also been designed and
synthesized to mimic the .beta.-loop regions of NGF, BDNF, NT3 and
NT-4/5. Certain monomers, dimers or polymers of these cyclic
peptides may have a three-dimensional structure which binds to
neurotrophin receptors under physiological conditions. All of these
structural analogues of neurotrophins that bind to nerve cell
surface receptors and are internalized can serve as the binding
agent B of the compound according to the present invention to
deliver the conjugated therapeutic moiety TM to the nervous
system.
[0096] Alternatively, peptidomimetics (or "peptide mimics") that
are synthesized by incorporating unnatural amino acids or other
organic molecules may serve as the binding agent B of the compound
according to the present invention to deliver the conjugated
therapeutic agent TM into the nerve cells. Peptide mimetics are
thus generally non-naturally occurring analogues of a peptide
which, because of protective groups at one or both ends of the
mimetic, or replacement of one or more peptide bonds with
non-peptide bonds, is less susceptible to proteolytic cleavage than
the peptide itself. For instance, one or more peptide bonds can be
replaced with an alternative type of covalent bond (e.g., a
carbon-carbon bond or an acyl bond).
[0097] Peptide mimetics can also incorporate amino-terminal or
carboxyl terminal blocking groups such as t-butyloxycarbonyl,
acetyl, alkyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl,
dansyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl,
methoxyazelayl, methoxyadipyl, methoxysuberyl, and
2,4,-dinitrophenyl, thereby rendering the mimetic less susceptible
to proteolysis. Non-peptide bonds and carboxyl- or amino-terminal
blocking groups can be used singly or in combination to render the
mimetic less susceptible to proteolysis than the corresponding
peptide. Additionally, substitution of D-amino acids for the normal
L-stereoisomer can be effected, e.g. to increase the half-life of
the molecule. These synthetic peptide mimics are capable of binding
to the nerve cell surface receptor and being internalized into the
cell.
[0098] Other binding moieties B suitable for use in the invention
include peptoids of nerve growth factors and antibodies that
specifically bind a nerve cell surface receptor. Peptoids are
protein-like molecules that contain an unnatural imino acids (see,
e.g., Simon, R. J. et al. (1992) Proc. Natl. Acad. Sci. USA
89:9367-9371) containing N-substituted glycine residues wherein the
substituents on the nitrogen atom are the alpha-position side
chains of amino acids. Because they are amino acids that do not
occur in nature, peptoid residues or peptides containing peptoid
residues have higher resistance to enzymatic attacks. In recent
years, peptoid residues have been widely used in the design and
synthesis of drugs and other peptide related biomaterials.
[0099] Alternatively, antibodies against nerve cell surface
receptors that are capable of binding to the receptors and being
internalized can also serve as the binding agent B. For example,
monoclonal antibody (MAb) 5C3 is specific for the NGF docking site
of the human p140 trkA receptor, with no cross-reactivity with
human trkB receptor. MAb 5C3 and its Fab mimic the effects of NGF
in vitro, and image human trk-A positive tumors in vivo (Kramer et
al., Eur. J. Cancer, 33, 2090-2091, (1997)). Molecular cloning,
recombination, mutagenesis and modeling studies of MAb 5C3 variable
region indicated that three or less of its complementarity
determining regions (CDRs) are relevant for binding to trkA. Assays
with recombinant CDRs and CDR-like synthetic polypeptides
demonstrated that they had agonistic bioactivities similar to
intact MAb 5C3. Monoclonal antibody MC192 against p75 receptor has
also been demonstrated to have neurotrophic effects.
[0100] Thus antibodies, derivatives of antibodies and antibody
fragments that can serve as the binding agent B include, but are
not limited to, anti-human trkA monoclonal antibody 5C3 (Kramer,
K., et al., Eur. J. Cancer 33: 2020-2091 (1997)), anti-human p75
monoclonal antibody MC192 (Maliatchouk, S. and Saragovi, H. U., J.
Neurosci. 17: 6031-7) and fragments and derivatives thereof that
retain antigen binding. Such can serve as the binding agent B of
the compound according to the present invention to deliver the
conjugated moiety M to or into the nerve cells.
[0101] It is noted that the identification and selection of
moieties that can serve as binding agents in the present invention
can be readily performed by attaching an imaging moiety IM to the
potential binding agent in order to detect whether the potential
binding agent is internalized by the nerve cells. In this regard,
combinatorial and mutagenesis approaches may be used to identify
analogues, derivatives and fragments of known binding moieties
which may also be used as binding moieties according to the present
invention.
[0102] The position of the linker within the binding agent is
selected so as to maintaining the ability of the binding agent to
bind to the desired nerve cell surface receptor. In general, it is
preferred that the residue to which the linker is attached is one
that is outside of the area of the protein that binds to the
corresponding receptor. For example, where the binding agent is NGF
or BDNF, the second most exposed lysine at residue 95 is within the
TrkA binding site, and thus is less preferred for attachment of a
linker. In some embodiments where the binding agent B is NGF or
BDNF, it may be most desirable to attach the linker to the lysine
corresponding to the most exposed lysine residue at position 74 of
the native NGF or native BDNF protein.
[0103] In general, the binding agent is linked to at least one
moiety via a linker. Normally, and particularly where the binding
agent is a neurotrophin (e.g., NGF, BDNF, NT-3, NT-4), the binding
agent has only one linker, and dimers of the conjugated binding
agents have two linker-moiety constructs attached.
[0104] 2. Moiety (M)
[0105] As noted above, the binding agent B is conjugated to a
moiety M, which may be either a non-cytoxic therapeutic moiety (TM)
or an imaging moiety (IM).
