Note: Descriptions are shown in the official language in which they were submitted.
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Compounds For Intracellular Deliverx,
Of Therapeutic Moieties To Nerve Cells
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to compounds which can be used
to selectively deliver moieties to nerve cells. More specifically, the
invention relates to compounds which include a therapeutic moiety and
facilitate absorption of the therapeutic moiety by nerve cells.
Description of Related Art
io Our understanding of the structure and function of the nervous
system has been greatly advanced owing to enormous progresses
made in field of neuroscience. Cellular and molecular mechanisms of
neuron growth and development and diseases associated with the
central and peripheral nervous systems are studied extensively by using
is rapidly growing techniques in molecular and cell biology. However, 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 due to infection by
2o viruses and other microorganisms (herpes simplex, HIV,
cytomegalovirus, parasites, fungi, prion, etc.).
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
2s between these agents and cells or tissues other than the targeted cells.
For example, steroid hormone cortisone and its derivatives are widely
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used to treat inflammation in the body including the nerve 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 hormone binds to its cognate nuclear
s hormone receptor and induces a cascade of cellular effects, including
programmed cell death of the neurons in the brain (Kawata M., et al., J.
Steroid Biochem. Mol. Biol. 65: 273-280 (1998)). Since steroid hormone
receptors, such as glucocorticord receptor for cortisone, distribute in a
wide variety of tissues and cells, nonspecific interactions of the hormone
io with its cognate receptor in different sites is unavoidable if the drug is
circulated systemically.
A need 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, but with only limited
is success. 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
2o 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
2s liposomes are coated with antibody molecules, liposomes are readily
phagocytosed by macrophages and removed from circulation before
reaching their target sites.
2
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SUMMARY OF THE INVENTION
Compounds of the present invention include compounds having
the general formula:
s
B-L-M
where:
B is a binding agent capable of selectively binding to a
io 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
L is a linker coupling B to M.
is
In one embodiment, the compounds have the general formula:
B-L-TM
2o 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;
TM is a therapeutic moiety which has a non-cytotoxic
2s therapeutic effect when absorbed by a nerve cell; and
L is a linker coupling B to TM.
In another embodiment, the compounds have the general
formula:
B-L-I M
3
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where:
B is a binding agent capable of selectively binding to a
nerve cell surface receptor and mediating absorption of the compound
s by the nerve cell;
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
L is a linker coupling B to IM.
to 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, antibodies and
antibody fragments. Examples of nucleic acids that can serve as the
binding agent B include, but are not limited to, DNA and RNA ligands
is that function as antagonists of nerve growth factors or inhibit binding of
other growth factors to nerve cell surface receptors. 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 NGF, BDNF, NT-3, NT-4, NT-6; derivatives, analogs, and
2o fragments of nerve growth factors such as recombinant molecules of
NGF and BDNF; and synthetic peptides that bind to nerve cell surface
receptors and have agonist or antagonist activities of nerve growth
factors.
Antibodies, derivatives of antibodies and antibody fragments can
2s 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 MC192.
The therapeutic moiety TM is selected to perform a non-cytotoxic
therapeutic function within nerve cells. Examples of non-cytotoxic
3o functions which the therapeutic moiety TM may perform include, but are
not limited to, the functions performed by adrenergic agents, adrenergic
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agonists, analgesics, anti-trauma agents, anti-viral agents, gene therapy
agents, and hormones (growth factors, interferons, etc.). Examples of
classes of therapeutic moieties include, but are not limited to, adrenergic
agents (e.g., epinephrine, norepinephrine, dopamine, etenolol),
s adrenergic agonists (e.g., phenylephrine, isoproterenol, and
noradrenaline), analgesics (e.g., opioids, codeine, oxycodone), anti-
trauma agents, anti-viral agents (e.g., acyclovir, gancyclovir, AZT, ddl,
ddC, etc.), gene therapy agents (e.g., DNAs or RNAs which introduce a
gene or replace a mutated gene), steroids (e.g., cortisone,
to progesterone, estrogen), and hormones (e.g., growth factors,
interferons).
In one particular embodiment, the therapeutic moiety TM is a
charged moiety. Cells have difficulty transporting charged molecules
across cell membranes. According to this embodiment, the binding
is agent B serves to facilitate transport of a charged therapeutic moiety TM
into a cell. Within the cell, the compound (i.e. the conjugate formed
between B and TM) is metabolized to form a metabolite product that
comprises the charged therapeutic moiety TM. The metabolite product
is less prone to being transported across the cell membrane out of the
2o cell relative to the conjugate because of the metabolism of the conjugate
resulting in the separation of the therapeutic moeity TM 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 therapeutic moiety due to the charge which the
2s therapeutic moiety carries.
