Cleavable polymer-drug conjugates for alzheimer's disease

Abstract

Described herein are cleavable and non-cleavable polymer-drug conjugates that can cross the blood-brain barrier. An example polymer-drug conjugate includes paclitaxel and a transcytosis peptide that can transport the polymer-drug conjugate across the blood-brain barrier of a subject.. Also described herein are multi-domain conjugates including the polymer-drug conjugate that further include a second cleavable peptide linker. Methods for modulating neurodegenerative conditions with the polymer-drug conjugates and multi-domain conjugates thereof are also described herein.

Description

CLEAVABLE POLYMER-DRUG CONJUGATES FOR ALZHEIMER’SDISEASECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 514,874, filed July 21, 2023, and U.S. Provisional Patent Application No. 63 / 515,303, filed July 24, 2023, each of which are incorporated by reference herein in its entirety.FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grants R41 CAI 56933 and R42CA156933 awarded by the National Cancer Institute of the National Institute of Health. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] This application was filed with a Sequence Listing XML in ST.26 XML format accordance with 37 C.F.R. § 1.831 and PCT Rule 13ter. The Sequence Listing XML file submitted in the USPTO Patent Center, “026389-0026-W001,” was created on July 21, 2024, contains 29 sequences, has a file size of 19.0 Kbytes, and is incorporated by reference in its entirety into the specification.TECHNICAL FIELD

[0004] The present disclosure describes cleavable and non-cleavable polymer-drug conjugates. The polymer-drug conjugates are designed to improve pharmacodynamic and pharmacokinetic properties of drugs, such as paclitaxel. These properties can include improving the aqueous solubility of the drug and targeting the central nervous system.INTRODUCTION

[0005] Alzheimer’s disease (AD) is an age-associated progressive neurodegenerative disorder affecting millions of people worldwide along the combined presence of two lesions in the brain: the extracellular amyloid-0 (A|3) plaques and the intraneuronal neurofibrillary tangles (NFTs) caused by tau protein.

[0006] Tau is an intracellular, microtubule-associated protein (MAPT) that is highly enriched in axons. As a microtubule-binding phosphoprotein, tau stably assembles with axonal microtubule bundles. When tau becomes hyperphosphorylated, the protein dissociates from the microtubules.As a result, tau becomes misfolded and begins to aggregate, which eventually forms the NFTs seen in AD patients. Given that tau pathology correlates better with cognitive impairments than do Ap lesions, tau-targeting strategy has received increased attention in recent years. Initially anti-tau therapies mainly focused on inhibition of kinases or tau aggregation, or on the stabilization of microtubules, but most of these approaches have been discontinued due to toxicity and / or lack of efficacy. Accordingly, new and improved anti-tau therapies would be beneficial for the treatment of AD.

[0007] Although paclitaxel (PTX) is widely used for the treatment of cancer, it also binds to tubulin on the inner surface of microtubules, promoting assembly, stabilizing microtubule dynamics, and preventing depolymerization, leading to inhibition of cell proliferation and induction of apoptosis. PTX has been shown to promote axonal regeneration and reduced inflammation. In addition to these effects, intranasal (IN) administration of low dose PTX has been shown to alter phenotypic features in 3xTg-AD mice including a reduction of tau pathology, a decrease neuroinflammation, improvement of axonal transport, as well as normalization of cognitive function. Despite these encouraging findings, PTX is considered not ideal for the treatment of AD due to its poor aqueous solubility and poor blood-brain barrier (BBB) permeability. The poor solubility of PTX has been previously addressed by formulating PTX with polyoxyethylated castor oil (Cremophor EL) and ethanol (1 : 1, v / v). The formulation of PTX with Cremophore EL has been reported to contribute to severe adverse events including peripheral neurotoxicity and hypersensitivity reactions.

[0008] A family of peptides, named Angiopeps, have emerged as new brain drug delivery targeting agents. Angiopeps target the low-density lipoprotein receptor-related protein (LRP1) and mediate transcytosis across the BBB due to the high expression of LRP1 on capillary endothelial cells at the blood brain barrier (BBB) surface. Angiopep-2 (AP2), a 19-amino-acid peptide (TFFYGGSRGKRNNFKTEEY) has been shown to improve therapeutic efficiency in brain targeting studies. There have been attempts to conjugate PTX to AP2; however, these conjugates suffer from limited pharmacokinetic properties such aqueous solubility and limited blood circulation time.

[0009] Therefore, what is needed are therapeutic solution to address the pharmacodynamic and pharmacokinetic limitations of PTX and related drugs. In particular, therapeutic solutions toaddress toxicity associated with PTX, limited solubility of PTX itself, and targeting of PTX to the blood brain barrier are needed.SUMMARY

[0010] One embodiment described herein is a conjugate, or a pharmaceutically acceptable salt thereof, comprising: a copolymer, the copolymer comprising a recurring hydrophilic unit, a recurring drug linking unit, and a recurring peptide linking unit; a plurality of drugs, each individual drug independently attached to a different and individual drug linking unit through a first cleavable peptide linker; and a plurality of transcytosis peptides, each individual transcytosis peptide independently attached to a different and individual peptide linking unit through a linker. In one aspect, the hydrophilic unit is derived from an N-substituted methacrylamide, an N,N- di substituted acrylamide, a hydrophilic ester of methacrylic or acrylic acid, an N-vinylpyrrolidone, an N-acryloylmorpholine, a sulfoethylmethacrylate, an acrylic acid, a methacrylic acid, or a combination thereof. In another aspect, the hydrophilic unit is of formula (I)(I). In another aspect, the drug linking unit is derived from an acrylate-based monomer. In another aspect, the drug linking unit is of formula (II)(II), wherein:L1is the first cleavable peptide linker, andQ is the drug. In another aspect, the peptide linking unit is derived from an acrylate-based monomer. In another aspect, the peptide linking unit is of formula (III)(Ill), wherein:L2is a peptide linkerL3is a non-cleavable linker;Z is a detectable moiety or a transcytosis peptide; and m is 0 to 1. In another aspect, the hydrophilic unit is included at about 50 mol% to about 95 mol % of the copolymer. In another aspect, the drug linking unit is included at about 1 mol% to about 10 mol% of the copolymer. In another aspect, the peptide linking is included unit at about 0.5 mol% to about 10 mol% of the copolymer. In another aspect, the copolymer has a number average molecular weight (Mn) of about 20,000 Daltons (Da) to about 200,000 Da. In another aspect, the copolymer is a random copolymer. In another aspect, the conjugate comprises less than 13 transcytosis peptides per copolymer. In another aspect, the conjugate comprises 2 to 8 transcytosis peptides per copolymer. In another aspect, each transcytosis peptide independently comprises an amino acid sequence selected from the group consisting of: TFFYGGSRGKRNNFKTEEY (AP2) (SEQ ID NO:24); TFFYGGCRGKRNNFKTEEY (SEQ ID NO:25); TFFYGGSRGKRNNFRTEEY (SEQ ID NO:26);HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO:27); TFFYGGSRGRRNNFRTEEY (SEQ ID NO:28); andYPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO:29). In another aspect, each transcytosis peptide independently comprises an amino acid sequence of TFFYGGSRGKRNNFKTEEY (AP2) (SEQ ID NO:24). In another aspect, the conjugate comprises the drug at about 1% to about 25% by weight of the conjugate. In another aspect, the drug is paclitaxel. In another aspect, the conjugate comprises paclitaxel at about 1% to about 15% by weight of the conjugate. In another aspect, the first cleavable peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 1-23. In another aspect, thelinker attaching each individual transcytosis peptide to a different and individual peptide linking unit is a maleimide-derived moiety, a click chemistry-derived moiety, or a peptide linker.

[0011] Another embodiment described herein is a multi-domain conjugate, or a pharmaceutically acceptable salt thereof, comprising: a plurality of conjugates described herein, wherein each conjugate is attached to at least one other conjugate through a second cleavable peptide linker. In one aspect, the multi-domain conjugate comprises two conjugates attached through the second cleavable peptide linker. In another aspect, the second cleavable linker is of formula (IV): -(AAI)-K-(AA2)- (IV), wherein AAi and AA2 are the same or different amino acid sequence comprising up to six amino acids, and K is lysine, ornithine, or a diamine. In another aspect, the second cleavable peptide linker comprises an amino acid sequence of GFLGKGFLG (SEQ ID NO: 23).

[0012] Another embodiment described herein is a pharmaceutical composition comprising a conjugate as described herein, a multi-domain conjugate as described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0013] Another embodiment described herein is a method of modulating neurodegeneration in a subject in need thereof, the method comprising administering to the subject an effective amount of a conjugate as described herein, a multi-domain conjugate as described herein, or a pharmaceutically acceptable salt thereof. In one aspect, neurodegeneration is associated with Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), ataxia, Huntington’s disease, motor neuron disease, multiple system atrophy, progressive supranuclear palsy, traumatic brain injury, or a combination thereof. In another aspect, neurodegeneration is associated with Alzheimer’s disease. In another aspect, the method decreases an amount of phosphor-tau positive cells in the subject. In another aspect, the method improves cognitive defects associated with Alzheimer’s disease in the subject. In another aspect, the conjugate, or a pharmaceutically acceptable salt thereof, is administered intravenously. In another aspect, the conjugate, or a pharmaceutically acceptable salt thereof, is administered at less than 20 mg / kg based on the amount of drug administered.

[0014] Another embodiment described herein is a method of delivering a drug to a brain of a subject in need thereof, the method comprising intravenously administering to the subject an effective amount of a conjugate as described herein, a multi-domain conjugate as described herein, or a pharmaceutically acceptable salt thereof, wherein following administration, the conjugate, ora pharmaceutically acceptable salt thereof, is transported through a blood-brain barrier of the subject.DESCRIPTION OF DRAWINGS

[0015] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0016] FIG. 1 shows the levels of LRP-1 expression on bEnd.3 and hCMEC / D3 cells.

[0017] FIG. 2 are microscope images showing the colocalization of AP2 and LRP-1.

[0018] FIG. 3 shows a dose-response curve for the binding of AP2 peptide to hCMEC / D3 cells.

[0019] FIG. 4 shows a Transwell in vitro blood-brain barrier (BBB) model used to measure transcytosis.

[0020] FIG. 5 shows the trans-epithelial electrical resistance (TEER) value over time in hCMEC / D3 cells.

[0021] FIG. 6 is a bar graph showing the transcytosis ratio of P-PTX-AP2 and P-PTX.

[0022] FIG. 7 shows a dose-response curve for the cytotoxicity of PTX and P-PTX- AP2 in b.ENE)3 cells.

[0023] FIG. 8 shows a dose-response curve for the cytotoxicity of PTX and P-PTX-AP2 in SH- SY5Y cells.

[0024] FIG. 9 shows a dose-response curve for the cytotoxicity of PTX and P-PTX-AP2 in hCMEC / D3 cells.

[0025] FIG. 10 shows the biodistribution of P-Cy5.5 and P-AP2-Cy5.5 in a mouse from 0-72 hours.

[0026] FIG. 11 shows a line graph of the fluorescence intensity of P-Cy5.5 and P-AP2-Cy5.5 0-72 hours.

[0027] FIG. 12 shows a mouse brain and the localization of P-AP2-Cy5.5 to the brain. P-Cy5.5 does not localize to the brain in the absence of AP2.

[0028] FIG. 13 shows a bar graph of the fluorescence intensity of P-Cy5.5 and P-AP2-Cy5.5.

[0029] FIG. 14 shows histological micrographs of Tau protein and microglia.

[0030] FIG. 15 shows radial water maze results for (i) female wild-type mice and transgenic 3xTg mice receiving either four treatments of either (ii) saline or (iii) P-PTX-AP2

[0031] FIG. 16 shows radial water maze results for (i) male and female wild-type mice receiving six treatments of vehicle; (ii) male and female wild-type mice receiving six treatments of P-PTX-AP2; (iii) Tg mice receiving six treatments of vehicle; (iv) Tg mice receiving six treatments of P-PTX-AP2.DETAILED DESCRIPTIONA. Definitions

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0033] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0034] The term “about” or “approximately” as used herein as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In certain aspects, the term “about” refers to a range of values that fall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Alternatively, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, theterm “about” can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

[0035] As used herein “or” can mean the conjunctive or disjunctive. For example, two actions separated by an “or” can be performed either simultaneously, sequentially, or separately.

[0036] “Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, the phrase "optionally with a fluorophore" means that the fluorophore can or cannot be included and that the description includes instances where fluorophore is present and fluorophore is absent.

[0037] A weight percent of a component, unless specifically stated to the contrary, is based on the total weight of the formulation, conjugate, or composition in which the component is included.

[0038] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms “polypeptide”, “protein,” and “peptide” are used interchangeably herein. “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains, for example, enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains. “Domains” are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Exemplary domains include domains with enzymatic activity or ligand binding activity. Typical domains are made up of sections of lesser organization such as stretches of beta-sheet and alpha-helices. “Tertiary structure” refers to the complete three- dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three- dimensional structure formed by the noncovalent association of independent tertiary units. A “motif’ is a portion of a polypeptide sequence and includes at least two amino acids. A motif may be 2 to 20, 2 to 15, or 2 to 10 amino acids in length. In some embodiments, a motif includes 3, 4, 5, 6, or 7 sequential amino acids. A domain may be comprised of a series of the same type of motif.

[0039] “Substantially identical” can mean that a first and second amino acid or polynucleotide sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 amino acids or nucleotides, respectively.

[0040] “Variant” with respect to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Variant may also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. Representative examples of “biological activity” include the ability to be bound by a specific antibody or polypeptide or to promote an immune response. Variant can mean a functional fragment thereof. Variant can also mean multiple copies of a polypeptide. The multiple copies can be in tandem or separated by a linker. A conservative substitution of an amino acid, for example, replacing an amino acid with a different amino acid of similar properties (for example, hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes may be identified, in part, by considering the hydropathic index of amino acids, as understood in the art (Kyte et al., J. Mol. Biol. 1982, 157, 105-132). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes may be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids may also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

[0041] “Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal that wants or is in need of the herein described kits or methods. The subject may be a human or a non-human. The subject may be a vertebrate. The subject may be a mammal. The mammal may be a primate or a non-primate. The mammal can bea non-primate including, but not limited to, for example, cow, pig, camel, llama, hedgehog, anteater, platypus, elephant, alpaca, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse. The mammal can be a primate such as a human. The mammal can be a non-human primate, including, but not limited to, for example, monkey, cynomolgus monkey, rhesus monkey, chimpanzee, gorilla, orangutan, and gibbon. The subject may be of any age or stage of development, such as, for example, an adult, an adolescent, or an infant. The subject may be male. The subject may be female. In some embodiments, the subject may have an autoimmune disorder, a neurodegenerative disorder, or a cancer affecting the central nervous system. The subject may be undergoing other forms of treatment.

[0042] A “patient in need thereof’ or a “subject in need thereof’ refers to a subject afflicted with at least one symptom associated with one or more diseases or disorders. In some embodiments, the disease or disorder is an autoimmune disorder, a neurodegenerative disorder, a cancer affecting the central nervous system, or a combination thereof. Example neurodegenerative disorders include, but is not limited to, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), ataxia, Huntington’s disease, motor neuron disease, multiple system atrophy, progressive supranuclear palsy, and traumatic brain injury (TBI). A patient may have been diagnosed with a need for treatment of one or more of the aforementioned diseases or disorders prior.

[0043] “Treatment” or “treating” or “treatment” when referring to protection of a subject from a disease, means suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. Preventing the disease involves administering a disclosed conjugate to a subject prior to onset of the disease. Suppressing the disease involves administering a disclosed conjugate to a subject after induction of the disease but before its clinical appearance. Repressing or ameliorating the disease involves administering a disclosed conjugate to a subject after clinical appearance of the disease.

