HSP90-TARGETING CONJUGATES AND FORMULATIONS THEREOF

Conjugates of an active agent attached to a targeting moiety, such as an HSP90 binding moiety, via a linker, and particles comprising such conjugates have been designed. Such conjugates and particles can provide improved temporospatial delivery of the active agent, improved biodistribution and penetration in tumor, and/or decreased toxicity. Methods of making the conjugates, the particles, and the formulations thereof are provided. Methods of administering the formulations to a subject in need thereof are provided, for example, to treat or prevent cancer.

FIELD OF THE DISCLOSURE

The invention generally relates to the field of targeting ligands, conjugates thereof, and particles for drug delivery. More particularly, the invention relates to the use of molecules targeting heat shock proteins including heat shock protein 90 (HSP90), e.g., for treating cancer.

BACKGROUND

Heat shock protein 90 (HSP90) is an intracellular chaperone protein that assists protein folding, stabilizes proteins against heat stress, and aids in protein degradation. It is upregulated in many types of cancer. Many Hsp90 client proteins are over-expressed in cancer, often in mutated forms, and are responsible for unrestricted cancer cell proliferation and survival. HSP90 is activated in cancer tissues and latent in normal tissues. HSP90 derived from tumour cells has higher binding affinity to HSP90 inhibitors than the latent form in normal cells, allowing specific targeting of HSP90 inhibitors to tumour cells with little inhibition of HSP90 function in normal cells. Further, HSP90 has also been recently identified as an important extracellular mediator for tumour invasion. Therefore, HSP90 is considered a major therapeutic target for anticancer drug development.

Nanoparticulate drug delivery systems are attractive for systemic drug delivery because they may be able to prolong the half-life of a drug in circulation, reduce non-specific uptake of a drug, and improve accumulation of a drug at tumors, e.g., through an enhanced permeation and retention (EPR) effect. There are limited examples of therapeutics formulated for delivery as nanoparticles, which include DOXIL® (liposomal encapsulated doxyrubicin) and ABRAXANE® (albumin bound paclitaxel nanoparticles).

The development of nanotechnologies for effective delivery of drugs or drug candidates to specific diseased cells and tissues, e.g., to cancer cells, in specific organs or tissues, in a temporospatially regulated manner potentially can overcome or ameliorate therapeutic challenges, such as systemic toxicity. However, while targeting of the delivery system may preferentially deliver drug to a site where therapy is needed, the drug released from the nanoparticle may not for example, remain in the region of the targeted cells in efficacious amounts or may not remain in the circulation in a relatively non-toxic state for a sufficient amount of time to decrease the frequency of treatment or permit a lower amount of drug to be administered while still achieving a therapeutic effect. Accordingly, there is a need in the art for improved drug targeting and delivery, including identification of targeting molecules that can be incorporated into particles and whose presence does not substantially interfere with efficacy of the drug.

SUMMARY

The present application provides a conjugate comprising an active agent coupled to an HSP90 targeting moiety by a linker and a pharmaceutical composition comprising such a conjugate.

Methods of making and using such conjugates are also provided.

DETAILED DESCRIPTION

Applicants have designed HSP90 targeting conjugates comprising an active agent and novel particles comprising such conjugates. Such targeting can, for example, improve the amount of active agent at a site and decrease active agent toxicity to the subject. HSP90 targeting conjugates of the present invention have deep and rapid tumor penetration and do not require receptor internalization. High accumulation and long retention time of HSP90 targeting conjugates enable the use of cytotoxic and non-cytotoxic payloads, such as chemotherapeutic agents, kinase inhibitors, or immuno-oncology modulators.

As used herein, “toxicity” refers to the capacity of a substance or composition to be harmful or poisonous to a cell, tissue organism or cellular environment. Low toxicity refers to a reduced capacity of a substance or composition to be harmful or poisonous to a cell, tissue organism or cellular environment. Such reduced or low toxicity may be relative to a standard measure, relative to a treatment or relative to the absence of a treatment.

Toxicity may further be measured relative to a subject's weight loss where weight loss over 15%, over 20% or over 30% of the body weight is indicative of toxicity. Other metrics of toxicity may also be measured such as patient presentation metrics including lethargy and general malaiase. Neutropenia or thrombopenia may also be metrics of toxicity.

Pharmacologic indicators of toxicity include elevated AST/ALT levels, neurotoxicity, kidney damage, GI damage and the like.

The conjugates are released after administration of the particles. The targeted drug conjugates utilize active molecular targeting in combination with enhanced permeability and retention effect (EPR) and improved overall biodistribution of the particles to provide greater efficacy and tolerability as compared to administration of targeted particles or encapsulated untargeted drug.

In addition, the toxicity of a conjugate containing an HSP90 targeting moiety linked to an active agent for cells that do not overexpress HSP90 is predicted to be decreased compared to the toxicity of the active agent alone. Without committing to any particular theory, applicants believe that this feature is because the ability of the conjugated active agent to be retained in a normal cell is decreased relative to a tumor cell.

It is an object of the invention to provide improved compounds, compositions, and formulations for temporospatial drug delivery.

It is further an object of the invention to provide methods of making improved compounds, compositions, and formulations for temporospatial drug delivery.

It is also an object of the invention to provide methods of administering the improved compounds, compositions, and formulations to individuals in need thereof.

Conjugates include an active agent or prodrug thereof attached to a targeting moiety, e.g., a molecule that can bind to HSP90, by a linker. The conjugates can be a conjugate between a single active agent and a single targeting moiety, e.g., a conjugate having the structure X-Y-Z where X is the targeting moiety, Y is the linker, and Z is the active agent.

In some embodiments the conjugate contains more than one targeting moiety, more than one linker, more than one active agent, or any combination thereof. The conjugate can have any number of targeting moieties, linkers, and active agents. The conjugate can have the structure X-Y-Z-Y-X, (X-Y)n-Z, X-(Y-Z)n, Xn-Y-Z, X-Y-Zn, (X-Y-Z)n, (X-Y-Z-Y)n-Z, where X is a targeting moiety, Y is a linker, Z is an active agent, and n is an integer between 1 and 50, between 2 and 20, for example, between 1 and 5. Each occurrence of X, Y, and Z can be the same or different, e.g., the conjugate can contain more than one type of targeting moiety, more than one type of linker, and/or more than one type of active agent.

The conjugate can contain more than one targeting moiety attached to a single active agent. For example, the conjugate can include an active agent with multiple targeting moieties each attached via a different linker. The conjugate can have the structure X-Y-Z-Y-X where each X is a targeting moiety that may be the same or different, each Y is a linker that may be the same or different, and Z is the active agent.

The conjugate can contain more than one active agent attached to a single targeting moiety. For example the conjugate can include a targeting moiety with multiple active agents each attached via a different linker. The conjugate can have the structure Z-Y-X-Y-Z where X is the targeting moiety, each Y is a linker that may be the same or different, and each Z is an active agent that may be the same or different.

A. Active Agents

A conjugate as described herein contains at least one active agent (a first active agent). The conjugate can contain more than one active agent, that can be the same or different from the first active agent. The active agent can be a therapeutic, prophylactic, diagnostic, or nutritional agent. A variety of active agents are known in the art and may be used in the conjugates described herein. The active agent can be a protein or peptide, small molecule, nucleic acid or nucleic acid molecule, lipid, sugar, glycolipid, glycoprotein, lipoprotein, or combination thereof. In some embodiments, the active agent is an antigen, an adjuvant, radioactive, an imaging agent (e.g., a fluorescent moiety) or a polynucleotide. In some embodiments the active agent is an organometallic compound.

In certain embodiments, the active agent of the conjugate comprises a predetermined molar weight percentage from about 1% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99% such that the sum of the molar weight percentages of the components of the conjugate is 100%. The amount of active agent(s) of the conjugate may also be expressed in terms of proportion to the targeting ligand(s). For example, the present teachings provide a ratio of active agent to ligand of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4; 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

In some embodiments, the active agent can be a cancer therapeutic. Cancer therapeutics include, for example, death receptor agonists such as the TNF-related apoptosis-inducing ligand (TRAIL) or Fas ligand or any ligand or antibody that binds or activates a death receptor or otherwise induces apoptosis. Suitable death receptors include, but are not limited to, TNFR1, Fas, DR3, DR4, DR5, DR6, LTβR and combinations thereof.

Cancer therapeutics such as chemotherapeutic agents, cytokines, chemokines, and radiation therapy agents can be used as active agents. Chemotherapeutic agents include, for example, alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. Such agents typically affect cell division or DNA synthesis and function. Additional examples of therapeutics that can be used as active agents include monoclonal antibodies and the tyrosine kinase inhibitors e.g. imatinib mesylate, which directly targets a molecular abnormality in certain types of cancer (e.g., chronic myelogenous leukemia, gastrointestinal stromal tumors).

The small molecule active agents used in this invention (e.g. antiproliferative (cytotoxic and cytostatic) agents) include cytotoxic compounds (e.g., broad spectrum), angiogenesis inhibitors, cell cycle progression inhibitors, PBK/m-TOR/AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperones inhibitors, HDAC inhibitors, PARP inhibitors, Wnt/Hedgehog signaling pathway inhibitors, RNA polymerase inhibitors and proteasome inhibitors. The small molecule active agents in some embodiments the active agent is an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof.

Broad spectrum cytotoxins include, but are not limited to, DNA-binding or alkylating drugs, microtubule stabilizing and destabilizing agents, platinum compounds, and topoisomerase I or II inhibitors.

Exemplary doxorubicin analogs include nemorubicin metabolite or analog drug moiety disclosed in US 20140227299 to Cohen et al., the contents of which are incorporated herein by reference in their entirety.

Exemplary CC-1065 analogs include duocarmycin SA, duocarmycin CI, duocarmycin C2, duocarmycin B2, DU-86, KW-2189, bizelesin, seco-adozelesin, and those described in U.S. Pat. Nos. 5,475,092; 5,595,499; 5,846,545; 6,534,660; 6,586,618; 6,756,397 and 7,049,316. Doxorubicin and its analogs include PNU-159682 and those described in U.S. Pat. No. 6,630,579 and nemorubicin metabolite or analog drugs disclosed in US 20140227299 to Cohen et al., the contents of which are incorporated herein by reference in their entirety.

Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine (vinorelbine). SuitableVincaalkaloids that can be used in the present invention are also disclosed in U.S. Publication Nos. 2002/0103136 and 2010/0305149, and in U.S. Pat. No. 7,303,749 B1, the disclosures of which are incorporated herein by reference in their entirety.

Exemplary cryptophycin compounds are described in U.S. Pat. Nos. 6,680,311 and 6,747,021, the disclosures of which are incorporated herein by reference in their entirety.

Exemplary topoisomerase II inhibitors include azonafide and etoposide.

Additional agents acting on DNA include Lurbinectedin (PM01183), Trabectedin (also known as ecteinascidin 743 or ET-743) and analogs as described in WO 200107711, WO 2003014127.

Angiogenesis inhibitors include, but are not limited to, MetAP2 inhibitors.

Exemplary cell cycle progression inhibitors include CDK inhibitors such as BMS-387032 and PD0332991; Rho-kinase inhibitors such as GSK429286; checkpoint kinase inhibitors such as AZD7762; aurora kinase inhibitors such as AZD1152, MLN8054 and MLN8237; PLK inhibitors such as BI 2536, BI6727 (Volasertib), GSK461364, ON-01910 (Estybon); and KSP inhibitors such as SB 743921, SB 715992 (ispinesib), MK-0731, AZD8477, AZ3146 and ARRY-520.

Receptor tyrosine kinases (RTK) are cell surface receptors which are often associated with signaling pathways stimulating uncontrolled proliferation of cancer cells and neoangiogenesis. Many RTKs, which over express or have mutations leading to constitutive activation of the receptor, have been identified, including, but not limited to, VEGFR, EGFR, FGFR, PDGFR, EphR and RET receptor family receptors. Exemplary RTK specific targets include ErbB2, FLT-3, c-Kit, c-Met, and HIF.

Exemplary kinase inhibitors include inhibitors of the kinases ATM, ATR, CHK1, CHK2, WEE1, and RSK.

In another embodiment, the drug used in the invention is a combination of two or more drugs, such as, for example, PI3 kinases and MEK inhibitors; broad spectrum cytotoxic compounds and platinum compounds; PARP inhibitors and platinum compounds; broad spectrum cytotoxic compounds and PARP inhibitors.

The active agent can be a cancer therapeutic. The cancer therapeutics may include death receptor agonists such as the TNF-related apoptosis-inducing ligand (TRAIL) or Fas ligand or any ligand or antibody that binds or activates a death receptor or otherwise induces apoptosis. Suitable death receptors include, but are not limited to, TNFR1, Fas, DR3, DR4, DR5, DR6, LTβR and combinations thereof.

The active agent can be a DNA minor groove binders such as lurbectidin and trabectidin

The active agent can be E3 ubiquitin ligase inhibitors, adeubiquitinase inhibitors or an NFkB pathway inhibitor.

The active agent can be an inhibitor of tumor metabolism, such as an inhibitor of GAPDH, GLUT1, HK II, PFK, GAPDH, PK, LDH or MCTs

The active agent can target Bcl-2, IAP, or fatty acid synthase.

The active agent can be an inorganic or organometallic compound containing one or more metal centers. In some examples, the compound contains one metal center. The active agent can be, for example, a platinum compound, a ruthenium compound (e.g., trans-[RuCl2(DMSO)4], or trans-[RuCl4(imidazole)2, etc.), cobalt compound, copper compound, or iron compounds.

In some embodiments, the active agent is a small molecule. In some embodiments, the active agent is a small molecule cytotoxin. In one embodiment, the active agent is cabazitaxel, or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof. In another embodiment, the active agent is mertansine (DM1) or DM4, or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof. DM1 or DM4 inhibits the assembly of microtubules by binding to tubulin. Structure of DM1 is shown below:

In some embodiments, the active agent Z is Monomethyl auristatin E (MMAE), or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof. Structure of MMAE is shown below:

In some embodiments, the active agent Z is a sequence-selective DNA minor-groove binding crosslinking agent. For example, Z may be pyrrolobenzodiazepine (PBD), a PBD dimer, or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof. Structures of PBD and PBD dimer are shown below:

In some embodiments, the active agent Z is a topoisomerase I inhibitor, such as camptothecin, irinotecan, SN-38, or an analog, derivative, prodrug, or pharmaceutically acceptable salt thereof.

Any cytotoxic moiety disclosed in WO2013158644, WO2015038649, WO2015066053, WO2015116774, WO2015134464, WO2015143004, WO2015184246, the contents of each of which are incorporated herein by reference in their entirety, such as bendamustine, VDA, doxorubicin, pemetrexed, vorinostat, lenalidomide, docetaxel, 17-AAG, 5-FU, abiraterone, crizotinib, KW-2189, BUMB2, DC1, CC-1065, adozelesin, or derivatives/analogs thereof, may be used as an active agent in conjugates of the present invention.