According to this embodiment, compounds are provided which
comprise a charged derivative of a therapeutic agent having a
therapeutic activity, the charged derivative being conjugated to a protein
having a biological activity of being transported across a cell membrane
3o into a cell, the cell metabolizing at least a portion of the protein to
form a
charged metabolite product that possesses the therapeutic activity of the
s
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therapeutic agent, 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 the therapeutic agent.
In one particular embodiment, the charged therapeutic moiety TM
is a quartinery alkyl amine derivative of a therapeutic moiety A
particular example of a quartinery alkyl amine derivative of a therapeutic
moiety TM is a quartinery alkyl amine of propoxycaine, shown in Table
3.
to The imaging moiety IM is a non-cytotoxic agent which can be
used to locate and optionally visualize a nerve cell or an internal
component of the nerve cell which has absorbed the imaging moiety.
Fluorescent dyes may be used as an imaging moiety IM. Radioactive
agents which are non-cytotoxic may also be an imaging moiety IM.
is In general, the linker may be any moiety which 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. The cleavable linker may
2o be cleaved by a chemical agent, enzymatically, due to a pH change, or
by being exposed to energy. Examples of forms of energy which may
be used include light, microwave, ultrasound, and radiofrequency.
The present invention also relates to a method for selectively
delivering a moiety into nerve cells comprising the steps of:
2s delivering to a patient a compound having the general formula:
B-L-M
where:
B is a binding agent capable of selectively binding
3o to a nerve cell surface receptor and mediating absorption of the
compound by the nerve cell;
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M is a moiety which performs a useful non-cytotoxic
function when absorbed by a nerve cell; and
L is a linker coupling B to M.
having the compound selectively bind to a nerve cell surface
receptor via the binding agent B; and
having the compound be absorbed by the nerve cell mediated by
the binding of the binding agent B to the nerve cell surface receptor.
In one embodiment, moiety M is a therapeutic moiety TM as
described herein and in another embodiment is an imaging moiety IM.
io 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.
The above method can be used to deliver therapeutic moieties for
treating pain when a therapeutic moiety TM for treating pain, such as an
is analgesic, is included as the therapeutic moiety TM in the compound.
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.
2o 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.
The above method can also be used to treat infected nerve cells
infected with viruses or immunize nerve cells from viruses when the
2s therapeutic moiety TM in the compound is an antiviral agent.
The above method can also be used to perform gene therapy
when the therapeutic moiety TM is a gene therapy agent.
The present invention also relates to a method for improving
intracellular administration of a therapeutic agent. The method
3o comprises contacting cells with a compound comprising a charged
derivative of a therapeutic agent having a therapeutic activity, the
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charged derivative being conjugated to a protein having a biological
activity of being transported across a cell membrane into a cell; and
having the cell transport the compound into the cell where the cell
metabolizes at least a portion of the protein to form a charged
s metabolite product that possesses the therapeutic activity of the
therapeutic agent, 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 the therapeutic agent.
io 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.
In one particular embodiment, the charged therapeutic moiety TM
is is a quartinery alkyl amine derivative of a therapeutic moiety A
particular example of a quartinery alkyl amine derivative of a therapeutic
moiety TM is a quartinery alkyl amine of propoxycaine, shown in Table
3.
2o DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to compounds which include a
binding agent which binds to a nerve cell surface receptor and facilitates
absorption of the compound by the nerve cell; and a moiety. DifFerent
2s 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 which can be used to image nerve cells which
absorb these compounds.
In one embodiment, compounds of the present invention have the
so general formula:
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B-L-TM
where:
B is a binding agent capable of selectively binding to a
s nerve cell surface receptor and mediating absorption of the compound
by the nerve cell;
TM is a therapeutic moiety which has a non-cytotoxic
therapeutic effect when absorbed by a nerve cell; and
L is a linker coupling B to TM.
1o
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. The binding agent B can be any
is molecule which can perform these two 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.
Examples of nucleic acids that can serve as the binding agent B
2o include, but are not limited to, DNA and RNA ligands that function as
antagonists of nerve growth factors or inhibit binding of other growth
factors to nerve cell surface receptors (Binkley, J., et al., Nucleic Acid
Res. 23: 3198-3205 (1995); Jellinek, D., et al., Biochem. 33:10450-
10456 (1994)).
2s 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 NGF, BDNF, NT-3, NT-4, NT-6, etc. (see
reviews: Frade, J. M., et al., Bioessays 20: 137-145 (1998); Shieh, P. B.,
Curr. Biol. 7: 8627-8630 (1997); Dechant, G., et al., Curr. Opin.