[0044] The terms "effective amount" or “therapeutically effective amount” refer to an amount sufficient to effect beneficial or desirable biological and / or clinical results to treat a disease or one or more of its symptoms and / or to prevent or reduce the risk of the occurrence or reoccurrence of the disease or disorder or symptom(s) thereof. In reference to neurodegenerative associateddiseases an effective or therapeutically effective amount can include an amount sufficient to, among other things, improve cognitive defects of the subject.

[0045] The term “attached” refers to two moieties being attached through a bond where there can be intervening moieties or molecules in between. For example, the peptide linking unit can be attached to the transcytosis peptide through a linker, where there is an intervening moiety (other than the linker) between the peptide linking unit and the transcytosis peptide. Attached can also include “directly attached,” which refers to two moieties being attached through a bond with no other intervening moieties or molecules.

[0046] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0047] The term “alkoxy,” as used herein, refers to a group -O-alkyl. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy and tertbutoxy.

[0048] The term “alkyl,” as used herein, means a straight or branched, saturated hydrocarbon chain. The term “lower alkyl” or “Ci-ealkyl” means a straight or branched chain hydrocarbon containing from 1 to 6 carbon atoms. The term “Cwalkyl” means a straight or branched chain hydrocarbon containing from 1 to 4 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n- pentyl, isopentyl, neopentyl, n-hexyl, 3 -methylhexyl, 2,2-dimethylpentyl, 2,3 -dimethylpentyl, n- heptyl, n-octyl, n-nonyl, and n-decyl.

[0049] The term “alkenyl,” as used herein, means a straight or branched, hydrocarbon chain containing at least one carbon-carbon double bond.

[0050] The term “alkoxyalkyl,” as used herein, refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.

[0051] The term “aminoalkyl” as used herein, means at least one amino group, as defined herein, is appended to the parent molecular moiety through an alkylene group, as defined herein.

[0052] The term “amino,” as used herein, means -NRxRy, wherein Rx and Ry may be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, alkenyl, or heteroalkyl. In the case of an aminoalkyl group or any other moiety where amino appends together two other moieties, amino may be -NRx-, wherein Rx may be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, alkenyl, or heteroalkyl.

[0053] The term “aryl,” as used herein, refers to a phenyl or a phenyl appended to the parent molecular moiety and fused to a cycloalkane group (e.g., the aryl may be indan-4-yl), fused to a 6-membered arene group (i.e., the aryl is naphthyl), or fused to a non-aromatic heterocycle (e g., the aryl may be benzo[d][l,3]dioxol-5-yl). The term “phenyl” is used when referring to a substituent and the term 6-membered arene is used when referring to a fused ring. The 6- membered arene is monocyclic (e.g., benzene or benzo). The aryl may be monocyclic (phenyl) or bicyclic (e.g., a 9- to 12-membered fused bicyclic system).

[0054] The term “cycloalkyl” or “cycloalkane,” as used herein, refers to a saturated ring system containing all carbon atoms as ring members and zero double bonds. The term “cycloalkyl” is used herein to refer to a cycloalkane when present as a substituent. A cycloalkyl may be a monocyclic cycloalkyl (e.g., cyclopropyl), a fused bicyclic cycloalkyl (e g., decahydronaphthalenyl), or a bridged cycloalkyl in which two non-adjacent atoms of a ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms (e.g., bicyclo[2.2.1]heptanyl). Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, and bicyclo[l. l.l]pentanyl.

[0055] The term “cycloalkenyl” or “cycloalkene,” as used herein, means a non-aromatic monocyclic or multicyclic ring system containing all carbon atoms as ring members and at least one carbon-carbon double bond and preferably having from 5-10 carbon atoms per ring. The term “cycloalkenyl” is used herein to refer to a cycloalkene when present as a substituent. A cycloalkenyl may be a monocyclic cycloalkenyl (e.g., cyclopentenyl), a fused bicyclic cycloalkenyl (e.g., octahydronaphthalenyl), or a bridged cycloalkenyl in which two non-adjacent atoms of a ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms (e.g.,bicyclo[2.2.1]heptenyl). Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl or cycloheptenyl. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl or cycloheptenyl.

[0056] The term “carbocyclyl” means a “cycloalkyl” or a “cycloalkenyl.” The term “carbocycle” means a “cycloalkane” or a “cycloalkene.” The term “carbocyclyl” refers to a “carbocycle” when present as a substituent.

[0057] The terms cycloalkylene and heterocyclylene refer to divalent groups derived from the base ring, i.e., cycloalkane, heterocycle. For illustration, an example cycloalkylene may be cyclohexene orand a heterocyclylene may be. Cycloalkylene and heterocyclylene include a geminal divalent groups such as l,l-C3-6cycloalkylene. A further example is 1,1- cyclopropylene.

[0058] The term “halogen” or “halo,” as used herein, means Cl, Br, I, or F.

[0059] The term “haloalkyl,” as used herein, means an alkyl group, as defined herein, in which one, two, three, four, five, six, seven or eight hydrogen atoms are replaced by a halogen.

[0060] The term “heteroaryl,” as used herein, refers to an aromatic monocyclic heteroatomcontaining ring (monocyclic heteroaryl) or a bicyclic ring system containing at least one monocyclic heteroaromatic ring (bicyclic heteroaryl). The term “heteroaryl” is used herein to refer to a heteroarene when present as a substituent. The monocyclic heteroaryl are five or six membered rings containing at least one heteroatom independently selected from the group consisting of N, O and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N). The five membered aromatic monocyclic rings have two double bonds, and the six membered aromatic monocyclic rings have three double bonds. The bicyclic heteroaryl is an 8- to 12-membered ring system and includes a fused bicyclic heteroaromatic ring system (i.e., IOTC electron system) such as a monocyclic heteroaryl ring fused to a 6-membered arene (e.g., quinolin-4-yl, indol-l-yl), a monocyclic heteroaryl ring fused to a monocyclic heteroarene (e.g., naphthyridinyl), and a phenyl fused to a monocyclic heteroarene (e.g., quinolin-5-yl, indol-4-yl). A bicyclic heteroaryl / heteroarene group includes a 9-membered fused bicyclic heteroaromatic ring system having four double bonds and at least one heteroatom contributing a lone electron pair to a fully aromatic I Ort electron system, such as ring systems with a nitrogen atom at the ring junction (e.g., imidazopyridine) or a benzoxadiazolyl. A bicyclic heteroaryl also includes a fused bicyclic ringsystem composed of one heteroaromatic ring and one non-aromatic ring such as a monocyclic heteroaryl ring fused to a monocyclic carbocyclic ring (e.g., 6,7-dihydro-5H- cyclopenta[b]pyridinyl), or a monocyclic heteroaryl ring fused to a monocyclic heterocycle (e.g., 2,3-dihydrofuro[3,2-b]pyridinyl). The bicyclic heteroaryl is attached to the parent molecular moiety at an aromatic ring atom. Other representative examples of heteroaryl include, but are not limited to, indolyl (e.g., indol-l-yl, indol-2-yl, indol-4-yl), pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl (e.g., pyrazol-4-yl), pyrrolyl, benzopyrazolyl, 1,2,3-triazolyl (e.g., triazol-4-yl), 1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl (e.g., thiazol-4-yl), isothiazolyl, thienyl, benzimidazolyl (e.g., benzimidazol-5-yl), benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzothienyl, benzofuranyl, isobenzofuranyl, furanyl, oxazolyl, isoxazolyl, purinyl, isoindolyl, quinoxalinyl, indazolyl (e.g., indazol-4-yl, indazol-5-yl), quinazolinyl, 1,2,4-triazinyl, 1,3,5- triazinyl, isoquinolinyl, quinolinyl, imidazo[l,2-a]pyridinyl (e.g., imidazo[l,2-a]pyridin-6-yl), naphthyridinyl, pyridoimidazolyl, thiazolo[5,4-b]pyridin-2-yl, and thiazolo[5,4-d]pyrimidin-2-yl.

[0061] The term “heterocycle” or “heterocyclic,” as used herein, means a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. The term “heterocyclyl” is used herein to refer to a heterocycle when present as a substituent. The monocyclic heterocycle is a three-, four-, five-, six-, seven-, or eight-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. The three- or four-membered ring contains zero or one double bond, and one heteroatom selected from the group consisting of O, N, and S. The five-membered ring contains zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S. The six-membered ring contains zero, one or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. The seven- and eight-membered rings contains zero, one, two, or three double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocyclyls include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1, 3 -di thiol any 1, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, 2-oxo-3-piperidinyl, 2-oxoazepan-3-yl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, oxepanyl, oxocanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. The bicyclic heterocycle is a monocyclic heterocycle fused to a 6- membered arene, or a monocyclic heterocycle fused to a monocyclic cycloalkane, or a monocyclic heterocycle fused to a monocyclic cycloalkene, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a monocyclic heterocycle fused to a monocyclic heteroarene, or a spiro heterocycle group, or a bridged monocyclic heterocycle ring system in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms. The bicyclic heterocyclyl is attached to the parent molecular moiety at a non-aromatic ring atom (e.g., indolin-l-yl). Representative examples of bicyclic heterocyclyls include, but are not limited to, chroman-4-yl, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzothien- 2-yl, l,2,3,4-tetrahydroisoquinolin-2-yl, 2-azaspiro[3.3]heptan-2-yl, 2-oxa-6-azaspiro[3.3]heptan- 6-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), azabicyclo[3.1.0]hexanyl (including 3-azabicyclo[3. 1.0]hexan-3-yl), 2,3 -dihydro- IH-indol-l-yl, isoindolin-2-yl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, tetrahydroisoquinolinyl, 7- oxabicyclo[2.2.1]heptanyl, hexahydro-2H-cyclopenta[b]furanyl, 2-oxaspiro[3.3]heptanyl, 3- oxaspiro[5.5]undecanyl, 6-oxaspiro[2.5]octan-l-yl, and 3-oxabicyclo[3.1.0]hexan-6-yl. Tricyclic heterocycles are exemplified by a bicyclic heterocycle fused to a 6-membered arene, or a bicyclic heterocycle fused to a monocyclic cycloalkane, or a bicyclic heterocycle fused to a monocyclic cycloalkene, or a bicyclic heterocycle fused to a monocyclic heterocycle, or a bicyclic heterocycle in which two non-adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2, 5 -epoxypentalene, hexahydro- 2H-2,5-methanocyclopenta[b]furan, hexahydro- 1H- 1 ,4-methanocyclopenta[c]furan, azaadamantane (l-azatricyclo[3.3.1.13,7]decane), and oxa-adamantane (2- oxatricyclo[3.3.1.13,7]decane). The monocyclic, bicyclic, and tricyclic heterocyclyls are connected to the parent molecular moiety at a non-aromatic ring atom.

[0062] The term “hydroxyl” or “hydroxy,” as used herein, means an -OH group.

[0063] Terms such as "alkyl," "cycloalkyl," "alkylene," etc. may be preceded by a designation indicating the number of atoms present in the group in a particular instance (e.g., "Cwalkyl," "C3- scycloalkyl," "Cwalkylene"). These designations are used as generally understood by those skilledin the art. For example, the representation "C" followed by a subscripted number indicates the number of carbon atoms present in the group that follows. Thus, "Csalkyl" is an alkyl group with three carbon atoms (i.e., n-propyl, isopropyl). Where a range is given, as in "Ci-4," the members of the group that follows may have any number of carbon atoms falling within the recited range. A "Ci-4alkyl," for example, is an alkyl group having from 1 to 4 carbon atoms, however arranged (i.e., straight chain or branched).

[0064] The term “substituted” refers to a group that may be further substituted with one or more non-hydrogen substituent groups. Substituent groups include, but are not limited to, halogen, =0 (oxo), =S (thioxo), cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, - COOH, ketone, amide, carbamate, and acyl.B. Conjugates

[0065] Described herein are drug delivery conjugates (e.g., polymer-drug conjugates) useful in the delivery of bioactive agents, such as pharmaceutical drugs (also referred to herein as “drug”). The conjugate can include a copolymer, a plurality of drugs, and a plurality of transcytosis peptides, where the plurality of drugs and the plurality of transcytosis peptides can be attached to the copolymer through different types of linkers.

[0066] In some embodiments, the drug modulates the activity of tubulin and / or microtubules. In some embodiments, the drug modulates the activity of tubulin. In some embodiments, the drug comprises a taxane functional group. In some embodiments, the drug comprises paclitaxel, docetaxel, cabazitaxel, salts thereof, or derivatives thereof. In some embodiments, the drug stabilizes the microtubules. In some embodiments, the drug is paclitaxel.

[0067] The copolymer can include different recurring units, which can be varied by the polymerization method. For example, the copolymer can include a recurring hydrophilic unit, a recurring drug linking unit, and a recurring peptide linking unit. The hydrophilic unit can be water- soluble and can make up the majority of the copolymer. The drug linking unit and the peptide linking unit can be used to attach drugs and peptides, respectively, to the copolymer.

[0068] The hydrophilic unit can be derived from any suitable monomer having hydrophilic and / or water-soluble properties. For example, the hydrophilic unit can be derived from a monomer including a hydrophilic group. In some embodiments, the hydrophilic unit contains at least one hydrophilic group that enhances the water-solubility of the conjugate. The hydrophilic groups can be present on one or more monomers used to produce the hydrophilic unit in the conjugate. Examples of such groups include, but are not limited to, a hydroxyl group, an amino group, a thiol group, a carboxyl group, a SO3H group, or a zwitterionic group.

[0069] Example monomers that the hydrophilic unit can be derived from include, but are not limited to, N-(2-hydroxypropyl)methacrylamide (HPMA), an N,N-disubstituted acrylamide, a hydrophilic ester of methacrylic or acrylic acid, an N- vinylpyrrolidone, an N-acryloylmorpholine, a sulfoethylmethacrylate, an acrylic acid, a methacrylic acid, salts thereof, derivatives thereof, or any a combination thereof.

[0070] In some embodiments, the copolymer comprises the hydrophilic unit at about 50 mol% to about 95 mol% of the copolymer. In one aspect, the copolymer comprises the hydrophilic unit at s about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, about 95 mol%, about or greater of the copolymer. In another aspect, about the copolymer comprises the hydrophilic at about 50-60 mol%, about 50-65 mol%, about 50-70 mol%, about 50-75 mol%, about 50-80 mol%, about 50- 85 mol%, about 50-90 mol%, about 50-95 mol%, about 55-60 mol%, about 55-65 mol%, about 55-70 mol%, about 55-75 mol%, about 55-80 mol%, about 55-85 mol%, about 55-90 mol%, about 55-95 mol%, about 60-75 mol%, about 60-80 mol%, about 60-85 mol%, about 60-90 mol%, about 60-95 mol%, about 70-80 mol%, about 70-85 mol%, about 70-90 mol%, about 70- 95 mol%, about 75-80 mol%, about 75-85 mol%, about 75-90 mol%, or about 75-95 mol% of the copolymer including all endpoints, integers and subranges within the disclosed ranges.

[0071] The copolymer may also include a drug linking unit. The drug linking unit can be derived from a monomer having formula (Ila):whereinR1is hydrogen or Ci-4alkyl;L1is a first cleavable peptide linker;Q is a drug; and w is 0 to 1.

[0072] The monomer of formula (Ila) once polymerized has formula (II) as described herein. In some embodiments, the peptide of L1is a first cleavable peptide linker. In some embodiments, the first cleavable peptide linker has about 2 to about 13 amino acid residues. By varying the amino acid or sequence of amino acids, it may be possible to design a first cleavable peptide linker so that it is cleaved under specific conditions. It is desirable that the first cleavable peptide linker be considerably stable in the bloodstream but degrade when it comes into contact with an enzyme.

