Neurotrophin receptor-binding conjugate composition, and method of use and preparation thereof.

Abstract

Neurotrophin receptor-binding conjugate compositions for delivering active agents to neuronal cells are provided. Conjugate compositions of the present disclosure, according to certain embodiments, comprise one or more active agent compounds (e.g., dyes or small molecule therapeutic agents) covalently attached to a protein, peptide, or peptidomimetic that selectively binds to a neurotrophin receptor. In composition embodiments, the average ratio of active agent compound to protein, peptide, or peptidomimetic in the conjugate is 5 or less. Dual-color imaging compositions are also described. Methods for selectively delivering active agent conjugates to neuronal cells (e.g., intraocular delivery) are provided. Methods for making compositions comprising neurotrophin receptor-binding conjugates are also included.

Description

Technical Field

[0001] Provided is a neurotrophin receptor-binding conjugate composition for delivering an active agent to nerve cells.

Background Art

[0002] Diseases of the peripheral nervous system (PNS) or central nervous system (CNS) can be caused by genetic defects in either the known low-affinity (p75 neurotrophin receptor, p75NTR) or high-affinity (tropomyosin kinase, Trk) receptors, or by chemical exposures such as chemotherapy, or trauma such as blast-induced eye injury or neurodegeneration due to optic nerve injury. Also, neurotrophins (NTs) can function outside the nervous system. Many cells of the immune system and glia express receptors of the Trk family, including TrkA, a high-affinity receptor that binds the NT nerve growth factor (NGF).

[0003] The effectiveness of NGF as a drug for treating neurotrophic keratitis is consistent with evidence showing that NGF promotes survival and acts as an immunotrophin in "complex bidirectional" functions, probably because the TrkA and TrkB receptors, which are high-affinity receptors that bind brain-derived neurotrophic factor (BDNF), are widely expressed throughout the immune system. This bidirectional interaction can explain new evidence that NGF plays a role in cross-talk between the neural, immune, and endocrine systems and also plays a complex role in modulating certain cancers. In cancer, several signaling pathways, including the Ras / MAPK pathway, PLCγ1 pathway, PI3K / Akt-mTOR pathway, and p75NTR-mediated signaling pathway, have been confirmed to be involved in many NT functions, and intervention in one or more of those pathways is thought to be able to improve the treatment of cancers of the nervous system and other organs.

Summary of the Invention

[0004] Aspects of this disclosure include neurotrophin receptor-binding conjugate compositions for delivering activators to nerve cells. In certain embodiments, the methods and compositions described herein include neurotrophin receptor-binding conjugate compounds that bind to high-affinity receptors. In some cases, the receptors may be physiologically and / or pathologically impaired. In some embodiments, the neurotrophin receptor-binding conjugate compound binds to Trk receptors, e.g., TrkA receptors. In some embodiments, the neurotrophin receptor-binding conjugate compound binds to Trk receptors in a manner sufficient to promote endocytosis. In some embodiments, the binding of the neurotrophin receptor-binding compound is sufficient to promote retrograde axonal transport of the dye or small molecule of the neurotrophin receptor-binding compound. In some embodiments, the binding of the neurotrophin receptor-binding compound is sufficient to promote anterograde axonal transport of the dye or small molecule of the neurotrophin receptor-binding compound. In some embodiments, the binding of the neurotrophin receptor-binding compound is sufficient to promote intracellular uptake of the dye or small molecule, e.g., uptake of the dye or small molecule into the nerve cell body. In some embodiments, the binding of the neurotrophin receptor conjugate compound is sufficient to promote the intracellular transport of the dye or small molecule agent. In some embodiments, the binding of the neurotrophin receptor conjugate compound is sufficient to promote the semi-intracellular transport of the dye or small molecule agent. In some embodiments, the semi-intracellular transport of the dye or small molecule agent includes transport along the myelin nerve sheath. In some embodiments, uptake into the nerve cell body is sufficient for the dye or small molecule agent to act intracellularly, for example, the dye to fluoresce from the nerve cell body, or the small molecule agent to exhibit pharmacological activity.In certain embodiments, the methods and compositions described herein include neurotrophin receptor conjugate compounds exhibiting one or more of the following: 1) binding to a receptor (e.g., TrkA receptor), 2) retrograde or anterograde axonal transport of the dye or small molecule of the neurotrophin receptor conjugate compound, and 3) uptake of the dye or small molecule of the neurotrophin receptor conjugate compound into the nerve cell body, or semineuronal transport of the dye or small molecule, for example, transport along the myelin nerve sheath.

[0005] A neurotrophin receptor-binding conjugate composition according to a particular embodiment comprises one or more activating compounds (e.g., dyes or small molecule therapeutics) covalently bound to a protein, peptide, or peptide mimetic that selectively binds to a neurotrophin receptor. In embodiments of the subject composition, the average ratio of the activating compound to the protein, peptide, or peptide mimetic in the conjugate is 5 or less. In some embodiments, the average ratio of the activating compound to the protein, peptide, or peptide mimetic in the conjugate is 3.2 or less. In a particular embodiment, the average ratio of the activating component to the protein, peptide, or peptide mimetic component is 2 or less, for example, about 0.95 to about 1.85. In some cases, 90% or more (e.g., 95% or more) of the activator conjugates in the composition have an average ratio of 5 or less between the activator component and the protein, peptide, or peptide mimetic component. For example, 90% or more (e.g., 95% or more) of the activator conjugates in the composition have an average ratio of 3.2 or less between the activator component and the protein, peptide, or peptide mimetic component. For example, 90% or more (e.g., 95% or more) of the activator conjugates in the composition have an average ratio of 2 or less between the activator component and the protein, peptide, or peptide mimetic component.

[0006] The embodiments also include methods for intraocular delivery of activators to nerve cells. Methods according to certain embodiments include contacting a subject's eye with a composition comprising an activator conjugate, the activator conjugate comprising one or more activator compounds covalently bound to a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors. The conjugate compositions of the Disclosure are contacted to the surface of the eye (e.g., topically applied to the corneal surface), or injected into the vitreous fluid or implanted into the eye (wherein injection or implantation is described as intravitreal or intraocular administration). Contact lenses and intravitreal implants containing the compositions of the Subject are also described. The compositions of the Subject are also intended to be chimeric proteins of components of NGF that bind to TrkA and components of brain-derived neurotrophic factor (BDNF) that bind to TrkB, and these chimeras are used for topical application to the eye to promote both transocular transport and binding to TrkB receptors expressed on the posterior side of the vitreous humor on retinal ganglion cells.

[0007] Aspects of the present disclosure further include compositions having two or more detectable label-biomolecular conjugates. A composition of the present disclosure in a particular embodiment includes a detectable label-biomolecular conjugate having a first detectable label covalently attached to a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors typically expressed on nerve tissue, and a detectable label-biomolecular conjugate having a second detectable label covalently attached to a biopolymer that selectively binds to neoplastic tissue. In a particular embodiment, the composition of the subject includes a nerve imaging conjugate configured to image or structurally visualize nerves and / or neurites, and a tumor imaging conjugate configured to image tumors. In a particular case, the composition of the subject is used during surgery (e.g., resection), and the nerve imaging conjugate and tumor imaging conjugate are administered to the subject simultaneously and can be used as a visual aid during surgery, thereby providing oncologists with a visual surgical aid to identify nerves and individual cancer cells.

[0008] In embodiments, the protein, peptide, or peptide mimetic component of the conjugate in the subject composition may vary and include, but are not limited to, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), neurotrophic factor 4 / 5 (NT-4 / 5), neurotrophic factor 6 (NT-6), neurotrophic factor 7 (NT-7), glial cell-derived neurotrophic factor (GDNF), ciliary body neurotrophic factor (CNTF), and derivatives or fragments thereof. In some embodiments, the protein, peptide, or peptide mimetic component of the subject conjugate is a mammalian protein, such as mammalian nerve growth factor, mammalian brain-derived neurotrophic factor, or mammalian neurotrophic factor. In certain cases, the protein, peptide, or peptide mimetic component of the subject conjugate is not a bacterial protein, such as Escherichia coli (E. coli) nerve growth factor, E. coli brain-derived neurotrophic factor, or E. coli neurotrophic factor. In certain embodiments, the conjugate in question comprises a combination of two or more protein, peptide, or peptide mimetic components, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), neurotrophic factor 4 / 5 (NT-4 / 5), neurotrophic factor 6 (NT-6), neurotrophic factor 7 (NT-7), glial cell-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and two or more components selected from derivatives or fragments thereof. In some embodiments, the composition in question comprises a combination of two or more different conjugates, where the dye fluoresces in a different spectrum from the neuro-specific dye NT conjugate, and the cancer-specific dye conjugate binds to specific non-Trk receptors using non-neurotrophins. In some embodiments, the protein, peptide, or peptide mimetic is a neurotrophic factor derived from a non-human animal. In other embodiments, the protein, peptide, or peptide mimetic is a recombinant human neurotrophic factor. In yet another embodiment, the protein, peptide, or peptide mimetic is a protein subunit, such as the β-subunit of NGF, rhNGF, or rhBDNF, or a chimeric combination of one or more NTs.In some cases, the protein, peptide, or peptide mimetic is a natural neurotrophic factor (e.g., natural NGF, natural BDNF). In certain cases, the natural neurotrophic factor does not include an additional peptide conjugate functionalized for conjugation to a payload compound (e.g., a dye or small molecule activator). As described in more detail below, the neurotrophin-binding conjugate compounds of the present disclosure in certain embodiments include natural nerve growth factor (NGF), natural brain-derived neurotrophic factor (BDNF), or natural neurotrophic factor conjugated to an activator compound by one or more amino acid residues (e.g., lysine residues) present in the natural neurotrophic factor. In other words, according to these embodiments, the neurotrophin-binding component does not include a non-natural or additional peptide sequence. In some cases, the protein, peptide, or peptide mimetic component of the subject conjugate does not include an additional C-terminal peptide sequence for conjugation to a payload, such as a dye or activator payload. In other cases, the subject conjugate protein, peptide, or peptide mimetic component does not include an additional N-terminal peptide sequence for conjugation to, for example, a dye or activator payload.

[0009] In certain embodiments, the protein, peptide, or peptide mimetic is a neurotrophic factor that binds to tropomyosin kinase A (TrkA). In certain embodiments, the protein, peptide, or peptide mimetic is a neurotrophic factor that exerts retrograde axonal transport of a payload compound (e.g., a dye or small molecule agent) conjugated to the protein, peptide, or peptide mimetic. In certain embodiments, the protein, peptide, or peptide mimetic is a neurotrophic factor that exerts intracellular uptake of a payload compound (e.g., a dye or small molecule agent) conjugated to the protein, peptide, or peptide mimetic into, for example, the nerve cell body, axon, or dendrite.

[0010] In certain cases, the activating component of the conjugate in the subject composition is a detectable label. Each detectable label may be a compound such as a fluorophore, chromophore, enzyme, redox label, radioactive label, sound-absorbing label, Raman (SERS) tag, mass tag, isotope tag, magnetic particle, fine particle, and nanoparticle. In some embodiments, each detectable label in the subject composition is, in certain cases, a dye, such as an organic dye or an inorganic dye. In certain cases, the dye is a luminescent dye, such as a fluorescent dye having an emission wavelength of 300 nm or more. Examples of dyes that can be used include, but are not limited to, body pie dyes, coumarin dyes, rhodamine dyes, acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes, chlorophyll-containing dyes, triarylmethane dyes, azo dyes, diazonium dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone-imine dyes, azine dyes, eurodin dyes, safranin dyes, indamine, indophenol dyes, fluorine dyes, oxazine dyes, oxazone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronin dyes, fluorine dyes, rhodamine dyes, phenanthoridine dyes, squalane, body pie, squaline oxytan, naphthalene, coumarin, oxadiazole, anthracene, pyrene, acridine, arylmethine or tetrapyrrole, and combinations thereof.In certain embodiments, the conjugate may contain two or more dyes selected from, for example, body pie dyes, coumarin dyes, rhodamine dyes, acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes, chlorophyll-containing dyes, triarylmethane dyes, azo dyes, diazonium dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone-imine dyes, azine dyes, eurodin dyes, safranin dyes, indamine, indophenol dyes, fluorine dyes, oxazine dyes, oxazone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronin dyes, fluorine dyes, rhodamine dyes, phenanthoridine dyes, squalane, body pie, squaline roxitan, naphthalene, coumarin, oxadiazole, anthracene, pyrene, acridine, arylmethine or tetrapyrrole, and combinations thereof. In some embodiments, the conjugate in the composition in question has an activating component containing the same or substantially the same dye as Alexa Fluor dyes, e.g., the same or substantially the same dye as Alexa Fluor 488 dye, or the same or substantially the same dye as Alexa Fluor 680 dye, or the same or substantially the same dye as Alexa Fluor 780 dye. In other embodiments, the conjugate in the composition in question has an activating component containing the same or substantially the same dye as Dyomics dyes, e.g., the same or substantially the same dye as Dyomics DY-800 dye. In yet another embodiment, the conjugate in the composition in question has an activating component containing the same or substantially the same dye as Curadel dyes, e.g., the same or substantially the same dye as ZW-800 dye. In some embodiments, the dye is the same or substantially the same Cy dye, e.g., Cy 7.5 or Cy 5.5. In certain cases, the dye is a compound having axial or plane symmetry.In some embodiments, the dye is a compound that facilitates the intracellular visualization of the dye (e.g., by spectrometer or with the naked eye), and intracellular visualization may include well-established axonal transport modes of one or both NT-Trk receptor vesicles in microtubules and / or NT-Trk receptors expressed in the myelin sheath. In some embodiments, axonal transport includes retrograde axonal transport. In some embodiments, axonal transport includes anterograde axonal transport. In these embodiments, the conjugate in question facilitates binding by neurons or components thereof, endocytosis, and intracellular transport (e.g., retrograde or anterograde axonal transport).

[0011] In other embodiments, the conjugate activating agent component in the subject composition is a small molecule therapeutic activator that acts intracellularly after the synthesis operation, after Trk receptor binding and endocytosis, and after intracellular or retrograde / anterograde axonal transport. In some cases, the small molecule therapeutic agent is an anticancer agent for the treatment of cancers of the central nervous system, including, for example, cancers selected from the group consisting of adult and pediatric gliomas, visual pathway gliomas, spinal cord tumors, neurofibromas, schwannomas, malignant peripheral schwannomas, malignant schwannomas, neurofibrosarcomas, and neurosarcomas. In some cases, the subject is diagnosed with a glioma (mild, severe, etc.). In some embodiments, the small molecule therapeutic agent is a mammalian target of a rapamycin (mTOR) inhibitor or a mitogen-activated protein kinase (MEK) inhibitor. In other embodiments, the small molecule therapeutic agent is a compound selected from glucocorticoids, heat shock protein inhibitors, checkpoint inhibitors, and / or chemokine / chemokine ligand inhibitors. For example, the glucocorticoid may be fluocinolone acetonide.

[0012] In some embodiments, biopolymers that selectively bind to neoplastic tissue are provided. The biopolymer that selectively binds to neoplastic tissue may be a polypeptide, nucleic acid, or polysaccharide. In certain embodiments, the biopolymer component is a nucleic acid, such as an oligonucleotide, DNA, or RNA. In other embodiments, the biopolymer component is a polypeptide, such as a peptide mimetic, protein, enzyme, or antibody. In certain embodiments, the biopolymer is an antibody that specifically binds to cancerous neoplastic tissue.

[0013] In some embodiments, the activating agent (e.g., a detectable label such as a fluorescent dye) is bound to the protein, peptide, or peptide mimetic component or biopolymer by direct binding. In certain embodiments, the activating agent is directly covalently bound to the protein, peptide, or peptide mimetic component or biopolymer. In some embodiments, the activating agent is conjugated to the protein, peptide, or peptide mimetic component by binding to an internal amino acid residue of the protein, peptide, or peptide mimetic component. In other embodiments, the activating agent may be bound to an intrachain amino acid residue along the surface of the protein, peptide, or peptide mimetic component. In yet other embodiments, the activating agent may be bound to a residue found at the binding site of the protein, peptide, or peptide mimetic component. In yet other embodiments, the activating agent residue may be bound to the protein, peptide, or peptide mimetic component by the N-terminal or C-terminal amino acid of the protein, peptide, or peptide mimetic component. In some embodiments, the activating agent is bound to a mutated amino acid (e.g., amino acid substitution, non-natural amino acid, etc.) of a native protein, peptide, or peptide mimetic component. In other embodiments, the activating agent is bound to a reactive amino acid of a native protein, peptide, or peptide mimetic component. For example, the linker may be bound to a lysine residue of the protein, peptide, or peptide mimetic. The activating component may be bound to a carbon atom or a non-carbon atom of the protein, peptide, or peptide mimetic. In one example, the activating component is bound to a carbon atom of the protein, peptide, or peptide mimetic. In another example, the activating component is bound to a non-carbon atom of the protein, peptide, or peptide mimetic, such as a nitrogen atom or a sulfur atom of the protein, peptide, or peptide mimetic.

[0014] In some embodiments, an active agent component (e.g., a small molecule therapeutic agent) and a protein, peptide, or peptide mimetic component or biopolymer may be linked to each other by one or more linkers. The linkers, if present, may be cleavable or non-cleavable linkers. In some cases, the linkers are cleavable linkers, such as acid-cleavable, base-cleavable, photocleavable, or enzymatically cleavable (e.g., peptidase, esterase) linkers. In certain cases, the linkers include a carbonate or carbamate moiety. In other cases, the linkers are non-cleavable linkers. The linkers may be zero-length cross-linkers, homobifunctional linkers, heterobifunctional linkers, or trifunctional cross-linkers. In some embodiments, the linkers may be used to conjugate the active agent component to the protein, peptide, or peptide mimetic component via internal amino acid residues of the protein, peptide, or peptide mimetic. In other cases, the linkers may be bound to intrachain amino acid residues along the surface of the protein, peptide, or peptide mimetic. In other cases, the linker may be bound to a residue found at the binding site of the protein, peptide, or peptide mimetic. In yet other cases, the linker may be used to conjugate the activating component to the protein, peptide, or peptide mimetic component via the N-terminal or C-terminal amino acid of the protein, peptide, or peptide mimetic. In some embodiments, the linker is bound to a mutated amino acid of the native protein, peptide, or peptide mimetic. In other embodiments, the linker is bound to a reactive amino acid of the native protein, peptide, or peptide mimetic. The type of bond used to bind the linker to the protein, peptide, or peptide mimetic may be an ether bond, a disulfide bond, or an amino bond. For example, the linker may be bound to a lysine residue of the protein, peptide, or peptide mimetic. The linker may be bound to a carbon atom or a non-carbon atom of the protein, peptide, or peptide mimetic. In one example, the linker is bound to a carbon atom of the protein, peptide, or peptide mimetic.In another example, the linker is bonded to an atom other than carbon in the protein, peptide, or peptide mimetic, such as a nitrogen or sulfur atom in the protein, peptide, or peptide mimetic.

