CA2436830A1 - Intracellular delivery of charged therapeutic moieties to nerve cells by means of targeting proteins - Google Patents
Intracellular delivery of charged therapeutic moieties to nerve cells by means of targeting proteins Download PDFInfo
- Publication number
- CA2436830A1 CA2436830A1 CA002436830A CA2436830A CA2436830A1 CA 2436830 A1 CA2436830 A1 CA 2436830A1 CA 002436830 A CA002436830 A CA 002436830A CA 2436830 A CA2436830 A CA 2436830A CA 2436830 A1 CA2436830 A1 CA 2436830A1
- Authority
- CA
- Canada
- Prior art keywords
- cell
- protein
- therapeutic agent
- nerve
- compound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/04—Centrally acting analgesics, e.g. opioids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/06—Antimigraine agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/08—Antiepileptics; Anticonvulsants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
Abstract
A method for improving intercellular administration of a therapeutic agent is provided comprising: contacting cells with a compound comprising a charged derivative of a therapeutic agent, the charged derivative being conjugated to a protein having a biological activity of being transported across a cell membrane into a cell; and having the transport the compound into the cell, where the cell metabolizes 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 relative to the compound and less prone to being transported across the cell membrane out of the cell relative to the therapeutic agent. For example, the target cells are nerve cells and the protein is selected from nerve growth factors, antibodies to nerve cell surface receptors, and members of the nerve growth factor family (e.g. MGF, BDMF, MT-4, NT-6).
Description
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 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.
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 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 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
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 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
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
4 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 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;
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 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:
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 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 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 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.
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 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-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 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, 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 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 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 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, 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.
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-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-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.
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.
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 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;
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 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:
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 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 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 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.
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 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-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 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, 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 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 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 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, 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.
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-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-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.
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.
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
N O
O O
i~N- B
AZT wq-OO
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.
TABLE 2-continued O
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.
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
N O
O O
i~N- B
AZT wq-OO
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.
TABLE 2-continued O
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.
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.
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.
Pain - Steroidal anti-inflammatory agents Betamethasone Dexamethasone Hz OH
Hz OH
H3 0 H ....OH
H ...OH H .,.~CH3 H CHs / / F ~H
F fi 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 ~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
Mol. Wt:331.35 TABLE 3 (cont.) Pain - Local anesthetic agents PfOpDXyCaiaO Quaternary Propoxycaine Derivative ~CH3 H3 ~~-CH3 _ CH3 _ ~CHa "
\ ~ 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
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 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 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 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
Pain - Steroidal anti-inflammatory agents Betamethasone Dexamethasone Hz OH
Hz OH
H3 0 H ....OH
H ...OH H .,.~CH3 H CHs / / F ~H
F fi 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 ~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
Mol. Wt:331.35 TABLE 3 (cont.) Pain - Local anesthetic agents PfOpDXyCaiaO Quaternary Propoxycaine Derivative ~CH3 H3 ~~-CH3 _ CH3 _ ~CHa "
\ ~ 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
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 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 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 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
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
2 hrs @ R.T.
Mal. Wt.: 439.38 H H
"' L I
I~HI~~(Lys), wNG F
f
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
2 hrs @ R.T.
Mal. Wt.: 439.38 H H
"' L I
I~HI~~(Lys), wNG F
f
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 Mol Wt 2.1 1.1 2.3 mmol0 C .
mmol mmol .
.
stir 2 hours acyclovir-monophosphate added 100 mg (ACV-MP) ice to
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 Mol Wt 2.1 1.1 2.3 mmol0 C .
mmol mmol .
.
stir 2 hours 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
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
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]:
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
BRAIN DERIVED NEUROTROPHIC FACTOR (BDNF) [SEQ ID N0: 2]:
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
NEUROTROPHIN-3 (NT-3) [SEQ ID N0: 3]:
TYR ALA GLU HIS LY 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 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
NEUROTROPIiIN-4 (NT-4) [SEQ ID NO: 4]:
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
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
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
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]:
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
BRAIN DERIVED NEUROTROPHIC FACTOR (BDNF) [SEQ ID N0: 2]:
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
NEUROTROPHIN-3 (NT-3) [SEQ ID N0: 3]:
TYR ALA GLU HIS LY 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 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
NEUROTROPIiIN-4 (NT-4) [SEQ ID NO: 4]:
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
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 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 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 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 Arg Arg Ala <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 Ser Glu Trp Val Thr Ala Ala Asp Lys Lys Thr Ala Val Asp Met Ser Gly Gly Thr Val Thr Val Leu Gly Lys Val Pro Val Ser Lys Gly Gln 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 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 Leu Thr Ile Lys Arg Gly Arg <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 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 Arg Thr <210> 4 <211> 130 <212> PRT
<213> Homo Sapiens <400> 4 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 Ala Cys Val Cys Thr Leu Leu Ser Arg Thr Gly Arg Ala
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 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 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 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 Arg Arg Ala <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 Ser Glu Trp Val Thr Ala Ala Asp Lys Lys Thr Ala Val Asp Met Ser Gly Gly Thr Val Thr Val Leu Gly Lys Val Pro Val Ser Lys Gly Gln 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 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 Leu Thr Ile Lys Arg Gly Arg <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 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 Arg Thr <210> 4 <211> 130 <212> PRT
<213> Homo Sapiens <400> 4 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 Ala Cys Val Cys Thr Leu Leu Ser Arg Thr Gly Arg Ala
Claims (24)
1. A method for improving intracellular administration of a therapeutic agent comprising:
contacting cells with a compound comprising 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 into a cell; and having the cell transport the compound into the cell where the cell metabolizes 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 relative to the compound and less prone to being transported across the cell membrane out of the cell relative to the therapeutic agent.