3o Neurobiol. 7: 413-418 (1997); Chao, M. V. and Hempstead, B. L.,
Trends Neurobiol. 18: 321-326 (1995)); and derivatives, analogs, and
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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
s 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)).
Examples of antibodies, derivatives of antibodies and antibody
to 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).
is According to this embodiment, 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 analgesics, anti-trauma agents, anti-viral agents, gene therapy
2o agents, and hormones (growth factors, interferons, etc.). Examples of
classes of therapeutic moieties include, but are not limited to, adrenergic
agents (e.g., epinephrine, norepinephrine, dopamine, etenolol),
adrenergic agonists (e.g., phenylephrine, isoproterenol, and
noradrenaline), analgesics (e.g., opioids, codeine, oxycodone), anti-
2s trauma agents, anti-viral agents (e.g., acyclovir, gancyclovir, AZT, ddl,
ddC, etc.), gene therapy agents (e.g., DNAs or RNAs which introduce a
gene or replace a mutated gene), steroids (e.g., cortisone,
progesterone, estrogen), and hormones (e.g., growth factors,
interferons).
so The linker L 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
to
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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.
Examples of classes of linkers that may be used to link the
s binding agent B to the therapeutic moiety TM include amide, alkylamine,
thiolether, alkyl, cycloalkyl, aryl linkages such as those described in
Hermanson, G.T., Bioconjugate Techniques (1996), Academic Press,
San Diego, CA.
In certain applications, it is desirable to release the therapeutic
to moiety TM once the compound has entered the nerve cell, resulting in a
release of the therapeutic moiety TM. Accordingly, in one variation, the
linker L is a cleavable linker. This enables the therapeutic moiety TM to
be released from the compound once absorbed by the nerve cell. This
may be desirable when the therapeutic moiety TM has a greater
is therapeutic effect when separated from the binding agent. 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
2o moiety TM can enter the intracellular compartment.
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
2s enzyme within the nerve cell which 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,
3o microwave and radiofrequency energy.
n
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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 US Patent No. 5,767,288 and No.
4,469,774. The linker L used to link the binding agent B to the
s therapeutic moiety TM may also be an acid labile linker. Examples of
acid labile linkers include linkers formed by using cis-aconitic acid, cis-
carboxylic alkatriene, polymaleic anhydride, and other acidlabile linkers,
such as those linkers described in US Patent Nos. 5,563,250 and 5,505,
931.
to 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.,
is 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 (Dynal, Inc., 5 Delaware Drive, Lake Success, NY 11042).
In another embodiment, compounds of the present invention
2o have the general formula:
B-L-IM
where:
2s 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;
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
3o L is a linker coupling B to IM.
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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 and radioisotopes which are non-cytotoxic.
The present invention also relates to a method for selectively
delivering a non-cytotoxic therapeutic moiety into nerve cells comprising
the steps of:
to delivering to a patient a therapeutic amount of a compound
having the general formula:
B-L-TM
is 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,
TM is a therapeutic moiety which has a non-
2o cytotoxic therapeutic effect when absorbed by a nerve cell, and
L is a linker coupling B to TM;
having the compound selectively bind to a nerve cell surface
receptor via the binding agent B; and
having the compound be absorbed by the nerve cell mediated by
2s the binding of the binding agent B to the nerve cell surface receptor.
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
3o mediated by the binding of the binding agent B to nerve cell surface
receptors. Once internalized, the therapeutic moiety can accumulate
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within the nerve cells where it has a therapeutic efFect. The ability to
selectively deliver the compound to nerve cells reduces the overall
amount of therapeutic moiety which needs to be administered.
Selective delivery of the therapeutic moiety to the nerve cell reduces the
s 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.
to 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
is may be used include proteins, antibiotics, adrenergic agents, adrenergic
agonists, anticonvulsants, nucleotide analogs, anti-trauma agents,
peptides and other classes of agents used to treat or prevent a
neurological disorders. For example, analgesics such as opioids,
codeine and oxycodone can be conjugated to the binding agent B and
2o 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. Steroid hormones such as
corticosteriods can also be conjugated with nerve cell-specific binding
2s 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.
The method according to the present invention can also be used
so to deliver agents that induce the production of nerve growth factor in the
target nerve cells, especially under conditions of pathogenic under-
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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
s Lett. 208: 258-262 (1986)), Schwann cells (Ohi, T. et al., Biochem. Int.
20:739-746 (1990)) with a variety of agents including cytokines, steriods,
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,
io forskolin, fellutamide A, gangliosides and quinone derivatives (Riaz, S.