[0015] Aspects of the present disclosure also include a method for delivering an activator conjugate to a neuron by contacting the neuron (e.g., in vitro or in vivo) with a composition comprising a neurotrophin receptor-binding conjugate having one or more activator compositions (e.g., dyes or small molecule therapeutic agents) covalently bound to proteins, peptides, or peptide mimetics that selectively bind to neurotrophin receptors on nerve cells. In one embodiment, the composition contacted with the nerve cell has an average ratio of activator compound to protein, peptide, or peptide mimetic in the conjugate of 5 or less. In some embodiments, the average ratio of activator component to protein, peptide, or peptide mimetic is 3.2 or less. In a particular embodiment, the average ratio of activator component to protein, peptide, or peptide mimetic component is 2 or less, for example, about 0.95 to about 1.85. In some cases, nerve cells come into contact with a composition in which 90% or more (e.g., 95% or more) of the activator conjugate has an average ratio of 5 or less between the activator component and the protein, peptide, or peptide mimetic component; for example, 90% or more (e.g., 95% or more) of the activator conjugate in the composition has an average ratio of 3.2 or less between the activator component and the protein, peptide, or peptide mimetic component; for example, 90% or more (e.g., 95% or more) of the activator conjugate in the composition has an average ratio of 2 or less between the activator component and the protein, peptide, or peptide mimetic component.

[0016] Aspects of this disclosure also include methods for treating a subject with one or more compositions of the subject. Methods according to certain embodiments include administering to a subject a composition comprising a neurotrophin receptor-binding conjugate having one or more active agent compounds (e.g., dyes or small molecule therapeutic agents) covalently bound to a protein, peptide or peptide mimetic that selectively binds to neurotrophin receptors on the subject's nerve cells. In some cases, the composition is administered to the subject topically intraocularly. In other cases, the composition is administered to the subject intracapsularly by injection. In yet other cases, the composition is administered to the subject intrathecally by injection. In yet other cases, the composition is administered to the subject intravitreously by intraocular injection or intravitreal implantation. In yet other cases, the composition is administered to the subject topically or transdermally. In other cases, the composition is administered to the subject by injection, for example, subcutaneous injection, intramuscular injection or intrathecal injection.

[0017] Aspects of this disclosure also include methods for preparing the subject composition. A method according to a particular embodiment includes contacting an activator compound with a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors to produce a composition having an activator conjugate containing one or more activator compounds covalently bound to the protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors, and isolating the activator conjugate from the composition (e.g., by reverse-phase high-performance liquid chromatography, RP-HPLC) having an average ratio of activator compounds to proteins, peptides, or peptide mimetics of 5 or less. In some embodiments, the average ratio of activator compounds to proteins, peptides, or peptide mimetics in the conjugate is 3.2 or less. In a particular embodiment, the composition prepared by the subject method has an average ratio of activator compounds to proteins, peptides, or peptide mimetics in the conjugate of 2 or less, for example, about 0.95 to about 1.85. In some cases, in compositions prepared by the method of the subject, 90% or more (e.g., 95% or more) of the activator conjugates have an average ratio of 5 or less between the activator component and the protein, peptide, or peptide mimetic component. For example, 90% or more (e.g., 95% or more) of the activator conjugates in the composition have an average ratio of 3.2 or less between the activator component and the protein, peptide, or peptide mimetic component. For example, 90% or more (e.g., 95% or more) of the activator conjugates in the composition have an average ratio of 2 or less between the activator component and the protein, peptide, or peptide mimetic component.

[0018] Aspects of this disclosure also include methods of contacting the tissue of a subject (e.g., a human or non-human animal subject) with one or more compositions of the subject. In certain embodiments, the tissue of the subject (e.g., a human or non-human animal subject) contacted with the compositions of the subject is neoplastic tissue. In other embodiments, the tissue of the subject contacted with the compositions of the subject is normal tissue. A method according to a particular embodiment involves administering to a subject a composition comprising a first detectable label-biomolecular conjugate having a first detectable label covalently bonded to a protein, peptide or peptide mimetic that selectively binds to neurotrophin receptors, and a second detectable label-biomolecular conjugate having a second detectable label covalently bonded to a biopolymer that selectively binds to neoplastic tissue. In some cases, the composition is administered intraocularly to the eye of the subject. In other cases, the composition is administered intracapsularly to the subject. In yet another case, the composition is administered intrathecally to the subject. In yet another case, the composition is administered intravitreally to the subject, may be administered after intravitreous injection, or implanted in the vitreous humor. In other cases, the composition is administered to the subject topically or transdermally. In other cases, the composition is administered to the subject by injection, such as subcutaneous, intramuscular, intravitreous, intracapsular, or intrathecal injection.

[0019] This invention is best understood by reading the following detailed description in conjunction with the accompanying drawings. The drawings include the following figures. [Brief explanation of the drawing]

[0020] [Figure 1A] This figure shows the fluorescence NGF in cell bodies after application of 10 ng / mL of NGF-Alexa Fluor 488 (NGF-488) to the distal end. Mouse-derived NGF was used here (mNGF). [Figure 1B] This figure shows the receptor-mediated activity when 1000 ng / mL of unlabeled NGF is added to 100 ng / mL of mNGF-488. [Figure 1C]A diagram showing the neuronal uptake of mNGF-488 according to a specific embodiment. [Figure 2A-2D] A diagram showing a construct having a Dyomics DY-800 Near InfraRed (NIR) dye directly conjugated to recombinant human NGF (rhNGF), demonstrating the ability to visualize cell bodies, and a diagram showing that the modified rhNGF compound was retrogradely transported. [Figure 3] After synthesis operations, the TrkB binding activities of two constructs, NIR800-rhBDNF in which the NIR dye was directly conjugated to rhBDNF and that in which fluocinonide acetonide (FA) of glucocorticoid was conjugated to rhBDNF using a heterobifunctional linker, were evaluated, and both showed that they maintained the survival of neurons. [Figure 4] A diagram showing an analysis using mass spectrometry (MS) to measure the activator peptide conjugate for the survival of neurons maintained for different ratios of the fluorescent dye in the 800 region (800-rhNGF). In that analysis, a similar MS ratio analysis used to describe the results of antibody-drug conjugates (ADCs) was adapted, and the dye-adduct ratio (DAR) or drug-adduct ratio (also DAR) was quantified. The leftmost column is the control, followed by F0 (mixed or average) = 1.64 DAR, F1 =.96 DAR, F2 = 1.1 DAR, and further F3 = 1.83 DAR. [Figure 5] A diagram showing a reverse-phase high-performance liquid chromatography (RP-HPLC) analysis of a synthetic activator-peptide conjugate composition according to a specific embodiment. [Figure 6] A diagram showing an analysis using mass spectrometry (MS) to measure the activator peptide conjugate for the survival of neurons maintained for different ratios of the fluorescent dye in the 800 region (800-rhBDNF).

Embodiments for Carrying Out the Invention

[0021] Main definition The following terms have the following meanings unless otherwise indicated. Undefined terms have the meanings recognized in their technical fields.

[0022] "Pharmaceutical composition" refers to at least one compound, which may further include a pharmaceutically acceptable carrier with which the compound is administered to a patient.

[0023] "Pharmaceutically acceptable salt" refers to a salt of the compound that retains the desired pharmacological activity of the compound. Such salts are (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid, or organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and mucic acid, or (2) salts formed when the acidic proton present in the compound is replaced by a metal ion such as an alkali metal ion, an alkaline earth ion, or an aluminum ion, or salts including a ligand with an organic base such as ethanolamine, diethanolamine, triethanolamine, and N-methylglucamine.

[0024] As used herein, the term “solvate” refers to a complex or aggregate formed of one or more molecules of a solute, such as a conjugate compound or a pharmaceutically acceptable salt thereof, and one or more molecules of a solvent. Such solvates are typically crystalline solids in which the molar ratio of solute to solvent is substantially fixed. Typical examples of solvents include water, methanol, ethanol, isopropanol, and acetic acid. When the solvent is water, the resulting solvate is a hydrate.

[0025] "Pharmaceutically acceptable carrier" means a diluent, adjuvant, excipient, or medium in which the compound is administered together with or contained therein.

[0026] "Prevention" or "prophylaxis" refers to reducing the risk of a certain condition, such as pain.

[0027] In any given condition, such as cancer, “treating” or “treatment” means, in a particular embodiment, improving the condition (i.e., preventing or reducing the onset of the condition). In a particular embodiment, “treating” or “treatment” means improving at least one physical parameter, which does not necessarily have to be recognizable by the patient. In a particular embodiment, “treating” or “treatment” means suppressing the condition physically (e.g., by stabilizing recognizable symptoms), physiologically (e.g., by stabilizing physical parameters), or both. In a particular embodiment, “treating” or “treatment” means delaying the onset of the condition.

[0028] The "therapeutic dose" refers to the amount of compound (e.g., conjugate) that, when administered to a patient, is sufficient to achieve the treatment in question. The therapeutic dose varies depending on the compound, as well as the patient's condition and its severity, age, weight, etc.

[0029] Detailed explanation Before further description of the present invention, it should be understood that the present invention is not limited to the specific embodiments described and is therefore subject to modification. Since the scope of the present invention is limited only by the appended claims, it should also be understood that the terms used herein are used solely to describe specific embodiments and are not intended to limit them.

[0030] It should be noted that, as used in this specification and the attached claims, the singular forms "a," "an," and "the" include the plural form unless the context clearly indicates otherwise. It should also be noted that claims may be drafted to exclude optional elements. This is therefore intended to serve as a prerequisite for using exclusive terms such as "only" and "only," or for using "negative" limitations, in describing elements of the claims.

[0031] As used herein, the term "a" or "an" entity should be understood to refer to one or more such entities. For example, "a compound" refers to one or more compounds. Thus, the terms "a" or "an," "one or more," and "at least one" are interchangeable. Similarly, the terms "comprising," "including," and "having" are interchangeable.

[0032] The publications described herein are presented solely for their disclosure prior to the filing date of this application. Nothing in this specification should be construed as acknowledging that the present invention has no prior rights on the grounds that such publications are prior inventions. Furthermore, the publication date of a presentation may differ from the actual publication date which may need to be independently verified.

[0033] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as those widely understood by those skilled in the art in which the present invention pertains. Methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of the present invention, but preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials to which the publications relating thereto are cited.

[0034] The compounds described herein can be purified by any chromatographic means known in the art, such as high-performance liquid chromatography (HPLC), preparative thin-layer chromatography, flash column chromatography, and ion exchange (IEX) chromatography. Any suitable stationary phase, including normal-phase, reverse-phase, and ionic resins, can be used. See, for example, Introduction to Modern Liquid Chromatography, 2nd edition, edited by L.R. Snyder and J.J. Kirkland, John Wiley and Sons, 1979, and Thin Layer Chromatography, edited by E. Stahl, Springer-Verlag, New York, 1969.

[0035] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described herein has individual components and features that can be readily separated or merged with features of any of several other embodiments without departing from the scope or spirit of the invention. Any described method may be carried out in the order of the described events or in any other logically possible order.

[0036] Apparatus and methods have been described, or will be described, in accordance with grammatical variations relating to functional descriptions. However, unless the claims are clearly drafted in accordance with Section 112 of the Patent Act, they should not necessarily be interpreted as being limited by the configuration of "means" or "processes." Rather, the meaning of the definitions presented in the claims and the full scope of equivalents should be granted in accordance with the doctrine of equivalents. If the claims are clearly drafted in accordance with Section 112 of the Patent Act, it should be clearly understood that full statutory equivalents should be granted in accordance with Section 112 of the Patent Act.

[0037] Typical Embodiments Next, various embodiments will be described in detail. It should be understood that the present invention is not limited to these embodiments. Conversely, the present invention is intended to include substitutes, modifications and equivalents that can be included in the spirit and scope of the permitted claims.

[0038] This disclosure provides conjugate compositions comprising an activator (e.g., a dye or small molecule therapeutic agent) conjugated directly (e.g., for detectable labels, e.g., for fluorescent dyes) or via a linker (e.g., for small molecule activators) to a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors, as well as methods for using and preparing them. In the compositions of the subject, the average ratio of the activator component to the protein, peptide, or peptide mimetic component in the conjugate is 5 or less (e.g., an average ratio of 3.2 or less, or an average ratio of 2 or less in the composition). Methods for using the conjugate compound composition to selectively bind and deliver the activator to nerve cells are also provided, for example, by administering a therapeutically effective dose to a subject. Methods for preparing compositions having a conjugate compound in which the average ratio of the activator component to the protein, peptide, or peptide mimetic component in the conjugate is 5 or less (e.g., an average ratio of 3.2 or less, e.g., an average ratio of 2 or less) are also described below.

[0039] This disclosure also provides a method for intraocular delivery of an activator to nerve cells by bringing a composition containing an activator conjugate into contact with a subject's eye, wherein the activator conjugate comprises one or more activator compounds covalently bound to a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors. This disclosure also provides a pharmaceutical composition comprising one or more of the subject conjugate compounds and a pharmaceutically acceptable carrier, as well as a device for delivering the subject composition to a subject's eye, such as an intravitreal implant or a contact lens device.

[0040] The disclosure also provides a composition having a detectable label-biomolecular conjugate having a first detectable label covalently attached to a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors typically expressed in the distal ends of nerves and / or nerve sheaths, including nerve tissue, and a second detectable label-biomolecular conjugate having a second detectable label covalently attached to a biopolymer that selectively binds to neoplastic tissue.

[0041] Activator conjugate composition As outlined above, aspects of the present disclosure include neurotrophin receptor-binding conjugate compositions for delivering activators to nerve cells. A neurotrophin receptor-binding conjugate composition according to one embodiment comprises one or more activator compounds (e.g., dyes or small molecule therapeutics, as described in more detail below) covalently bound to a protein, peptide or peptide mimetic so that the compound selectively binds to a neurotrophin receptor and enables intracellular transport by retrograde axonal transport and / or migration along the nerve sheath. In embodiments of the present disclosure, the composition in question has a predetermined average ratio of activator components to protein, peptide or peptide mimetic components. "Average ratio of activator components to protein, peptide or peptide mimetic components" means the average number of activators bound to each protein, peptide or peptide mimetic in the conjugate of the composition. In one embodiment, the composition in question has an average ratio of activator component to protein, peptide, or peptide mimetic component of 5 or less, for example 4.5 or less, for example 4.0 or less, for example 3.5 or less, for example 3.2 or less, for example 3.0 or less, for example 2.5 or less, for example 2.0 or less, for example 1.5 or less, for example 1.0 or less, and includes 0.5 or less. For example, in one example where the activator is a small molecule therapeutic agent, the average ratio of the small molecule therapeutic agent component to protein, peptide, or peptide mimetic component in the composition in question is 5.0 or less. In another example where the activator is a dye, the average ratio of the dye component to protein, peptide, or peptide mimetic component in the composition in question is 2.0 or less. In some embodiments, the average ratio of activator component to protein, peptide, or peptide mimetic component in the conjugate of the composition in question is in the range of 0.5 to 5.0, for example 0.75 to 4.5, for example 1.0 to 4.0, for example 1.25 to 3.5, and includes the range of 1.5 to 3.0. For example, in one instance, the average ratio of the activating agent component to the protein, peptide, or peptide mimetic component in the composition conjugate is approximately 0.95 to approximately 1.85.

[0042] In some embodiments, the composition in question is purified such that 75% or more of the activator conjugate in the composition has an average ratio of 5 or less between the activator component and the protein, peptide, or peptide mimetic component, for example, 80% or more of the activator conjugate in the composition, for example, 85% or more, for example, 90% or more, for example, 95% or more, for example, 97% or more, for example, 99% or more, for example, 99.9% or more, for example, 99.99% or more, and 99.999% or more, and the activator conjugate in these proportions has an average ratio of 5 or less between the activator component and the protein, peptide, or peptide mimetic component. In certain embodiments, 75% or more of the activator conjugates in the composition have an average ratio of activator component to protein, peptide, or peptide mimetic component of 3.2 or less, for example, 80% or more of the activator conjugates in the composition, for example, 85% or more, for example, 90% or more, for example, 95% or more, for example, 97% or more, for example, 99% or more, for example, 99.9% or more, for example, 99.99% or more, and 99.999% or more have an average ratio of activator component to protein, peptide, or peptide mimetic component of 3.2 or less. In certain embodiments, 75% or more of the activator conjugates in the composition have an average ratio of 2 or less between the activator component and the protein, peptide, or peptide mimetic component. For example, 80% or more of the activator conjugates in the composition, for example, 85% or more, for example, 90% or more, for example, 95% or more, for example, 97% or more, for example, 99% or more, for example, 99.9% or more, for example, 99.99% or more, and 99.999% or more have an average ratio of 2 or less between the activator component and the protein, peptide, or peptide mimetic component.

[0043] In certain cases, the activator is a small molecule therapeutic agent, and 75% or more of the conjugates in the composition have an average ratio of 5 or less between the small molecule therapeutic agent and a protein, peptide, or peptide mimetic. For example, conjugates containing 80% or more of the conjugates in the composition, for example 85% or more, for example 90% or more, for example 95% or more, for example 97% or more, for example 99% or more, for example 99.9% or more, for example 99.99% or more, and 99.999% or more have an average ratio of 5 or less between the small molecule therapeutic agent and a protein, peptide, or peptide mimetic. In other cases, the activator is a small molecule therapeutic agent, and 75% or more of the conjugates in the composition have an average ratio of the small molecule therapeutic agent to a protein, peptide, or peptide mimetic of 3.2 or less, for example, 80% or more of the conjugates in the composition, for example 85% or more, for example 90% or more, for example 95% or more, for example 97% or more, for example 99% or more, for example 99.9% or more, for example 99.99% or more, and 99.999% or more, where the average ratio of the small molecule therapeutic agent to a protein, peptide, or peptide mimetic is 3.2 or less. In other cases, the activator is a small molecule therapeutic agent, and 75% or more of the conjugates in the composition have an average ratio of 2 or less between the small molecule therapeutic agent and a protein, peptide, or peptide mimetic, for example, 80% or more of the conjugates in the composition, for example, 85% or more, for example, 90% or more, for example, 95% or more, for example, 97% or more, for example, 99% or more, for example, 99.9% or more, for example, 99.99% or more, and 99.999% or more, where the average ratio of the small molecule therapeutic agent to a protein, peptide, or peptide mimetic is 2 or less.