[0106] Therapeutic Moiety (TM)
[0107] An aspect of the present invention relates to the delivery
of compounds into nerve cells that are non-cytotoxic to the nerve
cells and perform a therapeutic function. Examples of therapeutic
functions include, but are not limited to, treatment of
neurological disorders, gene therapy, intracellular target imaging,
cell sorting, or separation schemes. In general, the therapeutic
moiety is not a nerve growth factor.
[0108] Examples of classes of therapeutic moieties include, but are
not limited to adrenergic agents such as epinephrine,
norepinephrine, dopamine, atenolol; analgesics such as opioids,
morphine, codeine, oxycodone; anti-trauma agents;
anti-inflammatories (steroidal-e.g., prednisolone,
methylprednisolone, betamethasone, dexamethason and
nonsteroidal-eg., piroxicam, meclofenamate, etodolac); anti-viral
agents such as acyclovir, ganciclovir, trifluridine, AZT, ddI, ddC,
trifluridine, and the like; gene therapy agents (e.g., DNAs,RNAs,
or nucleomimetics which introduce a gene or replace a mutated
gene); and hormones, including steroidal hormones (e.g., pregnanes,
estranes, androstanes, and specifically corticosteroids, including
cortisone, cortisone (4-pregnen-17.alpha., 21-diol-3,11,20-trione),
progestins (e.g,. progesterone), estrogen, and estranes, such as
estradiol) and non-steroidal hormones, such as growth factors; and
interferons. Such compounds may optionally also include an imaging
moiety, such as fluorescent moieties, or proteins, a luminophore;
or radioactive labels, for imaging intracellular components of the
nerve cells.
[0109] Examples of neuropharmaceuticals that may be used as, or
adapted for use as, therapeutic moieties include proteins,
antibiotics, adrenergic agents, anticonvulsants, analgesics,
anti-inflammatories, nucleotide analogues, anti-trauma agents,
peptides and other classes of agents used to treat or prevent a
neurological disorders. For example, analgesics such as opioids,
morphine, codeine, and oxycodone can be conjugated to the binding
agent B and specifically delivered to the nerve cells. Since the
same level of pain relief can be achieved using a smaller dosage of
analgesics, side effects such as respiratory depression or
potential drug addiction can be avoided or at least ameliorated.
Such can find particular use as therapeutic moieties in treatment
of a variety of neurological disorders including, but not limited
to, Alzheimer's disease, Parkinson's disease, multiple sclerosis,
neurodegenerative disease, epilepsy, seizure, migraine, trauma and
pain.
[0110] In one embodiment, the therapeutic moiety is a steroid
hormone or derivative thereof. As noted above, such find use in,
for example, treatment of inflammation of the nerves, which may
reduce the side effects associated with high doses of steroids,
such as weight gain, redistribution of fat, increase in
susceptibility to infection, and avascular necrosis of bone.
Corticosteroids contemplated by the invention include, inter alia,
cortisol (4-pregnen-11,17,21-triol-3,20-dione), cortisone
(4-pregnen-17,21-diol-3,11,20-trione), deoxycorticosterone
(4-pregnen-21-hydroxy-3,20-dione), prednisone
(1,4-pregnadien-17.alpha.,2- 1-diol--3,11,20-trione), prednisolone
(1,4-pregnadiene-11.beta.,17.alpha.,- 21-triol-3,20-dione),
methylprednisolone (1,4-pregnadiene,
9-Fluoro-1,16,17,21-trihydroxy-16.beta.-methyl-3,20-dione),
beclomethasone
(1,4-pregnadiene-9-chloro-11.beta.,17,21-triol-16.beta.-me-
thyl-3,20-dione-17,21-dipropionate), triamcinolone (1,
4-pregnadiene-9-fluoro-11.beta., 16.alpha.,
17,21-tetrahydroxy-3,20-dione- ), triamcinolone acetonide
(1,4-pregnadiene-9-fluoro-11.beta., 16.alpha.,
17,21-tetrahydroxy-3,20-dione cyclic 16,17-acetal with acetone),
desonide
(1,4-pregnadiene-3,20-dione,11.beta.,21-dihydroxy-16.alpha.,17-[(1-methyl-
ethylidene)bis(oxy)], alclometasone (typically as the dipropionate:
1,4-pregnadiene-7a-chloro-11.beta., 17, 21-trihydroxy,
16.alpha.-methyl, 3,20-dione, 17,21-dipropionate), flurandrenolide
(4-pregnene-3,20-dione, 6.alpha.-fluoro- 11.beta., 21
dihydroxy-16.beta.,17-[(1-methylethylidene) bis (oxy)]),
dexamethasone (1,4-pregnadiene-9-fluoro-11.beta.,17,21-trihy-
droxy-16.alpha.-methyl, 3,20-dione), desoximetasone
(1,4-pregnadiene-3,20-dione,9-fluoro-11.beta.,21-dihydroxy-16.alpha.-meth-
yl), flumethasone
(1,4-pregnadiene-3,20-dione-9.alpha.-fluoro-16.alpha.-me-
thyl-11.beta.,17,21-trihydroxy), and betamethasone
(1,4-pregnadiene,
9-Fluoro-1,16,17,21-trihydroxy-16.beta.-methyl-3,20-dione) and its
derivatives (such as the diprionate: 1,4-pregnadiene,
9-Fluoro-1,16,17,21-trihydroxy-16.beta.-methyl-3,20-dione
17,21-dipropionate).
[0111] Further therapeutic moieties for use in the conjugates
include moieties that induce the production of nerve growth factor
in the target nerve cells, especially under conditions of
pathogenic under-expression of NGFs (See Riaz, et al. Prog.