contacting cells with a compound comprising 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 into a cell; and having the cell transport the compound into the cell where the cell metabolizes 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 relative to the compound and less prone to being transported across the cell membrane out of the cell relative to the therapeutic agent.
2. A method according to claim 1 wherein the charged derivative of the therapeutic agent is a quartinery alkyl amine.
3. A method according to claim 1 wherein the therapeutic agent is propoxycaine and the charged derivative of the therapeutic agent is a quartinery alkyl amine of propoxycaine.
4. A method according to claim 1 wherein the therapeutic agent is propoxycaine and the charged derivative of the therapeutic agent is a quartinery alkyl amine of etorphine.
5. A method according to claim 1 wherein the protein is selected from the group consisting of a nucleic acid sequence, a peptide, a peptidomimetic, an antibody and an antibody fragment.
6. A method according to claim 1 wherein protein is selected from the group consisting of nerve growth factors and analogs, derivatives and fragments of nerve growth factors.
7. A method according to claim 1 wherein the protein is selected from the group consisting of antibodies and antibody fragments that selectively bind to nerve cell surface receptors.
5. A method according to claim 1 wherein the protein is a DNA or RNA ligand that functions as an antagonist of nerve growth factors or inhibits binding of other growth factors to nerve cell surface receptors.
9. A method according to claim 1 wherein the protein is a synthetic peptide that binds to nerve cell surface receptors and has agonist or antagonist activity of nerve growth factors.
10. A method according to claim 1 wherein the protein is selected from the group consisting of anti-human trkA monoclonal antibody 5C3 and anti-human p75 monoclonal antibody MC192.
11. A method according to claim 1 wherein the protein is selected from the group consisting of NGF, BDNF, NT-3, NT-4, and NT-6.
12. A method according to claim 1 wherein the protein is selected from the group consisting of NGF, a fragment or derivative of NGF, a protein capable of competing with NGF for binding to a NGF receptor, BDNF, a fragment or derivative of BDNF, and a protein capable of competing with BDNF for binding to a BDNF receptor.
13. A compound comprising:
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 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 relative to the compound and less prone to being transported across the cell membrane out of the cell relative to the therapeutic agent.
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 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 relative to the compound and less prone to being transported across the cell membrane out of the cell relative to the therapeutic agent.
14. A compound according to claim 13 wherein the charged derivative of the therapeutic agent is a quartinery alkyl amine.
15. A compound according to claim 13 wherein the therapeutic agent is propoxycaine and the charged derivative of the therapeutic agent is a quartinery alkyl amine of propoxycaine.
16. A compound according to claim 13 wherein the therapeutic agent is propoxycaine and the charged derivative of the therapeutic agent is a quartinery alkyl amine of etorphine.
17. A compound according to claim 13 wherein the protein is selected from the group consisting of a nucleic acid sequence, a peptide, a peptidomimetic, an antibody and an antibody fragment.
18. A compound according to claim 13 wherein protein is selected from the group consisting of nerve growth factors and analogs, derivatives and fragments of nerve growth factors.
19. A compound according to claim 13 wherein the protein is selected from the group consisting of antibodies and antibody fragments that selectively bind to nerve cell surface receptors.
20. A compound according to claim 13 wherein the protein is a DNA
or RNA ligand that functions as an antagonist of nerve growth factors or inhibits binding of other growth factors to nerve cell surface receptors.
or RNA ligand that functions as an antagonist of nerve growth factors or inhibits binding of other growth factors to nerve cell surface receptors.
21. A compound according to claim 13 wherein the protein is a synthetic peptide that binds to nerve cell surface receptors and has agonist or antagonist activity of nerve growth factors.
22. A compound according to claim 13 wherein the protein is selected from the group consisting of anti-human trkA monoclonal antibody 5C3 and anti-human p75 monoclonal antibody MC192.
23. A compound according to claim 13 wherein the protein is selected from the group consisting of NGF, BDNF, NT-3, NT-4, and NT-6.