[0044] In certain embodiments, the activator is a dye, and 75% or more of the conjugates in the composition have an average ratio of dye to protein, peptide, or peptide mimetic of 5 or less, for example, 80% or more of the conjugates in the composition, for example 85% or more, for example 90% or more, for example 95% or more, for example 97% or more, for example 99% or more, for example 99.9% or more, for example 99.99% or more, and 99.999% or more have an average ratio of dye to protein, peptide, or peptide mimetic of 5 or less. In other cases, the activator is a dye, and 75% or more of the conjugates in the composition have an average ratio of dye to protein, peptide, or peptide mimetic of 3.2 or less, for example, 80% or more of the conjugates in the composition, for example 85% or more, for example 90% or more, for example 95% or more, for example 97% or more, for example 99% or more, for example 99.9% or more, for example 99.99% or more, and 99.999% or more, where the average ratio of dye to protein, peptide, or peptide mimetic is 3.2 or less. In other cases, the activator is a dye, and 75% or more of the conjugates in the composition have an average ratio of dye to protein, peptide, or peptide mimetic of 2 or less, for example, 80% or more of the conjugates in the composition, for example, 85% or more, for example, 90% or more, for example, 95% or more, for example, 97% or more, for example, 99% or more, for example, 99.9% or more, for example, 99.99% or more, and 99.999% or more, where the average ratio of dye to protein, peptide, or peptide mimetic is 2 or less. For example, when the activator is a dye, 75% or more of the conjugates in the composition have an average ratio of dye to protein, peptide, or peptide mimetic in the range of about 0.5 to about 2.0, for example, about 0.6 to about 1.9, for example, about 0.7 to about 1.8, for example, about 0.8 to about 1.7, for example, about 0.9 to about 1.6, and include the range of about 1.0 to about 1.5.In certain cases, the activator is a dye, and 75% or more of the conjugates in the composition have an average ratio of dye to protein, peptide, or peptide mimetic in the range of approximately 0.95 to approximately 1.85, for example, 80% or more of the conjugates in the composition, for example 85% or more, for example 90% or more, for example 95% or more, for example 97% or more, for example 99% or more, for example 99.9% or more, for example 99.99% or more, and 99.999% or more, where the average ratio of dye to protein, peptide, or peptide mimetic is in the range of approximately 0.95 to approximately 1.85.

[0045] The conjugate compound in the subject composition comprises an activator covalently bonded to a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors. In a particular embodiment, the compound in question comprises a conjugate represented by formula XLB, where X is an activator, e.g., a small molecule therapeutic agent or dye, B is a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors, and L is an optional linker.

[0046] The target conjugate compound comprises a protein, peptide, or peptide mimetic that promotes one or more of the following actions by neurotrophin receptors: binding, endocytosis, and transport (e.g., retrograde axonal transport to the neuronal cell body). In some embodiments, the protein, peptide, or peptide mimetic is a component that promotes the binding of the conjugate compound to neurotrophin receptors. In other embodiments, the protein, peptide, or peptide mimetic is a component that promotes the binding and endocytosis of the conjugate compound by neurotrophin receptors. In yet another embodiment, the protein, peptide, or peptide mimetic is a component that promotes the binding, endocytosis, and transport (e.g., retrograde axonal transport, anterograde axonal transport) of the conjugate compound by neurotrophin receptors. Examples of target proteins, peptides, or peptide mimetic compounds include, but are not limited to, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), neurotrophic factor 4 / 5 (NT-4 / 5), neurotrophic factor 6 (NT-6), neurotrophic factor 7 (NT-7), glial cell-derived neurotrophic factor (GDNF), ciliary body neurotrophic factor (CNTF), their derivatives, analogs, and fragments, such as recombinant molecules of NGF, BDNF, GDNF, and CNTF, and synthetic peptides that bind to nerve cell surface receptors and have growth factor agonist or antagonist activity. The proteins, peptides, or peptide mimetic compounds may be derived from animals other than humans, and may be recombinant human forms expressed and produced in bacterial systems, such as Escherichia coli cells, or mammalian cell systems, such as CHO (Chinese hamster ovary) cells. In some embodiments, the protein, peptide, or peptide mimetic component of the subject conjugate is a mammalian protein, such as mammalian nerve growth factor, mammalian brain neurotrophic factor, or mammalian neurotrophic factor. In certain cases, the protein, peptide, or peptide mimetic component of the subject conjugate is not a bacterial protein, such as E. coli nerve growth factor, E. coli brain neurotrophic factor, or E. coli neurotrophic factor.In certain embodiments, the protein, peptide, or peptide mimetic is a subunit of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), neurotrophic factor 4 / 5 (NT-4 / 5), neurotrophic factor 6 (NT-6), neurotrophic factor 7 (NT-7), glial cell-derived neurotrophic factor (GDNF), or ciliary neurotrophic factor (CNTF), such as the β-subunit of NGF, rhNGF, or rhBDNF, or a chimeric combination of one or more NTs. In some cases, the protein, peptide, or peptide mimetic is a native neurotrophic factor (e.g., native NGF, native BDNF). In certain cases, the native neurotrophic factor does not include additional peptide conjugates functionalized for binding to a payload compound (e.g., a dye or small molecule activator). As described in more detail below, the neurotrophin-binding conjugate compounds of the present disclosure in certain embodiments include natural nerve growth factor (NGF), natural brain-derived neurotrophic factor (BDNF), or natural neurotrophic factor conjugated to an activator compound by one or more amino acid residues (e.g., lysine residues) present in natural proteins, such as natural nerve growth factor (NGF), natural brain-derived neurotrophic factor (BDNF), or natural neurotrophic factor. In other words, according to these embodiments, the neurotrophin-binding component does not contain any non-natural or additional peptide sequences. In some cases, the protein, peptide, or peptide mimetic component of the subject conjugate does not contain any additional C-terminal peptide sequences for conjugation to, for example, a dye or activator payload. In other cases, the protein, peptide, or peptide mimetic component of the subject conjugate does not contain any additional N-terminal peptide sequences for conjugation to, for example, a dye or activator payload.

[0047] In certain embodiments, the protein, peptide, or peptide mimetic is a neurotrophic factor that binds to tropomyosin kinase A (TrkA). In certain embodiments, the protein, peptide, or peptide mimetic is a neurotrophic factor that exerts retrograde axonal transport of a payload compound (e.g., a dye or small molecule agent) conjugated to the protein, peptide, or peptide mimetic. In certain embodiments, the protein, peptide, or peptide mimetic is a neurotrophic factor that exerts the uptake (e.g., uptake into the nerve cell body or cytoprocess) of a payload compound (e.g., a dye or small molecule agent) conjugated to the protein, peptide, or peptide mimetic.

[0048] The composition may contain one or more proteins, peptides, or peptide mimetic conjugate compounds, for example, two or more, for example, three or more (including five or more). For example, the composition may contain one or more NGF-containing conjugates, BDNF-containing conjugates, NT-3-containing conjugates, NT-4 / 5-containing conjugates, NT-6-containing conjugates, NT-7-containing conjugates, GDNF-containing conjugates, CNTF-containing conjugates, or conjugates containing, for example, NGF-BDNF chimeric constructs that retain Trk-binding activity after the synthesis operation.

[0049] The conjugate compound comprises one or more activators. The term "activator" is used herein to refer to a compound known to become active within a cell and be delivered to the target nerve cell, for example, by endocytosis and / or transport (e.g., retrograde axonal transport to the nerve cell body). In some embodiments, the activator is a small molecule compound, e.g., a small molecule therapeutic agent. In other embodiments, the activator is a dye. In some embodiments, the activator is a dye whose binding to nerve tissue in the posterior part of the eye can be used as a diagnostic method for retinal diseases, i.e., insufficient binding can indicate degeneration of retinal tissue. In certain embodiments, the activator in the target conjugate is not a biopolymer, e.g., a polysaccharide, protein, nucleic acid, or other biopolymer. In other embodiments, the activator in the target conjugate is not a polymer, e.g., a biopolymer. In yet another embodiment, the activator in the target conjugate has molecular weights including molecular weights of 2000 g / mol or less, for example 1500 g / mol or less, for example 1000 g / mol or less, for example 900 g / mol or less, for example 750 g / mol or less, for example 500 g / mol or less, and molecular weights of 250 g / mol or less.

[0050] In certain cases, the activating agent component in the subject composition is a detectable label. Each detectable label may be a compound, such as a fluorophore, chromophore, enzyme, redox label, radioactive label, sound-absorbing label, Raman (SERS) tag, mass tag, isotope tag, magnetic particle, fine particle, or nanoparticle. In some embodiments, each detectable label in the subject composition is, in certain cases, a dye, such as an organic dye or an inorganic dye. In certain cases, the dye is a luminescent dye, for example, a fluorescent dye having emission wavelengths including peak emission wavelengths of 200 nm or more, 250 nm or more, 300 nm or more, 350 nm or more, 400 nm or more, 450 nm or more, 500 nm or more, 550 nm or more, 600 nm or more, 650 nm or more, 700 nm or more, 750 nm or more, 800 nm or more, 850 nm or more, 900 nm or more, 950 nm or more, 1000 nm or more, and a peak emission wavelength of 1050 nm or more. For example, the dye may be a fluorescent dye with a peak emission wavelength range of 200 nm to 1200 nm, for example 300 nm to 1100 nm, for example 400 nm to 1000 nm, for example 500 nm to 900 nm, and includes a fluorescent dye with a peak emission wavelength range of 600 nm to 800 nm.

[0051] Examples of dyes that can be used include, but are not limited to, body pie dyes, coumarin dyes, rhodamine dyes, acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes, chlorophyll-containing dyes, triarylmethane dyes, azo dyes, diazonium dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone-imine dyes, azine dyes, eurodin dyes, safranin dyes, indamine, indophenol dyes, fluorine dyes, oxazine dyes, oxazone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronin dyes, fluorine dyes, rhodamine dyes, phenanthoridine dyes, squalane, body pie, squaline oxytan, naphthalene, coumarin, oxadiazole, anthracene, pyrene, acridine, arylmethine or tetrapyrrole, and combinations thereof. In certain embodiments, the conjugate may contain two or more dyes selected from, for example, body pie dyes, coumarin dyes, rhodamine dyes, acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes, chlorophyll-containing dyes, triarylmethane dyes, azo dyes, diazonium dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone-imine dyes, azine dyes, eurodin dyes, safranin dyes, indamine, indophenol dyes, fluorine dyes, oxazine dyes, oxazone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronin dyes, fluorine dyes, rhodamine dyes, phenanthoridine dyes, squalane, body pie, squaline roxitan, naphthalene, coumarin, oxadiazole, anthracene, pyrene, acridine, arylmethine or tetrapyrrole, and combinations thereof.

[0052] In some embodiments, the conjugate in the composition in question has an activating component containing the same or substantially the same dye as Alexa Fluor dyes, e.g., the same or substantially the same dye as Alexa Fluor 488 dye, or the same or substantially the same dye as Alexa Fluor 670 dye, or the same or substantially the same dye as Alexa Fluor 680 dye, or the same or substantially the same dye as Alexa Fluor 780 dye. In other embodiments, the conjugate in the composition in question has an activating component containing the same or substantially the same dye as Dyomics dyes, e.g., the same or substantially the same dye as Dyomics DY-800 dye. In yet another embodiment, the conjugate in the composition in question has an activating component containing the same or substantially the same dye as Curadel dyes, e.g., the same or substantially the same dye as ZW-800 dye. In some embodiments, the dye is the same or substantially the same as Cy dyes, e.g., Cy 7.5 or Cy 5.5. In certain cases, the dye is a compound having axial or plane symmetry. In some embodiments, the dye is a compound that facilitates the intracellular visualization of the dye (e.g., by spectrometer or naked eye). In these embodiments, the target conjugate promotes binding by neurons or their components, endocytosis, and intraneuronal transport (e.g., retrograde axonal transport to the neuronal cell body).

[0053] In some embodiments, the target conjugate compound includes a small molecule compound, such as a small molecule therapeutic agent. In some cases, the small molecule therapeutic agent is an anticancer agent. In some cases, the cancer is a central nervous system cancer, selected from the group consisting of adult and pediatric gliomas (low-grade or high-grade gliomas), visual pathway gliomas, spinal cord tumors, neurofibromas, schwannomas, malignant peripheral schwannomas, malignant schwannomas, neurofibrosarcomas, and neurosarcomas. In certain cases, the cancer is a visual pathway glioma. The anticancer agent may vary depending on the desired therapeutic effect and target symptom, and may be a mammalian target of a rapamycin (mTOR) inhibitor or a mitogen-activated protein kinase (MEK) inhibitor. In some embodiments, the anticancer agent is an mTOR inhibitor. For example, the anticancer agent may be sirolimus, temsirolimus, everolimus, and ridafololimus or a combination thereof. In other embodiments, the anticancer agent is a MEK inhibitor. For example, the anticancer drug may be trametinib, dabrafenib, cobimetinib, vemurafenib, binimetinib, selumetinib, or a combination thereof. In some cases, the anticancer drug is selumetinib. In some embodiments, the anticancer agent is a heat shock protein 90 (hsp90) inhibitor, such as albespinomycin, 17-N-allylamino-17-demethoxygeldanamycin (17AAG), luminespib (AUY-922, NVP-AUY922), ganetespib (STA-9090), onarespib (AT13387), NVP-BEP800, BIIB021, PF-04929113 (SNX-5422), SNX-2112 (PF-04928473), KW-2478, XL888, XL888, PU-H71, VER-49009, CH5138303, VER-50589, VER155008, and geldanamycin. In some embodiments, the anticancer agent is a checkpoint inhibitor. In some cases, checkpoint inhibitors are inhibitory compounds that target one or more of PD-1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFRβ, such as S7911, BMS202, and S8158.In some cases, checkpoint inhibitors are inhibitory compounds that target PD-1. In some embodiments, the anticancer agent is a chemokine 4 / chemokine ligand 12 (CX4 / CXCL12) inhibitor, e.g., burixafor, LY2510924, e.g., AMD3100 and AMD3465. In some embodiments, the compound is an imidazoquinoloneamine. In certain embodiments, the composition in question comprises one or more small molecule-containing conjugates, such as those described in International Patent Application PCT / US2019 / 42253, filed on 17 July 2019, the entire disclosure of which is incorporated herein by reference.

[0054] In some embodiments, the activator is an anti-inflammatory agent. In certain cases, the anti-inflammatory agent is a glucocorticoid, such as fluocinolone acetonide.

[0055] In other embodiments, the activator is an antimicrobial compound. Examples of antimicrobial agents include, but are not limited to, antibacterial agents, antifungal agents, antiviral agents, anthelmintic agents, and antimicrobial insecticides. For example, antimicrobial agents include fluoroquinolones, such as ciprofloxacin, norfloxacin, ofloxacin, enoxacin, perfloxacin, freloxacin, enrofloxacin, marbofloxacin, salafloxacin, orbifloxacin, danofloxacin; aminoglucosides, such as streptomycin, netylmycin, kanamycin, neomycin, tobramycin, amikacin, disomicin, ribostamycin, dibekacin, furamycetin, gentamicin; penicillin and aminopenicillins, such as penicillin, ampicillin, amoxicillin, nafcillin, oxacillin, and tica Examples include lucilin, cephalosporins such as ceftriaxone, cephalexin, cefadroxil, and ceftiofur, β-lactams such as clavulanic acid which can be used in combination with penicillin or aminopenicillin, macrolides such as clarithromycin and erythromycin, and other antibiotics such as dactinomycin, clindamycin, nalidixic acid, chloramphenicol, rifamopin, clofazimine, spectinomycin, polymyxin B, colistin, minocycline, vancomycin, hygromycin B or C, fusidic acid, trimethoprim, and cefotaxime. In some embodiments, antiviral compounds include, but are not limited to, ribavirin, fenretinide, favipiravir, brincidofovir, ZMapp, TKM-100802, BCX4430, interferon, amiodarone, atrovostatin, irbesartan, clomiphene, FX06, Zmab, tamoxifen, albendazole, AC-93253, toremifene, T-705, and GS-5734 (remdesivir).

[0056] In some embodiments, the activating agent (e.g., a detectable label such as a fluorescent dye) binds to the protein, peptide, or peptide mimetic component or biopolymer by direct binding. In certain embodiments, the activating agent is directly covalently bound to the protein, peptide, or peptide mimetic component or biopolymer. In some embodiments, the activating agent binds to the protein, peptide, or peptide mimetic component by binding to an internal amino acid residue of the protein, peptide, or peptide mimetic. In other embodiments, the activating agent may bind to an intrachain amino acid residue along the surface of the protein, peptide, or peptide mimetic. In yet other embodiments, the activating agent may bind to a residue found at the binding site of the protein, peptide, or peptide mimetic. In yet other embodiments, the activating agent residue may bind to the protein, peptide, or peptide mimetic component by the N-terminal or C-terminal amino acid of the protein, peptide, or peptide mimetic. In some embodiments, the activating agent binds to a mutant amino acid (e.g., a substituted amino acid or a non-natural amino acid) of a native protein, peptide, or peptide mimetic. In other embodiments, the activating agent binds to a reactive amino acid of a native protein, peptide, or peptide mimetic. For example, the activator may bind to a lysine residue of the protein, peptide, or peptide mimetic. The activating agent may be bonded to a carbon atom or a non-carbon atom of the protein, peptide, or peptide mimetic. In one example, the activating agent is bonded to a carbon atom of the protein, peptide, or peptide mimetic. In another example, the activating agent is bonded to a non-carbon atom of the protein, peptide, or peptide mimetic, such as a nitrogen or sulfur atom.

[0057] In certain embodiments, the activating component is conjugated to a protein, peptide, or peptide mimetic component by a linker. The linker may be any simple covalent bonding protocol, such as a zero-length crosslinker, a homobifunctional linker, a heterobifunctional linker, or a trifunctional crosslinker. The linker may preferably contain one or more functional groups, such as an amide, alkylamine, carbamate, carbonate, thiol ether, alkyl, cycloalkyl, or aryl moiety. In some embodiments, the linker contains a carbamate moiety. The linker may be used to conjugate the activating component to the protein, peptide, or peptide mimetic component by an N-terminal or C-terminal amino acid of the protein, peptide, or peptide mimetic. In some embodiments, the linker is conjugated to a mutant amino acid of the native protein, peptide, or peptide mimetic. The type of bond used to conjugate the linker to the protein, peptide, or peptide mimetic may be an ether bond, a disulfide bond, or an amino bond. For example, the linker may be conjugated to a lysine residue of the protein, peptide, or peptide mimetic. The linker may be bonded to a carbon atom or a non-carbon atom of the protein, peptide, or peptide mimetic. In one example, the linker is bonded to a carbon atom of the protein, peptide, or peptide mimetic. In another example, the linker is bonded to a non-carbon atom of the protein, peptide, or peptide mimetic, such as a nitrogen atom or a sulfur atom of the protein, peptide, or peptide mimetic.

[0058] In some embodiments, the linker is cleavable. The term “cleavable” is used herein to mean a linker that can be cleaved under certain conditions to break the bond between the activator and the binding site, in its conventional sense. For example, the linker may be an acid-cleavable linker, a base-cleavable linker, a photocleavable linker, or an enzymatically cleavable (e.g., peptidase, esterase) linker. An acid-cleavable linker is cleaved by bringing the conjugate compound to a pH of 7 or less, for example, 6.5 or less, for example, 6.0 or less, for example, 5.5 or less, for example, 5.0 or less, for example, 4.5 or less, for example, 4.0 or less, for example, 3.5 or less, for example, 3.0 or less, for example, 2.5 or less, for example, 2.0 or less, for example, 1.5 or less, and 1.0 or less. A base-cleaving linker cleaves when the conjugate compound is subjected to a pH of 7 or higher, for example, 7.5 or higher, for example, 8.0 or higher, for example, 8.5 or higher, for example, 9.0 or higher, for example, 9.5 or higher, for example, 10.0 or higher, for example, 10.5 or higher, for example, 11.0 or higher, for example, 11.5 or higher, for example, 12.0 or higher, for example, 12.5 or higher, and 13.0 or higher.