[0073] A peptide linker may be cleaved by an enzyme. In some embodiments, the first cleavable peptide linker is cleaved by a lysosomal enzyme. Lysosomal enzymes include a number of proteinases with the ability to hydrolyze peptide linkages (e.g., cathepsin B, L, D or K). The rate of lysosomal hydrolysis of the first cleavable peptide linker is dependent on both the number and the nature of the amino acid residues present in the linker. This is a reflection of both steric and structural factors. In some embodiments, the first cleavable peptide linker is a peptide having and amino acid sequence comprising any one of SEQ ID NO: 1-21. In some embodiments, the amino acid sequence comprises a peptide of Table 1. In some embodiment, the first cleavable peptide linker is SEQ ID NO: 13.Table 1. Listing of Example Cleavable Peptide LinkersAmino Acid SequencePeptide Number One-Letter Three-Letter(SEQ ID NO:) N-terminus- C-terminus) (N-terminus- C-terminus)SEQ ID NO: 1 G-P-NIe Gly-Pro-NleSEQ ID NO: 2 Cit-F Cit-PheSEQ ID NO: 3 KK Lys-LysSEQ ID NO: 4 FK Phe-LysSEQ ID NO: 5 RR Arg- ArgSEQ ID NO: 6 V-Cit Val-CitSEQ ID NO : 7 GFG Gly-Phe-GlySEQ ID NO: 8 GFF Gly-Phe-PheSEQ ID NO: 9 GLG Gly-Leu-GlySEQ ID NO: 10 GVA Gly-Val-AlaSEQ ID NO: 11 GFAGLF Gly-Phe-Ala-Gly-Leu-PheSEQ ID NO: 12 GLAAVA Gly-Leu-Ala-Ala-Val-AlaSEQ ID NO: 13 GFLG Gly-Phe-Leu-GlySEQ ID NO: 14 GFFL Gly-Phe-Phe-LeuSEQ ID NO: 15 GLLG Gly-Leu-Leu-GlySEQ ID NO: 16 GFYA Gly-Phe-Tyr-AlaSEQ ID NO: 17 GF GF Gly-Phe-Gly-PheSEQ ID NO: 18 AGVF Ala-Gly-Val-PheSEQ ID NO: 19 GFFG Gly-Phe-Phe-GlySEQ ID NO: 20 GFLGF Gly-Phe-Leu-Gly-PheSEQ ID NO: 21 GGFLGF Gly-Gly-Phe-Leu-Gly-PheSEQ ID NO: 22 QSFRFK Gln-Ser-Phe-Arg-Phe-LysSEQ ID NO: 23 GFLGKGFLG Gly-Phe-Leu-Gly-Lys-Gly-Phe-Leu-Phe

[0074] In some embodiments, the copolymer comprises the drug linking unit at about 1 mol% to about 10 mol% of the copolymer. In one aspect, the copolymer comprises the drug linking unit at about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about or greater. In another aspect, the copolymer comprises the drug linking unit at about 1-3 mol%, about 1-5 mol%, about 1-8 mol%, about 1-10 mol%, about 2-5 mol%, about 2-8 mol%, about 2-10 mol%, about 3-5 mol%, about 3-8 mol%, about 3-10 mol%, or about 5-10 mol% of the copolymer, including all endpoints, integers and subranges within the disclosed ranges.

[0075] The copolymer may also include a peptide linking unit. The peptide linking unit can be derived from a monomer having of formula (Illa):(Illa), wherein:R2is hydrogen or Ci-4alkyl;Z is a detectable moiety or targeting moiety;L2is a non-cleavable peptide orL3is a non-cleavable linker; and m is 0 to 1.

[0076] The monomer of formula (Illa) once polymerized has formula (III) as described herein. In some embodiments, the peptide linking unit of the conjugates OOcomprises a targeting moiety. The term “targeting moiety,” as used herein, refers to a moiety that can influence the transport and localization of drug in an organism. In some embodiments, the targeting moiety transports drugs across the blood brain barrier leading to localization of the drug in the central nervous system. In some embodiments, the targeting moiety is a transcytosis peptide. In some embodiments, the transcytosis peptide is selected from SEQ ID NO: 24-29. In some embodiments, the transcytosis is a member of the Angiopeps family. In some embodiments, the transcytosis peptide is Angiopep- 2 (AP2) (SEQ ID NO: 24).Table 2. Listing of Example Transcytosis PeptidesPeptide Number Amino Acid Sequence(SEQ ID NO.) (N-terminus^C-terminus)SEQ ID NO: 24 TFFYGGSRGKRNNFKTEEYSEQ ID NO: 25 TFFYGGCRGKRNNFKTEEYSEQ ID NO: 26 TFFYGGSRGKRNNFRTEEYSEQ ID NO: 27 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSSEQ ID NO: 28 TFFYGGSRGRRNNFRTEEYSEQ ID NO: 29 YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY

[0077] In some embodiments, the conjugate may optionally comprise a detectable moiety. A “detectable moiety” as used herein refers to a moiety that fluoresces, luminesces, or is radioactive. In some embodiments the detectable moiety is a radioisotope of hydrogen, carbon, iodine, or fluorine. In some embodiments, the detectable moiety is a fluorophore. Any fluorophore may be used with the methods described herein. Examples of fluorophores include, but are not limited to, rhodamine-based molecules, squarylium-based molecules, cyanine-based molecules, aromatic ring-based molecules, oxazine-based molecules, carbopyronine-based molecules and pyrromethene-based molecules. In some embodiments, the fluorophores may be selected from, for example, Alexa Fluor®-based molecules (registered trademark, manufactured by Invitrogen), BODIPY®-based molecules (registered trademark, manufactured by Invitrogen), Cy™-based molecules (registered trademark, manufactured by GE Healthcare), DY™-based molecules (registered trademark, Dyomics GmbH), HiLyte™-based molecules (registered trademark, manufactured by AnaSpec Inc.), DyLight®-based molecules (registered trademark, manufactured by Thermo Fisher Scientific K.K.), ATTO™-based dmolecules (registered trademark, manufactured by ATTO-TEC GmbH) and MFP™-based molecules (registered trademark, manufactured by Mobitec Co., Ltd.), among other well known fluorophores. The generic names of these molecules are designated based on the main structure (skeleton) or the registered trademark of the respective compounds. Those of ordinary skill in the art can properly understand the scope of fluorophores belonging to the respective generic names without having to bear undue experimentation.

[0078] Specific examples of rhodamine-based fluorophores may include, but are not limited to, 5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethyl rhodamine, X-rhodamine, Texas Red, Spectrum Red and LD700 PERCHLORATE.

[0079] Some specific examples of squarylium-based fluorophores include, but are not limited to, SRfluor 680-carboxylate, l,3-Bis[4-(dimethylamino)-2-hydroxyphenyl]-2,4- dihydroxycyclobutenediylium dihydroxide, bis, l,3-bis[4-(dimethylamino)phenyl]-2,4- dihydroxycyclobutenediylium dihydroxide, bis, 2-(4-(diethylamino)-2-hydroxyphenyl)-4-(4- (diethyliminio)-2-hydroxycyclohexa-2,5-dienylidene)-3-oxocyclobut-l-enolate, 2-(4-(dibutylamino)-2-hydroxyphenyl)-4-(4-(dibutyliminio)-2-hydroxycyclohexa-2,5-dienylidene)-3- oxocyclobut-1 -enolate and 2-(8-hydroxy-l,l,7,7-tetramethyl-l,2,3,5,6,7-hexahydropyrido[3,2,l-ij]quinolin-9-yl)-4-(8-hydroxy- 1,1,7, 7-tetram ethyl -2,3,6, 7-tetrahydro-l H-pyrido[3, 2,1- ij ]quinolinium-9(5H)-ylidene)-3 -oxocyclobut- 1 -enolate.

[0080] Specific examples of cyanine-based fluorophores include, but are not limited to, 1 -butyl - 2-[5-(l -butyl- l,3-dihydro-3, 3-dimethyl-2H-indol-2-ylidene)-penta-l, 3-dienyl]-3, 3-dimethyl- 3eiti-indolium hexafluorophosphate, l-butyl-2-[5-(l-butyl-3,3-dimethyl-l,3-dihydro-indol-2- ylidene)-3-chloro-penta-l,3-dienyl]-3,3-dimethyl-3H-indolium hexafluorophosphate and 3-ethyl- 2-[5-(3-ethyl-3H-benzothiazol-2-ylidene)-penta-l,3-dienyl]-benzothiazol -3-ium iodide.

[0081] Specific examples of aromatic ring-based fluorophores include, but are not limited to, N,N-bis-(2,6-diisopropylphenyl)-l,6,7,12-(4-tert-butylphenoxy)-perylen-3,4,9,10- tetracarbonaciddiimide, N,N'-bis(2,6-diisopropylphenyl)-l,6,7,12-tetraphenoxyperylene- 3,4:9, 10-tetracarboxdiimide, N,N'-bis(2,6-diisopropylphenyl)perylene-3,4,9,10-bis(dicarbimide), 16,N,N'-bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarboxylicdiimide, 4,4'-[(8,16-dihydro- 8, 16-dioxodibenzo[a,j ]perylene-2, 10-diyl)dioxy]dibutyric acid, 2, 10-dihydroxy- dibenzo[a,j ]perylene-8, 16-dione, 2, 10-bi s(3 -aminopropoxy)dibenzo[a,j ]perylene-8, 16-dione,3 ,3 '-[(8, 16-dihydro-8, 16-dioxodibenzo[a,j ]perylen-2, 10-diyl)dioxy]dipropylamine, 17- bis(octyloxy)anthra[9,l,2-cde-]benzo[rst]pentaphene-5-10-dione, octadecanoic acid, 5,10- dihydro-5,10-dioxoanthra[9,l,2-cde]benzo[rst]pentaphene-16,17-diylester, N,N'-di(nonyldecyl)- perylene-3,4,9,10-tetracarboxylic diimide and dihydroxydibenzanthrone.

[0082] Specific examples of oxazine-based fluorophores include, but are not limited to, Cresyl violet, Oxazine 170, EVOblue 30 and Nile Blue.

[0083] Specific examples of carbopyronine-based fluorophores include, but are not limited to, CARBOP YRONIN 149.

[0084] Specific examples of the pyrromethene-based fluorophores include, but are not limited to, PYRROMETHENE 650.

[0085] Specific examples of Alexa Fluor®-based fluorophores include, but are not limited to, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750.

[0086] Specific examples of BODIPY®-based fluorophores include, but are not limited to BODIPY FL, BODIPY TMR, BODIPY 493 / 503, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY564 / 570, BODIPY 576 / 589, BODIPY 581 / 591 , BODIPY 630 / 650 and BODIPY 650 / 665 (all of which are manufactured by Invitrogen).

[0087] Specific examples of Cy™-based fluorophores include, but are not limited to, Cy 3.5, Cy 5, Cy 5.5, and Cy7.

[0088] Specific examples of DY™-based fluorophores include, but are not limited to, DY-590, DY-610, DY-615, DY-630, DY-631, DY-632, DY-633 and DY-634.

[0089] Specific examples of HiLyte™-based fluorophores include, but are not limited to, HiLyte™ 594 and HiLyteFluor TR.

[0090] Specific examples of DyLight™-based fluorophores include, but are not limited to, DyLight 594 and DyLight 633.

[0091] Specific examples of ATTO™-based fluorophores include, but are not limited to, ATTO590, ATTO 610, ATTO 620, ATTO 633 and ATTO 655.

[0092] Specific examples of MFP™-based fluorophores include, but are not limited to, MFP 590 and MFP 631.

[0093] Examples of other fluorophores include, but are not limited to, C-phycocyanin, phycocyanin, APC (allophycocyanin), APC-XL and NorthernLights 637.

[0094] Further examples of fluorophores also include derivatives of the above-described dyes.

[0095] In some embodiments, L3of the peptide linking unit is a non-cleavable linker comprising a maleimide (maL) or maleimide derivative. In some embodiments, the non-cleavable linker has a formula IV:whereinG1is Ci-scarbocycle, a 6- to 12-membered aryl, or a 5- to 12-membered heteroaryl, wherein G1is optionally substituted with 1-6 substituents independently selected from the group consisting of Ci-4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi-2haloalkyl; andL4is Ci-ealkyl.

[0096] Tn some embodiments, G1of the non-cleavable linker is Ci-scarbocycle. In some embodiments, G1of the non-cleavable linker is Cecarbocycle. In some embodiments, L4of the non-cleavable linker is Cialkyl. In some embodiments the non-cleavable amino acid is Gly-Gly (G-G).In some embodiments, the copolymer comprises the peptide linking unit at about 0.5 mol% to about 10 mol%. In one aspect, the copolymer comprises the peptide linking unit at about 0.5 mol%, about 0.6 mol%, about 0.7 mol%, about 0.8 mol%, about 0.9 mol%, about 1 mol%, about 1.5 mol%, about 2 mol%, about 2.5 mol%, about 3 mol%, about 3.5 mol%, about 4 mol%, about 4.5 mol%, about 5 mol%, about 5.5 mol%, about 6 mol%, about 6.5 mol%, about 7 mol%, about 7.5 mol%, about 8 mol%, about 8.5 mol%, about 9 mol%, about 9.5 mol%, about 10 mol%, or greater. In another aspect, the copolymer comprises the peptide linking unit comprises about 0.5-1 mol%, about 0.5-1.5 mol%, about 0.5-2 mol%, about 0.5-2.5 mol%, about 0.5-3 mol%, about 0.5-3.5 mol%, about 0.5-4 mol%, about 0.5-4.5 mol%, about 0.5-5 mol%, about 0.5-5.5 mol%, about0.5-6 mol%, about 0.5-6.5 mol%, about 0.5-7 mol%, about 0.5-7.5 mol%, about 0.5-8 mol%, about 0.5-8.5 mol%, about 0.5-9 mol%, about 0.5-9.5 mol%, about 0.5-10 mol%, about 0.6-1 mol%, about 0.6-1.5 mol%, about 0.6-2 mol%, about 0.6-2.5 mol%, about 0.6-3 mol%, about0.6-3.5 mol%, about 0.6-4 mol%, about 0.6-4.5 mol%, about 0.6-5 mol%, about 0.6-5.5 mol%, about 0.6-6 mol%, about 0.6-6.5 mol%, about 0.6-7 mol%, about 0.6-7.5 mol%, about 0.6-8 mol%, about 0.6-8.5 mol%, about 0.6-9 mol%, about 0.6-9.5 mol%, about 0.6-10 mol%, about 0.7-1 mol%, about 0.7-1.5 mol%, about 0.7-2 mol%, about 0.7-2.5 mol%, about 0.7-3 mol%, about 0.7-3.5 mol%, about 0.7-4 mol%, about 0.7-4.5 mol%, about 0.7-5 mol%, about 0.7-5.5 mol%, about 0.7-6 mol%, about 0.7-6.5 mol%, about 0.7-7 mol%, about 0.7-7.5 mol%, about 0.7-8 mol%, about 0.7-8.5 mol%, about 0.7-9 mol%, about 0.7-9.5 mol%, about 0.7-10 mol%, about 0.8-1 mol%, about 0.8-1.5 mol%, about 0.8-2 mol%, about 0.8-2.5 mol%, about 0.8-3 mol%, about 0.8-3.5 mol%, about 0.8-4 mol%, about 0.8-4.5 mol%, about 0.8-5 mol%, about 0.8-5.5 mol%, about 0.8-6 mol%, about 0.8-6.5 mol%, about 0.8-7 mol%, about 0.8-7.5 mol%, about 0.8-8 mol%, about 0.8-8.5 mol%, about 0.8-9 mol%, about 0.8-9.5 mol%, about 0.8-10 mol%, about 0.9-2 mol%, about 0.9-3 mol%, about 0.9-4 mol%, about 0.9-5 mol%, about 0.9-6 mol%, about 0.9-7 mol%, about 0.9-8 mol%, about 0.9-9 mol%, about 0.9-10 mol%, about 1-3 mol%, about 1-5 mol%, about 1-8 mol%, about 1-10 mol%, about or 5-10 mol%, of the copolymer, including all endpoints, integers and subranges within the disclosed ranges.