Neurobiol. 49: 125-143 (1996)). NGF induction has been demonstrated
in a wide variety of cell types, such as fibroblasts (Furukawa, et
al., FEBS Lett. 247: 463-467(1989)), astrocytes (Furukawa, et al.,
FEBS Lett. 208: 258-262 (1986)), Schwann cells (Ohi, et al.,
Biochem. Int. 20:739-746 (1990)) with a variety of agents including
cytokines, steroids, vitamins, hormones, and unidentified
components of serum. Specific examples of agents known to induce
NGF, and thus specifically contemplated as therapeutic moieties in
the present invention, include 4-methylcatechol, clenbuterol,
isoprenaline, L-tryptophan, 1,25-dihydroxyvitamin D3, forskolin,
fellutamide A, gangliosides and quinone derivatives (Riaz, et al.
Prog. Neurobiol. 49: 125-143 (1996)).
[0112] In one embodiment of particular interest, the therapeutic
moiety is an antiviral agent. Conjugates of the invention having an
antiviral agent as a therapeutic moiety can be used to treat
diseases or symptoms caused by or associated with viral infection,
to eliminate viral spread to the nerves, and to inhibit infection
by such viruses. For example, antiviral drugs such as acyclovir,
ganciclovir, cidofovir, and trifluridine can be conjugated to the
binding agent and used to inhibit active or latent herpes simplex
viruses in the peripheral and central nervous system. Specific
delivery of the conjugate containing these antiviral drugs to the
nervous system can reduce the side effects associated with high
doses or long-term administration of these drugs, such as
headaches, rash and paresthesia.
[0113] The Table immediately below provides exemplary classes, and
exemplary compounds within the classes, of therapeutic moieties
that can be used or adapted for use in the conjugates of the
invention.
2 Table of Exemplary Therapeutic Moieties Antivirals Acyclovir
Enviroxime Ganciclovir Lamivudine (3TC) Trifluridine Zidovudine
(AZT) Steroidal Anti-inflammatories Betamethasone Dexamethasone
Flumethasone Fluocinolone Acetonide Methylprednisolone Prednisolone
Steroid Hormones Cortisone Estradiol Progesterone Testosterone
Non-Steroidal Anti-inflammatories Celecoxib Etodolac Meclofenamate
Piroxicam Analgesics Buprenorphine Butorphanol Etorphine
Levorphanol Morphine Naltrexone Oxymorphone Na+/K+/Ca++ Channel
Blockers Baclofen Gabapentin Mexilitine Procaine Propoxycaine
Anti-bacterials/infectives Ceftriaxone Eflornithine Minocycline
Pentamidine NMDA Antagonists/Modifiers Amantadine Dextromethorphan
Ketamine Phenformin Adrenergic agents Dopamine Epinephrine
Phentolamine Tranylcypromine Mitochondrial modifiers Creatine
Creatine Phosphate Anti-oxidants N-Acetyl-Cysteine
Tauroursodeoxycholic Acid Anti-arrthymic Adenosine Mexilitine Other
Compounds DNA oligomer - Antisense Hyoscyamine - Anticholinergic
N-Acetyl-Aspartic Acid - Myelin Lipid Acylator WHI-P131 - Jak3
inhibitor
[0114] Further examples of therapeutic agents suitable for use, or
suitable to be adapted for use, in the conjugates of the invention
are described in the Tables below.
[0115] Imaging Moiety (IM)
[0116] The method according to the present invention can also be
used to deliver marker compounds to identify, detect, and,
optionally, locate or visualize, a nerve cell or an internal
component of the nerve cell. Such is accomplished by specific
binding of the binding moiety B to the nerve cell and, in some
embodiments, internalize or absorb, the imaging moiety of the
conjugate. Imaging moieties of interest include but are not limited
to fluorescent dyes or proteins, a lumniphore; or radioactive
labels. In general, IM is not horse radish peroxidase (HRP). In one
embodiment, the imaging moiety that provides a detectable signal
that does not require the addition of a substrate for
detection.
[0117] A further aspect of the present invention relates to
compositions and methods for improving the intracellular delivery
of a imaging agent to a cell, particularly a nerve cell using a
charged imaging moiety in the conjugate. According to this
embodiment, the binding agent B facilitates transport of a charged
imaging moiety IM into a cell. Within the cell, the compound (i.e.
the conjugate formed between B and IM) is metabolized to form a
metabolite product that comprises the charged imaging moiety IM.
The charged metabolite product is less prone to being transported
across the cell membrane out of the cell relative to a non-charged
version of the imaging moiety.
[0118] In one particular embodiment, the charged imaging moiety IM
is Alexa Fluor 488.RTM., Molecular Probes, a fused heterocyclic 3
ring aromatic system with a pendant phenyl ring with an amino, a
quaternary amine, two sulfonic acid lithium salts, a carboxylic
acid, and one carboxylic acid N-hydroxy-succinnimidyl ester groups
attached. It is this last group that forms an amide crosslink to
the epsilon amino group of lysine. This compound is a highly
modified derivative of the imaging moiety fluorescein with very
similar absorption and emission spectra but with a much higher
extinction coefficient. In another embodiment, the charged imaging
moiety IM is a similarly modified derivative of Texas Red.RTM. but
of proprietary makeup, Alexa Fluor 647.RTM., Molecular Probes.