[0059] In certain embodiments, the conjugate compound in the subject composition comprises an enzymatically cleavable linker. In some cases, the enzymatically cleavable linker is cleaved by contacting the compound with a peptidase, such as trypsin or chymotrypsin. In other cases, the enzymatically cleavable linker is cleaved by contacting the compound with an esterase. In some embodiments, the linker in question includes the linkers described in U.S. Patents 5,767,288, 5,563,250, 5,505,931 and 4,469,774, the disclosures of which are incorporated herein by reference.

[0060] In other embodiments, the conjugate compound in the subject composition includes an incleavable linker. The term “incleavable” is used herein, in its conventional sense, to refer to a covalent bond that is stable under physiological conditions and does not release an activator from the binding site (for example, a dye or small molecule therapeutic agent maintains a covalent bond to a protein, peptide, or peptide mimetic that selectively binds to a neurotrophin receptor). In other words, a conjugate compound having an incleavable linker is not easily cleaved by acid, base, light, or enzymatic treatment. In these embodiments, 90% or more of the conjugate compounds in compositions treated with acid, base, light, or enzymes do not result in the release of an activator from the binding site (e.g., a protein, peptide, or peptide mimetic that selectively binds to nerve cells). For example, conjugate compounds in proportions of 95% or more, e.g., 97% or more, e.g., 98% or more, e.g., 99% or more, and 99.9% or more in compositions subjected to acid, base, light, or enzymatic treatment do not result in the release of an activator from the binding site.Examples of suitable non-cleaving linkers include maleimide-containing crosslinkers, such as N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidecaproic acid), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ-maleimidobutyrate N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimidate ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimidate ester (MBS), and N-(α-maleimidoacetoxy)-succinimidate. Examples include, but are not limited to, synimide esters [AMAS], succinimidyl-6-(β-maleimidopropionamide) hexanoate (SMPH), N-succinimidyl-4-(p-maleimidophenyl)-butyrate (SMPB), and N-(p-maleimidophenyl) isocyanate (PMPI), or haloacetyl-containing crosslinkers, such as N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyliodoacetate (SIA), N-succinimidylbromoacetate (SBA), and N-succinimidyl-3-(bromoacetamide)propionate (SBAP).

[0061] In certain embodiments, the composition in question includes a pharmaceutically acceptable carrier. A wide range of pharmaceutically acceptable excipients are known in the art and do not need to be discussed in detail herein. Pharmaceutically acceptable excipients are extensively described in various publications, such as A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) edited by HC Ansel et al., 7th edition, Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) edited by AH Kibbe et al., 3rd edition, Amer. Pharmaceutical Assoc. For example, one or more excipients may be sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate or calcium carbonate, binders (e.g., cellulose, methylcellulose, hydroxymethylcellulose, polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic, poly(ethylene glycol), sucrose or starch), disintegrants (e.g., starch, carboxymethylcellulose, hydroxypropyl starch, low-substituted hydroxypropylcellulose, sodium bicarbonate, calcium phosphate or calcium citrate), lubricants (e.g., stearin It may also contain magnesium sulfate, light anhydrous silicic acid, talc, or sodium lauryl sulfate, flavoring agents (e.g., citric acid, menthol, glycine, or orange powder), preservatives (e.g., sodium benzoate, sodium bisulfite, methylparaben, or propylparaben), stabilizers (e.g., citric acid, sodium citrate, or acetic acid), suspending agents (e.g., methylcellulose, polyvinylpyrrolidone, or aluminum stearate), dispersants (e.g., hydroxypropyl methylcellulose), diluents (e.g., water), and base waxes (e.g., cocoa butter, white petrolatum, or polyethylene glycol).

[0062] Conjugate compositions having an average ratio of 5 or less (e.g., 2 or less, e.g., 0.95 to 1.85) of an active agent component to a protein, peptide, or peptide mimetic component may be formulated in combination with a suitable pharmaceutically acceptable carrier or diluent to form a composition suitable for delivery to a subject or a composition suitable for contact with nerve cells, and may be formulated in solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols, and / or by lyophilization.

[0063] In certain cases, the composition is formulated for injection, for example, by subcutaneous, intramuscular, intravitreous, intracapsular, or intrathecal injection. In other cases, the composition is formulated for intraocular administration to a subject. In yet other cases, the composition is formulated for intracapsular administration to a subject. In yet other cases, the composition is formulated for intrathecal administration to a subject. In yet other cases, the composition is formulated for intravitreous administration to a subject. In yet other cases, the composition is formulated for topical or transdermal administration to a subject.

[0064] In some embodiments, the composition in question includes an aqueous buffer. Suitable aqueous buffers include, but are not limited to, acetate, succinate, citrate, and phosphate buffers, with concentrations ranging from about 5 mM to about 100 mM. In some embodiments, the aqueous buffer includes a reagent for forming an isotonic solution. Suitable reagents include, but are not limited to, sodium chloride and sugars, such as mannitol, dextrose, and sucrose. In some embodiments, the aqueous buffer further includes a nonionic surfactant, such as polysorbate 20 or 80. In some cases, the composition in question further includes a preservative. Suitable preservatives include, but are not limited to, benzyl alcohol, phenol, chlorobutanol, and benzalkonium chloride. In many cases, the composition is stored at about 4°C. The formulation may be freeze-dried, in which case the formulation generally includes an antifreeze agent, such as sucrose, trehalose, lactose, maltose, or mannitol. Freeze-dried formulations can be stored at room temperature for extended periods.

[0065] In some embodiments, the composition comprises other additives, such as lactose, mannitol, corn starch, or potato starch, along with a binder, such as crystalline cellulose, a cellulose derivative, acacia, corn starch, or gelatin, a disintegrant, such as corn starch, potato starch, or sodium carboxymethylcellulose, a lubricant, such as talc or magnesium stearate, and optionally diluents, buffers, wetting agents, preservatives, and flavoring agents.

[0066] When a composition is formulated for injection, the conjugate compounds can be formulated by dissolving, suspending, or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable oil or other similar oils, synthetic fatty acid glycerides, higher fatty acid esters, or propylene glycol, along with, optionally, conventional additives, such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0067] The dosage used to treat a subject (as described in more detail below) will vary depending on the clinical goal to be achieved, but the preferred dosage range for the conjugate compound is approximately 0.0001 mg to 5000 mg of the active ingredient per single dose, for example, approximately 1 mg to 25 mg, 25 mg to 50 mg, 50 mg to 100 mg, 100 mg to 200 mg, 200 mg to 250 mg, 250 mg to 500 mg, 500 mg to 1000 mg, or 1000 mg to 5000 mg. Those skilled in the art will readily understand that the dosage level may vary depending on the specific compound, route of administration, the severity of symptoms, and the subject's susceptibility to adverse reactions, if the effect of neurotrophin as the target portion is local and can affect the dosage.

[0068] In some embodiments, a suitable dose of the conjugate compound in the composition of the subject is in the range of about 1 mg to about 500 mg per kg of body weight, for example, about 5 mg to about 500 mg per kg of body weight, about 10 mg to about 500 mg per kg of body weight, about 20 mg to about 500 mg per kg of body weight, about 30 mg to about 500 mg per kg of body weight, about 40 mg to about 500 mg per kg of body weight, about 50 mg to about 500 mg per kg of body weight, about 60 mg to about 500 mg per kg of body weight, about 70 mg to about 500 mg per kg of body weight, about 80 mg to about 500 mg per kg of body weight, about 90 mg to about 500 mg per kg of body weight, about 100 mg to about 500 mg per kg of body weight, about 200 mg to about 500 mg per kg of body weight, about 300 mg to about 500 mg per kg of body weight, or about 400 mg to about 500 mg per kg of body weight.

[0069] In some embodiments, preferred doses of the compound in the subject composition are in the range of about 1 mg to about 5 mg per kg of body weight, about 5 mg to about 10 mg per kg of body weight, about 10 mg to about 20 mg per kg of body weight, about 20 mg to about 30 mg per kg of body weight, about 30 mg to about 40 mg per kg of body weight, about 40 mg to about 50 mg per kg of body weight, about 50 mg to about 100 mg per kg of body weight, or about 100 mg to about 500 mg per kg of body weight.

[0070] In some embodiments, the conjugate compound is administered as a single dose. In other embodiments, the conjugate compound is administered multiple times. When administered multiple times over a period of time, the conjugate compound is administered, for example, twice daily (qid), once daily (qd), every other day (qod), every three days, three times a week (tiw), or twice a week (biw) over a period of time. For example, the conjugate compound is administered qid, qd, qod, tiw, or biw over a period of time ranging from one day to approximately two years or more. For example, the conjugate compound is administered at any of the above frequencies over a period of one week, two weeks, one month, two months, six months, one year, or two years or more, depending on various factors.

[0071] The dosage units of this disclosure can be prepared using manufacturing methods available in the art and may be in various forms suitable for injection administration (including intracapsular, intrathecal, intravenous, intramuscular, subcutaneous, intravitreous, and intraocular injection), such as solutions, suspensions, lyophilized products, or emulsions. The dosage units may contain conventional components in pharmaceutical formulations, such as one or more carriers, binders, lubricants, excipients (to impart sustained-release properties in addition to any sustained-release effects that can be contributed by conjugating the composition with neurotrophin), pH adjusters, colorants, or further activators.

[0072] The dosage units provided as liquid dosage units can have a total weight of approximately 1 microgram to 1 gram, approximately 5 micrograms to 1.5 grams, approximately 50 micrograms to 1 gram, approximately 100 micrograms to 1 gram, 50 micrograms to 750 milligrams, or approximately 1 microgram to 2 grams.

[0073] The dosage unit can contain any relative amount of the component. For example, the dosage unit can contain about 0.1% to 99% by weight of the active component (i.e., the conjugate compound) per total weight of the dosage unit. In some embodiments, the dosage unit can contain 10% to 50% by weight, 20% to 40% by weight, or about 30% by weight of the active component per total weight of the dosage unit.

[0074] Dosage units can be provided in a variety of forms, and optionally in a form suitable for storage. For example, dosage units can be housed in containers suitable for containing pharmaceutical compositions. Containers may include, for example, bottles (e.g., bottles with closure devices, e.g., bottles with caps), vials, ampoules (for single-dose units), dispensing devices, thin films, tubes, and the like.

[0075] The container may include a cap (e.g., a screw cap) that is detachably attached to the container above an opening that allows access to the dosage units contained within the container.

[0076] The container may include a seal that can serve as an indicator of tampering and / or an anti-tampering element, the seal being destroyed upon contact with a dose unit contained within the container. Such a seal element may be a fragile element that breaks or deforms upon contact with a dose unit contained within the container, for example. An example of such a fragile seal element is a seal positioned above the container opening such that destruction of the seal (e.g., peeling and / or puncture of the seal) is required to contact the dose unit contained within the container. An example of a fragile seal element is a fragile ring attached to the cap around the container opening such that the ring breaks when the cap is opened to contact the dose unit inside the container. In certain embodiments, the composition is a lyophilized composition, and the subject conjugate compound in the composition is reconstituted at the time of treatment. In certain cases, the container is a pen-type syringe that is reconstituted at the time of treatment.

[0077] Liquid dosing units may be contained in containers (e.g., bottles or ampoules) of a size and configuration adapted to maintain the stability of the dosing units over the period during which they are dispensed into prescription drugs. For example, containers may be sized and configured to hold 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more single-dose liquid dosing units. Containers may be sealed or resealable. Containers may be packaged in cartons (e.g., for transport from the manufacturer to a pharmacy or other dispensing facility). The cartons may be boxes, tubes, or other configurations and may be made of any material (e.g., corrugated cardboard, plastic, etc.). The packaging system and / or the containers placed therein may have one or more affixed labels (for displaying information, e.g., lot number, type of dosing unit, manufacturer, etc.).

[0078] The container may include, for example, a moisture-proof and / or light-shielding material to facilitate the maintenance of the stability of the active ingredient in the dosage unit contained within the container. The container can be adapted to accommodate a single dosage unit or multiple dosage units. The container may include a dispensing control mechanism, such as a lockout mechanism to facilitate the maintenance of dosage.

[0079] Dosage units may be provided in containers in which they are contained, and may be provided as part of a packaging system (optionally with instructions for use). For example, dosage units containing different amounts of conjugate compounds may be provided in individual containers, and these containers may be housed in a larger container (for example, to facilitate the protection of dosage units for shipment). For example, one or more dosage units described herein may be provided in individual containers, in which case dosage units of different compositions may be provided in individual containers, and the individual containers may be housed in a dispensing package.

[0080] Two-color tissue imaging composition As outlined above, aspects of the present disclosure by particular embodiments also include compositions having two or more activator conjugate compounds, for example, two or more detectable label-biomolecular conjugates. In these embodiments, the composition includes a first detectable label-biomolecular conjugate having a first detectable label covalently bound to a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors. In some embodiments, the composition includes 1) a detectable label-biomolecular conjugate represented by formula XLA, for example, where X is a detectable label, A is a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors, and L is an optional linker; and 2) a detectable label-biomolecular conjugate represented by formula XLB, for example, where X is a detectable label, B is a biopolymer (e.g., an antibody) that selectively binds to one or more receptors (e.g., non-Trk receptors) in neoplastic tissue, and L is an optional linker.

[0081] Proteins, peptides, or peptide mimetic compounds that selectively bind to neurotrophin receptors according to a particular embodiment promote one or more of the neurotrophin receptor-mediated binding, endocytosis, and transport (e.g., retrograde axonal transport to the neuronal cell body), as described above. For example, examples of proteins, peptides, or peptide mimetic compounds in the activator conjugate compound of the subject dichromatic tissue imaging composition include, but are not limited to, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), neurotrophic factor 4 / 5 (NT-4 / 5), neurotrophic factor 6 (NT-6), neurotrophic factor 7 (NT-7), glial cell-derived neurotrophic factor (GDNF), ciliary body neurotrophic factor (CNTF), their derivatives, analogs, and fragments, such as recombinant molecules of NGF, BDNF, GDNF, and CNTF, and synthetic peptides that bind to neuronal cell surface receptors and have growth factor agonist or antagonist activity. The protein, peptide, or peptide mimetic may be derived from a non-human animal, or it may be a recombinant human protein, peptide, or peptide mimetic. In certain embodiments, the protein, peptide, or peptide mimetic is a subunit of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), neurotrophic factor 4 / 5 (NT-4 / 5), neurotrophic factor 6 (NT-6), neurotrophic factor 7 (NT-7), glial cell-derived neurotrophic factor (GDNF), or ciliary neurotrophic factor (CNTF), such as the β-subunit of NGF, rhNGF, or rhBDNF, or a chimeric construct thereof, such as rhNGF-rhBDNF.

[0082] The subject two-color tissue imaging composition comprises a detectable label-biomolecular conjugate that selectively binds to neoplastic tissue. The term "neoplastic tissue" is used herein in its conventional sense to refer to tissue exhibiting abnormal cell proliferation. In some embodiments, the neoplastic tissue is benign. In other embodiments, the neoplastic tissue is malignant. The tissue may be any body tissue capable of exhibiting neoplastic proliferation, including, but not limited to, brain and nervous system tissue, oral and esophageal tissue, nasal cavity tissue, muscle tissue, cardiac and pericardial tissue, vascular tissue, lung tissue, spleen tissue, liver tissue, gallbladder tissue, pancreatic tissue, gastrointestinal tissue, genitourinary tissue, and skin, bone, cartilage, and ligament tissue.

[0083] The term "biopolymer" refers to a polymer of one or more repeating units. Biopolymers are typically found in biological systems and include, in particular, polysaccharides (e.g., carbohydrates), peptides (the term is used to encompass polypeptides and proteins, whether or not they are bound to polysaccharides), and polynucleotides, as well as their analogues, such as amino acid analogues or non-amino acid groups, or nucleotide analogues or non-nucleotide groups, or compounds comprising or containing them. This includes polynucleotides in which the conventional backbone is replaced with a non-natural or synthetic backbone, and nucleic acids (or synthetic or natural analogues) in which one or more conventional bases are replaced with (natural or synthetic) groups capable of participating in Watson-Crick hydrogen bonding interactions. Polynucleotides include single-stranded or multi-stranded configurations, and one or more strands may or may not be perfectly aligned with each other. Specifically, "biopolymers" include DNA (including cDNA), RNA, and oligonucleotides, regardless of their origin. Thus, biomolecules may include polysaccharides, nucleic acids, and polypeptides. For example, nucleic acids may be oligonucleotides, truncated or full-length DNA, or RNA. In one embodiment, the oligonucleotide, shortened and full-length DNA or RNA is composed of nucleotide monomers comprising 10 or more nucleotide monomers, e.g., 15 or more, e.g., 25 or more, e.g., 50 or more, e.g., 100 or more, e.g., 250 or more nucleotide monomers, and 500 or more nucleotide monomers. For example, the oligonucleotide, shortened and full-length DNA or RNA in question may be in the range of 10 to 108 nucleotides, e.g., 102 to 107 nucleotides, including 103 to 106 nucleotides. In one embodiment, the biopolymer is not a single nucleotide or a short-chain oligonucleotide (e.g., less than 10 nucleotides). "Full length" means that the DNA or RNA is a nucleic acid polymer that contains 70% or more of its complete sequence (e.g., found in nature), e.g., 75% or more, e.g., 80% or more, e.g., 85% or more, e.g., 90% or more, e.g., 95% or more, e.g., 97% or more, e.g., 99% or more, and comprises 100% of the full-length sequence of the DNA or RNA (e.g., found in nature).

[0084] The polypeptide may, in certain cases, be a shortened or full-length protein, an enzyme, or an antibody. In one embodiment, the polypeptide, shortened and full-length protein, enzyme, or antibody is composed of amino acid monomers comprising 10 or more amino acid monomers, e.g., 15 or more, e.g., 25 or more, e.g., 50 or more, e.g., 100 or more, e.g., 250 or more amino acid monomers, and 500 or more amino acid monomers. For example, the polypeptide, shortened and full-length protein, enzyme, or antibody in question may have a length in the range of 10 to 108 amino acids, e.g., 102 to 107 amino acids, including 103 to 106 amino acids. In one embodiment, the biopolymer is not a single amino acid or a short-chain polypeptide (e.g., less than 10 amino acids). The term "full length" means that the protein, enzyme, or antibody is a polypeptide polymer that contains 70% or more of its complete sequence (as found in nature), e.g., 75% or more, e.g., 80% or more, e.g., 85% or more, e.g., 90% or more, e.g., 95% or more, e.g., 97% or more, e.g., 99% or more, and has 100% of the full length sequence of the protein, enzyme, or antibody (as found in nature).