[0097] Compounds of the present disclosure are set forth in the following numbered embodiments. The first embodiment is denoted El, another embodiment is denoted E2 and so forth.

[0098] El. A conjugate of formula (V), or a salt thereofwhereinR1, R2, and R3, at each occurrence, are independently hydrogen or C i 4al kyl ;L1, at each occurrence, is a first cleavable peptide linker;L2, at each occurrence, is a non-cleavable peptide orL3, at each occurrence, is a non-cleavable linker,,Or, wherein L4, at each occurrence, is Ci ealkyl; and G1is Ci- scarbocycle, a 6- to 12-membered aryl, or a 5- to 12-membered heteroaryl, wherein G1is optionally substituted with 1-6 substituents independently selected from the group consisting of Ci-4alkyl, Ci-2haloalkyl, halogen, cyano, -OCi-4alkyl, and -OCi-2haloalkyl;G3is a hydrophilic polymer;Q is a drug;Z, at each occurrence, is independently a detectable moiety or drug targeting moiety;T1and T2are terminal end units;x is 1 to 20 y is 1 to 20 n is 20 to 300 w is 0 to 1; and m is 0 to 1. It is noted that the recuring units described above for formula (V) can be included randomly or as blocks in the conjugate.

[0099] E2. The conjugate of El, wherein R1is Cialkyl.

[0100] E3. The conjugate of El or E2, wherein R2is Cialkyl.

[0101] E4. The conjugate of any one of E1-E3, wherein R3is Cialkyl.

[0102] E5. The conjugate of any one of E1-E4, wherein w is 1.

[0103] E6. The conjugate of any one of E1-E5, wherein the first cleavable peptide linker is— Gly-Phe— Leu-Gly—

[0104] E7. The conjugate of any one of E1-E6, wherein the non-cleavable peptide is Gly— Gly—

[0105] E8. The conjugate of any one of E1-E7, wherein m=l and L3is

[0106] E9. The conjugate of any one of E1-E8, wherein

[0107] E10. The conjugate of any one of E1-E10, wherein the drug is a microtubule stabilizing drug.

[0108] El l. The conjugate of any one of E1-E10, wherein the drug comprises a taxane.

[0109] E12. The conjugate of any one of El-El 1, wherein the drug is selected from the groups consisting of paclitaxel, docetaxel, cabazitaxel, salts thereof, and derivatives thereof.

[0110] El 3. The conjugate of any one of E1-E12, wherein the detectable moiety is a fluorophore.

[0111] E14. The conjugate of any one of E1-E13, wherein the drug directing moiety is angiopep-2 peptide.

[0112] E15. The conjugate of any one of E1-E14, wherein m=0 and L3is a non-cleavable linker.

[0113] El 6. The conjugate of any one of El-El 5, wherein T1 and T2, at each occurrence, are

[0114] In some embodiments, the copolymer has a molecular weight (e.g., number average - Mn) from about 20 kilo Daltons (kDa) to about 200 kilo Daltons (kDa). In one aspect, the molecular weight of the copolymer is about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 55 kDa, about 60 kDa, about 65 kDa, about 70 kDa, about 75 kDa, about 80 kDa, about 85 kDa, about 90 kDa, about 95 kDa, about 100 kDa, about 105 kDa, about 110 kDa, about 115 kDa, about 120 kDa, about 125 kDa, about 130 kDa, about 135 kDa, about 140 kDa, about 145 kDa, about 150 kDa, about 155 kDa, about 160 kDa, about 165 kDa, about 170 kDa, about 175 kDa, about 180 kDa, about 185 kDa, about 190 kDa, about 195 kDa, about 200 kDa, or greater.

[0115] In another aspect, the copolymer has a molecular weight from about 20-50 kDa, about 20-75 kDa, about 20-100 kDa, about 20-125 kDa, about 20-150 kDa, about 20-175 kDa, about 20-200 kDa, about 25-50 kDa, about 25-75 kDa, about 25-100 kDa, about 25-125 kDa, about 25-150 kDa, about 25-175 kDa, about 25-200 kDa, about 30-50 kDa, about 30-75 kDa, about 30-100 kDa, about 30-125 kDa, about 30-150 kDa, about 30-175 kDa, about 30-200 kDa, about 35-50 kDa, about 35-75 kDa, about 35-100 kDa, about 35-125 kDa, about 35-150 kDa, about 35-175 kDa, about 35-200 kDa, about 40-50 kDa, about 40-75 kDa, about 40-100 kDa, about 40-125 kDa, about 40-150 kDa, about 40-175 kDa, about 40-200 kDa, about 50-75 kDa, about 50-100 kDa, about 50-125 kDa, about 50-150 kDa, about 50-175 kDa, about 50-200 kDa, about 75-100 kDa, about 75-125 kDa, about 75-150 kDa, about 75-175 kDa, about 75-200 kDa, about 100-125 kDa, about 100-150 kDa, about 100-175 kDa, about 100-200 kDa, about 125-150 kDa,about 125-175 kDa, about 125-200 kDa, about or 150-200 kDa, including all endpoints, integers and subranges within the disclosed ranges.

[0116] weight of the copolymer can be measured by techniques known within the art, such as size exclusion chromatography (SEC), SEC combined with multi-angle light scattering, gel permeation chromatography, flash chromatography, rheometry, and the like.C. Multi-domain of Conjugates

[0117] The disclosed conjugates can be used to form a multi-domain conjugate As used herein, a “multi-domain conjugate” is a molecule having a formula (VI):. In some embodiments, Z and Y are conjugates described herein. In some embodiments, Z and Y are identical. In some embodiments, L6is a second cleavable linker, such as a second cleavable peptide linker. In some embodiments, the second cleavable peptide linker has about 2 to about 13 amino acid residues. By varying the amino acid or sequence of amino acids, it may be possible to design a second cleavable peptide linker so that it is cleaved under specific conditions. It is desirable that the second cleavable peptide linker be considerably stable in the bloodstream but degrade when it comes into contact with an enzyme.A peptide linker may be cleaved by an enzyme.

[0118] In some embodiments, the second cleavable peptide linker is cleaved by a lysosomal enzyme. Lysosomal enzymes include a number of proteinases with the ability to hydrolyze peptide linkages (e.g., cathepsin B, L, D or K). The rate of lysosomal hydrolysis of the second cleavable peptide linker is dependent on both the number and the nature of the amino acid residues present in the linker. This is a reflection of both steric and structural factors.

[0119] In some embodiments, the second cleavable linker has a formula (VII): -(AAi)-K- (AA2)-. In some embodiments, AAi and AA2 are the same or different amino acid sequence comprising up to six amino acids, and K is lysine, ornithine, or a diamine. In some embodiments, the second cleavable peptide linker is a peptide having the amino acid SEQ ID NO. 1-23. In some embodiments, the second cleavable peptide linker is a peptide having the amino acid sequence described in Table 2. In some embodiments, the second cleavable peptide linker has a peptide sequence of GFLGKGFLG (SEQ ID NO: 23).

[0120] In some embodiments, the multi-domain of conjugates has a molecular weight from about 20 kilo Daltons (kDa) to about 200 kilo Daltons (kDa). In one aspect, the molecular weight of the conjugate is about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 55 kDa, about 60 kDa, about 65 kDa, about 70 kDa, about 75 kDa, about 80 kDa, about 85 kDa, about 90 kDa, about 95 kDa, about 100 kDa, about 105 kDa, about110 kDa, about 115 kDa, about 120 kDa, about 125 kDa, about 130 kDa, about 135 kDa, about140 kDa, about 145 kDa, about 150 kDa, about 155 kDa, about 160 kDa, about 165 kDa, about170 kDa, about 175 kDa, about 180 kDa, about 185 kDa, about 190 kDa, about 195 kDa, about200 kDa, or greater.

[0121] In another aspect, the multi-domain of conjugates has molecular weight from about 20- 50 kDa, about 20-75 kDa, about 20-100 kDa, about 20-125 kDa, about 20-150 kDa, about 20- 175 kDa, about 20-200 kDa, about 25-50 kDa, about 25-75 kDa, about 25-100 kDa, about 25- 125 kDa, about 25-150 kDa, about 25-175 kDa, about 25-200 kDa, about 30-50 kDa, about 30- 75 kDa, about 30-100 kDa, about 30-125 kDa, about 30-150 kDa, about 30-175 kDa, about 30- 200 kDa, about 35-50 kDa, about 35-75 kDa, about 35-100 kDa, about 35-125 kDa, about 35- 150 kDa, about 35-175 kDa, about 35-200 kDa, about 40-50 kDa, about 40-75 kDa, about 40- 100 kDa, about 40-125 kDa, about 40-150 kDa, about 40-175 kDa, about 40-200 kDa, about SO- 75 kDa, about 50-100 kDa, about 50-125 kDa, about 50-150 kDa, about 50-175 kDa, about 50- 200 kDa, about 75-100 kDa, about 75-125 kDa, about 75-150 kDa, about 75-175 kDa, about 75- 200 kDa, about 100-125 kDa, about 100-150 kDa, about 100-175 kDa, about 100-200 kDa, about 125-150 kDa, about 125-175 kDa, about 125-200 kDa, about or 150-200 kDa, including all endpoints, integers and subranges within the disclosed ranges.D. Pharmaceutical Salts

[0122] The disclosed conjugates may exist as pharmaceutically acceptable salts. The term “pharmaceutically acceptable salt” refers to salts or zwitterions of the conjugates which are water or oil-soluble or dispersible, suitable for treatment of disorders without undue toxicity, irritation, and allergic response, commensurate with a reasonable benefit / risk ratio and effective for their intended use. The salts may be prepared during the final isolation and purification of the compounds or separately by reacting an amino group of the compounds with a suitable acid. For example, a conjugate may be dissolved in a suitable solvent, such as but not limited to methanol and water and treated with at least one equivalent of an acid, like hydrochloric acid. The resultingsalt may precipitate out and be isolated by filtration and dried under reduced pressure. Alternatively, the solvent and excess acid may be removed under reduced pressure to provide a salt. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3 -phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, thri chloroacetate, trifluoroacetate, glutamate, para-toluenesulfonate, undecanoate, hydrochloric, hydrobromic, sulfuric, phosphoric and the like. The amino groups of the conjugates may also be quaternized with alkyl chlorides, bromides, or iodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl and the like.

[0123] Basic addition salts may be prepared during the final isolation and purification of the disclosed conjugates by reaction of a carboxyl group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation such as lithium, sodium, potassium, calcium, magnesium, or aluminum, or an organic primary, secondary, or tertiary amine. Quaternary amine salts can be prepared, such as those derived from methylamine, dimethylamine, trimethylamine, tri ethyl amine, diethylamine, ethylamine, tributylamine, pyridine, A,A-dimethylaniline, N- methylpiperidine, A-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N- dibenzylphenethylamine, 1 -ephenamine and A', A'-dibenzyl ethylenedi amine, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.E. Modes of Administration

[0124] Methods of treatment may include any number of modes of administering a conjugate or multi-domain conjugate described herein. Modes of administration may include tablets, pills, dragees, hard and soft gel capsules, granules, pellets, aqueous, lipid, oily or other solutions, emulsions such as oil-in-water emulsions, liposomes, aqueous or oily suspensions, syrups, elixirs, solid emulsions, solid dispersions or dispersible powders. For the preparation of pharmaceutical compositions for oral administration, the agent may be admixed with commonly known and used adjuvants and excipients such as for example, gum arabic, talcum, starch, sugars (such as, e.g., mannitose, methyl cellulose, lactose), gelatin, surface-active agents, magnesium stearate, aqueous or non-aqueous solvents, paraffin derivatives, cross-linking agents, dispersants, emulsifiers, lubricants, conserving agents, flavoring agents (e.g., ethereal oils), solubility enhancers (e.g.,benzyl benzoate or benzyl alcohol) or bioavailability enhancers (e.g., Gelucire™). In the pharmaceutical composition, the agent may also be dispersed in a microparticle, e.g., a nanoparticulate composition.

[0125] For parenteral administration, the agent can be dissolved or suspended in a physiologically acceptable diluent, such as, e.g., water, buffer, oils with or without solubilizers, surface-active agents, dispersants or emulsifiers. As oils for example and without limitation, olive oil, peanut oil, cottonseed oil, soybean oil, castor oil and sesame oil may be used. More generally spoken, for parenteral administration, the agent can be in the form of an aqueous, lipid, oily or other kind of solution or suspension or even administered in the form of liposomes or nanosuspensions.The term “parenterally,” as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrathecal, subcutaneous and intraarticular injection and infusion.F. Pharmaceutical Compositions

[0126] The conjugates and their pharmaceutically acceptable salts may be formulated for administration by, for example, injection or oral administration. Techniques and formulations may generally be found in “Remington’s Pharmaceutical Sciences,” (Meade Publishing Co., Easton, Pa.). Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage.

[0127] The route by which the disclosed compounds are administered, and the form of the pharmaceutical composition will dictate the type of carrier to be used. The composition may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral or parenteral).

[0128] Carriers for systemic administration typically include at least one of diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, combinations thereof, and others. All carriers are optional in the compositions.

[0129] Suitable diluents include sugars such as glucose, lactose, dextrose, and sucrose; diols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols, such as glycerin; mannitol; and sorbitol. The amount of diluent(s) in a systemic or topical composition is typically about 50 to about 90%.

[0130] Suitable lubricants include silica, talc, stearic acid and its magnesium salts and calcium salts, calcium sulfate; and liquid lubricants such as polyethylene glycol and vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of Theobroma. The amount of lubricant(s) in a systemic or topical composition is typically about 5 to about 10%.

[0131] Suitable binders include polyvinyl pyrrolidone; magnesium aluminum silicate; starches such as corn starch and potato starch; gelatin; tragacanth; and cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylcellulose. The amount of binder(s) in a systemic composition is typically about 5 to about 50%.

[0132] Suitable disintegrants include agar, alginic acid and the sodium salt thereof, effervescent mixtures, croscarmellose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays, and ion exchange resins. The amount of disintegrant(s) in a systemic or topical composition is typically about 0.1 to about 10%.

[0133] Suitable colorants include a colorant such as an FD&C dye. When used, the amount of colorant in a systemic or topical composition is typically about 0.005 to about 0.1%.

[0134] Suitable flavors include menthol, peppermint, and fruit flavors. The amount of flavor(s), when used, in a systemic or topical composition is typically about 0.1 to about 1.0%.

[0135] Suitable sweeteners include aspartame and saccharin. The amount of sweetener(s) in a systemic or topical composition is typically about 0.001 to about 1%.

[0136] Suitable antioxidants include butylated hydroxyanisole (“BHA”), butylated hydroxytoluene (“BHT”), and vitamin E. The amount of antioxidant(s) in a systemic or topical composition is typically about 0.1 to about 5%.

[0137] Suitable preservatives include benzalkonium chloride, methyl paraben and sodium benzoate. The amount of preservative(s) in a systemic or topical composition is typically about 0.01 to about 5%.

[0138] Suitable glidants include silicon dioxide. The amount of glidant(s) in a systemic or topical composition is typically about 1 to about 5%.

[0139] Suitable solvents include water, isotonic saline, ethyl oleate, glycerine, hydroxylated castor oils, alcohols such as ethanol, and phosphate buffer solutions. The amount of solvent(s) in a systemic or topical composition is typically from about 0 to about 100%.

[0140] Suitable suspending agents include AVICEL RC-591 (from FMC Corporation of Philadelphia, PA) and sodium alginate. The amount of suspending agent(s) in a systemic or topical composition is typically about 1 to about 8%.