The PI3K/AKT/mTOR signaling network (PI3K pathway) controls most hallmarks of cancer: cell cycle, survival, metabolism, motility and genomic stability. The PI3K pathway is the most frequently altered pathway in human cancer. Activation of PI3K has been directly linked to cancer through mutations or amplifications of PIK3CA, and loss of function tumor suppressor PTEN. PIK3CA gene is the 2ndmost frequently mutated oncogene. PTEN is among the most frequently mutated tumor suppressor genes. Pathway inhibitors demonstrate antitumor efficacy in xenograft models, but toxicity limits clinical benefit in patients. Conjugating a PI3K inhibitor with a HSP90 targeting moiety provides a method to delivery PI3K inhibitors for sufficient PI3K inhibition in tumors with reduced toxicity.

Conjugates comprising PI3K inhibitors may be used to treat hematological malignancies and solid tumors. In some embodiments, conjugates comprising PI3K inhibitors are used to treat colorectal cancer, multiple myeloma, leukemia, lymphoma, colon cancer, gastric cancer, kidney cancer, lung cancer, or breast cancers including metastatic breast cancer. In some embodiments, conjugates comprising PI3K inhibitors are used to treat PIK3CA-altered cancers or HER2 positive cancers.

In some embodiments, the PI3K inhibitor may be an inhibitor of delta and gamma isoforms of PI3K. In some embodiments, the PI3K inhibitor is an inhibitor of alpha isoforms of PI3K. In other embodiments, the PI3K inhibitor is an inhibitor of one or more alpha, beta, delta and gamma isoforms of PI3K. Non-limiting examples of PI3K inhibitors include compounds disclosed in U.S. Pat. No. 9,546,180 (Infinity Pharmaceuticals), WO 2009088990 (Intellikine Inc.), WO 2011008302 (Intellikine Inc.), WO 2010036380 (Intellikine Inc.), WO 2010/006086 (Intellikine Inc.), WO 2005113556 (Icos Corp.), US 2011/0046165 (Intellikine Inc.), or US 20130315865 (Pfizer), the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, the PI3K inhibitor is selected from the group of Omipalisib (GSK458) or its derivatives/analogs, BAY 80-6946 (Copanlisib) or its derivatives/analogs, PF-04691502 or its derivatives/analogs, PI-103 or its derivatives/analogs, BGT226 (NVP-BGT226) or its derivatives/analogs, Apitolisib (GDC-0980, RG7422) or its derivatives/analogs, Duvelisib (IPI-145, INK1197) or its derivatives/analogs, AZD8186 or its derivatives/analogs, Pilaralisib (XL147) or its derivatives/analogs, and PIK-93 or its derivatives/analogs.

In particular, the conjugates of the present application may comprise an HSP90 targeting moiety connected to Omipalisib (GSK458) or its derivatives/analogs, BAY 80-6946 (Copanlisib) or its derivatives/analogs, PF-04691502 or its derivatives/analogs, or PI-103 or its derivatives/analogs.

PARP Inhibitors

Poly-(ADP ribose)polymerase (PARP) is a family of enzymes involved a number of cellular processes including the repair of single-stranded DNA breaks and programmed cell death. Some cancer cells, such as small cell lung cancer (SCLC) cells or BRCA mutant cancer cells, are more dependent on PARP than regular cells, making them uniquely sensitive to PARP inhibition. Most PARP inhibitors have two possible actions: inhibition of PARP function or trapping PARP on single-stranded DNA breaks.

Main toxicity of PARP inhibitors is hematological (thrombocytopenia), sometimes myelosuppression seen. HSP90 mediated PARP inhibitor delivery increases intratumor concentrations of PARP inhibitors and reduce hematological toxicity by improving tumor:plasma ratios. The sustained release of PARP inhibitors from the conjugates may provide continuous inhibition and yields greater efficacy than the PARP inhibitor alone.

Conjugates comprising PARP inhibitors may be used to treat hematological malignancies and solid tumors. BRCA mutant cancers are reliant on PARP as the sole mechanism of DNA repair, as double-stranded break repair mechanisms are impaired. Inhibition of PARP leads to double-strand DNA breaks and cell death in BRCA mutant cancers. Any cancer cell that is low in BRCA1/2 proteins may be sensitive to PARP inhibition. PARP is overexpressed in SCLC cells making SCLC cells more sensitive to PARP inhibiting. In some embodiments, conjugates comprising PARP inhibitors are used to treat SCLC, non-small cell lung cancer (NSCLC), breast cancers including triple negative breast cancers and BRCA mutant breast cancers, ovarian cancers, colorectal cancers, prostate cancers, melanoma, or metastatic cancers including metastatic breast cancer, metastatic ovarian cancer, and metastatic melanoma.

In some embodiments, the PARP inhibitor is selected from the group of olaparib or its derivatives/analogs and talazoparib or its derivatives/analogs. The conjugates of the present application may comprise an HSP90 targeting moiety connected to olaparib or its derivatives/analogs or talazoparib or its derivatives/analogs.

In some embodiments, the conjugates comprising PARP inhibitors or derivatives thereof are inactive as PARP inhibitors and require linker release for PARP inhibiting activity. Such conjugates may have maximum tolerated doses (MTD) greater than PARP inhibitors alone. Linkers may comprise a disulfide bond, which is cleaved in the reducing environment in cytosol to release the PARP inhibitors in the conjugates. For example, olaparib or its derivatives or talazoparib or its derivatives may be connected to a linker on the heterocycle to inactive the PARP inhibiting function. A conjugates comprising olaparib that is inactivated as a PARP inhibitor and a conjugate comprising talazoparib that is inactivated as a PARP inhibitor are shown below.

Some proteins in the PI3K pathway are unregulated following treatment with PARP inhibitors. PI3K inhibitors may increase DNA damage and sensitize cells (e.g., triple negative breast cancer cells and SCLC cells) to PARP inhibition. Therefore, a combination of PI3K inhibiting agents and PARP inhibiting agents has a synergistic effect and enhances the effect of either agent alone. In some embodiments, a combination of conjugates comprising PI3K inhibitors and conjugates comprising PARP inhibitors is administered. In some embodiments, the conjugates comprise more than one active agent, wherein the conjugates comprise at least one PARP inhibitor and at least one PI3K inhibitor.

Targeting ligands (also referred to as targeting moieties) as described herein include any molecule that can bind one or more HSP90 proteins. Such targeting ligands can be peptides, antibody mimetics, nucleic acids (e.g., aptamers), polypeptides (e.g., antibodies), glycoproteins, small molecules, carbohydrates, or lipids.

The targeting moiety, X, can be any HSP90 binding moiety such as, but not limited to, natural compounds (e.g., geldanamycin and radicicol), and synthetic compounds such as geldanamycin analogue 17-AAG (i.e., 17-allylaminogeldanamycin), a purine-scaffold HSP90 inhibitor series including PU24FC1 (He H., et al,J. Med. Chem., vol. 49:381 (2006), the contents of which are incorporated herein by reference in their entirety), BIIB021 (Lundgren K., et al,Mol. Cancer Ther., vol. 8(4):921 (2009), the contents of which are incorporated herein by reference in their entirety), 4,5-diarylpyrazoles (Cheung K. M., et al,Bioorg. Med. Chem. Lett., vol. 15:3338 (2005), the contents of which are incorporated herein by reference in their entirety), 3-aryl,4-carboxamide pyrazoles (Brough P. A., et al,Bioorg. Med. Chem. Lett., vol. 15: 5197 (2005), the contents of which are incorporated herein by reference in their entirety), 4,5-diarylisoxazoles (Brough P. A., et al,J. Med. Chem., vol. 51:196 (2008), the contents of which are incorporated herein by reference in their entirety), 3,4-diaryl pyrazole resorcinol derivative (Dymock B. W., et al,J. Med. Chem., vol. 48:4212 (2005), the contents of which are incorporated herein by reference in their entirety), thieno[2,3-d]pyrimidine (WO2005034950 to VERNALIS et al., the contents of which are incorporated herein by reference in their entirety), aryl triazole derivatives of Formula I in EP2655345 to Giannini et al., the contents of which are incorporated herein by reference in their entirety, or any other example of HSP90 binding ligands or their derivatives/analogs.

In some embodiments, the HSP90 binding moiety may be heterocyclic derivatives containing three heteroatoms. WO2009134110 to MATULIS et al., the contents of which are incorporated herein by reference in their entirety, discloses 4,5-diaryl thiadiazoles which demonstrate good HSP90 binding affinity. Even though they have rather modest cell growth inhibition, they may be used as HSP90 binding moiety in conjugates of the present invention. Another class of aza-heterocyclic adducts, namely triazole derivatives or their analogs, may be used as HSP90 binding moiety in conjugates of the present invention. For example, the 1,2,4-triazole scaffold has been profusely documented as possessing HSP90 inhibiting properties. WO2009139916 to BURLISON et al. (Synta Pharmaceuticals Corp.), the contents of which are incorporated herein by reference in their entirety, discloses tricyclic 1,2,4-triazole derivatives inhibiting HSP90 at high micromolar concentrations. Any tricyclic 1,2,4-triazole derivatives disclosed in WO2009139916 or their derivatives/analogs may be used as HSP90 binding moiety in conjugates of the present invention. Any trisubstituted 1,2,4-triazole derivatives disclosed in WO 2010017479 and WO 2010017545 (Synta Pharmaceuticals Corp.) or their derivatives/analogs, the contents of which are incorporated herein by reference in their entirety, may be used as HSP90 binding moiety in conjugates of the present invention. In another example, a triazolone-containing HSP90 inhibitor named ganetespib (previously referred as to STA-9090, or as its highly soluble phosphate prodrug STA-1474) disclosed in WO2006055760 (Synta Pharmaceuticals Corp.), the contents of which are incorporated herein by reference in their entirety, or its derivatives/analogs may be used as HSP90 binding moiety in conjugates of the present invention.

In some embodiments, ganetespib or its derivatives/analogs may be used a targeting moiety. Non-limiting examples of ganetespib derivatives/analogs are shown below.

In some embodiments, Onalespib (AT13387) or its derivatives/analogs may be used as a targeting moiety in the conjugates of the present invention. Onalespib and non-limiting examples of Onalespib derivatives/analogs are shown below.

Any HSP9U ligand or HSP9U inhibitor disclosed in WO2013158644, WO2015038649, WO2015066053, WO2015116774, WO2015134464, WO2015143004, WO2015184246, the contents of which are incorporated herein by reference in their entirety, or their derivatives/analogs may be used as HSP90 binding moiety in the conjugates of the present invention, such as:

wherein R1 may be alkyl, aryl, halide, carboxamide or sulfonamide; R2 may be alkyl, cycloalkyl, aryl or heteroaryl, wherein when R2 is a 6 membered aryl or heteroaryl, R2 is substituted at the 3- and 4-positions relative to the connection point on the triazole ring, through which a linker L is attached; and R3 may be SH, OH, —CONHR4, aryl or heteroaryl, wherein when R3 is a 6 membered aryl or heteroaryl, R3 is substituted at the 3 or 4 position;

wherein R1 may be alkyl, aryl, halo, carboxamido, sulfonamido; and R2 may be optionally substituted alkyl, cycloalkyl, aryl or heteroaryl. Examples of such compounds include 5-(2,4-dihydroxy-5-isopropylphenyl)-N-(2-morpholinoethyl)-4-(4-(morpholinomethyl)phenyl)-4H-1,2,4-triazole-3-carboxamide and 5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-(4-methylpiperazin-1-yl)phenyl)-N-(2,2,2-trifluoroethyl)-4H-1,2,4-triazole-3-carboxamide;

wherein X, Y, and Z may independently be CH, N, O or S (with appropriate substitutions and satisfying the valency of the corresponding atoms and aromaticity of the ring); R1 may be alkyl, aryl, halide, carboxamido or sulfonamido; R2 may be substituted alkyl, cycloalkyl, aryl or heteroaryl, where a linker L is connected directly or to the extended substitutions on these rings; R3 may be SH, OH, NR4R5 AND —CONHR6, to which an effector moiety may be connected; R4 and R5 may independently be H, alkyl, aryl, or heteroaryl; and R6 may be alkyl, aryl, or heteroaryl, having a minimum of one functional group to which an effector moiety may be connected; or

wherein R1 may be alkyl, aryl, halo, carboxamido or sulfonamido; R2 and R3 are independently C1-C5 hydrocarbyl groups optionally substituted with one or more of hydroxy, halogen, C1-C2 alkoxy, amino, mono- and di-C1-C2 alkylamino; 5- to 12-membered aryl or heteroaryl groups; or, R2 and R3, taken together with the nitrogen atom to which they are attached, form a 4- to 8-membered monocyclic heterocyclic group, of which up to 5 ring members are selected from O, N and S. Examples of such compounds include AT-13387.

The HSP90 targeting moiety may also be PU-H71, an HSP90 inhibitor that is124I radiolabeled for PET imaging or its derivatives/analogs.

In some embodiments, the HSP90 targeting moiety comprises a Sansalvamide A derivative. Sansalvamide A (San A) is a cyclic pentapeptide isolated from a marine fungus and binds to HSP90. Any Di-Sansalvamide A derivative (dimerized San A molecules) disclosed in Alexander et al.,J. Med Chem., vol.52(24):7927 (2009), the contents of which are incorporated herein by reference in their entirety, for example, the Di-San A molecules in FIG. 1 of Alexander, may be used as a targeting moiety of the conjugate of the current disclosure.

In certain embodiments, the targeting moiety or moieties of the conjugate are present at a predetermined molar weight percentage from about 0.1% to about 10%, or about 1% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99% such that the sum of the molar weight percentages of the components of the conjugate is 100%. The amount of targeting moieties of the conjugate may also be expressed in terms of proportion to the active agent(s), for example, in a ratio of ligand to active agent of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4; 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

The conjugates contain one or more linkers attaching the active agents and targeting moieties. The linker, Y, is bound to one or more active agents and one or more targeting ligands to form a conjugate. The linker Y is attached to the targeting moiety X and the active agent Z by functional groups independently selected from an ester bond, disulfide, amide, acylhydrazone, ether, carbamate, carbonate, and urea. Alternatively the linker can be attached to either the targeting ligand or the active drug by a non-cleavable group such as provided by the conjugation between a thiol and a maleimide, an azide and an alkyne. The linker is independently selected from the group consisting alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein each of the alkyl, alkenyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups optionally is substituted with one or more groups, each independently selected from halogen, cyano, nitro, hydroxyl, carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, wherein each of the carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, or heterocyclyl is optionally substituted with one or more groups, each independently selected from halogen, cyano, nitro, hydroxyl, carboxyl, carbamoyl, ether, alkoxy, aryloxy, amino, amide, carbamate, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl.