[0085] In some embodiments, the biopolymer is an antibody. The antibodies in question include, but are not limited to, monoclonal antibodies, polyclonal antibodies, bispecific antibodies, Fab antibody fragments, F(ab)2 antibody fragments, Fv antibody fragments (e.g., VH or VL), single-chain Fv antibody fragments, and dsFv antibody fragments. Furthermore, the antibody molecule may be a fully human antibody, a humanized antibody, or a chimeric antibody. The antibody may contain any mature or untreated antibody variable region bound to any immunoglobulin constant region. Minor changes in the amino acid sequence of an antibody or immunoglobulin molecule are included in this disclosure, provided that the changes in the amino acid sequence maintain 75% or more of the sequence, e.g., 80%, 90%, 95%, or 99% or more.

[0086] Antibody fragments can be a portion of an intact antibody, such as the antigen-binding region or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062 (1995)), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody yields two identical antigen-binding fragments, each with a single antigen-binding site, called "Fab" fragments, and a residual "Fc" fragment, a name reflecting its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment, which has two antigen-binding sites but is capable of cross-binding antigens. "Fv" is the smallest antibody fragment containing a complete antigen recognition site and an antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in a tightly covalently bound state. It is in this configuration that the three complementarity-determining regions (CDRs) of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. A total of six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of Fv containing only three CDRs specific to a particular antigen) has the ability to recognize and bind to an antigen, although its affinity is lower than that of the entire binding site. The "Fab" fragment also includes the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment in that it has several additional residues at the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' where the cysteine ​​residues in the constant domain have free thiol groups. The F(ab')2 antibody fragment was originally generated as a pair of Fab' fragments with hinge cysteines between them. Other chemical bonds in antibody fragments are also known. The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins can also be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chain.There are five major classes of immunoglobulins, namely IgA, IgD, IgE, IgG, and IgM, and some of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0087] A "single-stranded Fv" or "sFv" antibody fragment contains the VH and VL domains of an antibody present in a single polypeptide chain. In some embodiments, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, enabling the sFv to form a structure desirable for antigen binding. For an overview of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315 (1994).

[0088] Therefore, antibodies that can be used in connection with this disclosure may include monoclonal antibodies, polyclonal antibodies, bispecific antibodies, Fab antibody fragments, F(ab)2 antibody fragments, Fv antibody fragments (e.g., VH or VL), single-chain Fv antibody fragments, and dsFv antibody fragments. Furthermore, the antibody molecule may be a fully human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody molecule is a fully human monoclonal antibody.

[0089] Antibodies usable in connection with this disclosure may include any mature or untreated antibody variable region conjugated to any immunoglobulin constant region. The light chain variable region may be a kappa chain constant region, if conjugated to a constant region. The heavy chain variable region may be a human gamma 1, gamma 2, gamma 3, or gamma 4 constant region, more preferably gamma 1, gamma 2, or gamma 4, and even more preferably gamma 1 or gamma 4, if conjugated to a constant region.

[0090] Minor changes in the amino acid sequence of an antibody or immunoglobulin molecule are included in the present invention, provided that the change in amino acid sequence maintains at least 75% of the sequence, e.g., at least 80%, 90%, 95%, or 99%. In particular, conservative amino acid substitutions (as described herein, for example) are intended. Whether a functional peptide results from an amino acid change can be readily determined by assaying the specific activity of the polypeptide derivative. Fragments (or analogues) of antibody or immunoglobulin molecules can be readily prepared by those skilled in the art. Preferred amino and carboxyl termini of the fragment or analogue are located near the boundaries of the functional domain. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data with publicly available or proprietary sequence databases. Preferably, computer-based comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins with known structure and / or function. Methods are known to identify protein sequences that fold into known three-dimensional structures. Sequence motifs and structural conformations can be used to define structural and functional domains according to the present invention.

[0091] In a particular embodiment, the two-color imaging composition of the subject is adecatumumab, ascrimbakumab, sixtumumab, conatumumab, daratumumab, doroditumumab, durigotumab, durvalumab, dusigitumab, enfortumab, enoticumab, figitumumab, ganitumumab, glenbatumumab, intetumumab, ipilimumab, iratumumab, iculcumab, lexatumumab, lucatumumab, mapatumumab, nalunatumumab, nesitumumab, nesbakumab, ofatumumab, oraratumumab, panitumumab, patritumumab, pritumumab, radretumab Ramucirumab, rilotumumab, lobatumumab, cerivantumab, tarextumumab, teprotumumab, tobetumumab, vanchikutumumab, besencumumab, botumumab, zaltumumab, frambotumab, artumomab, anatumomab, alsitumomab, vectumomab, blinatumomab, detumomab, ibritumomab, minretumomab, mitumomab, moxetumomab, naptumomab, nofetumomab, pemtumomab, pintumomab, lakotumomab, satumomab, solitomab, tapritumomab, tenatumomab, tositumomab, tremelimumab, avagobomab, igobo Mab, olegobomab, capromab, edrecolomab, nacolomab, amatuximab, bavituximab, bevacizumab, brentuximab, cetuximab, delrotuximab, dinutuximab, encituximab, futuximab, gilentuximab, indatuximab, isatuximab, margetuximab, rituximab, siltuximab, ubrituximab, eclomeximab, abituzumab, alemtuzumab, vibatuzumab, brontuxuzumab, cantuzumab, sitatuzumab, cribatuzumab, dacetuzumab, demcizumab, darotuzumab, denintz Zumab, elotuzumab, emuctuzumab, emibetuzumab, enobrituzumab, etalasizumab, falletuzumab, ficratuzumab, gemtuzumab, imugatuzumab, inotuzumab, rabetuzumab, rifastuzumab, lintuzumab, rorbotuzumab, lumretuzumab, matsuzumab, miratuzumab, nimotuzumab, obinutuzumab, okalatuzumab, otreltuzumab, onartuzumab, oporutuzumab, panitumumab, pulsatuzumab, pertuzumab, pinatuzumab, polatuzumab, ranibizumab, sibrotuzumab, simtuzumab, takatuzumab,Tigatuzumab, trastuzumab, tucotzumab, bundletuzumab, vanucizumab, beltuzumab, borsetuzumab, sofituzumab, katumakisomab, erzmakisomab, depatuxizumab, ontuxizumab, bronzbetomab, tamzbetomab, bevacizumab, ranibizumab, trastuzumab, infliximab, adalimumab, efalizumab, gemtuzumab The present invention comprises a detectable labeled biomolecular conjugate having an antibody selected from zumab-ozogamisin, tositumomab, ibritumomab, tiuxetan, eculizumab, alemtuzumab, rituximab, abisiximab, cetuximab, daclizumab, basiliximab, gemtuzumab, natalizumab, omalizumab, and palivizumab, or their antigen-binding variants. As used herein, the term "variant" refers to an antibody that binds to a specific alloantigen but has fewer or more amino acids than the parent antibody, has one or more amino acid substitutions relative to the parent antibody, and is a single-chain variant of the parent antibody (e.g., scFv variant) or any combination thereof.

[0092] As described in detail above, the conjugates in the subject composition include detectable labels that are detectable portions or markers based on, for example, fluorescence emission, absorbance, fluorescence polarization, fluorescence lifetime, fluorescence wavelength, maximum absorbance, absorbance wavelength, Stokes shift, light scattering, mass, molecular weight, redox, sound absorption, Raman, magnetism, radio frequency, enzymatic reactions (including chemiluminescence and electrochemiluminescence), or combinations thereof. In the two-color imaging composition of the subject, the detectable label of each conjugate compound may be a fluorophore, chromophore, enzyme, redox label, radioactive label, sound absorption label, Raman (SERS) tag, mass tag, isotope tag (e.g., isotopically pure rare earth elements), magnetic particles, fine particles, and nanoparticles. In certain embodiments, the detectable label of each conjugate compound in the two-color imaging composition is a dye, for example, an organic dye or an inorganic dye (e.g., the dyes described in detail above).

[0093] Method for delivering activators to nerve cells As outlined above, aspects of the present disclosure also include methods for delivering an activator conjugate to neurons by contacting the neurons (e.g., in vitro or in vivo) with a composition comprising a neurotrophin receptor-binding conjugate having one or more activator compounds (e.g., dyes or small molecule therapeutics) covalently bound to a protein, peptide or peptide mimetic that selectively binds to neurotrophin receptors in neurons. As described above, the compositions of the subject matter are also intended to be chimeric proteins of NGF bound to TrkA and brain-derived neurotrophic factor (BDNF) bound to TrkB, and these chimeras are used for topical application to the eye to promote both transocular transport and binding to TrkB receptors expressed on the posterior side of the vitreous humor on retinal ganglion cells.

[0094] In some embodiments, the composition brought into contact with nerve cells has an average ratio of 5 or less of the activator compound to the protein, peptide, or peptide mimetic in the conjugate. In certain embodiments, the average ratio of the activator component to the protein, peptide, or peptide mimetic component is 3.2 or less, for example, 2 or less, for example, about 0.95 to about 1.85. In some cases, nerve cells come into contact with a composition in which 90% or more (e.g., 95% or more) of the activator conjugate has an average ratio of 5 or less of the activator component to the protein, peptide, or peptide mimetic component, for example, 90% or more (e.g., 95% or more) of the activator conjugate in the composition has an average ratio of 2 or less of the activator component to the protein, peptide, or peptide mimetic component.

[0095] Aspects of this disclosure also include methods for treating a subject with one or more compositions of the subject. In describing the methods of this disclosure, the term “subject” means a human or organism to which the conjugate compound is administered. Thus, subjects in this disclosure include, but are not limited to, mammals, e.g., humans and other primates, e.g., chimpanzees and other apes and monkey species, dogs, rabbits, cats and other domestic pets, and in certain embodiments, the subject is human. The term “subject” is also intended to include humans or organisms of any age, weight or other physical characteristics, and the subject may be an adult, child, infant or neonatal. A method according to a particular embodiment involves administering to a subject a composition comprising a neurotrophin receptor-binding conjugate having one or more activating compounds (e.g., dyes or small molecule therapeutic agents) covalently bound to a protein, peptide or peptide mimetic that selectively binds to neurotrophin receptors on the subject’s nerve cells. In some cases, the composition is administered intraocularly to the subject (e.g., topically or intravitreally to the surface or just below the surface of the eye). In other cases, the composition is administered intracapsularly to the subject. In yet another case, the composition is administered to the subject intrathecally. In yet another case, the composition is administered to the subject intravitreally. In yet another case, the composition is administered to the subject topically or transdermally. In yet another case, the composition is administered to the subject by injection, such as subcutaneous, intramuscular, intravitreous, intraocular, intracapsular, or intrathecal injection. In yet another case, once administered, the composition can exert a sustained-release effect in nerve tissue due to the pharmacological properties of the drug in the case of direct delivery to nerve cell bodies or signaling effects, and / or due to the biological properties of neurotrophin by direct delivery by one or two possible transport mechanisms and / or in a "steady state".

[0096] In certain embodiments, the protocol may include multiple dosing intervals. "Multiple dosing intervals" means that the conjugate compound composition is administered to the subject two or more times sequentially. In practicing the method of the present disclosure, the treatment regimen may include two or more dosing intervals, for example, three or more dosing intervals, for example, four or more dosing intervals, for example, five or more dosing intervals including ten or more dosing intervals.

[0097] The interval between doses in a multi-dose treatment protocol may vary depending on the subject's physiological condition or the treatment protocol determined by the medical professional. For example, the interval between doses in a multi-dose treatment protocol may be a predetermined time and may follow a regular interval. Therefore, the interval between doses may vary and may include periods of 1 day or more, e.g., 2 days or more, e.g., 4 days or more, e.g., 6 days or more, e.g., 8 days or more, e.g., 12 days or more, e.g., 16 days or more, and 24 days or more. In a particular embodiment, the multi-dose protocol may have an interval of 5 weeks or more, including 1 week or more, e.g., 2 weeks or more, e.g., 3 weeks or more, e.g., 4 weeks or more, e.g., 6 weeks or more.

[0098] In certain embodiments, the compositions of the present invention may be administered before, simultaneously with, or after other therapeutic agents for treating the same or distinct symptoms. When provided simultaneously with another therapeutic agent, the composition having the subject conjugate compound may be administered in the same or a different composition. Thus, the subject anti-cancer neurotrophin-binding conjugate composition and other therapeutic agents may be administered to a patient in combination therapy. "Combination therapy" ensures that the intended administration to a subject produces the therapeutic effect of the combination of substances in the subject receiving treatment. For example, combination therapy can be achieved by administering the anti-cancer neurotrophin-binding conjugate composition of the present invention together with at least one other agent, for example, a pharmaceutical composition having an anti-inflammatory agent, immunosuppressant, steroid, analgesic, anesthetic, antihypertensive agent, or chemotherapeutic agent, among other types of therapeutic agents, which together constitute a therapeutically effective dose in accordance with a particular administration regimen. Administration of individual pharmaceutical compositions may be carried out simultaneously or at different times (i.e., sequentially on the same day or on different days, in any order) as long as the therapeutic effect of the combination of these substances is produced in the subject receiving treatment.

[0099] Method for intraocular delivery of activator conjugates to nerve cells As outlined above, aspects of this disclosure also include methods for intraocular delivery of activating agent conjugates to nerve cells. A neurotrophin receptor-binding conjugate composition according to one embodiment comprises one or more activating agent compounds (e.g., the dyes or small molecule therapeutics described above) covalently bound to a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors. In practicing the subject method according to this embodiment, a composition having one or more of the conjugate compounds described herein is brought into contact with the eye of a subject. In some embodiments, the eye is brought into contact locally, for example, by bringing the composition into contact with the corneal surface of the eye. In some cases, the eye is brought into contact locally with the composition using a syringe, for example, during a surgical procedure. In other embodiments, the composition is brought into contact with the eye using a contact lens containing the subject composition. In these embodiments, the contact lens may contain a coating or it may contain the composition having one or more of the activating agent conjugates described herein as is. In other embodiments, the composition is implanted in the eye of a subject, for example, by intravitreal implantation. In these embodiments, the composition may be injected intravitreally into the eye, for example, using a needle and syringe. In other embodiments, the composition may be administered intravitreally during a surgical procedure, for example, during a vitrectomy. In other embodiments, the composition may be placed postoperatively, and the procedure may be an intravitreal procedure, a local procedure, or a combination thereof.

[0100] The composition may be in contact with the eyes over a period of time including, for example, 0.01 minutes or more, 0.05 minutes or more, 0.1 minutes or more, 0.5 minutes or more, 1 minute or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 30 minutes or more, 60 minutes or more, 2 hours or more, 4 hours or more, 6 hours or more, 12 hours or more, 24 hours or more, and 168 hours or more.

[0101] In some embodiments, the composition is a sustained-release composition configured to deliver an activating agent conjugate intraocularly to a subject's eye over a predetermined period, including, for example, 1 minute or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 30 minutes or more, 60 minutes or more, 2 hours or more, 4 hours or more, 6 hours or more, 12 hours or more, 24 hours or more, and 168 hours or more. In certain embodiments, the composition is formulated to deliver an activating agent conjugate intraocularly to a subject's eye over a period of 1 month or more.

[0102] In other embodiments, the composition is formulated as an immediate-release composition to release 50% or more of the activator conjugate or a pharmaceutically acceptable salt thereof within 10 minutes after administration of the composition to a subject, for example, 60% or more, e.g., 75% or more, e.g., 90% or more, e.g., 95% or more, and 99% or more within 10 minutes after administration of the composition to a subject. In specific cases, the composition is formulated to release 50% or more of the activator conjugate or a pharmaceutically acceptable salt thereof immediately after contact with the subject's eyes, for example, 60% or more, e.g., 75% or more, e.g., 90% or more, e.g., 95% or more, and 99% or more immediately after contact with the subject's eyes.

[0103] In some embodiments, the subject composition is formulated for delayed-immediate release, and its effect may be due to a combination of one or more factors, including but not limited to the formulation, direct or indirect delivery characteristics of the small molecule therapeutic agent, and / or pharmacokinetics related to the absorption rate of the neurotrophin-Trk component of the composition. "Delayed-immediate release" means that the composition is formulated so that the release of the activator conjugate is delayed over a predetermined period, after which the activator conjugate is released immediately. In some cases, the predetermined delay period may include periods of 5 minutes or more, e.g., 10 minutes or more, e.g., 15 minutes or more, e.g., 20 minutes or more, and 30 minutes or more. In some embodiments, the delayed-immediate release composition is formulated so that 20% or less of the activator conjugate in the composition is released about 20 minutes after contact with the subject's eye, and 75% or more of the activator conjugate in the composition is released about 30 minutes later. In these embodiments, less than 20% of the activator conjugate in the composition is released approximately 20 minutes after administration to a subject, for example, less than 15%, e.g., less than 10%, e.g., less than 5%, e.g., less than 3%, and less than 1%, of the activator conjugate is released approximately 20 minutes after administration to a subject. Delayed-release activator conjugates are formulated to release more than 75%, e.g., more than 80%, e.g., more than 85%, e.g., more than 90%, e.g., more than 95%, e.g., more than 97%, and more than 99%, of the activator conjugate after a predetermined delay period (e.g., 20 minutes) and then approximately 30 minutes later. In certain embodiments, the composition is formulated to release 100% of the activator conjugate after a predetermined delay period has elapsed after administration.

[0104] In these embodiments, the subject composition may be formulated to contain one or more components that give a desired release profile. In certain embodiments, the formulation contains one or more non-erosive polymers. In certain cases, the subject composition is coated with one or more polymers that give a desired release profile. In other embodiments, the formulation contains one or more bio-erosive polymers. For example, the formulation may contain one or more polymers, such as cellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, maltodextrin, sucrose, modified starch, alginate, soluble gum, carrageenan, polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone (PVPP), ethylcellulose, cellulose phthalate acetate, hydroxypropylmethylcellulose phthalate, insoluble gum, polymethacrylate, polyvinyl alcohol, shellac, polyvinyl phthalate acetate, and combinations thereof.

[0105] The dosage used to treat a subject will vary depending on the clinical goal to be achieved, but the preferred dosage range for conjugate compounds is approximately 0.0001 mg to approximately 5000 mg of the active ingredient per single dose, for example, approximately 1 mg to approximately 25 mg, approximately 25 mg to approximately 50 mg, approximately 50 mg to approximately 100 mg, approximately 100 mg to approximately 200 mg, approximately 200 mg to approximately 250 mg, approximately 250 mg to approximately 500 mg, approximately 500 mg to approximately 1000 mg, or approximately 1000 mg to approximately 5000 mg. It will be readily understood by those skilled in the art that dosage levels may vary depending on the specific compound, the severity of the symptoms, and the intended beneficial pharmacological effect, and / or the subject's susceptibility and / or sensitivity to intended or unintended side effects.

[0106] In some embodiments, preferred doses of the activator conjugate are in the range of approximately 1 mg to 500 mg per kg of body weight, for example, approximately 5 mg to 500 mg per kg, approximately 10 mg to 500 mg per kg, approximately 20 mg to 500 mg per kg, approximately 30 mg to 500 mg per kg, approximately 40 mg to 500 mg per kg, approximately 50 mg to 500 mg per kg, approximately 60 mg to 500 mg per kg, approximately 70 mg to 500 mg per kg, approximately 80 mg to 500 mg per kg, approximately 90 mg to 500 mg per kg, approximately 100 mg to 500 mg per kg, approximately 200 mg to 500 mg per kg, approximately 300 mg to 500 mg per kg, or approximately 400 mg to 500 mg per kg.