[0119] Also according to this embodiment, methods are provided
which comprise administering an imaging agent to a cell, or to a
subject, in a form where the imaging agent comprises a charge and
is conjugated to a protein that acts as a binding moiety to
facilitate transport of the conjugate across a cell membrane into a
cell. Once within the cell, the cell metabolizes at least a portion
of the compound to form a metabolite product that has the
detectable properties of the imaging agent. The metabolite product
is less prone to being transported across the cell membrane out of
the cell relative to the compound, because of the metabolism of the
compound resulting separation of the imaging moiety from the
protein, and is less prone to being transported across the cell
membrane out of the cell relative to an uncharged version of the
imaging moiety.
[0120] This method may be used in conjunction with the conjugates
of the present invention for selectively delivering a moiety to
nerve cells. However, it is noted that charged imaging moieties can
be used with binding agents that target cells other than nerve
cells.
[0121] 3. Linker (L)
[0122] According to the present invention, a binding agent B is
linked to a moiety M by a linker L. In general, any method of
linking a binding agent to a therapeutic moiety may be used and is
intended to fall within the scope of the present invention. The
linker L generally serves to link the binding agent B to the
therapeutic moiety TM. A wide variety of linkers are known in the
art for linking two molecules together, particularly, for linking a
moiety to a peptide or nucleic acid, all of which are included
within the scope of the present invention.
[0123] Examples of classes of linkers that may be used to link the
binding agent B to the therapeutic moiety TM include amide,
alkylamine, carbamate, phosphoramide, ester, ether, thioether,
alkyl, cycloalkyl, aryl, and heteroaryl linkages such as those
described in Hermanson, GT., Bioconjugate Techniques (1996),
Academic Press, San Diego, Calif.
[0124] Many different types of linkers have been developed for
cross linking proteins and conjugating proteins or peptides with
other agents. These linkers include zero-length cross linkers,
homobifunctional cross-linkers, heterobifunctional cross-linkers
and trifunctional cross-linkers. These linkers may have different
susceptibility to cleavage under certain conditions. Depending on a
particular application according to the present invention, an
appropriate linker may be chosen. When an intracellular release of
the agent from its conjugate is desired, a cleavable linker is
chosen which is susceptible to cleavage by external stimuli such as
light and heat, by intracellular enzymes, or by a particular
microenvironment inside the cell.
[0125] In one embodiment, the linker L has one of the following
general structures:
[0126] B--R--(CO)--NH--R-M
[0127] B--R--NH--R-M
[0128] B--R--S--R-M
[0129] B--R--(CH.sub.2).sub.n--R-M
[0130] B--NH-((PO)OH)--O-M
[0131] B--NH--(CO)--O-M
[0132] B--NH--(CO)-M
[0133] B--NH--(CO)--X--O-M
[0134] B--NH--(CO)--X--(CO)--O-M
[0135] B--NH--(CO)--X--(CO)--NH-M
[0136] B--NH--(SO.sub.2)-M
[0137] B--NH--X--S--X--NH--(CO)-M
[0138] B--NH--X--S--X--(CO)--NH-M
[0139] B--NH--X--S--X--NH--(CO)--O-M
[0140] B--NH--X--S--S--X--(CO)--NH-M
[0141] B--NH--X--S--S--X--(CO)--NH--X--(CO)--NH-M
[0142] wherein R and X are each independently chosen from an alkyl,
a heteroalkyl, an alkene, a heteroalkene, an aryl, a heteroaryl, a
cycloalkyl, a heterocycloalkyl, a cycloalkene or a
heterocycloalkene.
[0143] 4. Cleavable Linkers
[0144] One particular embodiment of the present invention relates
to compounds that include a cleavable linker L, which cleavable
linker may be used to join a binding agent with either a
therapeutic moiety or an imaging moiety. Use of a cleavable linker
may be more desirable where, for example, the therapeutic moiety TM
is more efficacious or potent when free from a carrier molecule
such as a binding agent. In such instances, it is desirable to
utilize a cleavable linker which allows the therapeutic moiety TM
to be released from the compound once inside the cell.
[0145] Many cleavable linker groups have been developed which are
susceptible to cleavage and by a wide variety of mechanisms. For
example, linkers have been developed which may be cleaved by
reduction of a disulfide bond, by irradiation of a photolabile
bond, by hydrolysis of derivatized amino acid side chain, by serum
complement-mediated hydrolysis, and by acid-catalyzed
hydrolysis.
[0146] For example, cleavage of the linker releasing the
therapeutic moiety may be as a result of a change in conditions
within the nerve cells as compared to outside the nerve cells, for
example, due to a change in pH within the nerve cell. Cleavage of
the linker may occur due to the presence of an enzyme within the
nerve cell that cleaves the linker once the compound enters the
nerve cell. Alternatively, cleavage of the linker may occur in
response to energy or a chemical being applied to the nerve cell.
Examples of types of energies that may be used to effect cleavage
of the linker include, but are not limited to light, ultrasound,
microwave and radiofrequency energy.
[0147] Preferably, the linker used is one that, following conjugate
production, links the binding agent B and the moiety M by only an
amide or a carbamate bond. Furthermore, it is also preferred that
the linker, upon cleavage following delivery into the nerve cell,
provides for a binding agent product and a moiety product that is
either modified only by the addition of a carboxylic acid group or
not modified relative to the binding agent or moiety prior to
conjugation (e.g., cleavage of the linker provides the "native"
starting materials of the binding agent and moiety prior to
conjugation, or the "native" starting material modified only by
addition of a carboxylic acid group). Use of such linkers also
provides that cleavage may results in production of carbon dioxide
as the by-product. This embodiment thus provides the advantage of
reduced cytotoxicity of the products of conjugate cleavage in the
nerve cell.
[0148] The linker L used to link the binding agent B to the
therapeutic moiety TM may be a photolabile linker. Examples of
photolabile linkers include those linkers described in U.S. Pat.