In some embodiments, the linker comprises a cleavable functionality that is cleavable. The cleavable functionality may be hydrolyzed in vivo or may be designed to be hydrolyzed enzymatically, for example by Cathepsin B. A “cleavable” linker, as used herein, refers to any linker which can be cleaved physically or chemically. Examples for physical cleavage may be cleavage by light, radioactive emission or heat, while examples for chemical cleavage include cleavage by re-dox-reactions, hydrolysis, pH-dependent cleavage or cleavage by enzymes. For example, the cleavable functionality may be a disulfide bond or a carbamate bond.

In some embodiments the alkyl chain of the linker may optionally be interrupted by one or more atoms or groups selected from —O—, —C(═O)—, —NR, —O—C(═O)—NR—, —S—, —S—S—. The linker may be selected from dicarboxylate derivatives of succinic acid, glutaric acid or diglycolic acid. In some embodiments, the linker Y may be X′—R1—Y′—R2—Z′ and the conjugate can be a compound according to Formula Ia:

wherein X is a targeting moiety defined above; Z is an active agent; X′, R1, Y′, R2and Z′ are as defined herein.

X′ is either absent or independently selected from carbonyl, amide, urea, amino, ester, aryl, arylcarbonyl, aryloxy, arylamino, one or more natural or unnatural amino acids, thio or succinimido; R1and R2are either absent or comprised of alkyl, substituted alkyl, aryl, substituted aryl, polyethylene glycol (2-30 units); Y′ is absent, substituted or unsubstituted 1,2-diaminoethane, polyethylene glycol (2-30 units) or an amide; Z′ is either absent or independently selected from carbonyl, amide, urea, amino, ester, aryl, arylcarbonyl, aryloxy, arylamino, thio or succinimido. In some embodiments, the linker can allow one active agent molecule to be linked to two or more ligands, or one ligand to be linked to two or more active agent molecule.

In some embodiments, the linker Y may be Amand the conjugate can be a compound according to Formula Ib:

wherein A is defined herein, m=0-20.

A in Formula Ia is a spacer unit, either absent or independently selected from the following substituents. For each substituent, the dashed lines represent substitution sites with X, Z or another independently selected unit of A wherein the X, Z, or A can be attached on either side of the substituent:

wherein z=0-40, R is H or an optionally substituted alkyl group, and R′ is any side chain found in either natural or unnatural amino acids.

In some embodiments, the conjugate may be a compound according to Formula Ic:

wherein A is defined above, m=0-40, n=0-40, x=1-5, y=1-5, and C is a branching element defined herein.

C in Formula Ic is a branched unit containing three to six functionalities for covalently attaching spacer units, ligands, or active drugs, selected from amines, carboxylic acids, thiols, or succinimides, including amino acids such as lysine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, glutamic acid, aspartic acid, and cysteine.

Non-Limiting Examples of Conjugates

DM1 as Active Agent

In some embodiments, the active agent Z is DM1 and the HSP90 targeting moiety X is Ganetespib or its derivatives/analogs, wherein the active agent Z and the targeting moiety X are connected with a cleavable linker. The cleavable linker may comprise a disulfide bond, which allows the active agent to be released in cytosol, which is a reducing environment. Non-limiting examples of the conjugates are Compounds 1, 2, 3, and 14.

MMAE as Active Agent

In some embodiments, the active agent Z is MMAE and the HSP90 targeting moiety X is Ganetespib or its derivatives/analogs, wherein the active agent Z and the targeting moiety X are connected with a cleavable linker. The cleavable linker may comprise a disulfide bond, which allows the active agent to be released in cytosol, which is a reducing environment. Non-limiting examples of the conjugates are Compounds 15 and 16.

PARP Inhibitors as Active Agents

In some embodiments, the active agent Z is a PARP inhibitor and the HSP90 targeting moiety X is Ganetespib or its derivatives/analogs, wherein the active agent Z and the targeting moiety X are connected with a cleavable linker. The PARP inhibitor may be olaparib or talazoparib. The cleave linker may comprise a disulfide bond.

Olaparib as Active Agent

In some embodiments, the active agent Z is olaparib or a derivative/analog thereof and the HSP90 targeting moiety X is Ganetespib or its derivatives/analogs. The general structure of the conjugate is shown below:

In some embodiments, the cleavable linker may comprise a disulfide bond. In some embodiments, the disulfide linker comprises a spacer and a carbamate group. The structure of the conjugate is shown below:

wherein X is hydrogen or a non-hydrogen substituent, and R is hydrogen or a non-hydrogen substituent. Not willing to be bound to any theory, the R groups adjacent to disulfide greatly affect plasma and tumor stability. When R is not hydrogen, e.g., when R is -Me, the slower-releasing disulfide linker provides a slow release profile. When the carbamate substituent X is not hydrogen, plasma half-life of the conjugate is improved. The spacer improves half-life and mass recovery of the conjugate in tumor cells. The spacer may be, but not limited to, —O—CH2CH2-,

In some embodiments, R is a methyl group and examples of the conjugates may have a structure of

wherein R′ is H or any other substituents, such as an alkyl group which may be substituted.

When R′=H, the conjugate is Compound 4 having a structure of

In some embodiments, R′ is not hydrogen and such conjugates may have greater stability than Compound 4. One non-limiting example of a conjugate where R′ is not hydrogen is Compound 5, wherein R′=−CH2CH2NMe2, having a structure of

In some embodiments, R is H and the conjugate has a structure of

wherein R′ is H or any other substituents, such as an alkyl group which may be substituted.

When R′=—CH2CH2NMe2, the conjugate has a structure of

In some embodiments, the cleavable linker may comprise a disulfide bond and a spacer:

When the spacer comprises

the conjugate has a structure of

When the spacer comprises

the conjugate has a structure of

Talazoparib as Active Agent

In some embodiments, the active agent Z is talazoparib or a derivative/analog thereof and the HSP90 targeting moiety X is Ganetespib or its derivatives/analogs. The general structure of the conjugate is shown below:

One limiting example of the conjugate has a structure of

PI3K Inhibitors as Active Agents

In some embodiments, the active agent Z is a PI3K inhibitor and the HSP90 targeting moiety X is Ganetespib or its derivatives/analogs, wherein the active agent Z and the targeting moiety X are connected with a linker. The PI3K inhibitor may be PI-103 or PF-04691502. The linker may be a cleavable linker comprising a disulfide bond. The conjugate may comprise a carbamate group which is cleavable. Non-limiting examples of the conjugates are Compounds 10, 11, 12, and 13, wherein Compounds 10, 12 and 13 comprises PI-103 and a derivative of ganetespib and Compound 11 comprises PF-04691502 and a derivative of ganetespib.

Copanlisib as Active Agent

In some embodiments, the conjugate comprises Copanlisib or a fragment/derivative/analog thereof as payload. The copanlisib fragment/derivative/analog may comprise a structure of

The targeting moiety may be a ganetespib derivative, such as but not limited to TM1, TM2, TM3, TM4, TM5, and TM8. The targeting moiety may also be an Onalespib derivative, such as but not limited to TM6 and TM7. Non-limiting examples of conjugates comprising Copanlisib or a derivative/analog thereof include:

Omipalisib as Active Agent

In some embodiments, the conjugate comprise Omipalisib or a fragment/derivative/analog thereof as payload. The omipalisib fragment/derivative/analog may comprise a structure of

The targeting moiety may be a ganetespib derivative, such as but not limited to TM1, TM2, TM3, TM4, TM5, and TM8. The targeting moiety may also be an Onalespib derivative, such as but not limited to TM6 and TM7. Non-limiting examples of conjugates comprising Copanlisib or a derivative/analog thereof include:

PI-103 as Active Agent

In some embodiments, the conjugate comprises PI-103 or a fragment/derivative/analog thereof as payload. The PI-103 fragment/derivative/analogy may comprise a structure of

The targeting moiety may be a ganetespib derivative, such as but not limited to TM1, TM2, TM3, TM4, TM5, and TM8. The targeting moiety may also be an Onalespib derivative, such as but not limited to TM6 and TM7. Non-limiting examples of conjugates comprising Copanlisib or a derivative/analog thereof include:

The disclosure also provides activatable compositions that include conjugates that are coupled to a masking moiety where the ability of the conjugate to bind to HSP90. Such conjugates are referred to as masked conjugates. The binding of the targeting moiety to HSP90 may be inhibited or hindered by the masking moiety. For example, the binding may be sterically hindered by the presence of the masking moiety or may be inhibited by the charge of the masking moiety.

Cleavage of the masking moiety, a conformation change, or a chemical transformation may unmask/activate the conjugate. The masking/unmasking process may be reversible or irreversible. When the masked conjugates are activated, the ability to bind to HSP90 is at least comparable to the corresponding, un-masked conjugate.

In some embodiments, the masking moiety contains a peptide sequence that includes a substrate for a protease. The protease may be produced by a tumor cell. Once the masking moiety is cleaved by the protease, the masking moiety no longer interferes with the binding of the conjugate to HSP90, thereby activating the conjugates of the present invention. The masking moiety prevents binding of the conjugates of the present invention at nontreatment sites. Such conjugates can further provide improved biodistribution characteristics.

In some embodiments, the masking moiety comprises a peptide that may be a substrate for an enzyme selected from the group consisting of MMP1, MMP2, MMP3, MMP8, MMP9, MMP14, plasmin, PSA, PSMA, CATHEPSIN D, CATHEPSIN K, CATHEPSIN S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. For example, the masking moiety may comprise a protease substrate such as a plasmin substrate, a caspase substrate or a matrix metalloprotease (MMP) substrate (e.g., a substrate of MMP-1, MMP-2, MMP-9, or MMP-14).

In some embodiments, the masking moiety is connected to any place of the conjugate by a cleavable linker that is cleaved in the chemical environment of the tumor, for example in the acidic or reducing environment of a tumor. The masked conjugates are stable in circulation, activated at intended sites of therapy and/or diagnosis, but not in normal tissues. For example, the cleavable linker may comprise a cysteine-cysteine pair capable of forming a reducible disulfide bond, which may be cleaved by a reducing agent. Reducing agents of particular interest include cellular reducing agents such as proteins or other agents that are capable of reducing a disulfide bond under physiological conditions, e.g., glutathione, thioredoxin, NADPH, flavins, and ascorbate. In another example, the masking moiety or the linker may be acid-cleavable and the conjugate becomes unmasked in the acidic tumor microenvironment.

The conjugates of the present invention may further comprise at least one external linker connected to a reacting group that reacts with a functional group on a protein or an engineered protein or derivatives/analogs/mimics thereof, or comprise at least one external linker connected to a pharmacokinetic modulating unit. The external linkers connecting the conjugates and the reacting group or the pharmacokinetic modulating units may be cleavable linkers that allow release of the conjugates. Hence, the conjugates may be separated from the protein or pharmacokinetic modulating units as needed.

Any reacting group or PMU (such as PMUs comprising polymers) disclosed in WO2017/197241, the contents of which are incorporated herein by reference in their entirety, may be attached to the conjugates of the present disclosure.

The conjugates of the present invention may further comprise at least one permeability modulating unit. In some embodiments, the permeability modulating unit is attached to the payload of the conjugate, wherein the permeablity modulating unit regulates the cell membrane permeability of the payload. In some embodiments, the permeability modulating unit reduces the permeability of the payload. Not willing to be bound by any theory, once the payload is released from the conjugate, the permability modulating unit that is attached to the payload reduces the cell membrane permability of the payload, increases the retention time of the payload in target cells, improves the intracellular accumulation of the payload, and improves its efficacy.

In some embodiments, the permeability modulation unit does not adversely impact the permeability of the conjugate or the binding capability of the targeting moiety. In some embodiments, the permeability modulation unit is active only after the payload is released from the conjugate, e.g., after the cleavable linker between the payload and the targeting moiety is cleaved.

In some embodiments, the permeability modulating unit is a functional group that is covalently attached to the payload of the conjugate. In some embodiments, the permeability modulating unit is an integral part of the payload.

In some embodiments, the permeability modulating unit is attached to the payload via an external linker. The external linker may be a non-cleavable linker.

The passive permeation of a payload through the biological cell membranes is strongly dependent on the molecule physicochemical properties. Important factors that influence cell membrane permeation include the acid-base character of the molecule (which influences the charge of the molecule at the specific pH), its lipophilicity (which affects its partition between aqueous and lipid environments), and its solubility. For a payload to be permeable, there should be an appropriate balance between the hydrophobicity and hydrophilicity. In some embodiments, the permeability moduclating unit is hydrophilic. In some embodiments, the permeability moduclating unit is hydrophobic. In some embodiments, the permeability moduclating unit is polar. In some embodiments, the permeability moduclating unit is charged at physiological pH. For example, the permeability modulating unit may be positively charged, negatively charged, or a combination of multiple charges.

Non-limiting examples of the permability modulating unit include a functional group that has at least one nitrogen, such as a piperazine functional group. For example, Compound 38 comprises a piperazine functional group. Not willing to be bound to any theory, once the amide bond of the linkers is cleaved and the payload is released, the piperazine group reduces the permeability of the copanlisib derivative payload.

Particles containing one or more conjugates can be polymeric particles, lipid particles, solid lipid particles, inorganic particles, or combinations thereof (e.g., lipid stabilized polymeric particles). In some embodiments, the particles are polymeric particles or contain a polymeric matrix. The particles can contain any of the polymers described herein or derivatives or copolymers thereof. The particles generally contain one or more biocompatible polymers. The polymers can be biodegradable polymers. The polymers can be hydrophobic polymers, hydrophilic polymers, or amphiphilic polymers. In some embodiments, the particles contain one or more polymers having an additional targeting moiety attached thereto.

The size of the particles can be adjusted for the intended application. The particles can be nanoparticles or microparticles. The particle can have a diameter of about 10 nm to about 10 microns, about 10 nm to about 1 micron, about 10 nm to about 500 nm, about 20 nm to about 500 nm, or about 25 nm to about 250 nm. In some embodiments the particle is a nanoparticle having a diameter from about 25 nm to about 250 nm. It is understood by those in the art that a plurality of particles will have a range of sizes and the diameter is understood to be the median diameter of the particle size distribution.