[0141] Suitable surfactants include lecithin, Polysorbate 80, and sodium lauryl sulfate, and the TWEENS from Atlas Powder Company of Wilmington, Delaware. Suitable surfactants include those disclosed in the C.T.F.A. Cosmetic Ingredient Handbook, 1992, pp.587-592; Remington’s Pharmaceutical Sciences, 15th Ed. 1975, pp. 335-337; and McCutcheon’s Volume 1, Emulsifiers & Detergents, 1994, North American Edition, pp. 236-239. The amount of surfactant(s) in the systemic composition typically about 0.1% to about 5%.

[0142] Although the amounts of components in the systemic compositions may vary depending on the type of systemic composition prepared, in general, systemic compositions include 0.01% to 50% of an active compound (e.g., a compound of formula (I)) and 50% to 99.99% of one or more carriers. Compositions for parenteral administration typically include 0.1% to 10% of actives and 90% to 99.9% of a carrier including a diluent and a solvent.

[0143] Compositions for oral administration can have various dosage forms. For example, solid forms include tablets, capsules, granules, and bulk powders. These oral dosage forms include a safe and effective amount, usually at least about 5%, and more particularly from about 25% to about 50% of actives. The oral dosage compositions include about 50% to about 95% of carriers, and more particularly, from about 50% to about 75%.

[0144] Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed. Tablets typically include an active component, and a carrier comprising ingredients selected from diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, glidants, and combinations thereof. Specific diluents include calcium carbonate, sodium carbonate, mannitol, lactose and cellulose. Specific binders include starch, gelatin, and sucrose. Specific disintegrants include alginic acid and croscarmellose. Specific lubricants include magnesium stearate, stearic acid, and talc. Specific colorants are the FD&C dyes, which can be added for appearance. Chewable tablets preferably contain sweeteners such as aspartame and saccharin, or flavors such as menthol, peppermint, fruit flavors, or a combination thereof.

[0145] Capsules (including implants, time release and sustained release formulations) typically include an active compound (e.g., a conjugates or multi-domain conjugates) and a carrier including one or more diluents disclosed above in a capsule comprising gelatin. Granules typically comprisea disclosed compound, and preferably glidants such as silicon dioxide to improve flow characteristics.

[0146] Implants can be of the biodegradable or the non-biodegradable type.

[0147] The selection of ingredients in the carrier for oral compositions depends on secondary considerations like taste, cost, and shelf stability, which are not critical for the purposes of this invention.

[0148] Solid compositions may be coated by conventional methods, typically with pH or timedependent coatings, such that a disclosed compound is released in the gastrointestinal tract in the vicinity of the desired application, or at various points and times to extend the desired action. The coatings typically include one or more components selected from the group consisting of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, EUDRAGIT® coatings (available from Evonik Industries of Essen, Germany), waxes and shellac.

[0149] Compositions for oral administration can have liquid forms. For example, suitable liquid forms include aqueous solutions, emulsions, suspensions, solutions reconstituted from non- effervescent granules, suspensions reconstituted from non-effervescent granules, effervescent preparations reconstituted from effervescent granules, elixirs, tinctures, syrups, and the like.

[0150] Liquid orally administered compositions typically include a disclosed compound and a carrier, namely, a carrier selected from diluents, colorants, flavors, sweeteners, preservatives, solvents, suspending agents, and surfactants. Peroral liquid compositions preferably include one or more ingredients selected from colorants, flavors, and sweeteners.

[0151] Other compositions useful for attaining systemic delivery of the subject compounds include sublingual, buccal and nasal dosage forms. Such compositions typically include one or more of soluble filler substances such as diluents including sucrose, sorbitol and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropyl methylcellulose. Such compositions may further include, but not be limited to, lubricants, colorants, flavors, sweeteners, antioxidants, and glidants.

[0152] The amount of the carrier employed in conjunction with a disclosed compound is sufficient to provide a practical quantity of composition for administration per unit dose of the compound. Techniques and compositions for making dosage forms useful in the methods of this invention are described in the following references: Modem Pharmaceutics, Chapters 9 and 10,Banker & Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976), which are incorporated herein by reference.

[0153] A carrier may include a single ingredient or a combination of two or more ingredients. Suitable topical carriers include, but are not limited to, one or more ingredients selected from phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, symmetrical alcohols, aloe vera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristyl propionate, dimethyl isosorbide, castor oil, combinations thereof, and the like. More particularly, carriers may include propylene glycol, dimethyl isosorbide, and water, and even more particularly, phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, and symmetrical alcohols.

[0154] Suitable emollients include stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, propane- 1,2-diol, butane- 1,3 -diol, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil, coconut oil, arachis oil, castor oil, acetylated lanolin alcohols, petroleum, mineral oil, butyl myristate, isostearic acid, palmitic acid, isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, myristyl myristate, and combinations thereof. The amount of emollient(s) in a skin-based topical composition is typically about 5% to about 95%.

[0155] Suitable propellants include propane, butane, isobutane, dimethyl ether, carbon dioxide, nitrous oxide, and combinations thereof.

[0156] Suitable solvents include water, ethyl alcohol, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethyl sulfoxide, dimethyl formamide, tetrahydrofuran, and combinations thereof. Specific solvents include ethyl alcohol and homotopic alcohols. The amount of solvent(s) in a pharmaceutical composition is typically about 0% to about 95%.

[0157] Suitable humectants include glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, gelatin, and combinations thereof. Specific humectants include glycerin. The amount of humectant(s) in a pharmaceutical composition is typically 0% to 95%.

[0158] The amount of thickener(s) in a pharmaceutical composition is typically about 0% to about 95%.

[0159] Suitable powders include beta-cyclodextrins, hydroxypropyl cyclodextrins, chalk, talc, fullers earth, kaolin, starch, gums, colloidal silicon dioxide, sodium polyacrylate, tetra alkyl ammonium smectites, trialkyl aryl ammonium smectites, chemically-modified magnesium aluminum silicate, organically-modified montmorillonite clay, hydrated aluminum silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethyl cellulose, ethylene glycol monostearate, and combinations thereof. The amount of powder(s) in a pharmaceutical composition is typically 0% to 95%.

[0160] The amount of fragrance in a pharmaceutical composition is typically about 0% to about 0.5%, particularly, about 0.001% to about 0.1%.

[0161] Suitable pH adjusting additives include HO or NaOH in amounts sufficient to adjust the pH of a pharmaceutical composition.A. Dosages

[0162] It will be appreciated that appropriate dosages of the conjugates, multi-domains of conjugates and pharmaceutical compositions thereof, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments described herein. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and / or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious sideeffects.

[0163] Administration in vivo can be administered in one dose, in multiple doses, continuously, or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of administration. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single ormultiple administrations can be carried out with the dose level and pattern being selected by the treating physician or scientist.

[0164] In one embodiment, ,the dose of a conjugate comprises from about 0.1 to about 10 mg / kg. In one aspect, the dose comprises about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.25 mg / kg, about 1.5 mg / kg, about 1.75 mg / kg, about 2 mg / kg, about 2.25 mg / kg, about 2.5 mg / kg, about 2.75 mg / kg, about 3 mg / kg, about 3.25 mg / kg, about3.5 mg / kg, about 3.75 mg / kg, about 4 mg / kg, about 4.25 mg / kg, about 4.5 mg / kg, about 4.75 mg / kg, about 5 mg / kg, about 5.25 mg / kg, about 5.5 mg / kg, about 5.75 mg / kg, about 6 mg / kg, about 6.25 mg / kg, about 6.5 mg / kg, about 6.75 mg / kg, about 7 mg / kg, about 7.25 mg / kg, about7.5 mg / kg, about 7.75 mg / kg, about 8 mg / kg, about 8.25 mg / kg, about 8.5 mg / kg, about 8.75 mg / kg, about 9 mg / kg, about 9.25 mg / kg, about 9.5 mg / kg, about 9.75 mg / kg, about 10 mg / kg, or greater.In another aspect, the dose of a conjugate comprises about 0.1-0.5 mg / kg, about 0.1-1 mg / kg, about 0.1-1.5 mg / kg, about 0.1-2 mg / kg, about 0.1-2.5 mg / kg, about 0.1-3 mg / kg, about 0.1-3.5 mg / kg, about 0.1-4 mg / kg, about 0.1-4.5 mg / kg, about 0.1-5 mg / kg, about 0.1-5.5 mg / kg, about 0.1-6 mg / kg, about 0.1-6.5 mg / kg, about 0.1-7 mg / kg, about 0.1 -7.5 mg / kg, about 0.1-8 mg / kg, about 0.1-8.5 mg / kg, about 0.1-9 mg / kg, about 0.1-9.5 mg / kg, about 0.1-10 mg / kg, about 0.2- 0.5 mg / kg, about 0.2-1 mg / kg, about 0.2-1.5 mg / kg, about 0.2-2 mg / kg, about 0.2-2.5 mg / kg, about 0.2-3 mg / kg, about 0.2-3.5 mg / kg, about 0.2-4 mg / kg, about 0.2-4.5 mg / kg, about 0.2-5 mg / kg, about 0.2-5.5 mg / kg, about 0.2-6 mg / kg, about 0.2-6.5 mg / kg, about 0.2-7 mg / kg, about 0.2-7.5 mg / kg, about 0.2-8 mg / kg, about 0.2-8.5 mg / kg, about 0.2-9 mg / kg, about 0.2-9.5 mg / kg, about 0.2-10 mg / kg, about 0.3-1 mg / kg, about 0.3-1.5 mg / kg, about 0.3-2 mg / kg, about 0.3-2.5 mg / kg, about 0.3-3 mg / kg, about 0.3-3.5 mg / kg, about 0.3-4 mg / kg, about 0.3-4.5 mg / kg, about 0.3-5 mg / kg, about 0.3 -5.5 mg / kg, about 0.3-6 mg / kg, about 0.3-6.5 mg / kg, about 0.3-7 mg / kg, about 0.3-7.5 mg / kg, about 0.3-8 mg / kg, about 0.3-8.5 mg / kg, about 0.3-9 mg / kg, about 0.3-9.5 mg / kg, about 0.3-10 mg / kg, about 0.4-1 mg / kg, about 0.4-1.5 mg / kg, about 0.4-2 mg / kg, about 0.4-2.5 mg / kg, about 0.4-3 mg / kg, about 0.4-3.5 mg / kg, about 0.4-4 mg / kg, about 0.4-4.5 mg / kg, about 0.4-5 mg / kg, about 0.4-5.5 mg / kg, about 0.4-6 mg / kg, about 0.4-6.5 mg / kg, about 0.4-7 mg / kg, about 0.4-7.5 mg / kg, about 0.4-8 mg / kg, about 0.4-8.5 mg / kg, about 0.4-9 mg / kg, about 0.4-9.5 mg / kg, about 0.4-10 mg / kg, about 0.5-1 mg / kg, about 0.5-1.5 mg / kg, about 0.5-2 mg / kg,about 0.5-2.5 mg / kg, about 0.5-3 mg / kg, about 0.5-3.5 mg / kg, about 0.5-4 mg / kg, about 0.5-4.5 mg / kg, about 0.5-5 mg / kg, about 0.5-5.5 mg / kg, about 0.5-6 mg / kg, about 0.5-6.5 mg / kg, about 0.5-7 mg / kg, about 0.5-7.5 mg / kg, about 0.5-8 mg / kg, about 0.5-8.5 mg / kg, about 0.5-9 mg / kg, about 0.5-9.5 mg / kg, about 0.5-10 mg / kg, about 0.6-1.5 mg / kg, about 0.6-2 mg / kg, about 0.6-2.5 mg / kg, about 0.6-3 mg / kg, about 0.6-3.5 mg / kg, about 0.6-4 mg / kg, about 0.6-4.5 mg / kg, about 0.6-5 mg / kg, about 0.6-5.5 mg / kg, about 0.6-6 mg / kg, about 0.6-6.5 mg / kg, about 0.6-7 mg / kg, about 0.6-7.5 mg / kg, about 0.6-8 mg / kg, about 0.6-8.5 mg / kg, about 0.6-9 mg / kg, about 0.6-9.5 mg / kg, about 0.6-10 mg / kg, about 0.7- 1.5 mg / kg, about 0.7-2 mg / kg, about 0.7-2.5 mg / kg, about 0.7-3 mg / kg, about 0.7-3.5 mg / kg, about 0.7-4 mg / kg, about 0.7-4.5 mg / kg, about 0.7-5 mg / kg, about 0.7-5.5 mg / kg, about 0.7-6 mg / kg, about 0.7-6.5 mg / kg, about 0.7-7 mg / kg, about 0.7-7.5 mg / kg, about 0.7-8 mg / kg, about 0.7-8.5 mg / kg, about 0.7-9 mg / kg, about 0.7-9.5 mg / kg, about 0.7-10 mg / kg, about 0.8-1.5 mg / kg, about 0.8-2 mg / kg, about 0.8-2.5 mg / kg, about 0.8-3 mg / kg, about 0.8-3.5 mg / kg, about 0.8-4 mg / kg, about 0.8-4.5 mg / kg, about 0.8-5 mg / kg, about 0.8-5.5 mg / kg, about 0.8-6 mg / kg, about 0.8-6.5 mg / kg, about 0.8-7 mg / kg, about 0.8-7.5 mg / kg, about 0.8-8 mg / kg, about 0.8-8.5 mg / kg, about 0.8-9 mg / kg, about 0.8-9.5 mg / kg, about 0.8-10 mg / kg, about 0.9-1.5 mg / kg, about 0.9-2 mg / kg, about 0.9-2.5 mg / kg, about 0.9-3 mg / kg, about 0.9-3.5 mg / kg, about 0.9-4 mg / kg, about 0.9-4.5 mg / kg, about 0.9-5 mg / kg, about 0.9-5.5 mg / kg, about 0.9-6 mg / kg, about 0.9-6.5 mg / kg, about 0.9-7 mg / kg, about 0.9-7.5 mg / kg, about 0.9-8 mg / kg, about 0.9-8.5 mg / kg, about 0.9-9 mg / kg, about 0.9-9.5 mg / kg, about 0.9-10 mg / kg, about 1-5 mg / kg, about 1-8 mg / kg, about 1-10 mg / kg, including all endpoints, integers and subranges within the disclosed ranges.

[0165] In another aspect, a suitable dose of a multi-domain conjugate may be in the range of about about 0.1 to about 10 mg / kg. In one aspect, the dose comprises about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 1.25 mg / kg, about 1.5 mg / kg, about 1.75 mg / kg, about 2 mg / kg, about 2.25 mg / kg, about 2.5 mg / kg, about 2.75 mg / kg, about 3 mg / kg, about 3.25 mg / kg, about 3.5 mg / kg, about 3.75 mg / kg, about 4 mg / kg, about 4.25 mg / kg, about4.5 mg / kg, about 4.75 mg / kg, about 5 mg / kg, about 5.25 mg / kg, about 5.5 mg / kg, about 5.75 mg / kg, about 6 mg / kg, about 6.25 mg / kg, about 6.5 mg / kg, about 6.75 mg / kg, about 7 mg / kg, about 7.25 mg / kg, about 7.5 mg / kg, about 7.75 mg / kg, about 8 mg / kg, about 8.25 mg / kg, about8.5 mg / kg, about 8.75 mg / kg, about 9 mg / kg, about 9.25 mg / kg, about 9.5 mg / kg, about 9.75 mg / kg, about 10 mg / kg, or greater.