[0107] In some embodiments, preferred doses of the activator conjugate are in the range of approximately 1 mg to 5 mg per kg of body weight, approximately 5 mg to 10 mg per kg of body weight, approximately 10 mg to 20 mg per kg of body weight, approximately 20 mg to 30 mg per kg of body weight, approximately 30 mg to 40 mg per kg of body weight, approximately 40 mg to 50 mg per kg of body weight, approximately 50 mg to 100 mg per kg of body weight, or approximately 100 mg to 500 mg per kg of body weight.

[0108] In some embodiments, the activator conjugate is administered as a single dose. In other embodiments, the conjugate compound is administered multiple times. When administered multiple times over a period of time, the conjugate compound is administered, for example, twice daily (qid), once daily (qd), every other day (qod), every three days, three times a week (tiw), or twice a week (biw) over a period of time. For example, the conjugate compound is administered qid, qd, qod, tiw, or biw over a period of time ranging from one day to approximately two years or more. For example, the conjugate compound is administered at any of the above frequencies over a period of one week, two weeks, one month, two months, six months, one year, or two years or more, depending on various factors.

[0109] In certain embodiments, the protocol may include multiple dosing intervals. "Multiple dosing intervals" means that the conjugate compound composition is administered to the subject two or more times sequentially. In practicing the methods of this disclosure, the treatment regimen may include two or more dosing intervals, e.g., three or more dosing intervals, e.g., four or more dosing intervals, e.g., five or more dosing intervals including ten or more dosing intervals.

[0110] The interval between doses in a multi-dose therapy protocol may vary depending on the physiological state of the subject or the therapy protocol determined by the medical professional. For example, the interval between doses in a multi-dose therapy protocol may be a predetermined time and follow a regular interval. Thus, the interval between doses may vary and may include periods of 1 day or more, e.g., 2 days or more, e.g., 4 days or more, e.g., 6 days or more, e.g., 8 days or more, e.g., 12 days or more, e.g., 16 days or more, and 24 days or more. In a particular embodiment, a multi-dose protocol may have intervals of 5 weeks or more, including 1 week or more, e.g., 2 weeks or more, e.g., 3 weeks or more, e.g., 4 weeks or more, e.g., 6 weeks or more. In a particular embodiment, the subject composition is formulated to deliver the activator conjugate to the subject (e.g., by implantation, e.g., intravitreal implantation) over a long period of 5 years or more, including e.g., 3 months or more, e.g., 6 months or more, e.g., 9 months or more, e.g., 1 year or more, e.g., 10 years or more.

[0111] Method for administering a two-color imaging composition Aspects of the present disclosure also include a method of contacting a subject's tissue with one or more of the two-color imaging compositions described above. In certain embodiments, the subject's tissue contacted with the subject's composition is neoplastic tissue. In other embodiments, the subject's tissue contacted with the subject's composition is normal tissue. In certain embodiments, the tissue contacted is ocular tissue. A method according to a particular embodiment involves administering to a subject a composition comprising a first detectable label-biomolecular conjugate having a detectable label covalently bonded to a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors, and a second detectable label-biomolecular conjugate having a second detectable label covalently bonded to a biopolymer that selectively binds to neoplastic tissue. In some cases, the composition is administered topically to the subject during surgery. In other cases, the composition may be administered intraocularly to the subject. In other cases, the composition is administered intracapsularly to the subject. In yet another case, the composition is administered intrathecally to the subject. In yet another case, the composition is administered intravitreally to the subject. In yet another case, the composition is administered topically or transdermally to the subject. In other cases, the composition is administered to the subject by injection, for example, subcutaneous injection, intramuscular injection, intravitreous injection, intracapsular injection or intrathecal injection.

[0112] In some embodiments, the subject composition enables two-color imaging of nerve tissue and neoplastic tissue, for example, during surgical procedures. In specific cases, the surgical procedure is a tissue resection procedure, such as tumor resection surgery. In certain embodiments, the method comprises administering a composition to a subject during surgery, comprising a first detectable label-biomolecular conjugate having a detectable label covalently bound to a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors, and a second detectable label-biomolecular conjugate having a second detectable label covalently bound to a biopolymer that selectively binds to receptors in neoplastic tissue, and detecting the nerve imaging component and the neoplastic tissue imaging component. The imaging components of the nerve imaging component and the neoplastic tissue imaging component may be visualized by the same or different protocols. For example, the imaging components of the nerve imaging component and the neoplastic tissue imaging component may each be visualized independently, either by the naked eye, by spectral detection, such as ultraviolet light detection, infrared or near-infrared (NIR) light detection, or in a fluorescence spectrum below the NIR spectrum.

[0113] In some embodiments, the composition is administered to the subject at times including, for example, 5 minutes or more before, 10 minutes or more before, 15 minutes or more before, 30 minutes or more before, 60 minutes or more before, and 120 minutes or more before a surgical procedure. In certain embodiments, the composition is administered to the subject at the time of the surgical procedure.

[0114] Method for preparing a conjugate compound composition Aspects of this disclosure also include methods for preparing the subject composition. A method according to a particular embodiment includes contacting an activator compound with a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors to produce a composition having an activator conjugate containing one or more activator compounds covalently bound to the protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors, and isolating the conjugate having an average ratio of the activator compound to the protein, peptide, or peptide mimetic of 5 or less. In a particular embodiment, the composition prepared by the method of the subject has an average ratio of the activator compound to the protein, peptide, or peptide mimetic in the conjugate of 2 or less, for example, about 0.95 to about 1.85. In some cases, in compositions prepared by the method of the subject, 90% or more (e.g., 95% or more) of the activator conjugates have an average ratio of 5 or less between the activator component and the protein, peptide, or peptide mimetic component. For example, 90% or more (e.g., 95% or more) of the activator conjugates in the composition have an average ratio of 2 or less between the activator component and the protein, peptide, or peptide mimetic component.

[0115] In certain embodiments, the method includes contacting an activator with a linker precursor to generate an activated activator, and contacting the activated activator with a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors. In some embodiments, the linker precursor may be a zero-length cross-linker precursor, a homobifunctional linker precursor, a heterobifunctional linker precursor, or a trifunctional cross-linker precursor. In certain embodiments, the activator is contacted with a bifunctional linker precursor, such as a homobifunctional or heterobifunctional linker precursor, to generate an activated activator. In some embodiments, the bifunctional linker precursor includes succinimide, for example, when the bifunctional linker precursor is N,N'-disuccinimidyl carbonate. The linker precursor is contacted with the activator and the reactive portion of the activator. The linker may be used to conjugate the activator component to the protein, peptide, or peptide mimetic component by an N-terminal or C-terminal amino acid of the protein, peptide, or peptide mimetic. In some embodiments, the linker is bound to a mutant amino acid of a native protein, peptide, or peptide mimetic. The type of bond used to attach the linker to a protein, peptide, or peptide mimetic may be an ether bond, a disulfide bond, or an amino bond. For example, the linker may be attached to a lysine residue of the protein, peptide, or peptide mimetic. The linker may be attached to a carbon atom or a non-carbon atom of the protein, peptide, or peptide mimetic. In one example, the linker is attached to a carbon atom of the protein, peptide, or peptide mimetic. In another example, the linker is attached to a non-carbon atom of the protein, peptide, or peptide mimetic, such as a nitrogen atom or a sulfur atom. The reactive group may be nucleophilic or electrophilic depending on the reactive group of the linker precursor. In some cases, the activator includes a nucleophilic reactive group, such as a hydroxyl group. In certain cases, the activator includes a primary hydroxyl group.A method according to a particular embodiment involves functionalizing an activator to include a reactive group, such as a nucleophilic reactive group (e.g., a hydroxyl group) for reacting with a linker precursor component. Linking the linker precursor with the activator generates an activated activator. The activator is then contacted with a protein, peptide, or peptide mimetic that selectively binds to nerve cells (e.g., neurotrophin receptors) to generate a conjugate compound of the composition of the subject.

[0116] A method for preparing the subject composition includes isolating the activator conjugate from the composition. In some embodiments, isolating the activator conjugate includes separating the activator conjugate of interest by reverse-phase high-performance liquid chromatography (RP-HPLC). In certain embodiments, the method includes isolating the activator conjugate from a composition in which the average ratio of the activator compound to a protein, peptide, or peptide mimetic is 5 or less. In some embodiments, the method includes purifying the composition to produce a composition having a conjugate in which a predetermined ratio of the activator compound to a protein, peptide, or peptide mimetic is, for example, 3.2 or less, for example, 2 or less, for example, about 0.95 to about 1.85. In some cases, the method includes purification to produce a composition in which 90% or more (e.g., 95% or more) of the activator conjugate have an average ratio of 5 or less between the activator component and the protein, peptide, or peptide mimetic component; for example, 90% or more (e.g., 95% or more) of the activator conjugate in the composition have an average ratio of 3.2 or less between the activator component and the protein, peptide, or peptide mimetic component; for example, 90% or more (e.g., 95% or more) of the activator conjugate in the composition have an average ratio of 2 or less between the activator component and the protein, peptide, or peptide mimetic component. The term “purified” is used in its conventional sense to refer to a composition in which at least some isolation or purification process, e.g., filtration or aqueous treatment of the reaction mixture, has been performed. In specific cases, purification includes liquid chromatography, recrystallization, distillation (e.g., azeotropic distillation) or other types of compound purification.