S. and Tomlinson, D. R. Prog. Neurobiol. 49: 125-143 (1996)).
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
is 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, gancyclovir, and Cifodovir can be conjugated to the binding
2o 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
2s 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 complexes, and other luminophores.
3o The method according to the present invention can also be used
to perForm gene therapy wherein nucleic acids (DNA or RNA) are
Is
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delivered to the nerve cells. These nucleic acids may serve to replace
genes which are either defective, absent or otherwise not properly
expressed by the patient's nerve cell genome.
The above and other features and advantages of the present
s invention will become more apparent in the following description of the
preferred embodiments in greater detail.
1. Binding Agent (B)
to According to the present invention, a compound with a binding
agent B is used to selectively deliver the conjugated therapeutic
moieties TM to nerve cells. At the nerve cell, the binding agent B
interacts with a receptor on the nerve cell and is absorbed by the nerve
cell mediated by this interaction. Any molecules possessing these two
is 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
2o synthetic peptides derived from these peptides or proteins.
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
2s or more of a related family of transmembrane receptors. Table I lists
several members of the neurotrophin family and their cognate
receptors.
As listed in Table 1, nerve growth factor (NGF) is the first
identified and probably the best characterized member of the
3o 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,
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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
s 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
to teleost fish and binds to p75 receptor.
As listed in Table 1 at least two classes of transmembrane
glycoproteins (trk and p75) have been identified which serve as
receptors for neurotrophins. The trk receptors (tyrosine kinase-
containing receptor) bind to neurotrophins with high affinity, whereas the
is p75 receptors possess lower affinity to neurotrophins. For example,
nerve growth factor (NGF) binds to a relatively small number of trkA
receptors with high affinity (Kp = 1 O-") and to more abundant p75 with
lower affinity (Ko = 10-9). The receptor-bound NGF is internalized with
membrane-bound vesicles and retrogradely transported the neuronal
2o cell body. Thus, native neurotropins may serve as the binding agent B
in the compound according the present invention to deliver the
conjugated therapeutic agent TM to the neuronal cell body.
Table 1 The Neurotrophin Family and Its Receptors.
~s
Receptor
Factor Kinase isoforms Nonkinase forms Responsive neurons
(examples)
NGF trkA p75 Cholinergic forebrain
neurons
Sympathetic ganglia
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DRG nociceptive
BDNF trkB p75~NrR Many CNS
populations
trkBT, Vestibular ganglia
trkBT2 Nodose ganglia
DRG
mechanoreceptors
NT-3 trkC p75~NrR Many CNS
populations
trkB and trkA Choclear ganglia
Nonpreferred trkCTK-113 DRG proprioceptive
trkCrTK-108
NT-4 trk B p75 Many CNS
populations
trkBT1 Nodose ganglia
trkBT2 Petrosalganglia
NT-6 trkA p75
2o In addition to the neurotrophins described above, analogs 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
2s 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
3o 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 ~i-hairpin loops was
formed by the NGF 29-35 region. Structure/function relationship studies
of NGF and NGF-related recombinant molecules demonstrated that
3s mutations in NGF region 25-36, along with other ~-hairpin loop and non-
is
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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.
s Chem. 270, 6564-6569, 1995). Dimers of cyclized peptides
corresponding to ~i-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
to also been designed and synthesized to mimic the ~i-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 analogs of neurotrophins that bind to nerve cell surface
is 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.
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,
2s (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
3o intact Mab 5C3. Monoclonal antibody MC192 against p75 receptor has
also been demonstrated to have neurotrophic effects. Therefore, these
19
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antibodies and their functionally equivalent fragments can also 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.
Alternatively, peptidomimetics 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. These synthetic peptide mimics are capable of binding to the
nerve cell surface receptor and being internalized into the cell.
to It is noted that the identification and selection of moieties which
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
is mutagenesis approaches may be used to identify analogs, derivatives
and fragments of known binding moieties which may also be used as
binding moieties according to the present invention.
2. Therapeutic Moiety (TM)
An aspect of the present invention relates to the delivery of
compounds into nerve cells which 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
2s therapy, intracellular target imaging, cell sorting, or separation schemes.
Examples of classes of therapeutic moieties include, but are not limited
to adrenergic agents such as epinephrine, norepinephrine, dopamine,
etenolol; adrenergic agonists such as phenylephrine, isoproterenol, and
noradrenaline, analgesics such as opioids, codeine, oxycodone; anti-
3o trauma agents; anti-viral agents such as acyclovir, gancyclovir, AZT,
ddl, ddC; gene therapy agents such as; steroids such as cortisone,
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progesterone, estrogen; and hormones such as growth factors and
interferons. Such compounds may optionally also include an imaging
moiety, such as fluorescent moieties, for imaging intracellular
components of the nerve cells.