In various embodiments, a particle may be a nanoparticle, i.e., the particle has a characteristic dimension of less than about 1 micrometer, where the characteristic dimension of a particle is the diameter of a perfect sphere having the same volume as the particle. The plurality of particles can be characterized by an average diameter (e.g., the average diameter for the plurality of particles). In some embodiments, the diameter of the particles may have a Gaussian-type distribution. In some embodiments, the plurality of particles have an average diameter of less than about 300 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, less than about 100 nm, less than about 50 nm, less than about 30 nm, less than about 10 nm, less than about 3 nm, or less than about 1 nm. In some embodiments, the particles have an average diameter of at least about 5 nm, at least about 10 nm, at least about 30 nm, at least about 50 nm, at least about 100 nm, at least about 150 nm, or greater. In certain embodiments, the plurality of the particles have an average diameter of about 10 nm, about 25 nm, about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 500 nm, or the like. In some embodiments, the plurality of particles have an average diameter between about 10 nm and about 500 nm, between about 50 nm and about 400 nm, between about 100 nm and about 300 nm, between about 150 nm and about 250 nm, between about 175 nm and about 225 nm, or the like. In some embodiments, the plurality of particles have an average diameter between about 10 nm and about 500 nm, between about 20 nm and about 400 nm, between about 30 nm and about 300 nm, between about 40 nm and about 200 nm, between about 50 nm and about 175 nm, between about 60 nm and about 150 nm, between about 70 nm and about 130 nm, or the like. For example, the average diameter can be between about 70 nm and 130 nm. In some embodiments, the plurality of particles have an average diameter between about 20 nm and about 220 nm, between about 30 nm and about 200 nm, between about 40 nm and about 180 nm, between about 50 nm and about 170 nm, between about 60 nm and about 150 nm, or between about 70 nm and about 130 nm. In one embodiment, the particles have a size of 40 to 120 nm with a zeta potential close to 0 mV at low to zero ionic strengths (1 to 10 mM), with zeta potential values between +5 to −5 mV, and a zero/neutral or a small −ve surface charge.

The particles contain one or more conjugates as described above. The conjugates can be present on the interior of the particle, on the exterior of the particle, or both. The particles may comprise hydrophobic ion-pairing complexes or hydrophobic ion-pairs formed by one or more conjugates described above and counterions.

Hydrophobic ion-pairing (HIP) is the interaction between a pair of oppositely charged ions held together by Coulombic attraction. HIP, as used here in, refers to the interaction between the conjugate of the present invention and its counterions, wherein the counterion is not H+or HO−ions. Hydrophobic ion-pairing complex or hydrophobic ion-pair, as used herein, refers to the complex formed by the conjugate of the present invention and its counterions. In some embodiments, the counterions are hydrophobic. In some embodiments, the counterions are provided by a hydrophobic acid or a salt of a hydrophobic acid. In some embodiments, the counterions are provided by bile acids or salts, fatty acids or salts, lipids, or amino acids. In some embodiments, the counterions are negatively charged (anionic). Non-limited examples of negative charged counterions include the counterions sodium sulfosuccinate (AOT), sodium oleate, sodium dodecyl sulfate (SDS), human serum albumin (HSA), dextran sulphate, sodium deoxycholate, sodium cholate, anionic lipids, amino acids, or any combination thereof. Without wishing to be bound by any theory, in some embodiments, HIP may increase the hydrophobicity and/or lipophilicity of the conjugate of the present invention. In some embodiments, increasing the hydrophobicity and/or lipophilicity of the conjugate of the present invention may be beneficial for particle formulations and may provide higher solubility of the conjugate of the present invention in organic solvents. Without wishing to be bound by any theory, it is believed that particle formulations that include HIP pairs have improved formulation properties, such as drug loading and/or release profile. Without wishing to be bound by any theory, in some embodiments, slow release of the conjugate of the invention from the particles may occur, due to a decrease in the conjugate's solubility in aqueous solution. In addition, without wishing to be bound by any theory, complexing the conjugate with large hydrophobic counterions may slow diffusion of the conjugate within a polymeric matrix. In some embodiments, HIP occurs without covalent confutation of the counterion to the conjugate of the present invention.

Without wishing to be bound by any theory, the strength of HIP may impact the drug load and release rate of the particles of the invention. In some embodiments, the strength of the HIP may be increased by increasing the magnitude of the difference between the pKa of the conjugate of the present invention and the pKa of the agent providing the counterion. Also without wishing to be bound by any theory, the conditions for ion pair formation may impact the drug load and release rate of the particles of the invention.

In some embodiments, any suitable hydrophobic acid or a combination thereof may form an HIP pair with the conjugate of the present invention. In some embodiments, the hydrophobic acid may be a carboxylic acid (such as but not limited to a monocarboxylic acid, dicarboxylic acid, tricarboxylic acid), a sulfinic acid, a sulfenic acid, or a sulfonic acid. In some embodiments, a salt of a suitable hydrophobic acid or a combination thereof may be used to form a HIP pair with the conjugate of the present invention. Examples of hydrophobic acids, saturated fatty acids, unsaturated fatty acids, aromatic acids, bile acid, polyelectrolyte, their dissociation constant in water (pKa) and log P values were disclosed in WO2014/043,625, the contents of which are incorporated herein by reference in their entirety. The strength of the hydrophobic acid, the difference between the pKa of the hydrophobic acid and the pKa of the conjugate of the present invention, log P of the hydrophobic acid, the phase transition temperature of the hydrophobic acid, the molar ratio of the hydrophobic acid to the conjugate of the present invention, and the concentration of the hydrophobic acid were also disclosed in WO2014/043,625, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, particles of the present invention comprising an HIP complex and/or prepared by a process that provides a counterion to form HIP complex with the conjugate may have a higher drug loading than particles without an HIP complex or prepared by a process that does not provide any counterion to form an HIP complex with the conjugate. In some embodiments, drug loading may increase 50%, 100%, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times.

In some embodiments, the particles of the invention may retain the conjugate for at least about 1 minute, at least about 15 minutes, at least about 1 hour, when placed in a phosphate buffer solution at 37° C.

In some embodiments, the weight percentage of the conjugate in the particles is at least about 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% such that the sum of the weight percentages of the components of the particles is 100%. In some embodiments, the weight percentage of the conjugate in the particles is from about 0.5% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99% such that the sum of the weight percentages of the components of the particles is 100%.

In some instances, a conjugate may have a molecular weight of less than about 50,000 Da, less than about 40,000 Da, less than about 30,000 Da, less than about 20,000 Da, less than about 15,000 Da, less than about 10,000 Da, less than about 8,000 Da, less than about 5,000 Da, less than about 3,000 Da, less than 2000 Da, less than 1500 Da, less than 1000 Da, or less than 500 Da. In some cases, the conjugate may have a molecular weight of between about 1,000 Da and about 50,000 Da, between about 1,000 Da and about 40,000 Da, in some embodiments between about 1,000 Da and about 30,000 Da, in some embodiments bout 1,000 Da and about 50,000 Da, between about 1,000 Da and about 20,000 Da, in some embodiments between about 1,000 Da and about 15,000 Da, in some embodiments between about 1,000 Da and about 10,000 Da, in some embodiments between about 1,000 Da and about 8,000 Da, in some embodiments between about 1,000 Da and about 5,000 Da, and in some embodiments between about 1,000 Da and about 3,000 Da. The molecular weight of the conjugate may be calculated as the sum of the atomic weight of each atom in the formula of the conjugate multiplied by the number of each atom. It may also be measured by mass spectrometry, NMR, chromatography, light scattering, viscosity, and/or any other methods known in the art. It is known in the art that the unit of molecular weight may be g/mol, Dalton (Da), or atomic mass unit (amu), wherein 1 g/mol=1 Da=1 amu.

The particles may contain one or more polymers. Polymers may contain one more of the following polyesters: homopolymers including glycolic acid units, referred to herein as “PGA”, and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as “PLA”, and caprolactone units, such as poly(ε-caprolactone), collectively referred to herein as “PCL”; and copolymers including lactic acid and glycolic acid units, such as various forms of poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide) characterized by the ratio of lactic acid:glycolic acid, collectively referred to herein as “PLGA”; and polyacrylates, and derivatives thereof. Exemplary polymers also include copolymers of polyethylene glycol (PEG) and the aforementioned polyesters, such as various forms of PLGA-PEG or PLA-PEG copolymers, collectively referred to herein as “PEGylated polymers”. In certain embodiments, the PEG region can be covalently associated with polymer to yield “PEGylated polymers” by a cleavable linker.

In certain embodiments, the hydrophobic polymer is an aliphatic polyester. In some embodiments, the hydrophobic polymer is poly(lactic acid), poly(glycolic acid), or poly(lactic acid-co-glycolic acid).

The particles can contain one or more biodegradable polymers. Biodegradable polymers can include polymers that are insoluble or sparingly soluble in water that are converted chemically or enzymatically in the body into water-soluble materials. Biodegradable polymers can include soluble polymers crosslinked by hydolyzable cross-linking groups to render the crosslinked polymer insoluble or sparingly soluble in water.

The particles can contain one or more amphiphilic polymers. Amphiphilic polymers can be polymers containing a hydrophobic polymer block and a hydrophilic polymer block. The hydrophobic polymer block can contain one or more of the hydrophobic polymers above or a derivative or copolymer thereof. The hydrophilic polymer block can contain one or more of the hydrophilic polymers above or a derivative or copolymer thereof. In some embodiments the amphiphilic polymer is a di-block polymer containing a hydrophobic end formed from a hydrophobic polymer and a hydrophilic end formed of a hydrophilic polymer. In some embodiments, a moiety can be attached to the hydrophobic end, to the hydrophilic end, or both. The particle can contain two or more amphiphilic polymers.

The particles may contain one or more lipids or amphiphilic compounds. For example, the particles can be liposomes, lipid micelles, solid lipid particles, or lipid-stabilized polymeric particles. The lipid particle can be made from one or a mixture of different lipids. Lipid particles are formed from one or more lipids, which can be neutral, anionic, or cationic at physiologic pH. The lipid particle, in some embodiments, incorporates one or more biocompatible lipids. The lipid particles may be formed using a combination of more than one lipid. For example, a charged lipid may be combined with a lipid that is non-ionic or uncharged at physiological pH.

The particle can be a lipid micelle. Lipid micelles for drug delivery are known in the art. Lipid micelles can be formed, for instance, as a water-in-oil emulsion with a lipid surfactant. An emulsion is a blend of two immiscible phases wherein a surfactant is added to stabilize the dispersed droplets. In some embodiments the lipid micelle is a microemulsion. A microemulsion is a thermodynamically stable system composed of at least water, oil and a lipid surfactant producing a transparent and thermodynamically stable system whose droplet size is less than 1 micron, from about 10 nm to about 500 nm, or from about 10 nm to about 250 nm. Lipid micelles are generally useful for encapsulating hydrophobic active agents, including hydrophobic therapeutic agents, hydrophobic prophylactic agents, or hydrophobic diagnostic agents.

The particle can be a liposome. Liposomes are small vesicles composed of an aqueous medium surrounded by lipids arranged in spherical bilayers. Liposomes can be classified as small unilamellar vesicles, large unilamellar vesicles, or multi-lamellar vesicles. Multi-lamellar liposomes contain multiple concentric lipid bilayers. Liposomes can be used to encapsulate agents, by trapping hydrophilic agents in the aqueous interior or between bilayers, or by trapping hydrophobic agents within the bilayer.

The lipid micelles and liposomes typically have an aqueous center. The aqueous center can contain water or a mixture of water and alcohol. Suitable alcohols include, but are not limited to, methanol, ethanol, propanol, (such as isopropanol), butanol (such as n-butanol, isobutanol, sec-butanol, tert-butanol, pentanol (such as amyl alcohol, isobutyl carbinol), hexanol (such as 1-hexanol, 2-hexanol, 3-hexanol), heptanol (such as 1-heptanol, 2-heptanol, 3-heptanol and 4-heptanol) or octanol (such as 1-octanol) or a combination thereof.

The particle can be a solid lipid particle. Solid lipid particles present an alternative to the colloidal micelles and liposomes. Solid lipid particles are typically submicron in size, i.e. from about 10 nm to about 1 micron, from 10 nm to about 500 nm, or from 10 nm to about 250 nm. Solid lipid particles are formed of lipids that are solids at room temperature. They are derived from oil-in-water emulsions, by replacing the liquid oil by a solid lipid.

Suitable solid lipids include, but are not limited to, higher saturated alcohols, higher fatty acids, sphingolipids, synthetic esters, and mono-, di-, and triglycerides of higher saturated fatty acids. Solid lipids can include aliphatic alcohols having 10-40, for example, 12-30 carbon atoms, such as cetostearyl alcohol. Solid lipids can include higher fatty acids of 10-40, for example, 12-30 carbon atoms, such as stearic acid, palmitic acid, decanoic acid, and behenic acid. Solid lipids can include glycerides, including monoglycerides, diglycerides, and triglycerides, of higher saturated fatty acids having 10-40, for example, 12-30 carbon atoms, such as glyceryl monostearate, glycerol behenate, glycerol palmitostearate, glycerol trilaurate, tricaprin, trilaurin, trimyristin, tripalmitin, tristearin, and hydrogenated castor oil. Suitable solid lipids can include cetyl palmitate, beeswax, or cyclodextrin.

D. Additional Active Agents

The particles can contain one or more additional active agents in addition to those in the conjugates. The additional active agents can be therapeutic, prophylactic, diagnostic, or nutritional agents as listed above. The additional active agents can be present in any amount, e.g. from about 0.5% to about 90%, from about 0.5% to about 50%, from about 0.5% to about 25%, from about 0.5% to about 20%, from about 0.5% to about 10%, or from about 5% to about 10% (w/w) based upon the weight of the particle. In one embodiment, the agents are incorporated in an about 0.5% to about 10% loading w/w.

The particles can contain one or more targeting moieties targeting the particle to a specific organ, tissue, cell type, or subcellular compartment in addition to the targeting moieties of the conjugate. The additional targeting moieties can be present on the surface of the particle, on the interior of the particle, or both. The additional targeting moieties can be immobilized on the surface of the particle, e.g., can be covalently attached to polymer or lipid in the particle. In some embodiments, the additional targeting moieties are covalently attached to an amphiphilic polymer or a lipid such that the targeting moieties are oriented on the surface of the particle.

F. Methods of Making Particles

In various embodiments, a method of making the particles includes providing any method disclosed in WO2014/106208 and WO2016/004043, the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, compositions are administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to the conjugate or particles comprising the conjugates to be delivered as described herein.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.

Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.

The conjugates or particles of the present invention can be formulated using one or more excipients to: (1) increase stability; (2) permit the sustained or delayed release (e.g., from a depot formulation of the monomaleimide); (3) alter the biodistribution (e.g., target the monomaleimide compounds to specific tissues or cell types); (4) alter the release profile of the monomaleimide compounds in vivo. Non-limiting examples of the excipients include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, and preservatives. Excipients of the present invention may also include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, hyaluronidase, nanoparticle mimics and combinations thereof. Accordingly, the formulations of the invention may include one or more excipients, each in an amount that together increases the stability of the monomaleimide compounds.

Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.

In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical compositions.

Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.

Administration

The conjugates or particles of the present invention may be administered by any route which results in a therapeutically effective outcome. These include, but are not limited to enteral, gastroenteral, epidural, oral, transdermal, epidural (peridural), intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection, (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), or in ear drops. In specific embodiments, compositions may be administered in a way which allows them cross the blood-brain barrier, vascular barrier, or other epithelial barrier.