No. 5,767,288 and No. 4,469,774.
[0149] Acid-labile linkers are preferred in the practice of the
present invention by taking advantage of a cell's receptor-mediated
endocytosis pathways. Receptors that are internalized by
receptor-mediated endocytosis pass through acidified compartments
known as endosomes or receptosomes. Since the interior of the
endosomal compartment is kept acidic (pH.about.6.0) by ATP-driven
H.sup.+ pumps in the endosomal membrane that pump H.sup.+ into the
lumen from the cytosol, a change in pH within the nerve cell can be
used to cause the acid-labile linker to be cleaved and release the
moiety.
[0150] Thus, in one embodiment of particular interest, the linker L
used to link the binding agent B to the moiety M, particularly
where the moiety is a therapeutic moiety TM, is an acid labile
linker. Examples of acid labile linkers include linkers formed by
using cis-aconitic acid, cis-carboxylic alkatriene, polymaleic
anhydride, and other acid labile linkers, such as those linkers
described in U.S. Pat. Nos. 5,563,250 and 5,505, 931.
[0151] Further examples of cleavable linkers include, but are not
limited to the linkers described in Lin, et al., J. Org. Chem.
56:6850-6856 (1991); Ph.D. Thesis of W.-C. Lin, U.C. Riverside,
(1990); Hobart, et al., J. Immunological Methods 153: 93-98 (1992);
Jayabaskaran, et al., Preparative Biochemistry 17(2): 121-141
(1987); Mouton, et al., Archives of Biochemistry and Biophysics
218: 101-108 (1982); Funkakoshi, et al., J. of Chromatography
638:21-27 (1993); Gildea, et al., Tetrahedron Letters 31: 7095-7098
(1990); WO 85/04674; and Dynabeads.RTM. (Dynal, Inc., 5 Delaware
Drive, Lake Success, N.Y. 11042).
[0152] 5. Examples of Compounds According to the Present
Invention
[0153] In one embodiment, the compound of the present invention is
a conjugated 4-pregnen-21-hydroxy or 1,4-pregnadiene-21-hydroxy
steroid, wherein the conjugant group pends from the steroid 21
hydroxyl group and comprises a neurotrophin or a neurotrophin
receptor-binding fragment thereof. Conjugated steroids such as
described above may have, for example, a 21-carbamate linkage to
the conjugant group, or a 21-phosphoramide linkage to the conjugant
group. The neurotrophin or neurotrophin fragment may pend
covalently, for example, through a lysine residue epsilon amino
group or through a thiolated lysine residue epsilon amino
group.
[0154] The conjugated 4-pregnen-21-hydroxy or
1,4-pregnadiene-21-hydroxy steroid may be a conjugated
corticosteroid such as cortisone, prednisolone, methylprednisolone,
betamethasone, dexamethasone, flumethasone, triamcinolone
acetonide, or fluocinolone acetonide.
[0155] The neurotrophin may be, for example, NGF, BDNF, NT-3, NT-4,
or NT-6, or a receptor-binding fragment or derivative thereof.
[0156] The neurotrophin fragment may be, for example, an NGF
fragment capable of binding to trkA receptors and being
internalized therewith. In certain embodiments, the NGF fragment is
capable of binding to trkA receptors, being internalized therewith,
and then being retrogradely transported to the nerve cell body.
[0157] In other embodiments, the compound may be a conjugated
4-pregnen-21-hydroxy or 1,4-pregnadiene-21-hydroxy steroid, wherein
the conjugant group pends from the steroid 21 hydroxyl group and
comprises BDNF or a BDNF fragment or derivative that is capable of
binding to trkB receptors and being internalized therewith,
optionally additionally being retrogradely transported to the cell
body therewith.
[0158] In a further embodiment, the compound may comprise
triamcinolone acetonide conjugated by a 21-carbamate linkage to
NGF, or to a receptor-binding fragment of NGF, which pends
covalently through a lysine residue epsilon amino group. In another
embodiment, the compound may comprise fluocinolone acetonide
conjugated by a 21-carbamate linkage to NGF, a receptor-binding
fragment of NGF, BDNF, a receptor-binding fragment of BDNF, or
another neurotrophin or receptor-binding fragment thereof, which
pends covalently through a lysine residue epsilon amino group.
[0159] Table 2 provides several compounds according to the present
invention. It is noted that in each instance, the particular
therapeutic moieties, binding moieties, and linkers shown may be
interchanged with other suitable therapeutic moieties, binding
moieties, and linkers. In this regard, the compounds shown in the
table are intended to illustrate the diversity of compounds
provided according to the present invention.
3TABLE 2 1 wherein B is selected from the group consisting of nerve
growth factors, NGF, BDNF, NT-3, NT-4, NT-6, and anti-neurotrophin
receptor antibodies MAb 5C3 and MAb MC192. 2 wherein B is selected
from the group consisting of nerve growth factors NGF, BDNF, NT-3,
NT-4, NT-6, anti-neurotrophin receptor antibodies MAb 5C3 and MAb
MC192. 3 wherein B is selected from the group consisting of nerve
growth factors NGF, BDNF, NT-3, NT-4, NT-6, anti-neurotrophin
receptor antibodies MAb 5C3 and MAb MC192. 4 wherein B is selected
from the group consisting of nerve growth factors NGF, BDNF, NT-3,
NT-4, NT-6, anti-neurotrophin receptor antibodies MAb 5C3 and MAb
MC192. 5 wherein B is selected from the group consisting of nerve
growth factors NGF, BDNF, NT-3, NT-4, NT-6, anti-neurotrophin
receptor antibodies MAb 5C3 and MAb MC192. 6 wherein B is selected
from the group consisting of nerve growth factors NGF, BDNF, NT-3,
NT-4, NT-6, anti-neurotrophin receptor antibodies MAb 5C3 and MAb
MC192.