24. A compound according to claim 13 wherein the protein is selected from the group consisting of NGF, a fragment or derivative of NGF, a protein capable of competing with NGF for binding to a NGF
receptor, BDNF, a fragment or derivative of BDNF, and a protein capable of competing with BDNF for binding to a BDNF receptor.
receptor, BDNF, a fragment or derivative of BDNF, and a protein capable of competing with BDNF for binding to a BDNF receptor.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US09/707,730 US6887861B1 (en) | 1998-12-21 | 2000-11-06 | Compounds for intracellular delivery of therapeutic moieties to nerve cells |
| US09/707,730 | 2000-11-06 | ||
| PCT/US2001/051151 WO2002047730A2 (en) | 2000-11-06 | 2001-11-02 | Intracellular delivery of charged therapeutic moieties to nerve cells by means of targeting proteins |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2436830A1 true CA2436830A1 (en) | 2002-06-20 |
Family
ID=24842931
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002436830A Abandoned CA2436830A1 (en) | 2000-11-06 | 2001-11-02 | Intracellular delivery of charged therapeutic moieties to nerve cells by means of targeting proteins |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP1349577A2 (en) |
| JP (1) | JP2005503994A (en) |
| AU (1) | AU2002243422A1 (en) |
| CA (1) | CA2436830A1 (en) |
| WO (1) | WO2002047730A2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1205191A1 (en) * | 2000-11-13 | 2002-05-15 | Centre National De La Recherche Scientifique | Targeted modification of intracellular compounds |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5087618A (en) * | 1982-05-18 | 1992-02-11 | University Of Florida | Redox carriers for brain-specific drug delivery |
| WO1995007092A1 (en) * | 1993-09-10 | 1995-03-16 | The University Of Medicine And Dentistry Of New Jersey | Blood-brain barrier transporters of neurological agents |
| US5994317A (en) * | 1996-04-09 | 1999-11-30 | Vical Incorporated | Quaternary cytofectins |
| US6652864B1 (en) * | 1998-12-21 | 2003-11-25 | Asilomar Pharmaceuticals, Inc. | Compounds for intracellular delivery of therapeutic moieties to nerve cells |
| WO2001091798A2 (en) * | 2000-06-01 | 2001-12-06 | Universite Catholique De Louvain | Tumor activated prodrug compounds |
-
2001
- 2001-11-02 CA CA002436830A patent/CA2436830A1/en not_active Abandoned
- 2001-11-02 JP JP2002549299A patent/JP2005503994A/en active Pending
- 2001-11-02 WO PCT/US2001/051151 patent/WO2002047730A2/en active Application Filing
- 2001-11-02 AU AU2002243422A patent/AU2002243422A1/en not_active Abandoned
- 2001-11-02 EP EP01989317A patent/EP1349577A2/en not_active Withdrawn
Also Published As
| Publication number | Publication date |
|---|---|
| WO2002047730A2 (en) | 2002-06-20 |
| WO2002047730A3 (en) | 2003-07-31 |
| WO2002047730A8 (en) | 2003-02-20 |
| AU2002243422A1 (en) | 2002-06-24 |
| JP2005503994A (en) | 2005-02-10 |
| EP1349577A2 (en) | 2003-10-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US7718605B2 (en) | Compounds for intracellular delivery of therapeutic moieties to nerve cells | |
| US8232251B2 (en) | Compounds for delivery of therapeutic and imaging moieties to nerve cells | |
| KR100818038B1 (en) | Nanoparticles made of protein with coupled apolipoprotein e for penetration of the blood-brain barrier and methods for the production thereof | |
| US5670477A (en) | Method to enhance permeability of the blood/brain blood/nerve bariers to therapeutic agents | |
| US6881829B2 (en) | Chimeric hybrid analgesics | |
| DK2360258T3 (en) | Aprotininpolypeptider to transport a compound of blood-brain-barrier | |
| JP2000509394A (en) | Polypeptide conjugates for transporting substances across cell membranes | |
| Friden | Receptor-mediated transport of therapeutics across the blood-brain barrier | |
| EP4079328A1 (en) | Cancer immunotherapeutic | |
| US5456908A (en) | Polyamine-polyamine and polyamine-protein transport inhibitor conjugates and their use as pharmaceuticals and in research relating to polyamine transport | |
| CA2436830A1 (en) | Intracellular delivery of charged therapeutic moieties to nerve cells by means of targeting proteins | |
| US7368431B2 (en) | Polypeptide, the conjugate thereof with doxorubicine and a pharmaceutical composition based thereon | |
| Sheehan et al. | A horseradish peroxidase-light and electron microscopic study of immunoliposomes utilized for intracellular delivery to the rat striatum | |
| Patel et al. | Role of Transferrin Receptors In Brain Targeting Delivery | |
| WO2020232638A1 (en) | Antibody conjugate and application of pharmaceutical composition thereof | |
| Smith | Identification and characterization of the neurotrophic properties of dopamine-releasing protein (DARP) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Dead |