The formulations described herein contain an effective amount of conjugates or particles in a pharmaceutical carrier appropriate for administration to an individual in need thereof. The formulations may be administered parenterally (e.g., by injection or infusion). The formulations or variations thereof may be administered in any manner including enterally, topically (e.g., to the eye), or via pulmonary administration. In some embodiments the formulations are administered topically.

The conjugates or particles can be formulated for parenteral delivery, such as injection or infusion, in the form of a solution, suspension or emulsion. The formulation can be administered systemically, regionally or directly to the organ or tissue to be treated.

Parenteral formulations can be prepared as aqueous compositions using techniques is known in the art. Typically, such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants. In some cases, an isotonic agent is included, for example, one or more sugars, sodium chloride, or other suitable agent known in the art.

Solutions and dispersions of the conjugates or particles can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, and combinations thereof.

The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent(s) or particles.

The formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers. If using 10% sucrose or 5% dextrose, a buffer may not be required.

Water soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.

Sterile injectable solutions can be prepared by incorporating the conjugates or particles in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized conjugates or particles into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation include vacuum-drying and freeze-drying techniques that yield a powder of the particle plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are known in the art.

Pharmaceutical formulations for parenteral administration can be in the form of a sterile aqueous solution or suspension of conjugates or particles formed from one or more polymer-drug conjugates. Acceptable solvents include, for example, water, Ringer's solution, phosphate buffered saline (PBS), and isotonic sodium chloride solution. The formulation may also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as 1,3-butanediol.

In some instances, the formulation is distributed or packaged in a liquid form. Alternatively, formulations for parenteral administration can be packed as a solid, obtained, for example by lyophilization of a suitable liquid formulation. The solid can be reconstituted with an appropriate carrier or diluent prior to administration.

Solutions, suspensions, or emulsions for parenteral administration may be buffered with an effective amount of buffer necessary to maintain a pH suitable for ocular administration. Suitable buffers are well known by those skilled in the art and some examples of useful buffers are acetate, borate, carbonate, citrate, and phosphate buffers.

Solutions, suspensions, or emulsions for parenteral administration may also contain one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents are well known in the art and some examples include glycerin, sucrose, dextrose, mannitol, sorbitol, sodium chloride, and other electrolytes.

Solutions, suspensions, or emulsions for parenteral administration may also contain one or more preservatives to prevent bacterial contamination of the ophthalmic preparations. Suitable preservatives are known in the art, and include polyhexamethylenebiguanidine (PHMB), benzalkonium chloride (BAK), stabilized oxychloro complexes (otherwise known as Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixtures thereof.

Solutions, suspensions, or emulsions for parenteral administration may also contain one or more excipients known art, such as dispersing agents, wetting agents, and suspending agents.

The conjugates or particles can be formulated for topical administration to a mucosal surface Suitable dosage forms for topical administration include creams, ointments, salves, sprays, gels, lotions, emulsions, liquids, and transdermal patches. The formulation may be formulated for transmucosal transepithelial, or transendothelial administration. The compositions contain one or more chemical penetration enhancers, membrane permeability agents, membrane transport agents, emollients, surfactants, stabilizers, and combination thereof. In some embodiments, the conjugates or particles can be administered as a liquid formulation, such as a solution or suspension, a semi-solid formulation, such as a lotion or ointment, or a solid formulation. In some embodiments, the conjugates or particles are formulated as liquids, including solutions and suspensions, such as eye drops or as a semi-solid formulation, to the mucosa, such as the eye or vaginally or rectally.

Suitable classes of penetration enhancers are known in the art and include, but are not limited to, fatty alcohols, fatty acid esters, fatty acids, fatty alcohol ethers, amino acids, phospholipids, lecithins, cholate salts, enzymes, amines and amides, complexing agents (liposomes, cyclodextrins, modified celluloses, and diimides), macrocyclics, such as macrocylic lactones, ketones, and anhydrides and cyclic ureas, surfactants, N-methyl pyrrolidones and derivatives thereof, DMSO and related compounds, ionic compounds, azone and related compounds, and solvents, such as alcohols, ketones, amides, polyols (e.g., glycols). Examples of these classes are known in the art.

Dosing

The present invention provides methods comprising administering conjugates or particles containing the conjugate as described herein to a subject in need thereof. Conjugates or particles containing the conjugates as described herein may be administered to a subject using any amount and any route of administration effective for preventing or treating or imaging a disease, disorder, and/or condition (e.g., a disease, disorder, and/or condition relating to working memory deficits). The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.

Compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

In some embodiments, compositions in accordance with the present invention may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, from about 25 mg/kg to about 50 mg/kg, from about 50 mg/kg to about 100 mg/kg, from about 100 mg/kg to about 125 mg/kg, from about 125 mg/kg to about 150 mg/kg, from about 150 mg/to about 175 mg/kg, from about 175 mg/kg to about 200 mg/kg, from about 200 mg/kg to about 250 mg/kg of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In some embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used.

The concentration of the conjugates or particles of the present invention may be between about 0.01 mg/mL to about 50 mg/mL, about 0.1 mg/mL to about 25 mg/mL, about 0.5 mg/mL to about 10 mg/mL, or about 1 mg/mL to about 5 mg/mL in the pharmaceutical composition.

As used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g, two or more administrations of the single unit dose. As used herein, a “single unit dose” is a dose of any therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event. As used herein, a “total daily dose” is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose. In one embodiment, the monomaleimide compounds of the present invention are administered to a subject in split doses. The monomaleimide compounds may be formulated in buffer only or in a formulation described herein.

Dosage Forms

A pharmaceutical composition described herein can be formulated into a dosage form described herein, such as a topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, and subcutaneous).

Liquid Dosage Forms

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art and may include suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed include, but are not limited to, water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of an active ingredient, it may be desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the monomaleimide compounds then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered monomaleimide compound may be accomplished by dissolving or suspending the monomalimide in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the monomaleimide compounds in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of monomaleimide compounds to polymer and the nature of the particular polymer employed, the rate of monomaleimide compound release can be controlled. Examples of other biodegradable polymers include, but are not limited to, poly(orthoesters) and poly(anhydrides). Depot injectable formulations may be prepared by entrapping the monomaleimide compounds in liposomes or microemulsions which are compatible with body tissues.

Pulmonary

Formulations described herein as being useful for pulmonary delivery may also be used for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration may be a coarse powder comprising the active ingredient and having an average particle from about 0.2 μm to 500 μm. Such a formulation may be administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.

General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

Coatings or Shells

VI. Methods of Using the Conjugates and Particles

The conjugates or particles as described herein can be administered to treat any hyperproliferative disease, metabolic disease, infectious disease, or cancer, as appropriate. Formulations may be administered by injection, orally, or topically, typically to a mucosal surface (lung, nasal, oral, buccal, sublingual, vaginally, rectally) or to the eye (intraocularly or transocularly).

In various embodiments, methods for treating a subject having a cancer are provided, wherein the method comprises administering a therapeutically-effective amount of the conjugates, salt forms thereof, or particles comprising such conjugates, as described herein, to a subject having a cancer, suspected of having cancer, or having a predisposition to a cancer. According to the present invention, cancer embraces any disease or malady characterized by uncontrolled cell proliferation, e.g., hyperproliferation. Cancers may be characterized by tumors, e.g., solid tumors or any neoplasm.

In some embodiments, the cancer is a solid tumor. Large drug molecules have limited penetration in solid tumors. The penetration of large drug molecules is slow. On the other hand, small molecules such as conjugates of the present invention may penetrate solid tumors rapidly and more deeply. Regarding penetration depth of the drugs, larger molecules penetrate less, despite having more durable pharmacokinetics. Small molecules such as conjugates of the present invention penetrate deeper. Dreher et al. (Dreher et al.,JNCI, vol. 98(5):335 (2006), the contents of which are incorporated herein by reference in their entirety) studied penetration of dextrans with different sizes into a tumor xenograft. As summarized inFIG. 6(seeFIG. 1of the present application) and Table 1 of Dreher, Dextrans with a molecular weight of 3.3 kDa or 10 kDa showed rapid deep penetration into the tumor tissue (>35 um from the vascular surface of the tumor). However, 40 kDa, 70 kDa or 2 mDa sized dextrans penetrated much less than the 3.3 kDa or 10 kDa dextran. The 70 kDa dextran reached only about 15 um from the vascular surface of the tumor. Conjugates of the present invention have a molecule weight comparable to the 3.3 kDa and 10 kDa dextrans, while antibody drug conjugates have a molecule weight at least as big as the 70 kDa dextran. Therefore, conjugates of the present invention may penetrate deep and rapidly into the core/center of the solid tumor.

In one embodiment, conjugates of the present invention reach at least about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 75 μm, about 100 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm, about 1000 μm, about 1100 μm, about 1200 μm, about 1300 μm, about 1400 μm or about 1500 μm into the solid tμmor from the vascular surface of the tμmor. Zero distance is defined as the vascular surface of the tμmor, and every distance greater than zero is defined as the distance measured in three dimensions to the nearest vascular surface.

In another embodiment, conjugates of the present invention penetrate to the core of the tumor. “Core” of the tumor, as used herein, refers to the central area of the tumor. The distance from any part of the core area of the tumor to the vascular surface of the tumor is between about 30% to about 50% of the length or width of the tumor. The distance from any part of the core area of the tumor to the center point of the tumor is less than about 20% of the length or width of the tumor. The core area of the tumor is roughly the center ⅓ of the tumor.

In another embodiment, conjugates of the present invention conjugates of the present invention penetrate to the middle of the solid tumor. “Middle” of the tumor, as sued herein, refers to the middle area of the tumor. The distance from any part of the middle area of the tumor to the vascular surface of the tumor is between about 15% and about 30% of the length or the width of the tumor. The distance from any part of the middle area of the tumor to the center point of the tumor is between about 20% to about 35% of the length or width of the tumor. The middle area of the tumor is roughly between the center ⅓ of the tumor and the outer ⅓ of the tumor.

In some embodiments, the subject may be otherwise free of indications for treatment with the conjugates or particles. In some embodiments, methods include use of cancer cells, including but not limited to mammalian cancer cells. In some instances, the mammalian cancer cells are human cancer cells.

In some embodiments, the conjugates or particles of the present teachings have been found to inhibit cancer and/or tumor growth. They may also reduce, including cell proliferation, invasiveness, and/or metastasis, thereby rendering them useful for the treatment of a cancer.

In some embodiments, the conjugates or particles of the present teachings may be used to prevent the growth of a tumor or cancer, and/or to prevent the metastasis of a tumor or cancer. In some embodiments, compositions of the present teachings may be used to shrink or destroy a cancer.

In some embodiments, the conjugates or particles provided herein are useful for inhibiting proliferation of a cancer cell. In some embodiments, the conjugates or particles provided herein are useful for inhibiting cellular proliferation, e.g., inhibiting the rate of cellular proliferation, preventing cellular proliferation, and/or inducing cell death. In general, the conjugates or particles as described herein can inhibit cellular proliferation of a cancer cell or both inhibiting proliferation and/or inducing cell death of a cancer cell. In some embodiments, cell proliferation is reduced by at least about 25%, about 50%, about 75%, or about 90% after treatment with conjugates or particles of the present invention compared with cells with no treatment. In some embodiments, cell cycle arrest marker phospho histone H3 (PH3 or PHH3) is increased by at least about 50%, about 75%, about 100%, about 200%, about 400% or about 600% after treatment with conjugates or particles of the present invention compared with cells with no treatment. In some embodiments, cell apoptosis marker cleaved caspase-3 (CC3) is increased by at least 50%, about 75%, about 100%, about 200%, about 400% or about 600% after treatment with conjugates or particles of the present invention compared with cells with no treatment.

Furthermore, in some embodiments, conjugates or particles of the present invention are effective for inhibiting tumor growth, whether measured as a net value of size (weight, surface area or volume) or as a rate over time, in multiple types of tumors.

In some embodiments the size of a tumor is reduced by about 60% or more after treatment with conjugates or particles of the present invention. In some embodiments, the size of a tumor is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, by a measure of weight, and/or area and/or volume.

In one embodiment, the conjugates or particles as described herein or formulations containing the conjugates or particles as described herein are used to treat small cell lung cancer. About 12%-15% of patients having lung cancer have small cell lung cancer. Survival in metastatic small cell lung cancer is poor. Survival rate is below 5% five years after diagnosis. US incidence of small cell lung cancer is about 26K-30K.

In some embodiments, the conjugates or particles as described herein or formulations containing the conjugates or particles as described herein are used to treat patients with tumors that express or over-express the HSP90.

A feature of conjugates or particles of the present invention is relatively low toxicity to an organism while maintaining efficacy at inhibiting, e.g. slowing or stopping tumor growth. As used herein, “toxicity” refers to the capacity of a substance or composition to be harmful or poisonous to a cell, tissue organism or cellular environment. Low toxicity refers to a reduced capacity of a substance or composition to be harmful or poisonous to a cell, tissue organism or cellular environment. Such reduced or low toxicity may be relative to a standard measure, relative to a treatment or relative to the absence of a treatment. For example, conjugates or particles of the present invention may have lower toxicity than the active agent moiety Z administered alone. For conjugates comprising DM1, their toxicity is lower than DM1 administered alone.

Toxicity may further be measured relative to a subject's weight loss where weight loss over 15%, over 20% or over 30% of the body weight is indicative of toxicity. Other metrics of toxicity may also be measured such as patient presentation metrics including lethargy and general malaiase. Neutropenia, thrombopenia, white blood cell (WBC) count, complete blood cell (CBC) count may also be metrics of toxicity. Pharmacologic indicators of toxicity include elevated aminotransferases (AST/ALT) levels, neurotoxicity, kidney damage, GI damage and the like. In one embodiment, conjugates or particles of the present invention do not cause a significant change of a subject's body weight. The body weight loss of a subject is less about 30%, about 20%, about 15%, about 10%, or about 5% after treatment with conjugates or particles of the present invention. In another embodiment, conjugates or particles of the present invention do not cause a significant increase of a subject's AST/ALT levels. The AST or ALT level of a subject is increased by less than about 30%, about 20%, about 15%, about 10%, or about 5% after treatment with conjugates or particles of the present invention. In yet another embodiment, conjugates or particles of the present invention do not cause a significant change of a subject's CBC or WBC count after treatment with conjugates or particles of the present invention. The CBC or WBC level of a subject is decreased by less than about 30%, about 20%, about 15%, about 10%, or about 5% after treatment with conjugates or particles of the present invention.

In some embodiments, conjugates of the present disclosure mask the activity of its payload. Each conjugate blocks the target activity of the respective payload until the linker moiety gets cleaved in the tumor and releases active payload. Through the HSP90 platform, toxicity is mitigated by masking the payload's active site until it can be delivered to the tumor. For example, in one embodiment, the payload inhibits PI3K activity. A conjugate comprising the payload has less PI3K inhibiting activity compared with the payload alone.