A further aspect of the present invention relates to compositions
and methods for improving the delivery of a therapeutic agent having a
therapeutic activity intracellularly. This is accomplished by using
therapeutic moieties which are charged. Cells have difficulty
transporting charged molecules across cell membranes. According to
to this embodiment, the binding agent B serves to facilitate transport of a
charged therapeutic moiety TM into a cell. Within the cell, the
compound (i.e. the conjugate formed between B and TM) is metabolized
to form a metabolite product that comprises the charged therapeutic
moiety TM. The metabolite product is less prone to being transported
is 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 therapeutic moeity TM 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
2o therapeutic moiety due to the charge which the therapeutic moiety
carries.
According to this embodiment, compounds are provided which
comprise a charged derivative of a therapeutic agent having a
therapeutic activity, the charged derivative being conjugated to a protein
2s having a biological activity of being transported across a cell membrane
into a cell, the cell metabolizing at least a portion of the compound to
form a charged metabolite product that possesses the therapeutic
activity of the therapeutic agent, the charged metabolite product being
less prone to being transported across the cell membrane out of the cell
3o relative to the compound and less prone to being transported across the
cell membrane out of the cell relative to the therapeutic agent.
21
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In one particular embodiment, the charged therapeutic moiety TM
is a quartinery alkyl amine derivative of a therapeutic moiety. A
particular example of a quartinery alkyl amine derivative of a therapeutic
moiety TM is a quartinery alkyl amine of propoxycaine, shown in Table
3.
Also according to this embodiment, methods are provided which
comprise administering a therapeutic agent to a patient in a form where
the therapeutic agent comprises a charge and is conjugated to a protein
having the biological activity of being transported across a cell
io membrane into a cell. Once within the cell, the cell metabolizes at least
a portion of the compound to form a metabolite product that possesses
the therapeutic activity of the therapeutic 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
is compound resulting separation of the therapeutic 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 therapeutic agent.
This method may be used in conjunction with the conjugates of
the present invention for selectively delivering a moiety to nerve cells.
2o However, it is noted that charged therapeutic moieties can be used with
binding agents that target cells other than nerve cells.
3. Linker (L)
2s According to the present invention, a binding agent B is linked to
a therapeutic moiety TM 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.
Many different types of linkers have been developed for cross
30 linking proteins and conjugating proteins or peptides with other agents.
These linkers include zero-length cross linkers, homobifunctional cross-
22
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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
s 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.
In one embodiment, the linker L has one of the following general
to structures:
B-R,-(CO)-IVH -R~-TM
B-Rg-hl H-R4-TM
1$
B-R3-S -R4-TM
B-R5-(CH2)~ -R6 TM
2o Wherein R,, R2, R3, R4, R5, and Rs are independently selected
from the group consisting of alkyls, aryls, heteroaryls, cycloalkyls,
cycloalkenes and heterocycloalkenes.
4. Cleavable Linkers
One particular embodiment of the present invention relates to
compounds which include a cleavable linker L. In some instances, 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
3o desirable to utilize a cleavable linker which allows the therapeutic moiety
TM to be released from the compound once inside the cell.
23
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Many cleavable tinker 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
s hydrolysis of derivatized amino acid side chain, by serum complement-
mediated hydrolysis, and by acid-catalyzed hydrolysis.
Examples of photolabile linkers that may be used include those
linkers described in U.S. Patent Nos. 5,767,288 and No. 4,469,774.
Acid-labile linkers are preferred in the practice of the present
to 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~6.0) by ATP-driven H+ pumps in the endosomal
is membrane that pump H+ 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 therapeutic moiety. Examples of acid labile
linkers which may be used include the cis-aconitic acid, cis-carboxylic
alkatriene, polymaleic anhydride, and other acid labile linkers described
2o in US Patent Nos. 5,563,250 and 5,505, 931.
5. Examples Of Compounds According To The Present
Invention
Table 2 provides several compounds according to the present
2s 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.
24
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TABLE 2
O
N NH O O
'N I N~N~CH2~Ni B
HO~O~ H H
Acyclovir
to 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.
O
s,N ~ NH O H O
O N N~H~(CHZYN~~~N S~ B
HO~ ~ O
O.
Acyclovir
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.
O
H3C
N O
O O
i~N- B
AZT wq-OO
N3 O
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.
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TABLE 2-continued
O
H3C
N O
O O
to AZT °n/-p~~ N- B
Ns ~~O
wherein
B is selected from the group consisting of nerve growth factors NGF, BDNF,
15 NT-3, NT-4, NT-6, anti-neurotrophin receptor antibodies MAb 5C3 and Mab
MC192.