[0160] 6. Examples of Compounds for Treating Pain
[0161] Table 3 provides several therapeutic moieties which may be
used in the compounds and methods of the present invention for
treating pain. It is noted that any of the various binding moieties
and linkers described herein may be employed with these therapeutic
agents. Indicated in the table below as * are reactive groups
presently preferred for attaching linkers to the therapeutic
moieties.
[0162] 7. Examples Of Linkers
[0163] Table 4 provides a series of linkers for linking different
therapeutic moieties and binding moieties together. As illustrated,
linkers are provided for attaching moieties that have thiol (--SH),
hydroxyl (--OH), carboxylic acid (--COOH), sulfonic acid (--SO3H)
and amino (--NH2) groups to the linkers. In these examples,
neurotrophin is shown as the binding agent. However, it is noted
that neurotrophin can be substituted with other binding moieties
described herein, and other linkers can be substituted for the
linkers in the exemplary compounds below. These compounds provided
below are intended to be exemplary only, and not limiting.
4TABLE 3 Pain - Steroidal anti-inflammatory agents 7 8 9 10 Pain -
Non-steroidal anti-inflammatory agent 11 Pain - Local anesthetic
agents 12 Pain - Narcotic Agonists 13 Pain - Channel blockers 14
Anti-neurodegenerative 15 Antiviral 16 17 Trifluridine conjugate
18
[0164]
5TABLE 4 Hydroxyl group conjugations e.g., Steroids, Piroxicam,
Acyclovir, Morphine 19 Amino group conjugations e.g., Propoxycaine,
Gabapentin, Tacrine 20 21 22 Phosphate group conjugations 23 24
[0165] 8. Synthetic Sequence for Attaching Acyclovir To NGF Via
PMPI
[0166] Illustrated below is a synthetic sequence for the attachment
of acyclovir to NGF via the linker PMPI. 25
[0167] 9. Synthetic Sequence for Attaching Acyclovir to NGF Via
Imidazole
[0168] Illustrated below is a synthetic sequence for the attachment
of acyclovir to NGF via an imidazole linker. 26
[0169] 10. Examples of Human Neurotrophins as the Binding Agent
(B)
[0170] Table 5 lists the amino acid sequences of human
neurotrophins (NGF, BDNF, NT-3, and NT-4) that are used as the
binding agent (B) of the present invention. Lysine residues that
may be used to attach to the linker (L) which in turn is conjugated
with the moiety (M) (which can be a therapeutic moiety or an
imaging moiety) are highlighted and underlined in Table 5.
[0171] In general, it is preferred that the residue to which the
linker is attached is one that is outside of the area of the
protein that binds to the corresponding receptor. For example, the
lysine at residue 95 is within the TrkA binding site, and thus is
less preferred for attachment of a linker.
[0172] Preferably, where the binding agent B is NGF or BDNF, the
linker is attached to the lysine residue at position 74 in the
protein.
6TABLE 5 Sequences of Examples of Human Neurotrophins NERVE GROWTH
FACTOR (NGF) (SEQ ID NO: 1): 1 SER SER SER HIS PRO ILE PHE HIS ARG
GLY GLU PHE SER VAL CYS ASP SER VAL SER VAL TRP VAL GLY ASP LYS THR
THR ALA THR ASP ILE LYS GLY LYS GLU VAL MET VAL LEU GLY GLU VAL ASN
ILE ASN ASN SER VAL PHE LYS GLN TYR PHE PHE GLU THR LYS CYS ARG ASP
PRO ASN PRO VAL ASP SER GLY CYS ARG GLY ILE ASP SER LYS* HIS TRP
ASN SER TYR CYS THR THR THR HIS THR PHE VAL LYS ALA LEU THR MET ASP
GLY LYS GLN ALA ALA TRP ARG PHE ILE ARG ILE ASP THR ALA CYS VAL CYS
VAL LEU SER ARG LYS ALA VAL 120 ARG ARG ALA *residue 74 BRAIN
DERIVED NEUROTROPHIC FACTOR (BDNF) (SEQ ID NO: 2): 1 HIS SER ASP
PRO ALA ARG ARG GLY GLU LEU SER VAL CYS ASP SER ILE SER GLU TRP VAL
THR ALA ALA ASP LYS LYS THR ALA VAL ASP MET SER GLY GLY THR VAL THR
VAL LEU GLU LYS VAL PRO VAL SER LYS GLY GLN LEU LYS GLN TYR PHE TYR
GLY THR LYS CYS ASN PRO MET GLY TYR THR LYS GLU GLY CYS ARG GLY ILE
ASP LYS* ARG HIS TRP ASN SER GLN CYS ARG THR THR GLN SER TYR VAL
ARG ALA LEU THR MET ASP SER LYS LYS ARG ILE GLY TRP ARG PHE ILE ARG
ILE ASP THR SER CYS VAL CYS THR LEU THR ILE LYS ARG 119 GLY ARG
*residue 74 NEUROTROPHIN-3 (NT-3) (SEQ ID NO: 3): 1 TYR ALA GLU HIS