In some embodiments, conjugates of the present disclosure do not cause a significant increase of the blood glucose level of the subject receiving the treatment. As used herein, ‘significant increase’ means an increase of over 25% compared to the level before treatment. In some embodiments, the blood glucose level of the subject receiving the treatment of conjugates of the present disclosure increases less than about 200%, about 150%, about 100%, about 75%, about 50%, about 40%, about 30%, about 20%, or about 10% compare to the level before the treatment.

In some embodiments, conjugates or particles of the present invention are combined with at least one additional active agent. The active agent may be any suitable drug. The conjugates and the at least one additional active agent may be administered simultaneously, sequentially, or at any order. The conjugates and the at least one additional active agent may be administered at different dosages, with different dosing frequencies, or via different routes, whichever is suitable. The additional active agent may be selected from any active agent described herein such as a drug for treating cancer. It may also be a cancer symptom relief drug. Non-limiting examples of symptom relief drugs include: octreotide or lanreotide; interferon, cypoheptadine or any other antihistamines. In some embodiments, conjugates or particles of the present invention do not have drug-drug interference with the additional active agent. In one embodiment, conjugates or particles of the present invention do not inhibit cytochrome P450 (CYP) isozymes. CYP isozymes may include CYP3A4 Midazolam, CYP3A4 Testosterone, CYP2C9, CYP2D6, CYP1A2, CYP2C8, CYP2B6, and CYP2C19. The additional active agent may be administered concomitantly with conjugates or particles of the present invention.

In another example, conjugates or particles of the present invention may be combined with a moderate dose of chemotherapy agents such as mitomycin C, vinblastine and cisplatin (see Ellis et al., Br J Cancer, vol. 71(2): 366-370 (1995), the contents of which are incorporated herein by reference in their entirety).

In yet another example, a patient may first receive a pharmaceutically effective dose of an unconjugated active agent, followed by a pharmaceutically effective dose of a conjugate comprising the same active agent.

The conjugates or particles as described herein or formulations containing the conjugates or particles as described herein can be used for the selective tissue delivery of a therapeutic, prophylactic, or diagnostic agent to an individual or patient in need thereof. For example, DM1 conjugates or particles of the present invention are used to deliver DM1 to selective tissues. These tissues may be tumor tissues. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic.

In various embodiments, a conjugate contained within a particle is released in a controlled manner. The release can be in vitro or in vivo. For example, particles can be subject to a release test under certain conditions, including those specified in the U.S. Pharmacopeia and variations thereof.

In various embodiments, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20% of the conjugate contained within particles is released in the first hour after the particles are exposed to the conditions of a release test. In some embodiments, less that about 90%, less than about 80%, less than about 70%, less than about 60%, or less than about 50% of the conjugate contained within particles is released in the first hour after the particles are exposed to the conditions of a release test. In certain embodiments, less than about 50% of the conjugate contained within particles is released in the first hour after the particles are exposed to the conditions of a release test.

With respect to a conjugate being released in vivo, for instance, the conjugate contained within a particle administered to a subject may be protected from a subject's body, and the body may also be isolated from the conjugate until the conjugate is released from the particle.

Thus, in some embodiments, the conjugate may be substantially contained within the particle until the particle is delivered into the body of a subject. For example, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the total conjugate is released from the particle prior to the particle being delivered into the body, for example, a treatment site, of a subject. In some embodiments, the conjugate may be released over an extended period of time or by bursts (e.g., amounts of the conjugate are released in a short period of time, followed by a periods of time where substantially no conjugate is released). For example, the conjugate can be released over 6 hours, 12 hours, 24 hours, or 48 hours. In certain embodiments, the conjugate is released over one week or one month.

V. Kits and Devices

The invention provides a variety of kits and devices for conveniently and/or effectively carrying out methods of the present invention. Typically kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments.

In one embodiment, the present invention provides kits for inhibiting tumor cell growth in vitro or in vivo, comprising a conjugate and/or particle of the present invention or a combination of conjugates and/or particles of the present invention, optionally in combination with any other active agents.

The kit may further comprise packaging and instructions and/or a delivery agent to form a formulation composition. The delivery agent may comprise a saline, a buffered solution, or any delivery agent disclosed herein. The amount of each component may be varied to enable consistent, reproducible higher concentration saline or simple buffer formulations. The components may also be varied in order to increase the stability of the conjugates and/or particles in the buffer solution over a period of time and/or under a variety of conditions.

The present invention provides for devices which may incorporate conjugates and/or particles of the present invention. These devices contain in a stable formulation available to be immediately delivered to a subject in need thereof, such as a human patient. In some embodiments, the subject has cancer.

Non-limiting examples of the devices include a pump, a catheter, a needle, a transdermal patch, a pressurized olfactory delivery device, iontophoresis devices, multi-layered microfluidic devices. The devices may be employed to deliver conjugates and/or particles of the present invention according to single, multi- or split-dosing regiments. The devices may be employed to deliver conjugates and/or particles of the present invention across biological tissue, intradermal, subcutaneously, or intramuscularly.

The term “compound”, as used herein, is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. In the present application, compound is used interechangably with conjugate. Therefore, conjugate, as used herein, is also meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.

Compounds of the present disclosure also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Examples prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

Compounds of the present disclosure also include all of the isotopes of the atoms occurring in the intermediate or final compounds. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium.

The compounds and salts of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.

The terms “treating” or “preventing”, as used herein, can include preventing a disease, disorder or condition from occurring in an animal that may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having the disease, disorder or condition; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

A “target”, as used herein, shall mean a site to which targeted constructs bind. A target may be either in vivo or in vitro. In certain embodiments, a target may be cancer cells found in leukemias or tumors (e.g., tumors of the brain, lung (small cell and non-small cell), ovary, prostate, breast and colon as well as other carcinomas and sarcomas). In still other embodiments, a target may refer to a molecular structure to which a targeting moiety or ligand binds, such as a hapten, epitope, receptor, dsDNA fragment, carbohydrate or enzyme. A target may be a type of tissue, e.g., neuronal tissue, intestinal tissue, pancreatic tissue, liver, kidney, prostate, ovary, lung, bone marrow, or breast tissue.

The “target cells” that may serve as the target for the method or conjugates or particles, are generally animal cells, e.g., mammalian cells. The present method may be used to modify cellular function of living cells in vitro, i.e., in cell culture, or in vivo, in which the cells form part of or otherwise exist in animal tissue. Thus, the target cells may include, for example, the blood, lymph tissue, cells lining the alimentary canal, such as the oral and pharyngeal mucosa, cells forming the villi of the small intestine, cells lining the large intestine, cells lining the respiratory system (nasal passages/lungs) of an animal (which may be contacted by inhalation of the subject invention), dermal/epidermal cells, cells of the vagina and rectum, cells of internal organs including cells of the placenta and the so-called blood/brain barrier, etc. In general, a target cell expresses at least one type of HSP90. In some embodiments, a target cell can be a cell that expresses an HSP90 and is targeted by a conjugate described herein, and is near a cell that is affected by release of the active agent of the conjugate. For example, a blood vessel expressing an HSP90 that is in proximity to a tumor may be the target, while the active agent released at the site will affect the tumor.

The term “therapeutic effect” is art-recognized and refers to a local or systemic effect in animals, particularly mammals, and more particularly humans caused by a pharmacologically active substance. The term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease, disorder or condition in the enhancement of desirable physical or mental development and conditions in an animal, e.g., a human.

The term “modulation” is art-recognized and refers to up regulation (i.e., activation or stimulation), down regulation (i.e., inhibition or suppression) of a response, or the two in combination or apart. The modulation is generally compared to a baseline or reference that can be internal or external to the treated entity.

“Parenteral administration”, as used herein, means administration by any method other than through the digestive tract (enteral) or non-invasive topical routes. For example, parenteral administration may include administration to a patient intravenously, intradermally, intraperitoneally, intrapleurally, intratracheally, intraossiously, intracerebrally, intrathecally, intramuscularly, subcutaneously, subjunctivally, by injection, and by infusion.

“Topical administration”, as used herein, means the non-invasive administration to the skin, orifices, or mucosa. Topical administration can be delivered locally, i.e., the therapeutic can provide a local effect in the region of delivery without systemic exposure or with minimal systemic exposure. Some topical formulations can provide a systemic effect, e.g., via adsorption into the blood stream of the individual. Topical administration can include, but is not limited to, cutaneous and transdermal administration, buccal administration, intranasal administration, intravaginal administration, intravesical administration, ophthalmic administration, and rectal administration.

“Enteral administration”, as used herein, means administration via absorption through the gastrointestinal tract. Enteral administration can include oral and sublingual administration, gastric administration, or rectal administration.

“Pulmonary administration”, as used herein, means administration into the lungs by inhalation or endotracheal administration. As used herein, the term “inhalation” refers to intake of air to the alveoli. The intake of air can occur through the mouth or nose.

The terms “sufficient” and “effective”, as used interchangeably herein, refer to an amount (e.g., mass, volume, dosage, concentration, and/or time period) needed to achieve one or more desired result(s). A “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement or prevention of at least one symptom or a particular condition or disorder, to effect a measurable enhancement of life expectancy, or to generally improve patient quality of life. The therapeutically effective amount is thus dependent upon the specific biologically active molecule and the specific condition or disorder to be treated. Therapeutically effective amounts of many active agents, such as antibodies, are known in the art. The therapeutically effective amounts of compounds and compositions described herein, e.g., for treating specific disorders may be determined by techniques that are well within the craft of a skilled artisan, such as a physician.

The terms “bioactive agent” and “active agent”, as used interchangeably herein, include, without limitation, physiologically or pharmacologically active substances that act locally or systemically in the body. A bioactive agent is a substance used for the treatment (e.g., therapeutic agent), prevention (e.g., prophylactic agent), diagnosis (e.g., diagnostic agent), cure or mitigation of disease or illness, a substance which affects the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.

The term “prodrug” refers to an agent, including a small organic molecule, peptide, nucleic acid or protein, that is converted into a biologically active form in vitro and/or in vivo. Prodrugs can be useful because, in some situations, they may be easier to administer than the parent compound (the active compound). For example, a prodrug may be bioavailable by oral administration whereas the parent compound is not. The prodrug may also have improved solubility in pharmaceutical compositions compared to the parent drug. A prodrug may also be less toxic than the parent. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. Harper, N.J. (1962) Drug Latentiation in Jucker, ed.Progress in Drug Research,4:221-294; Morozowich et al. (1977) Application of Physical Organic Principles to Prodrug Design in E. B. Roche ed.Design of Biopharmaceutical Properties through Prodrugs and Analogs, APhA; Acad. Pharm. Sci.; E. B. Roche, ed. (1977)Bioreversible Carriers in Drug in Drug Design, Theory and Application, APhA; H. Bundgaard, ed. (1985)Design of Prodrugs, Elsevier; Wang et al. (1999) Prodrug approaches to the improved delivery of peptide drug, Curr. Pharm. Design. 5(4):265-287; Pauletti et al. (1997) Improvement in peptide bioavailability: Peptidomimetics and Prodrug Strategies, Adv. Drug. Delivery Rev. 27:235-256; Mizen et al. (1998). The Use of Esters as Prodrugs for Oral Delivery of β-Lactam antibiotics, Pharm. Biotech. 11:345-365; Gaignault et al. (1996) Designing Prodrugs and Bioprecursors I. Carrier Prodrugs, Pract. Med. Chem. 671-696; M. Asgharnejad (2000). Improving Oral Drug Transport Via Prodrugs, in G. L. Amidon, P. I. Lee and E. M. Topp, Eds.,Transport Processes in Pharmaceutical Systems, Marcell Dekker, p. 185-218; Balant et al. (1990) Prodrugs for the improvement of drug absorption via different routes of administration,Eur. J. Drug Metab. Pharmacokinet.,15(2): 143-53; Balimane and Sinko (1999). Involvement of multiple transporters in the oral absorption of nucleoside analogues,Adv. Drug Delivery Rev.,39(1-3):183-209; Browne (1997). Fosphenytoin (Cerebyx),Clin. Neuropharmacol.20(1): 1-12; Bundgaard (1979). Bioreversible derivatization of drugs—principle and applicability to improve the therapeutic effects of drugs,Arch. Pharm. Chemi.86(1): 1-39; H. Bundgaard, ed. (1985)Design of Prodrugs, New York: Elsevier; Fleisher et al. (1996) Improved oral drug delivery: solubility limitations overcome by the use of prodrugs,Adv. Drug Delivery Rev.19(2): 115-130; Fleisher et al. (1985) Design of prodrugs for improved gastrointestinal absorption by intestinal enzyme targeting,Methods Enzymol.112: 360-81; Farquhar D, et al. (1983) Biologically Reversible Phosphate-Protective Groups,J. Pharm. Sci.,72(3): 324-325; Han, H. K. et al. (2000) Targeted prodrug design to optimize drug delivery,AAPS PharmSci.,2(1): E6; Sadzuka Y. (2000) Effective prodrug liposome and conversion to active metabolite,Curr. Drug Metab.,1(1):31-48; D. M. Lambert (2000) Rationale and applications of lipids as prodrug carriers,Eur. J. Pharm. Sci.,11 Suppl. 2:S15-27; Wang, W. et al. (1999) Prodrug approaches to the improved delivery of peptide drugs.Curr. Pharm. Des.,5(4):265-87.

The term “biocompatible”, as used herein, refers to a material that along with any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause any significant adverse effects to the recipient. Generally speaking, biocompatible materials are materials which do not elicit a significant inflammatory or immune response when administered to a patient.

The term “biodegradable” as used herein, generally refers to a material that will degrade or erode under physiologic conditions to smaller units or chemical species that are capable of being metabolized, eliminated, or excreted by the subject. The degradation time is a function of composition and morphology. Degradation times can be from hours to weeks.

The term “pharmaceutically acceptable”, as used herein, refers to compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio, in accordance with the guidelines of agencies such as the U.S. Food and Drug Administration. A “pharmaceutically acceptable carrier”, as used herein, refers to all components of a pharmaceutical formulation that facilitate the delivery of the composition in vivo. Pharmaceutically acceptable carriers include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.

The term “molecular weight”, as used herein, generally refers to the mass or average mass of a material. If a polymer or oligomer, the molecular weight can refer to the relative average chain length or relative chain mass of the bulk polymer. In practice, the molecular weight of polymers and oligomers can be estimated or characterized in various ways including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as the weight-average molecular weight (Mw) as opposed to the number-average molecular weight (Mn). Capillary viscometry provides estimates of molecular weight as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.

The term “small molecule”, as used herein, generally refers to an organic molecule that is less than 2000 g/mol in molecular weight, less than 1500 g/mol, less than 1000 g/mol, less than 800 g/mol, or less than 500 g/mol. Small molecules are non-polymeric and/or non-oligomeric.