[0117] The nature of this disclosure The aspects of the subject matter described herein, including embodiments, may be useful individually or in combination with one or more other aspects or embodiments. Certain non-limiting aspects of this disclosure, numbered 1 to 194, are described below, without limitation. Each of the individually numbered aspects may be used individually or in combination with any of the preceding or succeeding individually numbered aspects, as will be apparent to those skilled in the art by reading this disclosure. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects specified below. 1. A composition comprising an activator conjugate containing one or more activator compounds covalently bonded to a protein, peptide, or peptide mimetic that selectively binds to a neurotrophin receptor, is taken up, and enables intracellular translocation (e.g., retrograde axonal transport), A composition in which the average ratio of the activator compound to the protein, peptide, or peptide mimetic in the activator conjugate is 5 or less. 2. The composition according to claim 1, wherein 95% or more of the activator conjugates in the composition have an average ratio of 5 or less between the activator compound and a protein, peptide, or peptide mimetic. 3. The composition according to any one of 1 to 2, wherein the average ratio of the activator compound to the protein, peptide, or peptide mimetic in the activator conjugate in the composition is 3.2 or less. 4. The composition according to any one of 1 to 2, wherein the average ratio of the activator compound to the protein, peptide, or peptide mimetic in the activator conjugate in the composition is 2 or less. 5. The composition according to 4, wherein the ratio of the activator compound to the protein, peptide, or peptide mimetic in the activator conjugate is about 0.95 to about 1.85. 6. The composition according to 4, wherein 90% or more of the activator conjugates in the composition have an average ratio of 2 or less between the activator compound and a protein, peptide, or peptide mimetic. 7. The composition according to 4, wherein 95% or more of the activator conjugates in the composition have an average ratio of 2 or less between the activator compound and a protein, peptide, or peptide mimetic. 8. A composition according to any one of 1 to 7, wherein the protein, peptide, or peptide mimetic is selected from the group consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), neurotrophic factor 4 / 5 (NT-4 / 5), neurotrophic factor 6 (NT-6), neurotrophic factor 7 (NT-7), glial cell-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and derivatives or fragments thereof. 9. The composition according to 8, wherein the protein, peptide, or peptide mimetic is a neurotrophic factor derived from an animal other than a human. 10. The composition according to 8, wherein the protein, peptide, or peptide mimetic is a recombinant human neurotrophic factor. 11. The composition according to 8, wherein the protein, peptide, or peptide mimetic comprises the β-subunit of NGF, rhNGF, or rhBDNF, or a chimeric construct of rhNGF and rhBDNF, or a chimeric construct of rhNGF and any other neurotrophic factor, or a chimeric construct between any two other neurotrophic factors. 12. A composition according to any one of 1 to 11, wherein the activator is a dye. 13. The composition according to 12, wherein the activator is an organic dye or an inorganic dye. 14. The composition according to 12, wherein the dye is selected from the group consisting of body pie dyes, coumarin dyes, rhodamine dyes, acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes, chlorophyll-containing dyes, triarylmethane dyes, azo dyes, diazonium dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone-imine dyes, azine dyes, eurodin dyes, safranin dyes, indamine, indophenol dyes, fluorine dyes, oxazine dyes, oxazone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronin dyes, fluorine dyes, rhodamine dyes, phenanthoridine dyes, squalane, body pie, squaline roxitan, naphthalene, coumarin, oxadiazole, anthracene, pyrene, acridine, arylmethine or tetrapyrrole, and combinations thereof. 15. The composition according to 12, wherein the dye has an emission wavelength of 300 nm or more. 16. The composition according to 12, wherein the dye is Alexa Fluor 488. 17. The composition according to 12, wherein the dye is Dynamics DY-800. 18. The composition according to 12, wherein the dye is ZW-800. 19. The composition according to 12, wherein the dye is a compound having axial symmetry. 20. The composition according to 12, wherein the dye is a compound having a plane of symmetry. 21. The composition according to 12, wherein the dye is a compound that promotes the visualization of the dye within a neuronal cell, and the visualization within the neuronal cell is enabled by binding of Trk receptors in microtubules and / or in the nerve sheath surrounding the nerve, endocytosis and retrograde axonal transport. 22. The composition according to any one of 1 to 11, wherein the activator is a small molecule activator. 23. A composition according to any one of 1 to 11, wherein the activator is an anticancer agent. 24. The composition according to any one of 1 to 11, wherein the activator is a mammalian target of a rapamycin (mTOR) inhibitor or a mitogen-activated protein kinase (MEK) inhibitor. 25. The composition according to any one of 1 to 11, wherein the activator is a compound selected from the group consisting of glucocorticoids, heat shock protein inhibitors, checkpoint inhibitors, and chemokines / chemokine ligand inhibitors. 26. The composition according to 25, wherein the glucocorticoid is fluocinolone acetonide. 27. The composition according to any one of 1 to 11, wherein the activator is a compound for the treatment of glioma, skin cancer, or perineural invasion. 28. The composition according to any one of 1 to 27, wherein the activator conjugate further comprises a linker that covalently bonds the activator compound to a protein, peptide, or peptide mimetic. 29. The composition according to 28, wherein the linker is selected from the group consisting of a bifunctional linker, a homobifunctional linker, and a heterobifunctional linker. 30. A composition according to any one of 1 to 29, wherein a protein, peptide, or peptide mimetic is bound to an activator by internal amino acid residues. 31. The composition according to 30, wherein the above amino acid residues are present on the surface of a protein, peptide, or peptide mimetic. 32. The composition according to any one of 1 to 29, wherein the activator is bound to the N-terminal or C-terminal amino acid of a protein, peptide, or peptide mimetic. 33. The composition according to any one of 28 to 32, wherein the linker is configured to bind to a mutant amino acid (e.g., a substituted amino acid or a non-natural amino acid) in a protein, peptide, or peptide mimetic, or to a reactive side chain of an amino acid in a natural protein, peptide, or peptide mimetic. 34. The composition according to any one of 28 to 33, wherein the linker is configured to bind to a protein, peptide or peptide mimetic by an ether bond, ester bond, carbamate bond, amide bond, disulfide bond or amino bond. 35. The composition according to any one of 1 to 34, wherein the activating compound is covalently bonded to a lysine residue of a protein, peptide, or peptide mimetic. 36. The composition according to any one of 1 to 34, wherein the linker is covalently bonded to the activator by the carbon of the activator. 37. The composition according to any one of 1 to 34, wherein the linker is covalently bonded to the activator by a non-carbon of the activator. 38. The composition according to 37, wherein the linker is covalently bonded to the activator by the nitrogen of the activator. 39. The composition according to any one of 1 to 34, wherein the linker is covalently bonded to the activator by the sulfur of the activator. 40. The composition according to any one of 1 to 39, wherein the activator conjugate is functionally capable of dimerization of a native protein, enabling endocytosis of the dye and visualization within nerve cells. 41. The composition according to any one of claims 1 to 40, wherein the activator conjugate promotes the binding of a protein, peptide, or peptide mimetic that selectively binds to a neurotrophin receptor. 42. A composition according to any one of claims 1 to 40, wherein the activator conjugate promotes endocytosis. 43. A composition according to any one of claims 1 to 40, wherein the activator conjugate promotes intracellular transport of nerve cells. 44. A method for delivering an activator conjugate to nerve cells, comprising contacting the nerve cells with a composition described in any of 1 to 43. 45. A method for selectively delivering an activator conjugate to nerve cells, comprising administering a composition according to any one of 1 to 43 to a subject. 46. ​​The method of 45, wherein the composition is administered intraocularly to a subject. 47. The method of 45, wherein the composition is administered intracapsularly to a subject. 48. The method of 45, wherein the composition is administered intrathecally to a subject. 49. The method of 45, wherein the composition is administered intravitreally to a subject. 50. The method according to 45, wherein the composition is administered topically. 51. The method according to 45, wherein the composition is administered by subcutaneous injection, intramuscular injection, intravitreous injection, intracapsular injection, intrathecal injection, or a combination thereof. 52. To produce a composition comprising an activator conjugate containing one or more activator compounds covalently bound to a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors, by contacting an activator compound with the protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors, is endocytotic, and enables intracellular translocation (e.g., retrograde axonal transport), and To isolate an activator conjugate from a composition in which the average ratio of the activator compound to a protein, peptide, or peptide mimetic is 5 or less. A method that includes this. 53. The method according to 52, comprising isolating an activator conjugate from a composition in which the average ratio of the activator compound to a protein, peptide, or peptide mimetic is 0.5 to 5. 54. The method according to any one of 52 to 53, wherein 95% or more of the activator conjugate in the composition has an average ratio of 5 or less between the activator compound and a protein, peptide, or peptide mimetic. 55. The method according to any one of 52 to 53, wherein the average ratio of the activator compound to the protein, peptide, or peptide mimetic in the activator conjugate in the composition is 3.2 or less. 56. The method according to any one of 52 to 54, comprising isolating an activator conjugate from a composition in which the average ratio of the activator compound to a protein, peptide, or peptide mimetic is 2 or less. 57. The method according to 56, comprising isolating an activator conjugate from a composition in which the average ratio of the activator compound to a protein, peptide, or peptide mimetic is about 0.95 to about 1.85. 58. The method according to any one of 56 to 57, wherein 90% or more of the activator conjugates in the isolated composition have an average ratio of 2 or less between the activator compound and a protein, peptide, or peptide mimetic. 59. The method according to any one of 56 to 57, wherein 95% or more of the activator conjugates in the isolated composition have an average ratio of 2 or less between the activator compound and a protein, peptide, or peptide mimetic. 60. The method according to any one of 52 to 59, wherein the protein, peptide, or peptide mimetic is selected from the group consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), neurotrophic factor 4 / 5 (NT-4 / 5), neurotrophic factor 6 (NT-6), neurotrophic factor 7 (NT-7), glial cell-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and derivatives or fragments thereof. 61. The method according to 60, wherein the protein, peptide, or peptide mimetic is a neurotrophic factor derived from a non-human animal. 62. The method according to 60, wherein the protein, peptide, or peptide mimetic is a recombinant human neurotrophic factor. 63. The method according to 60, wherein the protein, peptide, or peptide mimetic comprises the beta-subunit of NGF, rhNGF, or rhBDNF, or a chimeric construct of NGF and BDNF, or a chimeric construct of rhNGF and any other neurotrophic factor, or a chimeric construct between any two other neurotrophic factors. 64. The method according to any of 52-63, wherein the activator is a dye. 65. The method according to 64, wherein the activator is an organic dye or an inorganic dye. 66. The method according to 64, wherein the dye is selected from the group consisting of body pie dyes, coumarin dyes, rhodamine dyes, acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes, chlorophyll-containing dyes, triarylmethane dyes, azo dyes, diazonium dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone-imine dyes, azine dyes, eurodin dyes, safranin dyes, indamine, indophenol dyes, fluorine dyes, oxazine dyes, oxazone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronin dyes, fluorine dyes, rhodamine dyes, phenanthoridine dyes, squalane, body pie, squaline roxitan, naphthalene, coumarin, oxadiazole, anthracene, pyrene, acridine, arylmethine or tetrapyrrole, and combinations thereof. 67. The method according to 64, wherein the dye has an emission wavelength of 300 nm or more. 68. The method described in 64, wherein the dye is Alexa Fluor 488. 69. The method according to 64, wherein the dye is Dynamics DY-800. 70. The method described in 64, wherein the dye is ZW-800. 71. The method according to 64, wherein the dye is a compound having axial symmetry. 72. The method according to 64, wherein the dye is a compound having a plane of symmetry. 73. The method according to 64, wherein the dye is a compound that promotes the intracellular visualization of the dye, and the intracellular visualization is made possible by binding of Trk receptors in microtubules and / or in nerve sheaths surrounding nerves, endocytosis and retrograde axonal transport. 74. The method according to any of 52-63, wherein the activator is a small molecule activator. 75. The method according to any of 52-63, wherein the active agent is an anticancer drug. 76. The method according to any one of 52-63, wherein the activator is a mammalian target of a rapamycin (mTOR) inhibitor or a mitogen-activated protein kinase (MEK) inhibitor. 77. The method according to any one of 52 to 63, wherein the activator is a compound selected from the group consisting of glucocorticoids, heat shock protein inhibitors, checkpoint inhibitors, and chemokines / chemokine ligand inhibitors. 78. The method according to 77, wherein the glucocorticoid is fluocinolone acetonide. 79. The method according to any one of 52 to 63, wherein the activator is a compound for the treatment of glioma, skin cancer, or perineural invasion. 80. The method according to any one of 52 to 79, wherein the activator conjugate further comprises a linker that covalently bonds the activator compound to a protein, peptide, or peptide mimetic. 81. The method according to 80, wherein the linker is selected from the group consisting of a bifunctional linker, a homobifunctional linker, and a heterobifunctional linker. 82. The method according to any one of 52 to 81, wherein a protein, peptide, or peptide mimetic is bound to an activator by internal amino acid residues. 83. The method according to 82, wherein amino acid residues are present on the surface of a protein, peptide, or peptide mimetic. 84. The method according to any one of 52 to 83, wherein the activator is bound to the N-terminal or C-terminal amino acid of a protein, peptide, or peptide mimetic. 85. The method according to any one of 81 to 84, wherein the linker is configured to bind to a reactive side chain of a mutant amino acid in a protein, peptide, or peptide mimetic, or to an amino acid in a native protein, peptide, or peptide mimetic. 86. The method according to any one of 81 to 85, wherein the linker is configured to bind to a protein, peptide or peptide mimetic via an ether bond, ester bond, carbamate bond, amide bond, disulfide bond or amino bond. 87. The method according to any one of 52 to 86, wherein the activating compound is covalently bonded to a lysine residue of a protein, peptide, or peptide mimetic. 88. The method according to any one of 52 to 86, wherein the linker is covalently bonded to the activator by the carbon of the activator. 89. The method according to any one of 52 to 86, wherein the linker is covalently bonded to the activator by the non-carbon of the activator. 90. The method according to 89, wherein the linker is covalently bonded to the activator by the nitrogen of the activator. 91. The method according to any one of 52 to 86, wherein the linker is covalently bonded to the activator by the sulfur of the activator. 92. The method according to any one of 52-91, wherein the activator conjugate functionally enables intrinsic protein dimerization, which allows for endocytosis and intracellular visualization of the dye. 93. The method according to any one of 52 to 86, wherein the activator conjugate promotes the binding of a protein, peptide, or peptide mimetic that selectively binds to a neurotrophin receptor. 94. The method according to any one of 52-86, wherein the activator conjugate promotes endocytosis. 95. The method according to any one of 52-86, wherein the activator conjugate promotes intracellular transport of nerve cells. 96. A method for intraocular delivery of an activator to nerve cells, comprising bringing a subject's eye into contact with a composition containing an activator conjugate, wherein the activator conjugate comprises one or more activator compounds covalently bound to a protein, peptide or peptide mimetic that selectively binds to neurotrophin receptors, is endocytotic, and enables intracellular translocation (e.g., retrograde axonal transport). 97. The method according to 96, wherein the composition is brought into local contact with the surface of the eye. 98. The method according to 97, wherein the composition is brought into local contact with the corneal surface of the eye. 99. The method of 96, wherein the composition is implanted into the eye of a subject. 100. The method according to 99, wherein the composition is implanted intravitreally into the eye of a subject. 101. The method according to any one of 96 to 100, wherein the composition is formulated for the sustained release of an activator conjugate. 102. The method according to 101, wherein the composition is formulated for intraocular release of an activator conjugate over a period of seven days or more. 103. The method according to 101, wherein the composition is formulated for intraocular release of an activator conjugate over a period of more than one month. 104. The method according to any one of 96 to 100, wherein the composition is formulated for the delayed release of an activator conjugate. 105. The method according to any one of 96 to 100, wherein the composition is formulated for the delayed immediate release of an activator conjugate. 106. The method according to any one of 101 to 105, wherein the composition is formulated together with a non-erosive polymer. 107. The method according to any one of 101 to 105, wherein the composition is formulated together with a bio-erosive polymer. 108. The method according to any one of 96 to 107, wherein the protein, peptide, or peptide mimetic is selected from the group consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), neurotrophic factor 4 / 5 (NT-4 / 5), neurotrophic factor 6 (NT-6), neurotrophic factor 7 (NT-7), glial cell-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and derivatives or fragments thereof. 109. The method according to 108, wherein the protein, peptide, or peptide mimetic is a neurotrophic factor derived from a non-human animal. 110. The method according to 109, wherein the protein, peptide, or peptide mimetic is a recombinant human neurotrophic factor. 111. The method according to 109, wherein the protein, peptide, or peptide mimetic comprises the β-subunit of NGF, rhNGF, or rhBDNF, or a chimeric construct of NGF and BDNF, or a chimeric construct of rhNGF and any other neurotrophic factor, or a chimeric construct between any two other neurotrophic factors. 112. The method according to any one of 96-111, wherein the protein, peptide, or peptide mimetic is a neurotrophic factor that binds to tropomyosin kinase A (TrkA). 113. The method according to any of 96-112, wherein the activator is a dye. 114. The method according to 113, wherein the activator is an organic dye or an inorganic dye. 115. The method according to 114, wherein the dye is selected from the group consisting of body pie dyes, coumarin dyes, rhodamine dyes, acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes, chlorophyll-containing dyes, triarylmethane dyes, azo dyes, diazonium dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone-imine dyes, azine dyes, eurozine dyes, safranin dyes, indamine, indophenol dyes, fluorine dyes, oxazine dyes, oxazone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronin dyes, fluorine dyes, rhodamine dyes, phenanthoridine dyes, squalane, body pie, squaline roxitan, naphthalene, coumarin, oxadiazole, anthracene, pyrene, acridine, arylmethine or tetrapyrrole, and combinations thereof. 116. The method according to 115, wherein the dye has an emission wavelength of 300 nm or more. 117. The method described in 115, wherein the dye is Dynamics DY-800. 118. The method described in 115, wherein the dye is Cy7.5. 119. The method described in 115, wherein the dye is Cy7.0. 120. The method described in 115, wherein the dye is Cy5.5. 121. The method according to any one of 115-121, wherein the dye is a compound that promotes intraocular visualization of the dye that binds to nerve cells, and intraocular visualization is made possible by binding of Trk receptors in microtubules and / or nerve sheaths surrounding nerves, endocytosis and retrograde axonal transport. 122. The method according to any of 96-112, wherein the activator is a small molecule activator. 123. The method according to 122, wherein the activator is an anti-inflammatory agent. 124. The method according to 123, wherein the anti-inflammatory agent is a glucocorticoid. 125. The method according to 124, wherein the glucocorticoid is fluocinolone acetonide. 126. The method according to 122, wherein the activator is an antimicrobial compound. 127. The method according to 126, wherein the antimicrobial compound is an antiviral compound. 128. The method according to 122, wherein the activator is an anticancer drug. 129. The method according to 122, wherein the activator is a neuroprotective compound. 130. The method according to any of 96-129, further comprising determining the health of the retinal ganglion cells of the subject's eye. 131. The method according to any one of 96 to 130, wherein the activator conjugate further comprises a linker that covalently bonds the activator compound to a protein, peptide, or peptide mimetic. 132. The method according to 131, wherein the linker is selected from the group consisting of a bifunctional linker, a homobifunctional linker, and a heterobifunctional linker. 133. The method according to any one of 96 to 132, wherein a protein, peptide, or peptide mimetic is bound to an activator by internal amino acid residues. 134. The method according to 133, wherein amino acid residues are present on the surface of a protein, peptide, or peptide mimetic. 135. The method according to any one of 96 to 134, wherein the activator is bound to the N-terminal or C-terminal amino acid of a protein, peptide, or peptide mimetic. 136. The method according to any one of 131 to 134, wherein the linker is configured to bind to a reactive side chain of a mutant amino acid in a protein, peptide, or peptide mimetic, or to an amino acid in a native protein, peptide, or peptide mimetic. 137. The method according to any one of 131 to 134, wherein the linker is configured to bind to a protein, peptide or peptide mimetic via an ether bond, ester bond, carbamate bond, amide bond, disulfide bond or amino bond. 138. The method according to any one of 96 to 137, wherein the activating compound is covalently bonded to a lysine residue of a protein, peptide, or peptide mimetic. 139. The method according to any one of 131 to 138, wherein the linker is covalently bonded to the activator by the carbon of the activator. 140. The method according to any one of 131 to 138, wherein the linker is covalently bonded to the activator by the non-carbon of the activator. 141. The method according to 140, wherein the linker is covalently bonded to the activator by the nitrogen of the activator. 142. The method according to any one of 131 to 138, wherein the linker is covalently bonded to the activator by the sulfur of the activator. 143. The method according to any one of 96 to 142, wherein the activator compound is covalently bonded to a protein, peptide, or peptide mimetic via a disuccinimidyl carbonate linker. 144. A composition for use in any of the methods described in 96 to 143, comprising an activator conjugate, wherein the activator conjugate comprises one or more activator compounds covalently bound to a protein, peptide, or peptide mimetic that selectively binds to a neurotrophin receptor. 145. Contact lenses comprising the composition described in 144. 146. A first detectable label-biomolecular conjugate comprising a first detectable label covalently attached to a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors, is endocytosed, and enables intracellular translocation (e.g., retrograde / anterograde axonal transport), and A second detectable label-biomolecular conjugate containing a second detectable label covalently bonded to a biopolymer that selectively binds to neoplastic tissue. A composition containing the following: 147. The composition according to 146, wherein the protein, peptide, or peptide mimetic is selected from the group consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), neurotrophic factor 4 / 5 (NT-4 / 5), neurotrophic factor 6 (NT-6), neurotrophic factor 7 (NT-7), glial cell-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and derivatives or fragments thereof. 148. The composition according to 147, wherein the protein, peptide, or peptide mimetic is a neurotrophic factor derived from a non-human animal. 149. The composition according to 148, wherein the protein, peptide, or peptide mimetic is a recombinant human neurotrophic factor. 150. The composition according to 147, wherein the protein, peptide, or peptide mimetic comprises a β-subunit of NGF, rhNGF, or rhBDNF, a chimeric construct of rhNGF and rhBDNF, a chimeric construct of rhNGF and any other neurotrophic factor, or a chimeric construct of any two other neurotrophic factors. 151. A composition according to any one of 146 to 150, wherein the protein, peptide, or peptide mimetic is a neurotrophic factor that binds to tropomyosin kinase A (TrkA). 152. The composition according to any one of 146 to 151, wherein each detectable label is independently selected from the group consisting of fluorophores, chromophores, enzymes, redox labels, radioactive labels, sound-absorbing labels, Raman (SERS) tags, mass tags, isotope tags, magnetic particles, fine particles, and nanoparticles. 153. The composition according to 152, comprising a compound in which each detectable label is a dye. 154. The composition according to 153, wherein each detectable label is an organic dye or an inorganic dye. 155. The composition according to 154, wherein the dye is selected from the group consisting of body pie dyes, coumarin dyes, rhodamine dyes, acridine dyes, anthraquinone dyes, arylmethane dyes, diarylmethane dyes, chlorophyll-containing dyes, triarylmethane dyes, azo dyes, diazonium dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinone-imine dyes, azine dyes, eurozine dyes, safranin dyes, indamine, indophenol dyes, fluorine dyes, oxazine dyes, oxazone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronin dyes, fluorine dyes, rhodamine dyes, phenanthoridine dyes, squalane, body pie, squaline roxitan, naphthalene, coumarin, oxadiazole, anthracene, pyrene, acridine, arylmethine or tetrapyrrole, and combinations thereof. 156. The composition according to 155, wherein the dye has an emission wavelength of 300 nm or more. 157. The composition according to 155, wherein the dye is Alexa Fluor 680. 158. The composition according to 155, wherein the dye is Dyomics DY-800. 159. The composition according to 155, wherein the dye is Cy7.5. 160. The composition according to 155, wherein the dye is Cy5.5. 161. The composition according to any one of 146 to 160, wherein the first detectable label is a compound that promotes peripheral nerve visualization. 162. The composition according to any one of 146 to 160, wherein the first detectable label is a compound that promotes intracellular visualization of a neuron, and intracellular visualization is enabled by binding of Trk receptors in microtubules and / or in nerve sheaths surrounding nerves, endocytosis and retrograde axonal transport. 163. The composition according to any one of 146 to 162, wherein the biopolymer that selectively binds to neoplastic tissue is a compound selected from the group consisting of polypeptides, nucleic acids, and polysaccharides. 164. The composition according to 163, wherein the polypeptide is a peptide mimetic, a protein, an enzyme, or an antibody. 165. The composition according to 164, wherein the biopolymer that selectively binds to neoplastic tissue is an antibody. 166. The composition according to 165, wherein the biopolymer that selectively binds to neoplastic tissue is a monoclonal antibody. 167. The composition according to any one of 146 to 166, wherein the first detectable label-biomolecular conjugate further comprises a linker that covalently attaches the detectable label to a protein, peptide, or peptide mimetic. 168. The composition according to any one of 146 to 167, further comprising a second detectable label-biomolecular conjugate which further comprises a linker that covalently binds the detectable label to a biopolymer that selectively binds to neoplastic tissue. 169. The composition according to any one of 167 to 168, wherein the linker is selected from the group consisting of a bifunctional linker, a homobifunctional linker, and a heterobifunctional linker. 170. A composition according to any one of 146 to 169, wherein a protein, peptide, or peptide mimetic is bound to an activator by internal amino acid residues. 171. The composition according to 170, wherein amino acid residues are present on the surface of a protein, peptide, or peptide mimetic. 172. The composition according to any one of 167 to 171, wherein the linker is configured to bind to the N-terminal or C-terminal amino acid of a protein, peptide, or peptide mimetic. 173. The composition according to any one of 167 to 172, wherein the linker is configured to bind to a mutant amino acid of a protein, peptide, or peptide mimetic. 174. The composition according to any one of 167 to 172, wherein the linker is configured to bind to a protein, peptide or peptide mimetic by an ether bond, ester bond, carbamate bond, amide bond, disulfide bond or amino bond. 175. The composition according to any one of 146 to 174, wherein the first detectable label is covalently bonded to a lysine residue of a protein, peptide, or peptide mimetic. 176. The composition according to any one of 146 to 175, wherein the first detectable label is covalently bonded to the protein, peptide, or peptide mimetic by the carbon of the protein, peptide, or peptide mimetic. 177. The composition according to any one of 146 to 176, wherein the first detectable label is covalently bonded to the protein, peptide, or peptide mimetic by a non-carbon element of the protein, peptide, or peptide mimetic. 178. The composition according to 177, wherein the first detectable label is covalently bonded to the protein, peptide, or peptide mimetic by the nitrogen of the protein, peptide, or peptide mimetic. 179. The composition according to 177, wherein the first detectable label is covalently bonded to the protein, peptide, or peptide mimetic by the sulfur of the protein, peptide, or peptide mimetic. 180. The composition according to any one of 168 to 179, wherein a second detectable label-biomolecular conjugate comprises an antibody that selectively binds to neoplastic tissue, and a linker is configured to bind to the N-terminal or C-terminal amino acid of the antibody. 181. A method comprising contacting the tissue of a subject with a composition described in any of 146 to 180. 182. The method according to 181, wherein the composition is administered to a subject. 183. The method according to 182, wherein the composition is administered by local administration, local administration during surgery, intravenous administration, intraocular administration, intracapsular administration, intrathecal administration, intravitreous administration, or subcutaneous injection, intramuscular injection, intravitreous injection, intracapsular injection, intrathecal injection, or a combination thereof. 184. The method according to any one of 181 to 183, wherein the composition is brought into contact with the subject's tissue during surgery. 185. The method of 184, wherein the surgery involves the excision of at least a portion of the neoplastic tissue. 186. A neuroimaging agent comprising a first fluorescent compound covalently bound to a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors, and A tumor imaging agent containing a second fluorescent compound covalently bound to an antibody that selectively binds to malignant neoplasm tissue. A composition containing the following: 187. The composition according to 186, wherein the protein, peptide, or peptide mimetic is selected from the group consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), neurotrophic factor 4 / 5 (NT-4 / 5), neurotrophic factor 6 (NT-6), neurotrophic factor 7 (NT-7), glial cell-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and derivatives or fragments thereof. 188. The composition according to any one of 186 to 187, wherein the first fluorescent compound, the second fluorescent compound, or a combination thereof is selected from the group consisting of Alexa Fluor 680, Dyomics DY-800, Cy7.5, and Cy5.5. 189. A composition according to any one of 186 to 188, wherein the antibody is panitumumab. 190. Tissue of the subject undergoing surgery A neuroimaging agent comprising a first fluorescent compound covalently bound to a protein, peptide, or peptide mimetic that selectively binds to neurotrophin receptors, and A tumor imaging agent comprising an antibody or antibody fragment that selectively binds to malignant neoplasm tissue, or a second fluorescent compound covalently bound to such an antibody fragment. A method comprising contacting a composition containing the following: A method comprising nerve imaging agents and tumor imaging agents configured to provide visual aids during surgery. 191. The method according to 190, wherein the surgery is a procedure for removing malignant neoplasm tissue from a subject. 192. The method according to any one of 190 to 191, wherein the protein, peptide, or peptide mimetic is selected from the group consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), neurotrophic factor 4 / 5 (NT-4 / 5), neurotrophic factor 6 (NT-6), neurotrophic factor 7 (NT-7), glial cell-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and derivatives or fragments thereof. 193. The method according to any one of 190 to 192, wherein the first fluorescent compound, the second fluorescent compound, or a combination thereof is selected from the group consisting of Alexa Fluor 680, Dyomics DY-800, Cy7.5, and Cy5.5. 194. The method described in any of 190-193, wherein the antibody is panitumumab. 195. The method according to any of 190-193, wherein the antibody is folate receptor alpha (folate receptor-α). [Examples]

[0118] The following examples are provided to those skilled in the art to give a complete disclosure and explanation of how the embodiments are prepared and used, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent that the following experiments are all or only experiments performed. Efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantity, temperature, etc.), but some experimental error and deviation should be taken into consideration. Unless otherwise stated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is Celsius, and pressure is around atmospheric pressure. Standard abbreviations may also be used.