O
N
20 H~ ~ N~ H
H2N N O ~N- B
~O ' ~'O
O
Acyclovir
wherein
25 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.
26
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6. Examples Of Compounds For Treating Pain
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
s described herein may be employed with these therapeutic agents.
Indicated in the table below as * are preferred moieties for attaching
linkers to the therapeutic moieties.
7. Examples Of Linkers
to Table 4 provides a series of linkers for linking different
therapeutic moieties and binding moieties together. As illustrated,
tinkers are provided for attaching moieties which have thiol (-SH),
hydroxyl (-OH), and amino (-NH2) groups to the linkers. In these
examples, neurotrohin is shown as the binding agent. However, it is
is noted that neurotrohin and these examples are intended to be
exemplary only. Other linkers may also be used and are intended as
part of the present invention.
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TABLE 3
Pain - Steroidal anti-inflammatory agents
Betamethasone Dexamethasone
Hz OH
Hz OH
H3 0
H3 0 H ....OH
H ...OH H .,.~CH3
H CHs
/ / F ~H
F fi
0
CuHtsFDs
CxxHxsF~s
Mol. Wt.: 392.47
Mol. Wt.: 392.47
Fluocinolone acetonide
Triamcinolone acetonide *
Hz OH
H3 0 ~CHa
H H ..., ~C~CHa
F Fi
0 /
~xaW FDs DxaHaoFxDs
Mol. Wt,: 434.51 Mol. Wt.: 452.50
Pain - Non-steroidal anti-inflamatory agent
Piroxicam
* H
\ \ /
H
S~N CHa
O"'O
C 15H 13N J 04S
Mol. Wt:331.35
28
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TABLE 3 (cont.)
Pain - Local anesthetic agents
PfOpDXyCaiaO Quaternary Propoxycaine Derivative
~CH3 H3 ~~-CH3
H3
_ CH3
_ ~CHa "
H2
\ ~ 0 0
C~~Hz~NzO=
Cts H zsN z0 z
Mol. Wt.: 293.42
Mol. Wt: 278.39
Pain - Narcotic Agonists
Etorphine Quaternary Etorphine Derivative
-CHa CHa I-J" CHa CHa
Y
H3
4~...
f.
OH ~'~ OH
HO O~' OCH3 HO O OCHa
~ssH~aN~ a C1GH3GN~ a
Mol. Wt.: 411.54 Mol. Wt.: 426.57
Pain - Channel blockers
Gabapentin Carbamazepine
* NH2 OOH
C=O
/ HZ N/
C~H t~NO Z C H N O
is tz a
~Iol. Wt.: 171.24 Mol. Wt.: 236.27
29
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TABLE 3 (cont.)
Anti-neurodegenerative
Tacrine HCl
* Hz ~ HCl
Ct3Ht4N2 HCl
Mol. Wt.: 234.73
Antiviral
Cidofovir
Hz
O
HO y 2H20
Hz
* HD~O
OH
C$H~-0N30~P 2H20
Mol. Wt.: 315.22
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Table 4
H~ di roxyl giroup conjugations
e.g., Steroids, Piroxicam, Acyclovir, Etorphines
PMPI
HS~nrtNJ~(Lys),.nrNeurdraph~
\ ~~O
HO~~ Drug
~O
C~~H~N20~ MoLWt.:214.18 Spacer Arm=8.7d
Amino group conjugations
e.g., Propoxycaines, Gabapentin, Carbemazepine, Tacrine
LC-SPDP
Ne uratroph itw(Lys )x~~~~ ~SH Hz N,v~Drug
N S' ~ Ow
S VV
H
C~gHZ~N~OSSz Mol. Wt.:425.52 Spacer Arm=15.74
Sulfo-LC-SMPT
Hi NM Drug
Newdraphinnrx(Lys~~SH
O
O' ~~O
W
~O Na
S v
C=SH=eN~NaOHS~~o~~t.:G17.G9 Spacer Arm=20.Od
Neurotrophif~'x(Lysp~~~~'NHN'SH
\I ~/
s
HZ N~~ Drug
C~BH~6N=O~ S= Mol. Wt.: 388.dG Spacer Arm =11.8 d
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Table 4 (coat.)