[0166] In another aspect, the dose of the multi-domain conjugate comprises about 0. 1-0.5 mg / kg, about 0.1-1 mg / kg, about 0.1-1.5 mg / kg, about 0.1-2 mg / kg, about 0.1-2.5 mg / kg, about 0.1-3 mg / kg, about 0.1-3.5 mg / kg, about 0.1-4 mg / kg, about 0.1-4.5 mg / kg, about 0.1-5 mg / kg, about 0. 1-5.5 mg / kg, about 0. 1-6 mg / kg, about 0. 1-6.5 mg / kg, about 0. 1-7 mg / kg, about 0.1-7.5 mg / kg, about 0.1-8 mg / kg, about 0.1-8.5 mg / kg, about 0.1-9 mg / kg, about 0.1-9.5 mg / kg, about 0.1-10 mg / kg, about 0.2-0.5 mg / kg, about 0.2-1 mg / kg, about 0.2-1.5 mg / kg, about 0.2-2 mg / kg, about 0.2-2.5 mg / kg, about 0.2-3 mg / kg, about 0.2-3.5 mg / kg, about 0.2-4 mg / kg, about 0.2-4.5 mg / kg, about 0.2-5 mg / kg, about 0.2-5.5 mg / kg, about 0.2-6 mg / kg, about 0.2-6.5 mg / kg, about 0.2-7 mg / kg, about 0.2-7.5 mg / kg, about 0.2-8 mg / kg, about 0.2-8.5 mg / kg, about 0.2-9 mg / kg, about 0.2-9.5 mg / kg, about 0.2-10 mg / kg, about 0.3-1 mg / kg, about 0.3-1.5 mg / kg, about 0.3-2 mg / kg, about 0.3-2.5 mg / kg, about 0.3-3 mg / kg, about 0.3-3.5 mg / kg, about 0.3-4 mg / kg, about 0.3-4.5 mg / kg, about 0.3-5 mg / kg, about 0.3-5.5 mg / kg, about 0.3-6 mg / kg, about 0.3-6.5 mg / kg, about 0.3-7 mg / kg, about 0.3-7.5 mg / kg, about 0.3-8 mg / kg, about 0.3-8.5 mg / kg, about 0.3-9 mg / kg, about 0.3-9.5 mg / kg, about 0.3-10 mg / kg, about 0.4-1 mg / kg, about 0.4- 1.5 mg / kg, about 0.4-2 mg / kg, about 0.4-2.5 mg / kg, about 0.4-3 mg / kg, about 0.4-3.5 mg / kg, about 0.4-4 mg / kg, about 0.4-4.5 mg / kg, about 0.4-5 mg / kg, about 0.4-5.5 mg / kg, about 0.4-6 mg / kg, about 0.4-6.5 mg / kg, about 0.4-7 mg / kg, about 0.4-7.5 mg / kg, about 0.4-8 mg / kg, about 0.4-8.5 mg / kg, about 0.4-9 mg / kg, about 0.4-9.5 mg / kg, about 0.4-10 mg / kg, about 0.5—1 mg / kg, about 0.5-1.5 mg / kg, about 0.5-2 mg / kg, about 0.5-2.5 mg / kg, about 0.5-3 mg / kg, about 0.5-3.5 mg / kg, about 0.5-4 mg / kg, about 0.5-4.5 mg / kg, about 0.5-5 mg / kg, about 0.5-5.5 mg / kg, about 0.5-6 mg / kg, about 0.5-6.5 mg / kg, about 0.5-7 mg / kg, about 0.5-7.5 mg / kg, about 0.5-8 mg / kg, about 0.5-8.5 mg / kg, about 0.5-9 mg / kg, about 0.5-9.5 mg / kg, about 0.5-10 mg / kg, about 0.6-1.5 mg / kg, about 0.6-2 mg / kg, about 0.6-2.5 mg / kg, about 0.6-3 mg / kg, about 0.6-3.5 mg / kg, about 0.6-4 mg / kg, about 0.6-4.5 mg / kg, about 0.6-5 mg / kg, about 0.6-5.5 mg / kg, about 0.6-6 mg / kg, about 0.6-6.5 mg / kg, about 0.6-7 mg / kg, about 0.6-7.5 mg / kg, about 0.6-8 mg / kg, about 0.6-8.5 mg / kg, about 0.6-9 mg / kg, about 0.6-9.5 mg / kg, about 0.6-10 mg / kg, about 0.7-1.5 mg / kg, about 0.7-2 mg / kg, about 0.7-2.5 mg / kg, about 0.7-3 mg / kg, about 0.7-3.5 mg / kg, about 0.7-4 mg / kg, about 0.7-4.5 mg / kg, about 0.7-5 mg / kg, about 0.7-5.5 mg / kg, about 0.7-6 mg / kg, about 0.7-6.5 mg / kg, about 0.7-7 mg / kg, about 0.7-7.5 mg / kg, about 0.7-8 mg / kg, about 0.7-8.5 mg / kg, about 0.7-9 mg / kg, about0.7-9.5 mg / kg, about 0.7-10 mg / kg, about 0.8— 1 .5 mg / kg, about 0.8-2 mg / kg, about 0.8-2.5 mg / kg, about 0.8-3 mg / kg, about 0.8-3.5 mg / kg, about 0.8-4 mg / kg, about 0.8-4.5 mg / kg, about 0.8-5 mg / kg, about 0.8-5.5 mg / kg, about 0.8-6 mg / kg, about 0.8-6.5 mg / kg, about 0.8-7 mg / kg, about 0.8-7.5 mg / kg, about 0.8-8 mg / kg, about 0.8-8.5 mg / kg, about 0.8-9 mg / kg, about 0.8-9.5 mg / kg, about 0.8-10 mg / kg, about 0.9- 1.5 mg / kg, about 0.9-2 mg / kg, about 0.9-2.5 mg / kg, about 0.9-3 mg / kg, about 0.9-3.5 mg / kg, about 0.9-4 mg / kg, about 0.9-4.5 mg / kg, about 0.9-5 mg / kg, about 0.9-5.5 mg / kg, about 0.9-6 mg / kg, about 0.9-6.5 mg / kg, about 0.9-7 mg / kg, about 0.9-7.5 mg / kg, about 0.9-8 mg / kg, about 0.9-8.5 mg / kg, about 0.9-9 mg / kg, about 0.9-9.5 mg / kg, about 0.9-10 mg / kg, about 1-5 mg / kg, about 1-8 mg / kg, about 1-10 mg / kg, including all endpoints, integers and subranges within the disclosed ranges.

[0167] It will be apparent to those of ordinary skill in the relevant art that suitable modifications and adaptations to the methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the methods and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary methods described herein may substitute any component disclosed herein, or include any component disclosed elsewhere herein. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.

[0168] Various embodiments and aspects of the inventions described herein are summarized by the following clauses:Clause 1. A conjugate, or a pharmaceutically acceptable salt thereof, comprising: a copolymer, the copolymer comprising a recurring hydrophilic unit, a recurring drug linking unit, and a recurring peptide linking unit; a plurality of drugs, each individual drug independently attached to a different and individual drug linking unit through a first cleavable peptide linker; anda plurality of transcytosis peptides, each individual transcytosis peptide independently attached to a different and individual peptide linking unit through a linker.Clause 2. The conjugate of clause 1, or a pharmaceutically acceptable salt thereof, wherein the hydrophilic unit is derived from an N-substituted methacrylamide, an N,N-disubstituted acrylamide, a hydrophilic ester of methacrylic or acrylic acid, an N-vinylpyrrolidone, an N- acryloylmorpholine, a sulfoethylmethacrylate, an acrylic acid, a methacrylic acid, or a combination thereof.Clause 3. The conjugate of clause 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the hydrophilic unit is of formula (I)Clause 4. The conjugate of any one of clauses 1-3, or a pharmaceutically acceptable salt thereof, wherein the drug linking unit is derived from an acrylate-based monomer.Clause 5. The conjugate of any one of clauses 1-4, or a pharmaceutically acceptable salt thereof, wherein the drug linking unit is of formula (II)(II), wherein:L1is the first cleavable peptide linker, and Q is the drug.Clause 6. The conjugate of any one of clauses 1 -5, or a pharmaceutically acceptable salt thereof, wherein the peptide linking unit is derived from an acrylate-based monomer.Clause 7. The conjugate of any one of clauses 1-6, or a pharmaceutically acceptable salt thereof, wherein the peptide linking unit is of formula (III)(III), wherein:L2is a peptide linkerL3is a non-cleavable linker;Z is a detectable moiety or a transcytosis peptide; and m is 0 to 1.Clause 8. The conjugate of any one of clauses 1-7, or a pharmaceutically acceptable salt thereof, comprising the hydrophilic unit at about 50 mol% to about 95 mol% of the copolymer.Clause 9. The conjugate of any one of clauses 1 -8, or a pharmaceutically acceptable salt thereof, comprising the drug linking unit at about 1 mol% to about 10 mol% of the copolymer.Clause 10. The conjugate of any one of clauses 1-9, or a pharmaceutically acceptable salt thereof, comprising the peptide linking unit at about 0.5 mol% to about 10 mol% of the copolymer.Clause 11. The conjugate of any one of clauses 1-10, or a pharmaceutically acceptable salt thereof, wherein the copolymer has a number average molecular weight (Mn) of about 20,000 Daltons (Da) to about 200,000 Da.Clause 12. The conjugate of any one of clauses 1-11, or a pharmaceutically acceptable salt thereof, wherein the copolymer is a random copolymer.Clause 13. The conjugate of any one of clauses 1-12, or a pharmaceutically acceptable salt thereof, wherein the conjugate comprises less than 13 transcytosis peptides per copolymer.Clause 14. The conjugate of any one of clauses 1-13, or a pharmaceutically acceptable salt thereof, wherein the conjugate comprises 2 to 8 transcytosis peptides per copolymer.Clause 15. The conjugate of any one of clauses 1-14, or a pharmaceutically acceptable salt thereof, wherein each transcytosis peptide independently comprises an amino acid sequence selected from the group consisting of:TFFYGGSRGKRNNFKTEEY (AP2) (SEQ ID NO: 24);TFFYGGCRGKRNNFKTEEY (SEQ ID NO: 25);TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 26);HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 27);TFFYGGSRGRRNNFRTEEY (SEQ ID NO: 28); andYPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 29).Clause 16. The conjugate of any one of clauses 1-15, or a pharmaceutically acceptable salt thereof, wherein each transcytosis peptide independently comprises an amino acid sequence of TFFYGGSRGKRNNFKTEEY (AP2) (SEQ ID NO:24).Clause 17. The conjugate of any one of clauses 1-16, or a pharmaceutically acceptable salt thereof, wherein the conjugate comprises the drug at about 1% to about 25% by weight of the conjugate.Clause 18. The conjugate of any one of clauses 1-17, or a pharmaceutically acceptable salt thereof, wherein the drug is paclitaxel.Clause 19. The conjugate of any one of clauses 1-18, or a pharmaceutically acceptable salt thereof, comprising paclitaxel at about 1% to about 15% by weight of the conjugate.Clause 20. The conjugate of any one of clauses 1-19, or a pharmaceutically acceptable salt thereof, wherein the first cleavable peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 1-23.Clause 21. The conjugate of any one of clauses 1-20, or a pharmaceutically acceptable salt thereof, wherein the linker attaching each individual transcytosis peptide to a different and individual peptide linking unit is a maleimide-derived moiety, a click chemistry-derived moiety, or a peptide linker.Clause 22. A multi-domain conjugate, or a pharmaceutically acceptable salt thereof, comprising: a plurality of conjugates according to any one of clauses 1-21, wherein each conjugate is attached to at least one other conjugate through a second cleavable peptide linker.Clause 23. The multi-domain conjugate of clause 22, or a pharmaceutically acceptable salt thereof, comprising two conjugates attached through the second cleavable peptide linker.Clause 24. The multi-domain conjugate of clauses 22 or 23, or a pharmaceutically acceptable salt thereof, wherein the second cleavable peptide linker is of formula (IV)-(AAI)-K-(AA2)-(IV), wherein AAi and AA2 are the same or different amino acid sequence comprising up to six amino acids, and K is lysine, ornithine, or a diamine.Clause 25. The multi-domain conjugate of any one of clauses 22-24, wherein the second cleavable peptide linker comprises an amino acid sequence of GFLGKGFLG (SEQ ID NO:23).Clause 26. A pharmaceutical composition comprising the conjugate of any one of clauses 1 - 25, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.Clause 27. A method of modulating neurodegeneration in a subject in need thereof, the method comprising administering to the subject an effective amount of a conjugate according to any one of clauses 1-25, or a pharmaceutically acceptable salt thereof.Clause 28. The method of clause 27, wherein the neurodegeneration is associated with Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), ataxia, Huntington’s disease, motor neuron disease, multiple system atrophy, progressive supranuclear palsy, traumatic brain injury, or a combination thereof.Clause 29. The method of clause 28, wherein the neurodegeneration is associated with Alzheimer’s disease.Clause 30. The method of clause 29, wherein the method decreases an amount of phosphor- tau positive cells in the subject.Clause 31. The method of clause 29 or 30, wherein the method improves cognitive defects associated with Alzheimer’s disease in the subject.Clause 32. The method of any one of clauses 28-31, wherein the conjugate, or a pharmaceutically acceptable salt thereof, is administered intravenously.Clause 33. The method of clause 32, wherein the conjugate, or a pharmaceutically acceptable salt thereof, is administered at less than 20 mg / kg based on the amount of drug administered.Clause 34. A method of delivering a drug to a brain of a subject in need thereof, the method comprising intravenously administering to the subject an effective amount of a conjugate according to any one of clauses 1-25, or a pharmaceutically acceptable salt thereof, whereinfollowing administration, the conjugate, or a pharmaceutically acceptable salt thereof, is transported through a blood-brain barrier of the subject.EXAMPLESExample 1; Preparation of Polymerized PTX Conjugated to Angionpep-2 (P-PTX-AP2) with HPMA, APMA, and MA-GFLG-PTX

[0169] AP2 (TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 1) was prepared by solid phase peptide synthesis. The N-terminus of the AP2 sequence was either appended with a cysteine residue for subsequent conjugation to polymer backbone via reaction with pendent maleimide groups of a polymer precursor, or modified with MA-GG-OH to generate a polymerizable derivative MA-GG-AP2, that is MA-AP2. This derivative was then copolymerized with HPMA and MA-GFLG-PTX (a polymerizable derivative of PTX) to produce P-PTX-AP2. The first approach is described below.