The term “hydrophilic”, as used herein, refers to substances that have strongly polar groups that readily interact with water.

The term “hydrophobic”, as used herein, refers to substances that lack an affinity for water; tending to repel and not absorb water as well as not dissolve in or mix with water.

The term “lipophilic”, as used herein, refers to compounds having an affinity for lipids.

The term “amphiphilic”, as used herein, refers to a molecule combining hydrophilic and lipophilic (hydrophobic) properties. “Amphiphilic material” as used herein refers to a material containing a hydrophobic or more hydrophobic oligomer or polymer (e.g., biodegradable oligomer or polymer) and a hydrophilic or more hydrophilic oligomer or polymer.

The term “targeting moiety”, as used herein, refers to a moiety that binds to or localizes to a specific locale. The moiety may be, for example, a protein, nucleic acid, nucleic acid analog, carbohydrate, or small molecule. The locale may be a tissue, a particular cell type, or a subcellular compartment. In some embodiments, a targeting moiety can specifically bind to a selected molecule.

The term “reactive coupling group”, as used herein, refers to any chemical functional group capable of reacting with a second functional group to form a covalent bond. The selection of reactive coupling groups is within the ability of those in the art. Examples of reactive coupling groups can include primary amines (—NH2) and amine-reactive linking groups such as isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters. Most of these conjugate to amines by either acylation or alkylation. Examples of reactive coupling groups can include aldehydes (˜COH) and aldehyde reactive linking groups such as hydrazides, alkoxyamines, and primary amines. Examples of reactive coupling groups can include thiol groups (˜SH) and sulfhydryl reactive groups such as maleimides, haloacetyls, and pyridyl disulfides. Examples of reactive coupling groups can include photoreactive coupling groups such as aryl azides or diazirines. The coupling reaction may include the use of a catalyst, heat, pH buffers, light, or a combination thereof.

The term “protective group”, as used herein, refers to a functional group that can be added to and/or substituted for another desired functional group to protect the desired functional group from certain reaction conditions and selectively removed and/or replaced to deprotect or expose the desired functional group. Protective groups are known to the skilled artisan. Suitable protective groups may include those described in Greene and Wuts, Protective Groups in Organic Synthesis, (1991). Acid sensitive protective groups include dimethoxytrityl (DMT), tert-butylcarbamate (tBoc) and trifluoroacetyl (tFA). Base sensitive protective groups include 9-fluorenylmethoxycarbonyl (Fmoc), isobutyrl (iBu), benzoyl (Bz) and phenoxyacetyl (pac). Other protective groups include acetamidomethyl, acetyl, tert-amyloxycarbonyl, benzyl, benzyloxycarbonyl, 2-(4-biphεnylyl)-2-propy!oxycarbonyl, 2-bromobenzyloxycarbonyl, tert-butyl7tert-butyloxycarbonyl, 1-carbobenzoxamido-2,2.2-trifluoroethyl, 2,6-dichlorobenzyl, 2-(3,5-dimethoxyphenyl)-2-propyloxycarbonyl, 2,4-dinitrophenyl, dithiasuccinyl, formyl, 4-methoxybenzenesulfonyl, 4-methoxybenzyl, 4-methylbenzyl, o-nitrophenylsulfenyl, 2-phenyl-2-propyloxycarbonyl, α-2,4,5-tetramethylbenzyloxycarbonyl, p-toluenesulfonyl, xanthenyl, benzyl ester, N-hydroxysuccinimide ester, p-nitrobenzyl ester, p-nitrophenyl ester, phenyl ester, p-nitrocarbonate, p-nitrobenzylcarbonate, trimethylsilyl and pentachlorophenyl ester.

The term “activated ester”, as used herein, refers to alkyl esters of carboxylic acids where the alkyl is a good leaving group rendering the carbonyl susceptible to nucleophilic attack by molecules bearing amino groups. Activated esters are therefore susceptible to aminolysis and react with amines to form amides. Activated esters contain a carboxylic acid ester group —CO2R where R is the leaving group.

In some embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30for straight chains, C3-C30for branched chains), 20 or fewer, 12 or fewer, or 7 or fewer. Likewise, in some embodiments cycloalkyls have from 3-10 carbon atoms in their ring structure, e.g., have 5, 6 or 7 carbons in the ring structure. The term “alkyl” (or “lower alkyl”) as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a hosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.

It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include halogen, hydroxy, nitro, thiols, amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), —CF3, —CN and the like. Cycloalkyls can be substituted in the same manner.

The term “heteroalkyl”, as used herein, refers to straight or branched chain, or cyclic carbon-containing radicals, or combinations thereof, containing at least one heteroatom. Suitable heteroatoms include, but are not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. Heteroalkyls can be substituted as defined above for alkyl groups.

The term “alkylthio” refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In some embodiments, the “alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl, and —S-alkynyl. Representative alkylthio groups include methylthio, and ethylthio. The term “alkylthio” also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups. “Arylthio” refers to aryl or heteroaryl groups. Alkylthio groups can be substituted as defined above for alkyl groups.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, and tert-butoxy. An “ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of —O-alkyl, —O-alkenyl, and —O-alkynyl. Aroxy can be represented by —O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined below. The alkoxy and aroxy groups can be substituted as described above for alkyl.

The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula:

wherein R9, R10, and R′10each independently represent a hydrogen, an alkyl, an alkenyl, —(CH2)m—R8or R9and R10taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R8represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In some embodiments, only one of R9or R10can be a carbonyl, e.g., R9, R10and the nitrogen together do not form an imide. In still other embodiments, the term “amine” does not encompass amides, e.g., wherein one of R9and R10represents a carbonyl. In additional embodiments, R9and R10(and optionally R′10) each independently represent a hydrogen, an alkyl or cycloalkly, an alkenyl or cycloalkenyl, or alkynyl. Thus, the term “alkylamine” as used herein means an amine group, as defined above, having a substituted (as described above for alkyl) or unsubstituted alkyl attached thereto, i.e., at least one of R9and R10is an alkyl group.

The term “amido” is art-recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula:

The term “aralkyl”, as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).

The term “carbocycle”, as used herein, refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.

The term “carbonyl” is art-recognized and includes such moieties as can be represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R11represents a hydrogen, an alkyl, a cycloalkyl, an alkenyl, a cycloalkenyl, or an alkynyl, R′11represents a hydrogen, an alkyl, a cycloalkyl, an alkenyl, a cycloalkenyl, or an alkynyl. Where X is an oxygen and R11or R′11is not hydrogen, the formula represents an “ester”. Where X is an oxygen and R11is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when R11is a hydrogen, the formula represents a “carboxylic acid”. Where X is an oxygen and R′11is hydrogen, the formula represents a “formate”. In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a “thiocarbonyl” group. Where X is a sulfur and R11or R′11is not hydrogen, the formula represents a “thioester.” Where X is a sulfur and R11is hydrogen, the formula represents a “thiocarboxylic acid.” Where X is a sulfur and R′11is hydrogen, the formula represents a “thioformate.” On the other hand, where X is a bond, and R11is not hydrogen, the above formula represents a “ketone” group. Where X is a bond, and R11is hydrogen, the above formula represents an “aldehyde” group.

The term “monoester” as used herein refers to an analog of a dicarboxylic acid wherein one of the carboxylic acids is functionalized as an ester and the other carboxylic acid is a free carboxylic acid or salt of a carboxylic acid. Examples of monoesters include, but are not limited to, to monoesters of succinic acid, glutaric acid, adipic acid, suberic acid, sebacic acid, azelaic acid, oxalic and maleic acid.

The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Examples of heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium. Other useful heteroatoms include silicon and arsenic.

As used herein, the term “nitro” means —NO2; the term “halogen” designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term “hydroxyl” means —OH; and the term “sulfonyl” means —SO2-.

Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is understood that “substitution” or “substituted” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation, for example, by rearrangement, cyclization, or elimination.

The term “copolymer” as used herein, generally refers to a single polymeric material that is comprised of two or more different monomers. The copolymer can be of any form, for example, random, block, or graft. The copolymers can have any end-group, including capped or acid end groups.

The term “mean particle size”, as used herein, generally refers to the statistical mean particle size (diameter) of the particles in the composition. The diameter of an essentially spherical particle may be referred to as the physical or hydrodynamic diameter. The diameter of a non-spherical particle may refer to the hydrodynamic diameter. As used herein, the diameter of a non-spherical particle may refer to the largest linear distance between two points on the surface of the particle. Mean particle size can be measured using methods known in the art such as dynamic light scattering. Two populations can be said to have a “substantially equivalent mean particle size” when the statistical mean particle size of the first population of particles is within 20% of the statistical mean particle size of the second population of particles; for example, within 15%, or within 10%.

The terms “monodisperse” and “homogeneous size distribution”, as used interchangeably herein, describe a population of particles, microparticles, or nanoparticles all having the same or nearly the same size. As used herein, a monodisperse distribution refers to particle distributions in which 90% of the distribution lies within 5% of the mean particle size.

The terms “polypeptide,” “peptide” and “protein” generally refer to a polymer of amino acid residues. As used herein, the term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of corresponding naturally-occurring amino acids or are unnatural amino acids. The term “protein”, as generally used herein, refers to a polymer of amino acids linked to each other by peptide bonds to form a polypeptide for which the chain length is sufficient to produce tertiary and/or quaternary structure. The term “protein” excludes small peptides by definition, the small peptides lacking the requisite higher-order structure necessary to be considered a protein.

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably to refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form. These terms are not to be construed as limiting with respect to the length of a polymer. The terms can encompass known analogs of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones). In general and unless otherwise specified, an analog of a particular nucleotide has the same base-pairing specificity; i.e., an analog of A will base-pair with T. The term “nucleic acid” is a term of art that refers to a string of at least two base-sugar-phosphate monomeric units. Nucleotides are the monomeric units of nucleic acid polymers. The term includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in the form of a messenger RNA, antisense, plasmid DNA, parts of a plasmid DNA or genetic material derived from a virus. An antisense nucleic acid is a polynucleotide that interferes with the expression of a DNA and/or RNA sequence. The term nucleic acids refers to a string of at least two base-sugar-phosphate combinations. Natural nucleic acids have a phosphate backbone. Artificial nucleic acids may contain other types of backbones, but contain the same bases as natural nucleic acids. The term also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids.

A “functional fragment” of a protein, polypeptide or nucleic acid is a protein, polypeptide or nucleic acid whose sequence is not identical to the full-length protein, polypeptide or nucleic acid, yet retains at least one function as the full-length protein, polypeptide or nucleic acid. A functional fragment can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well-known in the art. Similarly, methods for determining protein function are well-known. For example, the DNA binding function of a polypeptide can be determined, for example, by filter-binding, electrophoretic mobility shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays or complementation, e.g., genetic or biochemical. See, for example, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No. 5,585,245 and PCT WO 98/44350.

The term “pharmaceutically acceptable salt(s)” refers to salts of acidic or basic groups that may be present in compounds used in the present compositions. Compounds included in the present compositions that are basic in nature are capable of forming a variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to sulfate, citrate, malate, acetate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds included in the present compositions that include an amino moiety may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above. Compounds included in the present compositions, that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.

The term “bioavailable” is art-recognized and refers to a form of the subject invention that allows for it, or a portion of the amount administered, to be absorbed by, incorporated to, or otherwise physiologically available to a subject or patient to whom it is administered.

It will be appreciated that the following examples are intended to illustrate but not to limit the present invention. Various other examples and modifications of the foregoing description and examples will be apparent to a person skilled in the art after reading the disclosure without departing from the spirit and scope of the invention, and it is intended that all such examples or modifications be included within the scope of the appended claims. All publications and patents referenced herein are hereby incorporated by reference in their entirety.

EXAMPLES

Example 1: Synthesis of the Conjugates

The conjugates of the invention may be prepared using any convenient methodology. In a rational approach, the conjugates are constructed from their individual components, targeting moiety, in some cases a linker, and active agent moiety. The components can be covalently bonded to one another through functional groups, as is known in the art, where such functional groups may be present on the components or introduced onto the components using one or more steps, e.g., oxidation reactions, reduction reactions, cleavage reactions and the like. Functional groups that may be used in covalently bonding the components together to produce the pharmaceutical conjugate include: hydroxy, sulfhydryl, amino, and the like. The particular portion of the different components that are modified to provide for covalent linkage will be chosen so as not to substantially adversely interfere with that components desired binding activity, e.g., for the active agent moiety, a region that does not affect the target binding activity will be modified, such that a sufficient amount of the desired drug activity is preserved. Where necessary and/or desired, certain moieties on the components may be protected using blocking groups, as is known in the art, see, e.g., Green & Wuts, Protective Groups in Organic Synthesis (John Wiley & Sons) (1991).

Alternatively, the conjugate can be produced using known combinatorial methods to produce large libraries of potential conjugates which may then be screened for identification of a bifunctional, molecule with the pharmacokinetic profile. Alternatively, the conjugates may be produced using medicinal chemistry and known structure-activity relationships for the targeting moiety and the active agent moiety. In particular, this approach will provide insight as to where to join the two moieties to the linker.

Synthesis of Compound 3

To a solution of 2,4-dihydroxy-5-isopropyl-benzenecarbodithioic acid (3.20 g, 14.0 mmol) in DMF (50 mL) was added sodium 2-chloroacetate (2.61 g, 22.4 mmol) and sodium carbonate (4.45 g, 42.0 mmol), and the solution degassed by bubbling nitrogen through the solution. The mixture was stirred at room temperature for 3 h, then a solution of tert-butyl 4-(4-aminobenzyl)piperazine-1-carboxylate (4.08 g, 14.0 mmol) in DMF (10 mL) was added. The resulting mixture was stirred at 80° C. for 3 h. The reaction mixture was poured into ice water, and extracted with ethyl acetate (3×100 ml). The combined organic layers were washed with brine, dried with sodium sulfate, and the solvent removed in vacuo to give 3A (5.20 g, 10.7 mmol, 76% yield).

To a solution of 3A (5.20 g, 10.7 mmol) in THF (80 mL) was added carbonyldiimidazole (2.00 g, 13.9 mmol). The reaction was stirred at room temperature for 2 h, then poured into a solution of saturated ammonium chloride (200 ml), and extracted with ethyl acetate (3×50 mL). The combined organic layers were washed with brine (50 mL), dried with sodium sulfate, and the solvent removed in vacuo to give 3B (4.30 g, 8.37 mmol, 78% yield) which was used without purification in the next step. LCMS M/Z=512.3 (M+1).

To a solution of 3B (4.30 g, 8.37 mmol) in ethanol (50 mL) was added hydrazine hydrate (1.26 g, 25.1 mmol). The mixture was stirred at room temperature for 16 h, and the solvent removed in vacuo. Ethanol (20 mL) was added to the remaining residue, the resulting solid was filtered off, washed with ethanol (10 mL), and dried to give 3C (2.86 g, 5.61 mmol, 67% yield). LCMS M/Z=510.2 (M+1).