Phospate group conjugate ions
Imidazolide Linker
acyctovir-monophosphate (ACV-MP)
1 " Iw
N /
H ~~ + \ ~ I +
~ ~~ S2
HO- ~~NHz CsH~sN C3H~Nz
C HN S I ~ /
Mol. Wt.: 101.19 1o s x z
D MF Mol. Wt.: 68.08
CRH~xN5O6P ' Mol.Wt.:220.32 CxsH,sF
15 ml 35 NI 84 mg
Mol. Wt.: 305.18 its 0.26 mmol 1.2 mmol 108mg Mol. Wt.: 262.29
25°C 0.49 mmoi
75 mg 129 mg
0.26 mmol 0.49 mmol
pptproductin
157 ml of acetone: Centrifuge 4 min Q 500D rpm
ether:EtxN (66:30:4) Wash and refuge in acetone,
containing 0.1 % NoCI04 then ether. Rotovap dry.
O
NGF dimer
dialysis in 250 ug H
_ H Slide-A-Lyzer 13 hDa
I ~5MWC0 ~19.~2-mol~c ~ I N NHz
~~ 8H
NeurotrophirrLys-NH- - f~NHz o.lml
~H ~ 1X15 min, 0.1 M Cv H~aN~OsF
2X30 min, pH 9.5
PBS butter NaHCO 3 Mol. Wt.: 355.25
7,4 24 hr
37° C
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8. Synthetic Sequence For Attaching Acyclovir To NGF Via
PMPI
Illustrated below is a synthetic sequence for the attachment of
acyclovir to NGF via the linker PMPI.
PMPI
Acyclovir
H,
+ ~I H
H ~~NH2
C11 H6N20~ C, H7N0
Mol. W t.: 214.18 Mol. Wt: 73.09 CsH 11 N 50~
50 mg Mol. Wt.: 225.21
0..223 mmoles NGF dimer, 250 ug,
40 mg 13 kDa, 19.2 nmoles
AcyclovirvN-(p-Maleimidophenylcarbamate)(ACV-PMPC) 0.1 Smmol H,~(Lys)"w~NGF
Traut's Reagent
0.1 ml, TEA Buffer pH 8.0
w
" , I H ~ 2 hrs @ R.T.
HS~~(Lys), wNGF
\ N"x dialysis in ido-A-Lyzer 3.5 MWCO
1X15 min, 2X30 min,
PBS buffer, '~Ii 7.4
+ H S~nnt"J~vr~(Lys), wNG F
C H N O ~ 0.1 ml, PBS Buffer pH 7A
19 17 7 6
2 hrs @ R.T.
Mal. Wt.: 439.38
H H
"' L I
I~HI~~(Lys), wNG F
f
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9. Synthetic Sequence For Attaching
Acyclovir To NGF Via Imidazole
Illustrated below is a synthetic sequence for the attachment of
s acyclovir to NGF via an imidazole linker.
H acyclovir
\
~2.3
\ + '
I-~ 1 I ~ NH
CI
CI + I-I~~H + HsC-CN I ~~
HO--1 ~ N"NH
POCK Hx0 CsHsN CsH~N ~ x
Mol. Mol. Mol. Mol
Wt.: Wt.: Wt.: : 41.05
153.33 18.02 79.10 Wt
. CftH11N5~7
.
200 20 200 W 1.0 ml
W W : 225
21
Mol
Wt
2.1 1.1 2.3 mmol0 C .
mmol mmol .
.
stir 2
hours
0
acyclovir-monophosphate added 100 mg
(ACV-MP) ice to
10 ml
stir 0.5 O.d4 mmol
hour
adjust Rotovnp dry.
to pH Redissolve in
2.35 n minimum
O-P ~ filter vof HxO. Adjust
through to pH 4.5. Apply
tol ml
v ' column of Bio-Rad
H AGl-X8 resin,
2.4 I ~~ lOg Charcoal:
O ~ 2g Cclite
~ Wash with
50 ml
H x O
formate
salt.,
~ 0.5
gram.
Wash
column
- ~ l with 5 mL 0.1,
HO- w ith GO 1 and 2 M formic
NHx El acid.
t t
I m 1M fraction contains
6 u ACV-MP.
e w
OH
0-NH
EtOH
H
' a
H -
x
0~~7.7 ~H (10:9:1)
N (30:27:3)
O
P
Mol. Wt.: 305.18
acyclovir-monophosphate (ACV-MP) \
H I/
1.~+C~+ I /I +
[JH
0 ~~ I ~,~~ N Sx \ \
w
HO-~- N~NHx CsH~sN CaHaNx /
bH C,°HaNx~x
DMF Mol. Wt.: 101.19 Mol. Wt.: 68.08
CsH,ZN506P > Mol. Wt.: 220.32 C~sH~sP
15 ml 35 W 84 mg
Mol. Wt.: 305.15 11~ 0.26 mmol 1.2 mmol lOBmg Mol. Wt.: 262.29
25°C 0.49 mmol
75 mg 129 mg
0.26 mmol 0.49 mmol
pptproductin
157 ml of acetone: Centrifuge 4 min Q 5000 rpm
ether:Et~N (66:30:4) Wash and refuge in acetone,
containing 0.1 % NaCI04v then ether. Rotovap dry.