[0170] P-PTX-AP2 was synthesized in two steps: first a copolymer of HPMA, APMA (N-(3- aminopropyljmethacrylamide), and a polymerizable derivative of PTX (MA-GFLG-PTX, MA is methacryloyl) was synthesized by RAFT copolymerization; in the second step the amino groups at copolymer side chain termini were converted to maleimide groups using SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate) followed by attachment of cysteine modified AP2 (Cys-AP2). The unreacted maleimide groups were capped with 2-mercaptoethanol.AbbreviationsMA is methacryloyl; PTX is paclitaxel; GFLG is Gly-Phe-Leu-Gly peptide; maL is maleimide; DMAP is 4-dimethylaminopyridine; DMF is dimethylformamide; DCC is N,N- dicyclohexylcarbodiimide; DCU is 1,3 -dicyclohexyl urea; EtOAc is ethyl acetate; KHSO4 is potassium bisulfate; CH3OH is methanol; HPMA or N-HPMA is N-(2- hydroxypropyl)methacrylamide; APMA is (A-(3-aminopropyl)methacrylamide; DMSO is dimethyl sulfoxide; DIPEA is N,N-diisipropylethylamine; EDTA is ethylenediaminetetraacetic acid; SMCC is succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate; NMR is nuclear magnetic resonance; aq. is aqueous; eq. or equiv. is equivalents; min or min. is minute(s); h or hr. is hour(s); rt, RT, or r.t. is room temperature; and sat’d or sat. is saturated.Scheme 1. Example Synthesis of MA-GFLG-PTXMA-GFLG-PTX

[0171] MA-GFLG-PTX: Under nitrogen atmosphere, MA-GFLG-OH (129 mg, 0.28 mmol), paclitaxel (200 mg, 0.23 mmol) and DMAP (50 mg, 0.41 mmol) were dissolved in anhydrous DMF (1.0 mL). The solution was stirred for 15 min at 0 °C., DCC (200 mg, 0.97 mmol) in DMF (1.0 mL) was added dropwise and the solution was stirred at 4 °C. for 24 h. The reaction solvent was removed by rotary evaporation at reduced pressure. EtOAc (50 mL) was added, DCU was removed by filtration, and the solution was washed with KHSO4 aq. (0.1 M), NaHCOs (1.0 M) and then NaCl aq, dried over MgSCh. The solution was removed by rotary evaporation. The crude product was purified by column chromatography (silica gel 60 A, 200-400 mesh, EtOAc: CH3OH, 15: 1) to white solid in 68% yield (206 mg, 1.5 mmol). The structure was confirmed by 'H NAIR spectroscopy and matrix assisted laser desorption ionization mass spectroscopy.Scheme 2. Preparation of Polymer Precursor P-PTX-NH2.[00172J P-PTX-NH2: RAFT copolymerization was used to synthesize P-PTX-NH2 following the synthetic route shown in scheme 2. As an example, HPMA (267 mg, 1.86 mmol), APMA (15 mg, 0.084 mmol) and MA-GFLG-PTX (73 mg, 0.056 mmol) were added into a 5-mL ampoule, then dissolved in 0.5 mL degassed DMSO / H+(0.3% acetic acid) under N2 atmosphere. RAFT agent, 4-cyanopentanoic acid dithiobenzoate and initiator V65 were added via syringe. The solution was bubbled with N2 for 10 min. The ampoule was sealed and stirred in 50 °C for 20 h. The polymer solution was drop-wise precipitated in cold acetone, and isolated by centrifugation and then dried under N2 to generate a light pink polymer powder with yield of 185 mg (~52%). The average molecular weight of the polymer was estimated by AKTA FPLC system equipped with a UV detector (GE Healthcare) and Superose 6 HR10 / 300 column that was calibrated with a series of HPMA homopolymer standards. Sodium acetate buffer (pH 6.5) containing 30% acetonitrile served as mobile phase and flow rate was 0.4 mL / min. Next, the end dithiobenzoate group on a polymer chain was removed by reaction with 40-fold excess V-65 in 2 mL DMSO at 55 °C for 2 h, and precipitation in acetone. The white polymer powder was obtained following dissolution-precipitation in methanol-ether three times. The final molecular weight and poly dispersity of the P-PTX-NH2 were determined and calculated based on HPMA standard curve as 62.5 kDa, and poly dispersity index (Mw / Mn) was 1.09. The content of amino group on side chain of the polymer was determined using Ninhydrin assay. It was found 312.7 nmol amino group / mg polymer, suggesting 19.6 NH2 per chain. PTX content was determined by incubation ofthe conjugate with papain, an enzyme mimic cathpsin B activity. PTX was cleaved from the polymer during the incubation of polymer solution in Mcllvaine’s buffer (pH 6) with 2 mM EDTA and 5 mM glutathione and papain. Cleaved PTX was quantified using HPLC. The content of PTX was calculated as 5% (wt).Table 3. Synthetic feed ratios of the HPMA, MA-GFLG-PTX, and APMA and the polymer characterization‘ Ninhydrin assay (for primary amine determination**Modified Eilman assay (for maleimide content determination)Scheme 3. Example Synthesis of Optional Fluorophore Attachment to P-PTX-NH2

[0173] To label polymer with fluorophore, side chain amino groups were reacted with Cy7- NHS ester (Lumiprobe). 49.9 mg of P-PTX-NH2 was dissolved in 650 pL of fresh dimethylformamide. 1.6 mg of Cy7-NHS ester (2.1 pmol, 2.8: 1 dye:polymer feed ratio) was then dissolved in 50 pL of DMF and added dropwise to the polymer solution under vigorous stirring. 16 pmol of diisopropylethylamine (DIPEA) was drop-wise added to the solution. After 2 h reaction, the product was precipitated in cold ether, washed twice, and dried under vacuum. The crude product was then redissolved in 0.5 mL DI water and applied to a PD10 column and remaining free dye was removed. The product was then lyophilized to yield 37.4 mg of Cy7-P- PTX-NH2.Scheme 4. Example Synthesis of P-PTX-maL with Optional Fluorophore

[0174] P-PTX-maL: Maleimide functionalized polymer precursor (P-PTX-maL) was then obtained by reaction of P-PTX-NH2 with SMCC (succinimidyl-4-(A- maleimidomethyl)cyclohexane-l -carboxylate), a heterobifunctional reagent in DMF in the presence of a N, A-di isopropyl ethyl amine (DIPEA) at room temperature for 2 h. Afterwards the product was isolated by precipitation of the solution into cold ether. Following centrifugation, the pellet was dried under N2. Further purification was performed by re-dissolving in methanol and re-precipitation in acetone twice. The maleimide content of the pure P-PTX-maL was measured by modified Ellman’s assay as 300 nmol / mg (>90% conversion).Scheme 5. Example Synthesis of P-PTX-AP2 with an Optional FluorophoreP«PTX-AP2

[0175] P-PTX-AP2: Two different batches of P-PTX-AP2 were preprared. AP2 conjugation was achieved by attaching Cys-AP2 to P-PTX-maL with the variable molar ratio of [Cys-AP2] to [maleimide] to generate the P-PTX-AP2 (Table 4). The reaction was performed in 10 mM PBS (pH 6.5) and kept stirring at room temperature for 3 h. At the end, 100-fold of 2-merpcaptoethanol was used to cap any unreacted / unhydrolyzed maleimide groups. Unreacted Cys-AP2 was removed by ultrafitration (30,000 Da cut-off) with 4 times DI water wash and freeze-dried. The content of AP2 in P-PTX-AP2 was determined using bicinchoninic acid (BCA) protein assay (Pierce) whilepure AP2 was used as standard for calibration curve. Two P-PTX-AP2 were obtained, containing 6 or 13 AP2 per polymer chain, respectively.>Table 4. Synthetic feed ratios of Cys-AP2>Monomers [S-H]:[Malemide]>Cys-AP2 #1 F3Cys-AP2 #2 1 : 1Example 2; Preparation of Polymerized PTX Conjugated to Angionpep-2 (P-PTX-AP2) with HPMA, MA-GG-AP2, and MA-GFLG-PTXAbbreviationsMA is methacryloyl; PTX is paclitaxel; GFLG is Gly-Phe-Leu-Gly peptide; GG is Gly-Gly; HPMA or N-HPMA is N-(2-hydroxypropyl)methacrylamide; DMSO is dimethyl sulfoxide; aq. is aqueous; eq. or equiv. is equivalents; min or min. is minute(s); h or hr. is hour(s); rt, RT, or r.t. is room temperature; and sat’d or sat. is saturated.Scheme 6. Example Synthesis of P-PTX-AP2 with HPMA, MA-GG-AP2, and MA-GFLG-PTX

[0176] P-PTX-AP2: HPMA, MA-GG-AP2 and MA-GFLG-PTX were added into a 1-mL ampoule with a stirring bar and then connected to a Schlenk line. After three vacuum-nitrogencycles to remove oxygen, 0.2 mb degassed DMSO / H+(acidified DMSO using 0.3% acetic acid) was added into the ampoule via syringe, followed by addition of the RAFT agent 4-cyanopentanoic acid dithiobenzoate (CTA) in DMSO and initiator V65. The ampoule was sealed, and polymerization was performed at 50 °C for 20 h. The polymer was isolated by precipitation into acetone. After purification (re-dissolving / re-precipitation) and end-modification with excess of V- 65, the average molecular weight and poly dispersity of the product was determined by sizeexclusion chromatography (SEC) on AKTA FPLC system equipped with Superose 6 HR10 / 30 column (GE Healthcare) and UV (280 nm) / OptilabREX detectors. Sodium acetate buffer containing 30% acetonitrile (pH 6.5) was used as eluent and flow rate was 0.4 mL / min.

[0177] Two batches of P-PTX-AP2 were synthesized. The number average molecular weight, the content of PTX and AP-2 were determined as described in Example 1. The results are summarized in the Table 5 below.Table 5. Synthetic feed ratios of the HPMA, MA-GFLG-PTX, and MA-AP2 and the polymer characterizationExample 3: Preparation of Backbone degradable 2P-PTX-AP2AbbreviationsMA is methacryloyl; PTX is paclitaxel; GFLG is Gly-Phe-Leu-Gly peptide; GG is Gly-Gly; HPMA or N-HPMA is N-(2-hydroxypropyl)methacrylamide; mAL is maleimide; DMSO is dimethyl sulfoxide; aq. is aqueous; eq. or equiv. is equivalents; min or min. is minute(s); h or hr. is hour(s); rt, RT, or r.t. is room temperature; and sat’d or sat. is saturated.Scheme 7. Example Synthesis of 2P-PTX-AP2 from HPMA, MA-GG-AP2, and MA-GFLG-PTX

[0178] 2P-PTX-AP2: Backbone degradable HPMA copolymer-paclitaxel conjugates containing AP2 was synthesized following similar procedure but by addition of the RAFT Pep2CTA. The ampoule was sealed and polymerization was performed at 50 °C for 20 h. The polymer was isolated by precipitation into acetone. After purification (re-dissolving / re- precipitation) and end-modification with 100 folds of V-65, 62 mg polymer precursor was obtained. Yield -60%. The average molecular weight and poly dispersity of the polymer precursorwere determined by size-exclusion chromatography (SEC) on AKTA FPLC system equipped with Superose 6 HR10 / 30 column (GE Healthcare) and UV (280 nm). Sodium acetate buffer containing 30% acetonitrile (pH 6.5) was used as eluent and flow rate was 0.4 mL / min. The number average molecular weight Mn of 2P-PTX-AP2 was calculated using polyHPMA standard curve as 114 kDa, Mw / Mn=1.29.Scheme 8. Example Synthesis of 2P-PTX-AP2 from P-PTX-NH2

[0179] 2P-PTX-AP2: Backbone degradable HPMA copolymer-paclitaxel conjugates containing pendant amino groups (2P-PTX-NH2) were synthesized via RAFT copolymerization according to the procedure described in Example but using different chain transfer agent, Pep2CTA. The full synthetic scheme is presented in Scheme 8. Molecular weight and poly dispersity of the fractionated polymer product (2P-PTX-NH2) was then characterized via size exclusion chromatography as described in Example 1.

[0180] Pendent amine were converted to maleimide groups using SMCC. The maleimide functionalized copolymer-paclitaxel conjugate (2P-PTX-Mal) was conjugated with Cys-AP2 as described in Example 1. The extent of conversion was determined by the modified Ellman's assay. The results were listed below.Table 6. Extent of NH2 to maleimide conversionExample 4: Characterization of P-PTX-AP2 ConjugatesMaterial and Methods

[0181] Cytotoxicity Studies. Cells were seeded in a 96-well plate at a density of 10,000 cells / well. Culture the cells in a CO2 incubator at 37°C for 24 hours. Various concentrations of substances (500, 250, 125, 62.5, 31.25, 15.625, 7.81, 3.9, 1.95 mg / mL). 10 pL of CCK-8 solution was added to each well of the plate using a repeating pipettor after 24 hour, and the plate was measured at the absorbance of 450 nm using a microplate reader.

[0182] 3xTg-AD mice. All procedures were conducted in accordance with the animal care guidelines issued by the National Institutes of Health and by the Institutional Animal Care and Use Committee. Mice were kept on a 12-h light / 12-h dark cycle with ad libitum access to water and food before and during experimental procedures and were randomly assigned to receive drug or vehicle. We bred and enrolled 124 3xTg-AD mice, which harbor a Psenl PS1M146V mutation and the co-injected APPSwe and tauP301L transgenes (Tg(APPSwe,tauP301L)lLfa)) (common name: 3xTg-AD on C57BL / 6J). Of these, 64 mice were 3xTg-AD mice and the other 60 were wild-type mice. These two groups were comprised of almost equal numbers of males and females. The 124 mice were randomly sorted into treatment groups. One arm (young) received seven bimonthly treatments from age of 2 months to 6.5 months. The other arm (aged) received treatment or vehicle starting at 50 weeks of age (4 groups: 3xTg-AD + treatment (n=23, 9 male), 3xTg-AD + vehicle only (n=16, 5 male), WT + Treatment (n=16, 6 male), WT + vehicle only, (n=16, 7 male).

[0183] Drug Treatment Studies. In vivo experiments in 3xTg-AD mice, P-PTX-AP2 (0.6 mg mg / kg of PTX equivalent dose) was administered intravenously every 2 weeks for 6 (aged) or 7 (young) doses.

[0184] Histology and Immunostaining. Mice were euthanized using isoflurane in a bell jar until respiratory arrest and then transcardially perfused with saline followed by 4% formaldehyde. Brains were removed and postfixed for two hours in 4% paraformaldehyde in PB S at 4°C, followed by 24 hours in 20% sucrose in PBS, and then 24 hours in 30% sucrose. Coronal sections were cut at 50 pm thickness, embedded in OCT (Tissue-Tek, Torrance, CA) with a CM1850UV cryostat (Leica, Buffalo Grove, IL). Free-floating sections were permeabilized (1 hour, room temperature, 0.2% Triton X-100 and 0.01% sodium azide in PBS) followed by three 5-minute washes in PBS. Antigen retrieval was performed by heating at 80°C for 30 minutes in 50mM sodium citrate (pH 6.0). Sections were then cooled to room temperature, washed in PBS (3 times for 5 minutes each) and blocked in PBS with 5% normal goat serum (NGS) and 0.1% Triton X-100 for two hours at room temperature with gentle agitation on an orbital shaker (10 rpm). Sections were incubated in primary antibody solution (PBS, 5% NGS, 0.1% Triton X-100; 200 pL per section) at 4 °C overnight with chicken anti-glial fibrillary acidic protein (GFAP, EMDMillipore, Temecula, CA), a reactive astroglial marker, or rabbit anti-Iba-1 (Wako, Richmond, VA), a microglial marker. Tau phosphorylation: sections were incubated with the AT8 antibody (Ser202; ThermoFisher, Grand Island, NY). Goat secondary antibodies labeled with Alexa 488, Alexa 633, and DyLight 549 were obtained from ThermoFisher, Rockford, IL (Alexa conjugates) or Abeam Cambridge, MA (DyLight conjugate). Nuclei were stained with DAPI fluorescent dye (ThermoFisher, Grand Island, NY) using the manufacturer's recommended conditions. Floating tissue sections were coverslipped with a drop of Prolong gold antifade solution without DAPI (Life Technologies, Grand Island, NY). Within an experiment, all directly compared sections / slides were identically and simultaneously prepared.

[0185] Laser scanning confocal imaging was conducted using a Leica TCS SP2 confocal / multiphoton hybrid microscope with tunable emission gating and using sequential, between stack, single photon excitation at 488, 543, and 633. Z-plane images acquired the full volume thickness of each slice imaged (i.e., 50 pm for floating sections and 5 pm for paraffin- embedded sections) using system optimized stepping. Within an experiment, all directly compared sections / slides were identically and simultaneously prepared.

[0186] Behavior Studies. Spatial memory and anxiety were tested in WT and 3XTg-AD mice using the radial water tread (RWT) maze and elevated-plus maze.