LYS SER HIS ARG GLY GLU TYR SER VAL CYS ASP SER GLU SER LEU TRP VAL
THR ASP LYS SER SER ALA ILE ASP ILE ARG GLY HIS GLN VAL THR VAL LEU
GLY GLU ILE LYS THR GLY ASN SER PRO VAL LYS GLN TYR PHE TYR GLU THR
ARG CYS LYS GLU ALA ARG PRO VAL LYS ASN GLY CYS ARG GLY ILE ASP ASP
LYS HIS TRP ASN SER GLN CYS LYS THR SER GLN THR TYR VAL ARG ALA LEU
THR SER GLU ASN ASN LYS LEU VAL GLY TRP ARG TRP ILE ARG ILE ASP THR
SER CYS VAL CYS ALA LEU SER ARG LYS ILE GLY 119 ARG THR
NEUROTROPHIN-4 (NT-4) (SEQ ID NO: 4): 1 GLY VAL SER GLU THR ALA PRO
ALA SER ARG ARG GLY GLU LEU ALA VAL CYS ASP ALA VAL SER GLY TRP VAL
THR ASP ARG ARG THR ALA VAL ASP LEU ARG GLY ARG GLU VAL GLU VAL LEU
GLY GLU VAL PRO ALA ALA GLY GLY SER PRO LEU ARG GLN TYR PHE PHE GLU
THR ARG CYS LYS ALA ASP ASN ALA GLU GLU GLY GLY PRO GLY ALA GLY GLY
GLY GLY CYS ARG GLY VAL ASP ARG ARG HIS TRP VAL SER GLU CYS LYS ALA
LYS GLN SER TYR VAL ARG ALA LEU THR ALA ASP ALA GLN GLY ARG VAL GLY
TRP ARG TRP ILE ARG ILE ASP THR 130 ALA CYS VAL CYS THR LEU LEU SER
ARG THR GLY ARG ALA
[0173] 11. Conjugates
[0174] Table 6 lists exemplary conjugation products indicative of
the present invention and is not intended to be exhaustive,
organized by type of linker chemistry; the structures of some of
the various therapeutic moieties follows thereafter. NT=member of
the neurotrophin family (e.g., NGF, BDNF, NT-3, NT-4, NT-6), with
NGF and BDNF being of particular interest.
7TABLE 6 Functional OH OH OH COOH or SO.sub.3H NH.sub.2 Group
Neuro- NT NT Thiolated NT NT Thiolated NT trophin Chemistry
POCl.sub.3/IM EDC or CDI PMPI EDC or CDI EMCS or Sulfo-EMCS Drug
Acyclovir Acyclovir Acyclovir Etodolac Tranylcypromine (Anti-viral)
(Anti-viral) (Anti-viral) (COX2 Inhibitor) (MAOI) Ganciclovir
Ganciclovir Ganciclovir Meclofenamate Propoxycaine (Na.sup.+
(Anti-viral) (Anti-viral) (Anti-viral) COX1&2, 5-LOX- channel
blocker) inhibitor) Dexamethasone Dexamethasone Dexamethasone
Tauroursodeoxy-cholic (Steroid) (Steroid) (Steroid) Acid
(Anti-oxidant) Flumethasone Flumethasone Flumethasone
N-Acetyl-Cysteine Procaine (Na.sup.+ (Steroid) (Steroid) (Steroid)
(Anti-oxidant) channel blocker) Piroxicam Piroxicam Piroxicam
N-Acetyl-Aspartic (COX1&2 COX1&2 COX1&2 Acid (Myelin
Lipid inhibitor) inhibitor) inhibitor) Acylator) Phentolamine
Phentolamine Phentolamine Creatine Mexilitine (Na.sup.+ (alpha
(alpha (alpha (Mitochondrial channel blocker) Adrenergic Adrenergic
Adrenergic Modulator) antagonist) antagonist) antagonist)
Minocycline Minocycline Minocycline Creatine Phosphate Gabapentin
(Na.sup.+, Ca.sup.2+ (Anti-bacterial) (Anti-bacterial)
(Anti-bacterial) (Mitochondrial channel blocker) Energetic)
Hyoscyamine Hyoscyamine Hyoscyamine- Ceftriaxone Baclofen
(Ca.sup.2+ (Anti- (Anti- (Anti- (Anti-bacterial) channel blocker)
cholinergic) cholinergic) cholinergic) Zidovudine Zidovudine
Zidovudine Ketamine (NMDA (AZT) (AZT) (AZT) antagonist)
(Anti-retroviral) (Anti-retroviral) (Anti-retroviral) Lamivudine
Lamivudine Lamivudine Phenformin (NMDA (3TC) (Anti- (3TC) (Anti-
(3TC) (Anti- channel subunit retroviral) retroviral) retroviral)
modifier) Enviroxime Enviroxime Enviroxime- Pentamidine (Anti-
(Anti- (Anti- (Anti-infective) enteroviral) enteroviral)
enteroviral) Adenosine Adenosine Adenosine- Eflorinithine
(Adenosine (Adenosine (Adenosine (Anti-infective) receptor receptor
receptor agonist) agonist) agonist) WHI-P131-* WHI-P131 WHI-P131-
(JAK3 (JAK3 (JAK3 inhibitor) inhibitor) inhibitor) Fluorescent
Alexa Fluor Alexa Fluor Alexa Fluor Dyes 488 488 488 succinimidyl
succinimidyl C.sub.5 maleimide- ester- ester- (Fluorescent
(Fluorescent (Fluorescent probe) probe) probe) Alexa Fluor Alexa
Fluor Alexa Fluor 647 647 647 succinimidyl succinimidyl C.sub.5
maleimide- ester- ester- (Fluorescent (Fluorescent (Fluorescent
probe) probe) probe) *WHI-P131 is also know as
4-(4'Hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P131). For
a description of WHI-P131 and its uses, see e.g., Uckun et al.
Blood. 2002 Jun. 1; 99(11): 4192-9.