[0119] The present invention will be described with reference to its specific embodiments, but it will be understood by those skilled in the art that various modifications can be made and equivalents can be substituted without departing from the actual spirit and scope of the invention. Furthermore, many modifications may be made to adapt specific circumstances, materials, compositions, processes, or single or more process steps to the object, spirit and scope of the invention. Any such modifications are intended to be included in the claims appended herein.

[0120] Neurotrophins as neuronal cell targeting vectors The neurotrophins (NTs) described herein include soluble peptides, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor 3 (NT-3), neurotrophic factors 4 / 5 (NT-4 / 5), neurotrophic factor 6 (NT-6), neurotrophic factor 7, glial cell-derived neurotrophic factor (GDNF), and ciliary body neurotrophic factor (CNTF). In developing mammals, NTs may be responsible for the development and differentiation of specific tissues and organs of the nervous system. For example, retinal development is regulated by NGF, BDNF, and NT-3. NTs promote cell proliferation, survival, and differentiation in several types of nerve cells. NTs have been identified as being associated with neuronal survival. Each of the NT families is selective for specific high-affinity receptors. Trk receptors are encoded by TrkA, TrkB, and TrkC of the Trk gene. All NTs also bind to the low-affinity p75 neurotrophin receptor, i.e., p75-NTR. In this embodiment, NT was selected as a neuronal delivery vector based in part on its known selectivity and transport properties. NT is expressed at the distal ends (e.g., dendrites) of specific neuronal populations and binds to high-affinity tropomyosin kinase receptors (TrkA for NGF, TrkB for BDNF, and TrkC for NT-3) that are widely expressed by immune system cells, thereby being selectively endocytized by neurons at the surrounding injection site or local application site. A strong hypothesis is that the NT / Trk complex travels within neurons via microtubule-mediated signaling endosomes through retrograde axonal transport to the neuronal cell body, e.g., the dorsal root ganglia (DRG) in the peripheral nervous system (PNS). It is also understood that the NT / Trk complex can travel in the opposite direction via anterograde axonal transport. When administered externally, NT is considered safe because it is well tolerated at low doses. NT has been found to be non-immunogenic, which is a distinguishing feature from viral vectors. NT is localized to nerve cells (e.g., dendrites) associated with the extracorporeal administration site and peripheral proximal regions. NGF is degraded by lysosomal proteases. The compounds of the compositions disclosed herein do not transsynaptic travel from the PNS to the brain.

[0121] The subjective compositions presented herein demonstrate that the target conjugates can be localized to specific neuronal populations, and that their effects, through localization and the resulting reduction in required doses, improve the therapeutic index of otherwise systemically toxic agents, such as fluocinolone acetonide (FA), a glucocorticoid with systemic dose-limiting toxicity, selectively treat pathological defects, namely Trk defects affecting neurons, and / or treat microtubules and / or other molecular pathological processes, namely signaling endosomes involved in retrograde or anterograde axonal transport in specific neuronal populations. The following experiments consider the following: • Structural considerations regarding neurotrophins and the selectivity of neurotrophins due to their high affinity for specific receptors expressed at the distal ends of specific neuronal cell populations. • Clinical safety of rhNGF and rhBDNF, • Mechanism of intracellular delivery of neurons by retrograde axonal transport. The mechanism of anterograde axonal transport is well known and will not be discussed herein. • Fluorescent dyes that can be considered for binding, and Evidence of the limits of NT-small molecule compound (SMC, NT-SMC represents the ratio of SMC to protein) conjugates for achieving binding, endocytosis, and retrograde axonal transport.

[0122] Neurotrophins: A Structural Examination The crystal structure of NGF is well understood. The precursor form of NGF (pro-NGF) is activated into the biologically functional NGF after cleavage, forming a single chain of approximately 13 kDa. This monomeric form spontaneously forms a 26,518 Da dimer through non-covalent bonding. BDNF undergoes a similar process to become a biologically functional mature form of a non-covalent dimer of approximately 26 kDa.

[0123] The structure of NGF includes amino acid residues that are physically distant from the region involved in binding to the TrkA receptor. The cellular events required for NGF binding and retrograde transport necessitate the high-affinity receptor TrkA. BDNF has approximately 55% homology to NGF, with five of its 11 lysine residues being conserved, and only two of its 11 lysine residues being physically close to the TrkB site. It is a dimer of two identical 119 amino acid subunits of 28 kDa, linked by a strong hydrophobic interaction. When BDNF binds to TrkB at its distal end, the BDNF / TrkB complex is taken up into signaling endosomes, which are transported retrogradely along axonal microtubules by the dynein motor. Similar to NGF-TrkA, the BDNF-TrkB complex is transported retrogradely to the interstitial and central (motor) neuron cell bodies. There is also other evidence suggesting that NGF-TrkA and BDNF-TrkB complexes can be transported by anterograde axonal transport. Structural considerations and mutagenicity data suggest that BDNF interacts with TrkB in a manner very similar to how NGF interacts with TrkA.

[0124] NTs, in a phenomenon known as specific distribution, are localized to populations of neurons that bind to adjacent Trk receptor groups. NTs are selective due to their Trk receptor distribution.

[0125] Clinical safety of neurotrophins rhNGF and rhBDNF Unmodified NT is non-immunogenic. rhNGF can be formulated for topical ocular administration (e.g., eye drops administered at 20 μg / ml six times daily for 8 weeks) to treat neurotrophic keratitis. rhNGF was well tolerated at subcutaneous doses (SQ) up to the maximum tolerated dose (MTD) of 1 μg / kg. rhNGF has been evaluated as a therapeutic agent in intraventricular (ICV) administration for Alzheimer's disease, multiple sclerosis, pain associated with multiple sclerosis, and other forms of systemic pain. rhBDNF has been evaluated subcutaneously and intrathecally for the treatment of amyotrophic lateral sclerosis (ALS), depression, post-traumatic stress disorder (PTSD), and glaucoma.

[0126] Compositions of nerve growth factor (NGF)-fluocinolone acetonide conjugate and composition of brain-derived neurotrophic factor (BDNF)-fluocinolone acetonide conjugate As an example of intracellularly active small molecule compounds (SMCs), when the glucocorticoid fluocinolone acetonide (FA) was conjugated to rhNGF and rhBDNF using a heterobifunctional linker, both constructs exhibited analgesic (anti-inflammatory) effects in vivo. The dorsal root ganglia (DRGs) play a fundamental role in chronic pain in general in peripheral nervous system (PNS) diseases. NGF and BDNF localize due to their selectivity for distinctly different peripheral nerve cell populations by binding to TrkA and TrkB, which are expressed on nociceptors and autoreceptors, respectively. In the treatment of forms of pain involving DRGs, NGF and / or BDNF should intracellularly deliver active FA to the spinal dorsal horn via axonal transport through their central synaptic terminals to subpopulations of sensory neuron cell bodies in the DRG. Since TrkB is highly expressed in subpopulations of low-threshold mechanosensory neurons expressing neurofilament-1 in the DRG, peripherally injected BDNF delivers FA to the DRG. In the treatment of pain, FA can act on nearby inflammatory cells, such as microglial cells, astrocytes, or perineuritis, by altering the excitability of nerve cells and being released or diffused. rhNGF is stable in plasma for up to 4 days at 37°C.

[0127] To enable evaluation of both survival and Trk-binding activity after the synthetic operation in conjugate generation, an in vitro system was used employing primary neurons expressing Trk receptors at their distal end.

[0128] Confirmation of Trk receptor binding.The fluorescent dye Alexa Fluor 488 (488-mNGF or fNGF), conjugated to mouse NGF, was endocytotically administered to the distal ends of peripheral (superior cervical ganglion, SCG) neurons via a TrkA-mediated mechanism. Receptor binding activity was measured when unmodified mNGF (100X or less) was administered simultaneously with a Trk binder. Figure 1A shows 488-mNGF in the cell body after binding to TrkA, endocytosis, and transport to the central chamber of the in vitro system. Figure 1B confirms that TrkA competes with 100 ng / ml of 488-mNGF for the same TrkA receptor upon addition of 1000 ng / ml of unmodified mNGF. In Figure 1C, neurons in fractionated cultures were contaminated in the distal axon fraction with either approximately 10 ng / mL of fNGF (determined by neuronal survival bioassay) or 10 ng / mL of fNGF plus 100X excess unlabeled NFG (1000 ng / mL). Fluorescence images of the cell bodies were acquired after 24 hours (Figure 1C). Fluorescence accumulation in the cell bodies was effectively inhibited when 100X excess unlabeled NGF was added in addition to fNGF. fNGF uptake is a specific TrkA receptor-mediated process. The requirement for Alexa Fluor is <4 DARs per mouse NGF monomer. Due to its size and hydrophobicity, the fluorescent dye Alexa Fluor 488, bound to NGF without the use of spacers of any length, binds to TrkA, is endocytotic after binding, and is retrogradely transported to the neuronal cell body. Figure 4 shows in vitro results for 1.64 “mixed DAR” 800-rhNGF, expressed as dye molecules with an average of 1 to 4 molecules per monomer, using standard analytical methods such as mass spectrometry (MS) or high-performance liquid chromatography (HPLC), where the involved NTs are dimers. Both Figure 4 and Figure 1C demonstrate that 488-mouse NGF maintained neuronal survival in this assay using superior cervical ganglion (SCG).

[0129] Survival.Superior cervical ganglion (SCG) neurons from primary cultures were first used in fractionated cultures in established assays, then adapted and grown in microfluidic devices for testing. SCG neurons were collected from juvenile E-21 Sprague Dolly rats. In all tests conducted in microfluidic devices, labeled NGF, whether 800-rhNGF or FA-rhNGF, exhibited similar bioavailability to unlabeled NGF. Specifically, the TrkA binding site remained intact after the synthesis process.

[0130] Labeled BDNF test materials, in both the case of 800-rhBDNF and FA-rhBDNF, were evaluated for their ability to maintain neuronal cell viability by plating the conjugate onto retinal ganglion cells (RGCs). RGCs were isolated from P6 rats by sequential immunopanning. The retina was isolated, enzymatically digested with papain, and then mechanically dissociated to form single-cell suspensions. The cells were negatively panned to remove macrophages and other nonspecific adherent cells, and then positively panned against the RGCs using the Thy1.1 T11D7 hybridoma supernatant. Labeled rhBDNF exhibited similar activity to unlabeled BDNF; that is, the TrkB binding remained intact.

[0131] Decreased solubility of protein conjugates due to enhanced conjugation.Constructs with a larger molecular weight of the activator and lower levels of hydrophobic Dyomics DY-800 Near InfraRed (NIR) dye were directly conjugated to recombinant human NGF (rhNGF) (Figure 2). In subsequent tests, DAR variants were purified using reverse-phase HPLC (RP-HPLC) (Waters column). Each DAR variant showed changes in the solubility level of the protein conjugate as the conjugation level increased, resulting in a significant increase in precipitation. Figure 2A shows superior cervical ganglion (SCG) neurons isolated from juvenile E-21 Sprague Dolly rats and plated on one side of a Xona microfluidic chamber. Culture was maintained until the axons extended through the channels in the microfluidic chamber to the unplated side of the cells. Figure 2B shows the addition of 30 ng / mL of 800-rhNGF to wells with terminal (distal) axons. 800-rhNGF was added to the distal end of the left chamber, and the chamber was imaged with a LI-COR Odyssey scanner 48 hours later. It was possible to visualize 800 Near InfraRed (NIR) labeled cell bodies, indicating that the modified rhNGF compound was transported retrogradely. Figures 2C and 2D show bright-field images of the wells containing cell bodies, demonstrating improved visualization of cell aggregates for comparison with images containing 800NIR cell bodies.

[0132] Confirmation of intact TrkB binding activity after synthesis by neuronal cell survival. Figure 3 shows that, even after the synthesis procedure, two constructs, NIR800-rhBDNF, in which NIR is directly bound to rhBDNF, and FA-rhNGF, in which the glucocorticoid fluocinolone acetonide (FA) is conjugated to rhBDNF using a heterobifunctional linker, exhibited TrkB binding activity, and both maintained neuronal survival in primary neurons of retinal ganglion cells (RGCs).

[0133] Ratio of activator to protein in conjugate composition 8 Analysis of 00-rhNGF DAR Although lysine is physically distinct from the Trk receptor, in this embodiment, regardless of the number of lysine molecules available for protein labeling and / or crosslinker binding, only NT dyes with an average activator-to-NT ratio (DAR) of 3.2 or less per monomer were able to maintain neuronal cell survival. DAR analysis was performed using reversed-phase high-performance liquid chromatography (RP-HPLC) and / or mass spectrometry (MS). The results of MS DAR analysis across the entire 800 region of fluorescent dyes (800-rhNGF) bound to rhNGF are shown in Figure 4, from left to right, with the control on the far left, followed by F0 (mixed or average) DAR = 1.64, F1 = 0.96 DAR, F2 = 1.1 DAR, and F3 = 1.83 DAR. All of these DAR types maintained neuronal cell survival. Furthermore, the F0 "mixed DAR" of 1.64 successfully localized to target neurons in vivo and allowed for imaging of target neurons. Higher DARs, F4=3.6 and F5=3.66, could not sustain neuronal survival. Higher DARs could not be synthesized and were "crashed out" before conjugation, as shown by reverse-phase high-performance liquid chromatography analysis. (Figure 5)

[0134] Analysis of 800-rhBDNF High-performance liquid chromatography and mass spectrometry were used to quantify the number of dye molecules bound to or cross-bound to rhBDNF (i.e., the ratio of fluorescent dye to rhBDNF in the 800 region). In this embodiment, the average ratio per monomer was approximately 3.1 (see Figure 6), indicating Trk binding, intracellular uptake in neurons, and sustained neuronal survival (see Figure 3).

[0135] Although the aforementioned invention has been described in some detail with illustrations and examples for the purpose of clarity of understanding, those skilled in the art will readily understand that, in light of the teachings of the present invention, some changes and modifications may be made to the invention without departing from the spirit or scope of the appended claims.

[0136] Therefore, the above merely illustrates the principles of the present invention. Those skilled in the art will understand that various configurations embodying the principles of the present invention and falling within the spirit and scope of the invention can be devised, although not expressly described or presented herein. Furthermore, all examples and conditional language described herein are intended primarily to help the reader understand the principles of the present invention and the concepts to which the inventors contribute to the advancement of the art, and should be interpreted as not limiting the reader to such specifically described examples and conditions. In addition, any descriptions relating to the principles, aspects and embodiments of the present invention, as well as specific examples of the present invention, are intended to include both structural and functional equivalents thereof. It is also intended that such equivalents include both currently known equivalents and equivalents to be developed in the future, i.e., any elements to be developed that perform similar functions regardless of structure. Furthermore, nothing disclosed herein is intended to be made available to the public, whether or not such disclosures are expressly stated in the claims.

[0137] Accordingly, the scope of the present invention is not intended to be limited to the embodiments presented and described herein. Rather, the scope and spirit of the present invention are embodied in the appended claims. In the claims, Sections 112(f) and 112(6) of the Patent Act are explicitly defined to be exercised only if the precise phrase "means of" or "process of" is placed at the beginning of the limitation in the claims, and if the precise phrase is not used in the limitation in the claims, Sections 112(f) or 112(6) of the Patent Act are not exercised.

Claims

1. A composition comprising a dye-protein conjugate containing a dye covalently bound to a protein that selectively binds to neurotrophin receptors, wherein the average ratio of dye to protein monomer in the dye-protein conjugate in the composition is 0.75 to 3.0, and the dye-protein conjugate is 1) Alexa Fluor 488 (488-NGF) covalently bound to nerve growth factor; 2) Alexa Fluor 488 (488-BDNF) covalently bound to brain-derived neurotrophic factor; 3) Dyomics DY-800 (800-NGF) covalently bound to nerve growth factor; and 4) Dyomics DY-800 (800-BDNF) covalently bound to brain-derived neurotrophic factor A composition selected from the group consisting of the following.

2. The composition according to claim 1, wherein the average ratio of dye to protein monomer in the dye-protein conjugate in the composition is 0.95 to 1.

85.

3. The composition according to claim 1 or 2, wherein the dye-protein conjugate further comprises a linker that covalently bonds the dye to the protein, and the linker is a bifunctional linker.

4. The dye-protein conjugate is a) Endocytosis, b) Axonal transport, and c) Pharmacological activity in nerve cell bodies A composition according to any one of claims 1 to 3, which promotes one or more of the following.

5. To produce a composition containing a dye-protein conjugate in which one or more dyes are covalently bound to a protein that selectively binds to neurotrophin receptors by contacting a dye with a protein that selectively binds to neurotrophin receptors, and To isolate a dye-protein conjugate from a composition in which the average ratio of dye to protein monomer is 0.75 to 3.

0. It contains a dye-protein conjugate, 1) Alexa Fluor 488 (488-NGF) covalently bound to nerve growth factor; 2) Alexa Fluor 488 (488-BDNF) covalently bound to brain-derived neurotrophic factor; 3) Dyomics DY-800 (800-NGF) covalently bound to nerve growth factor; and 4) Dyomics DY-800 (800-BDNF) covalently bound to brain-derived neurotrophic factor A method for preparing a composition selected from the group consisting of the following.

6. The method according to claim 5, comprising isolating a dye-protein conjugate from a composition having an average ratio of dye to protein monomer of 0.95 to 1.85.

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