[0187] The mouse cohorts treated from 2-6 months were not tested in the maze because cognitive deficits in 3XTg-AD mice are subtle before 6 months of age. The RTW maze protocol and application to a mouse model of TBI has been published previously (Cline, M.M. et al. Brain Res, 2017, 1657)(Pettan-Brewer, C. et al, Pathobiol Aging Age Relat Dis, 2013. 3). The RTW tested required no swimming and capitalized on the natural tendency of mice to avoid open areas in favor of hugging the edges of an apparatus (thigmotaxis). In brief, the RWT maze consists of a 32-inch galvanized steel tub with nine holes placed at equal intervals around the device, roughly 1 i inch above the apparatus floor. Of these, eight terminate after roughly 1 inch (decoy exits), and one leads to a safety box hidden behind a 90-degree angle bend to prevent visual confirmation of escape route. The safety box consists of a dark plastic container (30” L xl5” W xl5” H) heated externally via electronic heating pad. Five large unique visual cues line the sides of the apparatus for spatial orientation. Testing was conducted in a brightly lit room. Before testing, the maze was sanitized with 70% ethanol and filled with 1 inch of 12-14 °C water. Water was changed between mice, but not between trials, and the temperature was monitored to ensure the desired range. The mouse was placed in the center and allowed 180 seconds to find the escape hole. If the mouse was unable to escape within the time allowed, the mouse was guided to the exit and the trial was recorded as 180 seconds. If a mouse attempted to enter a decoy hole and did not voluntarily reenter the maze after five seconds, they were returned to the center of the maze by hand. Once inside the safety box, the mouse was given a food treat and allowed to remain in the box for 60 seconds. The safety box and maze were sprayed with 70% ethanol between trials to prevent olfactory cues. Mice were given three trials a day for 4 days (acquisition period), followed by a short-term memory test consisting of three trials on day five. Mice were unexposed to the maze until day 12, at which time they underwent three trials (long-term memory test).

[0188] Mice were tested via elevated-plus-maze (EPM) at 65 weeks of age. The elevated-plus maze measures anxiety, exploration and activity levels in mice by taking advantage of their innate tendency to avoid open and elevated areas. The maze is elevated 45 cm above the floor and consists of a central square (5 x 5 cm), from which radiate four arms (5 x 30 cm). Two of the arms have transparent plexiglass walls (15 cm high) around the edge (closed arms), whereas the other two arms do not have walls (open arms), but do have a 0.25 cm-high edge to prevent the mice fromfalling. A videotracking system (Noldus Ethovision, Noldus IT, Wageningen, The Netherlands) was used to measure entries and duration in the center, open arm, and closed arm zones. The test was performed in 100 lux white lighting, and mice were given a full hour to acclimate before testing commenced. During testing, mice were individually placed in the central square of the apparatus, facing an open arm, and allowed to explore the apparatus for 5 min while data were collected.

[0189] Statistical Analysis. Imaging statistical analyses are described in the imaging methods section. For behavioral studies, one-factor between-group analysis of variance (ANOVA) or Student t-tests were used to compare paclitaxel-3XTg-AD, saline-3 XTg-AD and WT groups. All reported p values were based on two-tailed critical values (p<0.05). Radial water tread maze data were analyzed by ANOVA with training or test days as a repeated measures within-subjects factor and treatment (drug / no drug) as abetween-subjects factor. Following the ANOVA we tested the a priori hypothesis that saline-treated 3XTg-AD mice would be significantly different from both WT and paclitaxel-treated 3XTg-AD mice was performed using a Helmert individual comparisons test (Field, A. et al, Discovering Statistics Using R, SAGE Publications)(Winer, B.J. et al, Statistical Principles in Experimental Design, McGraw-Hill). Statistical analyses were carried out using SPSS software (IBM, Armonk, NY, USA). In vitro values were compiled for analysis in Microsoft Excel and SPSS (IBM, North Castle, NY, USA), and significance difference between treatments was analyzed by Student’s t-test with equal or unequal variances at a 95% confidence interval.In vitro and In vivo Pharmacokinetic Activity of P-PTX-AP2

[0190] To evaluate the safety profile of P-PTX-AP2 compared to free PTX neurotoxicity assays were carried out on human neuroblastoma SH-SY5Y cells, human hCMEC / D3 endothelial cells, and mouse bEnd.3 endothelial cells. Additionally, the effectiveness of AP2 in mediating BBB transcytosis was quantified using both in vitro Transwell BBB models (Cy5-labeled conjugates) and in vivo mouse models (Cy5.5-labeled conjugates for tracking and quantification). Transendothelial Electrical Resistance (TEER) value was employed to confirm the integrity of in vitro BBB models. The expression of LRP-1 in bEnd.3 and hCMEC / D3 cell lines was confirmed (FIG. 1), and AP2 binding to the cell surface on the LRP-1 was assessed via confocal microscopy (FIG. 2) and flow cytometry (FIG. 3).

[0191] An in vitro transcytosis model utilized the hCMEC.D3 cell line. Trans-epithelial electrical resistance (TEER) confirmed cell layer integrity. On day 5, both batches of P-PTX-AP2 were introduced into the apical compartment, and the basolateral to apical concentration ratio (B / A ratio) was determined (FIG. 4). The 6 AP2 / chain conjugate demonstrated superior in vitro transcytosis compared to the 13 AP2 / chain conjugate.

[0192] The data show that P-PTX-AP2 has significantly lower neurotoxicity than free PTX, with ICso values indicating improved safety profiles in bEnd.3, Sh-SY5Y, and hCMEC / D3 cell lines. Specifically, IC50 values for PTX alone in each cell line were 32 pM, 33 nM, and 153 nM, respectively (FIG. 7-9). The ICso values for P-PTX-AP2 in each cell line were 88 pM, 148 nM, and 4 pM, respectively (FIG. 7-9).

[0193] AP2's role in enhancing brain delivery was corroborated through a biodistribution in vivo study using fluorescent imaging, showing higher intensity of conjugates in the brains of mice administered P-AP2-Cy5.5 compared to P-Cy5.5 (FIG. 10-13).

[0194] Histological analysis showed a marked reduction in phospho-tau positive cells in the brains of treated mice, underscoring the efficacy of P-PTX-AP2 in targeting and mitigating tau pathology (FIG. 14).Behavioral Response to P-PTX-AP2

[0195] PTX is typically administered intravenously at a dose of 20 mg / kg for various mice tumor models, which corresponds to a human dose of 62 mg / m2. In the clinic PTX is given at dose of 150 mg / m2as IV infusion for 3 h or longer. However, PTX at these high doses has been was reported to produce unwanted side effects, such as hyper-stabilized microtubules. Additionally, neutropenia and peripheral neuropathy are common clinical side effects in response to these concentrations of PTX.

[0196] A radial tread water maze test was used to assess cognitive decline in mice. The RTW maze protocol and application to a mouse model of TBI has been described previously. RTW requires no swimming and capitalizes on the natural tendency of mice to avoid open areas in favor of hugging the edges of an apparatus (thigmotaxis). The RWT maze consists of a 32-inch galvanized steel tub with nine holes placed at equal intervals around the device, roughly 1 ! inch above the apparatus floor. Of these, eight terminate after roughly 1 inch (decoy exits), and one leads to a safety box hidden behind a 90-degree angle bend to prevent visual confirmation of escape route. For each group the graph shows the mean time (seconds) to find the escape box. Thus, lower escape latencies denote improved spatial memory across the training / testing interval.

[0197] Two in vivo mice studies were performed with different cohorts of mice. The first cohort of mice were 3xTg-AD female mice on a B6;129 genetic background (Stock No. 004807 Jackson Labs) B6;129-Tg(APPSwe,tauP301L)lLfa PsenltmlMpm / Mmjax. Four intravenous drug treatments of administration were performed. The mice were 13 months at time of maze testing. Three test groups (treated and non-treated transgenic and only non-treated wild-type) were used. The non-treated group of mice were given saline injections.

[0198] The functional outcome of the preliminary conjugate P-PTX-AP2, on 3xTg-AD mice using Radial Water Tread (RWT) Maze Performance, which tests short- and long-term spatial memory, was tested. FIG. 15 shows a learning / acquisition phase (Days 1-4), short-term memory(Day 5), and long-term memory (Day 12) of mice. The non-treated wild-type (dashed line / triangles) and 3xTg-AD mice received either saline treatments (solid / circles) or four treatments of P-PTX-AP2 treatments (solid / squares), respectively.

[0199] P-PTX-AP2 treatment with IV administration alleviated AD-induced cognitive impairment in aged (13 mo.) 3xTg-AD mice after 4 treatments spaced 2 weeks apart. A two-factor between / within subjects (group by days) ANOVA revealed a significant difference between groups (F[2,32]=4.131, p= 0.025), as well as a significant group by days interaction (F[2,32]=3.823 p<0.032), indicating the benefits of the P-PTX-AP2 conjugate. A Helmert a priori individual comparisons test further confirmed that saline-treated 3xTg-AD mice (n=l 1) performed significantly worse than the P-PTX-AP2-treated (n=12) and WT (n=12) (p<0.007) and the P-PTX- AP2-treated was not significantly different from WT (p<0.921, n.s.).

[0200] The second cohort (n=63) of mice included both male and female transgenic mice on a C57BL / 6J-congenic background (Stock No. 033930 in Jackson Labs) B6.Cg-Tg(APPSwe,tauP301L)lLfa PsenltmlNIpml2i). Control mice were included wild-type male and female mice. The age of the mice at drug treatment was 11-14 months, and the age of mice at maze test 15 months. Treatment groups received six drug treatment administrations (same dose and same dose intervals as the first cohort with the additional two treatments). The non-treated Tg and WT mice were given injections of the polymer / peptide without PTX. Altogether, a total of four groups were tested: (i) P-AP2-PTX-treated transgenic-3XTg-AD mice; (ii) vehicle-treated transgenic 3XTg-AD mice; (iii) P-AP2-PTX -treated non-transgenic wild-type (WT) mice; and (iv) vehicle-treated non-transgenic WT mice (N= 23, 16, 16 and 16, respectively). The gender breakdown of the second cohort included a male-to-female ratio of N=M / F: 9 / 14, 5 / 11, 6 / 10 and 7 / 9.

[0201] A mixed between / within 3-Factor (Group X Sex X Day) analysis of variance (ANOVA) was carried. This revealed a statistically significant difference among Groups (F[3,63]=5.261, p< 0.003). The Sex and Group X Sex interaction factors were not statistically significant (F[l,63]=0.159, n.s. and F[3,63]=1.707, n.s., respectively). To control for multiple comparisons and to specifically test the hypothesis that: (a) vehicle-treated mice possessing the AD transgene would perform more poorly than the other three groups; and (b) that performance among PTX- treated transgenic-AD mice would be statistically comparable to both vehicle and PTX-treated non-transgenic control groups. The Tukey test analyses fully supported this conclusion. To further confirm that sex did not influence the efficacy of PTX in ameliorating AD-gene mediated memory impairment, we pooled males and females within their corresponding groups and carried out a mixed between / within 2-factor (Group X Day) ANOVA. Supporting the above findings there was a statistically significant difference between groups (F[3,67]=4.328, p< 0.008) (FIG. 16)

Claims

CLAIMSWhat is claimed:

1. A conjugate, or a pharmaceutically acceptable salt thereof, comprising: a copolymer, the copolymer comprising a recurring hydrophilic unit, a recurring drug linking unit, and a recurring peptide linking unit; a plurality of drugs, each individual drug independently attached to a different and individual drug linking unit through a first cleavable peptide linker; and a plurality of transcytosis peptides, each individual transcytosis peptide independently attached to a different and individual peptide linking unit through a linker.

2. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the hydrophilic unit is derived from an N-substituted methacrylamide, an N,N-disubstituted acrylamide, a hydrophilic ester of methacrylic or acrylic acid, an N-vinylpyrrolidone, an N- acryloylmorpholine, a sulfoethylmethacrylate, an acrylic acid, a methacrylic acid, or a combination thereof.

3. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the hydrophilic unit is of formula (I)(I).

4. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the drug linking unit is derived from an acrylate-based monomer.

5. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the drug linking unit is of formula (II)(II), wherein:L1is the first cleavable peptide linker, and Q is the drug.

6. The conjugate of claim 1 , or a pharmaceutically acceptable salt thereof, wherein the peptide linking unit is derived from an acrylate-based monomer.

7. The conjugate of claim 1 , or a pharmaceutically acceptable salt thereof, wherein the peptide linking unit is of formula (III)wherein:L2is a peptide linkerL3is a non-cleavable linker;Z is a detectable moiety or a transcytosis peptide; and m is 0 to 1.

8. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, comprising the hydrophilic unit at about 50 mol% to about 95 mol% of the copolymer.

9. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, comprising the drug linking unit at about 1 mol% to about 10 mol% of the copolymer.

10. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, comprising the peptide linking unit at about 0.5 mol% to about 10 mol% of the copolymer.

11. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the copolymer has a number average molecular weight (Mn) of about 20,000 Daltons (Da) to about 200,000 Da.

12. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the copolymer is a random copolymer.

13. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the conjugate comprises less than 13 transcytosis peptides per copolymer.

14. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the conjugate comprises 2 to 8 transcytosis peptides per copolymer.

15. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein each transcytosis peptide independently comprises an amino acid sequence selected from the group consisting of:TFFYGGSRGKRNNFKTEEY (AP2) (SEQ ID NO:24);TFFYGGCRGKRNNFKTEEY (SEQ ID NO:25);TFFYGGSRGKRNNFRTEEY (SEQ ID NO:26);HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO:27);TFFYGGSRGRRNNFRTEEY (SEQ ID NO:28); andYPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO:29).

16. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein each transcytosis peptide independently comprises an amino acid sequence of TFFYGGSRGKRNNFKTEEY (AP2) (SEQ ID NO:24).

17. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the conjugate comprises the drug at about 1% to about 25% by weight of the conjugate.

18. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the drug is paclitaxel.

19. The conjugate of claim 18, or a pharmaceutically acceptable salt thereof, comprising paclitaxel at about 1% to about 15% by weight of the conjugate.

20. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the first cleavable peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-23.

21. The conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the linker attaching each individual transcytosis peptide to a different and individual peptide linking unit is a maleimide-derived moiety, a click chemistry-derived moiety, or a peptide linker.

22. A multi-domain conjugate, or a pharmaceutically acceptable salt thereof, comprising: a plurality of conjugates according to claim 1, wherein each conjugate is attached to at least one other conjugate through a second cleavable peptide linker.

23. The multi-domain conjugate of claim 22, or a pharmaceutically acceptable salt thereof, comprising two conjugates attached through the second cleavable peptide linker.

24. The multi-domain conjugate of 22, or a pharmaceutically acceptable salt thereof, wherein the second cleavable peptide linker is of formula (VII)-(AAI)-K-(AA2)-(VII), wherein AAi and AA2are the same or different amino acid sequence comprising up to six amino acids, and K is lysine, ornithine, or a diamine.

25. The multi-domain conjugate of claim 22, wherein the second cleavable peptide linker comprises an amino acid sequence of GFLGKGFLG (SEQ ID NO: 23).

26. A pharmaceutical composition comprising the conjugate of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

27. A method of modulating neurodegeneration in a subject in need thereof, the method comprising administering to the subject an effective amount of a conjugate according to claim 1, or a pharmaceutically acceptable salt thereof.

28. The method of claim 27, wherein the neurodegeneration is associated with Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), ataxia, Huntington’s disease, motor neuron disease, multiple system atrophy, progressive supranuclear palsy, traumatic brain injury, or a combination thereof.

29. The method of claim 28, wherein the neurodegeneration is associated with Alzheimer’s disease.

30. The method of claim 29, wherein the method decreases an amount of phosphor-tau positive cells in the subject.

31. The method of claim 29, wherein the method improves cognitive defects associated with Alzheimer’s disease in the subject.

32. The method of claim 28, wherein the conjugate, or a pharmaceutically acceptable salt thereof, is administered intravenously.

33. The method of claim 32, wherein the conjugate, or a pharmaceutically acceptable salt thereof, is administered at less than 20 mg / kg based on the amount of drug administered.

34. Amethod of delivering a drug to a brain of a subject in need thereof, the method comprising intravenously administering to the subject an effective amount of a conjugate according to claim 1, or a pharmaceutically acceptable salt thereof, wherein following administration, the conjugate, or a pharmaceutically acceptable salt thereof, is transported through a blood-brain barrier of the subject.

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