[0175] 27
[0176] The Alexa Fluor dyes are available from Molecular Probes
Inc., Eugene Oreg. 2829303132
[0177] 12. Methods for Using Compounds of the Present Invention
[0178] Described below are several methods for formulating and
administering the compounds of the present invention. The compounds
of the present invention may be employed in these and other
applications.
[0179] a. Pharmaceutical Formulations Utilizing Compositions Of The
Present Invention
[0180] The compounds of the present invention may be incorporated
into a variety of pharmaceutical compositions including, but not
limited to: a sterile injectable solution or suspension; hard or
soft gelatin capsules; tablets; emulsions; aqueous suspensions,
dispersions, and solutions; suppositories. In general, the
conjugate is formulated with an appropriate pharmaceutically
acceptable carrier, and, where desired, with other additives such
as stabilizers, buffers, and the like.
[0181] Other pharmaceutically suitable formulations for delivering
the compounds of the present invention to nerve cells may also be
used and are intended to fall within the scope of the present
invention.
[0182] b. Routes of Administration
[0183] The compounds according to the present invention can be
administered orally, by subcutaneous or other injection,
intravenously, intracerebrally, intramuscularly, parenterally,
transdermally, nasally or rectally. The form in which the compound
is administered depends at least in part on the route by which the
compound is administered.
[0184] While the present invention is disclosed with reference to
preferred embodiments and examples detailed above, it is to be
understood that these examples are intended in an illustrative
rather than limiting sense, as it is contemplated that
modifications will readily occur to those skilled in the art, which
modifications will be within the spirit of the invention and the
scope of the appended claims. The patents, papers, and books cited
in this application are to be incorporated herein in their
entirety.
Sequence CWU 1
1
4 1 120 PRT H. sapiens 1 Ser Ser Ser His Pro Ile Phe His Arg Gly
Glu Phe Ser Val Cys Asp 1 5 10 15 Ser Val Ser Val Trp Val Gly Asp
Lys Thr Thr Ala Thr Asp Ile Lys 20 25 30 Gly Lys Glu Val Met Val
Leu Gly Glu Val Asn Ile Asn Asn Ser Val 35 40 45 Phe Lys Gln Tyr
Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val 50 55 60 Asp Ser
Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys 65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln 85
90 95 Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val
Leu 100 105 110 Ser Arg Lys Ala Val Arg Arg Ala 115 120 2 119 PRT
H. sapiens 2 His Ser Asp Pro Ala Arg Arg Gly Glu Leu Ser Val Cys
Asp Ser Ile 1 5 10 15 Ser Glu Trp Val Thr Ala Ala Asp Lys Lys Thr
Ala Val Asp Met Ser 20 25 30 Gly Gly Thr Val Thr Val Leu Glu Lys
Val Pro Val Ser Lys Gly Gln 35 40 45 Leu Lys Gln Tyr Phe Tyr Glu
Thr Lys Cys Asn Pro Met Gly Tyr Thr 50 55 60 Lys Glu Gly Cys Arg
Gly Ile Asp Lys Arg His Trp Asn Ser Gln Cys 65 70 75 80 Arg Thr Thr
Gln Ser Tyr Val Arg Ala Leu Thr Met Asp Ser Lys Lys 85 90 95 Arg
Ile Gly Trp Arg Phe Ile Arg Ile Asp Thr Ser Cys Val Cys Thr 100 105
110 Leu Thr Ile Lys Arg Gly Arg 115 3 119 PRT H. sapiens 3 Tyr Ala
Glu His Lys Ser His Arg Gly Glu Tyr Ser Val Cys Asp Ser 1 5 10 15
Glu Ser Leu Trp Val Thr Asp Lys Ser Ser Ala Ile Asp Ile Arg Gly 20
25 30 His Gln Val Thr Val Leu Gly Glu Ile Lys Thr Gly Asn Ser Pro
Val 35 40 45 Lys Gln Tyr Phe Tyr Glu Thr Arg Cys Lys Glu Ala Arg
Pro Val Lys 50 55 60 Asn Gly Cys Arg Gly Ile Asp Asp Lys His Trp
Asn Ser Gln Cys Lys 65 70 75 80 Thr Ser Gln Thr Tyr Val Arg Ala Leu
Thr Ser Glu Asn Asn Lys Leu 85 90 95 Val Gly Trp Arg Trp Ile Arg
Ile Asp Thr Ser Cys Val Cys Ala Leu 100 105 110 Ser Arg Lys Ile Gly
Arg Thr 115 4 130 PRT H. sapiens 4 Gly Val Ser Glu Thr Ala Pro Ala
Ser Arg Arg Gly Glu Leu Ala Val 1 5 10 15 Cys Asp Ala Val Ser Gly
Trp Val Thr Asp Arg Arg Thr Ala Val Asp 20 25 30 Leu Arg Gly Arg
Glu Val Glu Val Leu Gly Glu Val Pro Ala Ala Gly 35 40 45 Gly Ser
Pro Leu Arg Gln Tyr Phe Phe Glu Thr Arg Cys Lys Ala Asp 50 55 60
Asn Ala Glu Glu Gly Gly Pro Gly Ala Gly Gly Gly Gly Cys Arg Gly 65
70 75 80 Val Asp Arg Arg His Trp Val Ser Glu Cys Lys Ala Lys Gln
Ser Tyr 85 90 95 Val Arg Ala Leu Thr Ala Asp Ala Gln Gly Arg Val
Gly Trp Arg Trp 100 105 110 Ile Arg Ile Asp Thr Ala Cys Val Cys Thr
Leu Leu Ser Arg Thr Gly 115 120 125 Arg Ala 130
* * * * *