O NGF dime 'r
dialysis in 250ug ~ ~NH
Slide-A-Lyzer 13 kDa , I ~
H ~,~r~~t9.2nmoles ~N_ -~ ~ N' _NHx
~~ OH
v
NGFLys-NH-~- t~NHx 0.1 ml
~H ~ 1X15 min, 0.1 M C,~H,.,N.,OSP
2X30 min, pH 9.5
PBS buffer NaHCOs Mol. Wt.: 355.25
7.4 24 hr
37° C
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10. Examples of Human Neurotrophins as the Binding Agent (B)
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
s the present invention. Lysine residues that may be used to attach to the
linker (L) which in turn is conjugated with the therapeutic moiety (TM)
are highlighted and underlined in Table 5.
Table 5. Sec~uenees 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
BRAIN DERIVED NEUROTROPHIC FACTOR (BDNF) [SEQ ID N0: 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 LY GLN TYR
PHE TYR GLU THR LYS CYS ASN PRO MET GLY TYR THR LYS
GLU GLY CYS ARG GLY ILE ASP LY ARG HIS TRP ASN SER
GLN CYS ARG THR THR GLN SER TYR VAL ARG ALA LEU THR
MET ASP SER LYS LY ARG ILE GLY TRP ARG PHE ILE ARG
ILE ASP THR SER CYS VAL CYS THR LEU THR ILE LYE. ARG
119 GLY ARG
NEUROTROPHIN-3 (NT-3) [SEQ ID N0: 3]:
TYR ALA GLU HIS LY SER HIS ARG GLY GLU TYR SER VAL
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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 LY 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
NEUROTROPIiIN-4 (NT-4) [SEQ ID NO: 4]:
1 GLY VAL SER GLU THR ALA PRO ALA SER ARG GLU
ARG GLY
LEU ALA VAL CYS ASP ALA VAL SER GLY VAL ASP
TRP THR
ARG ARG THR ALA VAL ASP LEU ARG GLY GLU GLU
ARG VAL
VAL LEU GLY GLU VAL PRO ALA ALA GLY SER LEU
GLY PRO
AR.G GLN TYR PHE PHE GLU THR ARG CYS ALA ASN
LY ASP
ALA GLU GLU GLY GLY PRO GLY ALA GLY GLY CYS
GLY GLY
Y VAL ASP ARG ARG HIS TRP VAL GLU LYS
SER CYS
ARG GL
ALA LY GLN SER TYR VAL ARG ALA LEU ALA ALA
THR ASP
GLN GLY ARG VAL GLY TRP ARG TRP ILE ILE THR
ARG ASP
130 ALA CYS VAL CYS THR LEU LEU SER ARG THR GLY ARG ALA
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11. Methods For Using Compounds Of The Present Invention
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
s applications.
a. Pharmaceutical Formulations Utilizing
Compositions Of The Present Invention
io 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. Other pharmaceutically suitable formulations
is 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.
b. Routes of Administration
The compounds according to the present invention can be
administered orally, by subcutaneous or other injection, intravenously,
intracerebrally, intramuscularly, parenternally, transdermally, nasally or
rectally. The form in which the compound is administered depends at
2s least in part on the route by which the compound is administered.
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
3o than limiting sense, as it is contemplated that modifications will readily
occur to those skilled in the art, which modifications will be within the
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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.
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SEQUENCE LISTING
<110> Hill, Craig
Kahl, Steve
Webb, Robert R.
McKee, Constance A.
<120> COMPOUNDS FOR INTRACELLULAR DELIVERY OF THERAPUETIC MOIETIE
S TO NERVE CELLS
<130> 16778-709
<150> US 09/217,037
<151> 1998-12-21
<150> US 09/707,730
<151> 2000-11-06
<160> 4
<170> PatentIn version 3.1
<210> 1
<211> 120
<212> PRT
<213> Homo sapiens
<400> 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
Page 1
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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
<210> 2
<211> 119
<212> PRT
<213> Homo sapiens
<400> 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 Gly 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
g5 90 95
Arg Ile Gly Trp Arg Phe Ile Arg Ile Asp Thr Ser Cys Val Cys Thr
100 105 110
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Leu Thr Ile Lys Arg Gly Arg
115
<210> 3
<211> 119
<212> PRT
<213> Homo Sapiens
<400> 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
<210> 4
<211> 130
<212> PRT
<213> Homo Sapiens
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<400> 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
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