To a solution of 3C (2.86 g, 5.61 mmol) in methanol (20 mL) was added a solution of 4N HCl in MeOH (5 mL). The solution was stirred at room temperature for 16 h, the solvent removed under vacuum, and the resulting solid washed with methanol (2×5 mL) and dried to give 3D hydrochloride salt (1.90 g, 4.26 mmol, 75% yield). LCMS M/Z=410.1 (M+1).

Synthesis of Compound 17

A vial was charged with 17A (10.0 mg, 19.6 umol) and dissolved in DMF (1 mL). 17B (8.2 mg, 23.5 umol) was added and solution was stirred at room temperature for 30 mins, then DIPEA (10.2 μL, 3.00 equiv) was added and stirred for 2 h.

Synthesis of Compound 17D

4-mercaptobenzoic acid (10.0 g, 64.9 mmol) was added to a solution of lithium aluminum hydride (3.69 g, 97.3 mmol) in THF (500 ml) at 0° C. The mixture was then warmed to room temperature and stirred for 16 h, then quenched with 2M HCl to pH=2. The aqueous solution was extracted with diethyl ether (3×500 mL), the combined organic layers dried with sodium sulfate, and the solvent removed in vacuo to give 17D, which was used without further purification (6.70 g, 43% purity, 20.6 mmol, 31% yield).

To a solution of 17D (6.00 g, 43% purity, 18.4 mmol) in methanol (80 mL) was added 2,2′-dithiodipyridine (11.3 g, 51.4 mmol). The reaction was stirred at room temperature for 3 h, then the solvent removed in vacuo. The resulting mixture was purified by silica gel chromatography (8:1 to 6:1 petroleum ether:ethyl acetate) to give 17E (2.00 g, 8.02 mmol, 43% yield). LCMS M/Z=250 (M+1).

Synthesis of Compound 9

17E (1.00 g, 4.01 mmol) was dissolved in dichloromethane (10 mL) and triethylamine (410 mg, 4.00 mmol). This solution was added dropwise to a solution of triphosgene (476 mg, 1.60 mmol) in dichloromethane (5 mL) at 0° C. This solution was then warmed to room temperature, and stirred for 3 h. A solution of HOBt (542 mg, 4.01 mmol) in dichloromethane (10 mL) and triethylamine (410 mg, 4.00 mmol) was added slowly, and this mixture stirred at room temperature for 16 h. The reaction mixture was washed with 2N HCl (20 ml), water (3×20 mL), and brine (20 mL), the organic layer dried with sodium sulfate, and concentrated in vacuo to give 17B (850 mg, 2.07 mmol, 51% yield). LCMS M/Z=411.0 (M+1).

Synthesis of Compound 18

CDI (9.2 mg, 57 umol) was charged in a vial and dissolved in DMF (1 mL). 18A (30.0 mg, 57 umol) and DIPEA (7.4 mg, 57 umol, 10 uL) were added, and the reaction stirred for 2 hours. BT-1132 (26.3 mg, 57 umol) was added as a solid to the vial, and the reaction was stirred overnight at room temperature. The reaction was diluted with ethyl acetate (30 mL) and the organic phase was washed with 1M HCl in water (2×20 mL) and dried over anhydrous sodium sulfate. The solvent was removed under vacuum and the residue was resuspended in DMF and loaded to reverse phase column (5-60% acetonitrile/water-0.2% AcOH). Pure fractions were pooled, frozen and lyophilized. 18 (15.1 mg, 24% yield) was isolated as an off-white solid.

Synthesis of Compound 19

Synthesis of Compound 20

Synthesis of Compound 21

Synthesis of Compound 22

Synthesis of Compound 22A

Synthesis of Compound 23

Synthesis of Compound 23A

BT-1132 (50.00 mg, 108.33 umol) was charged in a vial and dissolved in DMF (3.00 mL). DIPEA (56.00 mg, 433.32 umol, 75.47 uL) followed by DSC (30.53 mg, 119.16 umol) were added and stirred at room temperature for 30 minutes. DMAP (13.23 mg, 108.33 umol) and tert-butyl 2-aminoacetate (36.32 mg, 216.66 umol, HCl) were added. The reaction was heated to 60° C. and stirred for 1 hour before it was cooled to room temperature. The reaction was diluted with MTBE (40 mL) and 1N HCl in water (40 mL). The organic phase was collected, and the aqueous was extracted with MTBE (2×20 mL). The organic phases were, washed with saturated sodium bicarbonate (3×20 mL), washed with brine (1×20 mL), dried over sodium sulfate, and evaporated to dryness. The crude material (3.23m, M+H=619 m/z) was moved directly onto the next step.

The crude was resuspended in trifluoroacetic acid (2.98 g, 26.14 mmol, 2.00 mL) and stirred at room temperature for 1 hour before being evaporated to dryness. The residue was resuspended in toluene (2×5 mL) and evaporated to dryness. The remaining residue was reconstituted in DMF and loaded to prep HPLC (ACN/water w/0.1% TFA). The fractions containing pure material were collected and evaporated to yield 15.4 mg of Compound 23A (25%) as an off-white powder (2.62m, M+H=563).

Synthesis of Compound 24

24B (87 mg, 126 umol) was charged in a vial, then dissolved in 2 mL of 20% piperidine in DMF solution. Solution was stirred at room temperature for 90 mins, then purified by reverse phase chromatography (5-30% acetonitrile in water with 0.1% trifluoroacetic acid). Pure fractions were pooled, frozen and lyophilized. 48 mg (81%) of Compound 24A was obtained as a white lyophilized powder.

Synthesis of Compound 25

Synthesis of Compound 26

Synthesis of Compound 27

Synthesis of Compound 27A

Triphenylphosphine (1.86 g, 7.08 mmol) was charged to a round bottom flask under nitrogen atmosphere and dissolved in dry tetrahydrofuran (40 mL). The solution was cooled to −40° C. via an acetonitrile/dry ice bath. After 30 minutes of cooling, DIAD (1.14 g, 7.08 mmol, 1.11 mL) was added dropwise over the course of about 30 minutes. The solution became a slurry of white precipitate and was allowed to stir for 10 minutes at −40° C. before 27B (500 mg, 1.42 mmol) and tert-butyl 4-(3-hydroxypropyl)piperazine-1-carboxylate (1.04 g, 4.25 mmol) were added. The reaction continued to stir for 1 hour before the the reaction was pulled from the cooling bath and allowed to warm to room temperature and stir for 3 days. The boc-protected product was precipitated from the reaction with water (150 mL) and isolated by vacuum filtration. The solid was resuspended in TFA (7.45 g, 65.34 mmol, 5 mL) and dichlormethane (5 mL) and stirred overnight. The product was precipitated with MTBE (100 mL) and isolated by filtration to yield 211 mg of Compound 27A (42%) as an off-white solid (2.23m, M+H=480)

Synthesis of Compound 28

Synthesis of Compound 28A

Compound 27A (200 mg, 282.65 umol, 2TFA) and Compound 28B (125.34 mg, 282.65 umol) were dissolved in DMF (5 mL). Hunig's base (73.06 mg, 565.30 umol, 98.46 uL) was added dropwise over the course of 1 minute. The reaction stirred at room temperature for 15 minutes before it was quenched with water (10 mL). The product precipitated, and it was filtered to isolate boc-protected Compound 28A. The solid was redissolved in Trifluoroacetic acid (5.96 g, 52.27 mmol, 4 mL) and stirred at room temperature for 1 hour. The reaction was diluted with toluene (2×5 mL) and evaporated. The remaining residue was resuspended in MeOH (2 mL) and Compound 28A was precipitated with MTBE (20 mL). 230 mg of Compound 28A (89%) was isolated as a light brown solid.

Synthesis of Compound 28B

Tert-butyl 4-(4-hydroxyphenyl)piperazine-1-carboxylate (5 g, 17.96 mmol) and (4-nitrophenyl) carbonochloridate (1.81 g, 8.98 mmol) were charged in a round bottom flask and dissolved in THF (17.96 mL) at room temperature. Once completely dissolved, Hunig's base (6.96 g, 53.89 mmol, 9.39 mL) was added dropwise. The reaction remained clear for 30 minutes. After 1 hour, a precipitate began to form. The solution was filtered to remove the precipitate, and the solvent was evaporated. The product was isolated by silica gel column (0-35% B, heptane/ethyl acetate) to yield 2.01 g of Compound 28B (25%) as a yellow crystalline solid (3.78m, M+H=444)

Synthesis of Compound 29

Synthesis of Compound 30

Synthesis of Compound 30A

A vial was charged with (4-nitrophenyl) carbonochloridate (43.87 mg, 217.64 umol) and tert-butyl 4-hydroxybenzoate (48.31 mg, 248.74 umol) and were dissolved in anhydrous Tetrahydrofuran (2.00 mL). At room temperature, Hunig's base (80.37 mg, 621.84 umol, 108.31 uL) was added dropwise to the solution over 1 minute. After 30 additional minutes of stirring, an additional aliquot of Hunig's base (80.37 mg, 621.84 umol, 108.31 uL) was added followed by a slow addition of a slurry of 27A (100 mg, 124.37 umol, 2TFA) in DMF (5 mL). The reaction stirred overnight and was quenched with water (20 mL) the next day. The tert-butyl protected product precipitated and the supernatant was decanted. The remaining residue was dissolved in ACN (8 mL) and was reprecipitated with water (12 mL). The solid was filtered and left to dry on the filter paper for 30 minutes. The solid was then dissolved in Trifluoroacetic acid (2.98 g, 26.14 mmol, 2 mL) and stirred for 2 hours. The solvent was removed in vacuo and the product was triturated with MTBE (10 mL) to give 79.9 mg of Compound 30A (85%) as an off-white solid (2.52m, M+H=644).

Synthesis of Compound 31

Synthesis of Compound 32

Synthesis of Compound 32A

32B (69.71 mg, 211.99 umol, HCl) and DSC (52.50 mg, 204.92 umol) were charged in a vial and dissolved in DMF (5 mL). Hunig's base (91.33 mg, 706.63 umol, 123.08 uL) was added dropwise over 1 minute and the reaction stirred at room temperature overnight. Separately, T-2026 (100 mg, 141.33 umol, 2TFA) was dissolved in DMF (1 mL) and added dropwise to the preactivated solution. The reaction continued to stir at room temperature overnight. The reaction was diluted with DI water (15 mL) and the boc-protected product precipitated. The solid was filtered and dried under vacuum. The residue was resuspended in Trifluoroacetic acid (1.99 g, 17.42 mmol, 1.33 mL) and stirred at room temperature for 2 hours. The reaction was diluted to 20 mL with MTBE and stirred for 3 days. The precipitated product was filtered and placed under vacuum to obtain 143.7 mg of Compound 32A (68%) as a yellow oil.

Synthesis of Compound 32B

Tert-butyl piperazine-1-carboxylate (1 g, 5.37 mmol) and 4-hydroxybenzaldehyde (1.31 g, 10.74 mmol) were charged in a round bottom flask and suspended in THF (30 mL) and Acetic Acid (6.45 g, 107.38 mmol, 6.14 mL). The imine formation stirred at room temperature for 1 hour before the addition of Sodium Triacetonxyborohydride (5.69 g, 26.85 mmol). The reaction was heated to 40° C. and stirred overnight. The reaction was neutralized with saturated sodium bicarbonate (50 mL) and extracted with MTBE (50 mL). The organic phase was washed with brine and dried over anhydrous sodium sulfate before evaporation. The remaining residue was dissolved in methanol and precipitated with 2M HCl in diethyl ether (5 mL). The product was filtered and placed under vacuum. The solid was resuspended in DCM with a few drops of MeOH and loaded to silica gel column (0-8% DCM/MeOH). The pure fractions were pooled and evaporated to yield 1.15 g of 32B (65%) as an off-white solid.

Synthesis of Compound 33

Synthesis of Compound 33A

33B (64.95 mg, 211.99 umol, HCl) and DSC (52.50 mg, 204.9 umol, TFA) were charged in a vial and dissolved in DMF (5 mL). Hunig's base (91.33 mg, 706.63 umol, 123.08 uL) was added dropwise over 1 minute and the activation stirred at room temperature overnight. Separately, 27A (100 mg, 141.33 umol, 2TFA) was dissolved in DMF (1 mL) and added dropwise over 1 minute, and the reaction continued at room temperature overnight. The reaction was diluted water (14 mL) and the boc-protected product precipitated. The solid was filtered and resuspended in trifluoroacetic acid (1.99 g, 17.42 mmol, 1.33 mL). The deprotection continued for 2 hours before the product was precipitated with MTBE (20 mL). The solvent was decanted, and the solid was placed under vacuum to remove residual solvent. 142.4 mg of Compound 33A was (76%) as an off-white solid (2.26m, M+H=713).

Synthesis of Compound 33B

Tert-butyl piperazine-1-carboxylate (1.00 g, 5.37 mmol) and 4-hydroxy-3-methyl-benzaldehyde (1.46 g, 10.74 mmol) were charged in a round bottom flask and suspended in THF (30 mL) and acetic acid (6.45 g, 107.40 mmol, 6.14 mL). The imine formation proceeded at room temperature for 1 hour before the addition of Sodium Triacetonxyborohydride (5.69 g, 26.85 mmol). The reaction was heated to 40° C. and stirred overnight. The reaction was neutralized with saturated sodium bicarbonate (50 mL) and extracted with MTBE (50 mL). The organic phase was washed with brine and dried over anhydrous sodium sulfate before evaporation. The remaining residue was dissolved in methanol and precipitated with 2M HCl in diethyl ether (5 mL). The product was filtered and placed under vacuum to dry. The solid was resuspended in DCM with a few drops of methanol and loaded to a silica gel column (0-9% MeOH in DCM). Pure fractions were pooled and evaporated to yield 536 mg of Compound 33B (29%) as an off-white solid (2.82m, M+H=307).

Synthesis of Compound 34

Synthesis of Compound 34A

Tert-butyl N-[(4-hydroxy-3,5-dimethyl-phenyl)methyl]carbamate (61.01 mg, 211.99 umol, HCl) and DSC (52.50 mg, 204.92 umol) were charged in a vial and dissolved in DMF (5 mL). Hunig's base (91.33 mg, 706.63 umol, 123.08 uL) was added and the reaction stirred overnight at room temperature. Separately, T-2026 (100 mg, 141.33 umol, 2TFA) was dissolved in DMF (1 mL) and added dropwise over 1 minute, and the reaction continued stirring at room temperature overnight. The reaction was diluted with water (14 mL) and the product precipitated. The solid was filtered and resuspended in Trifluoroacetic acid (1.99 g, 17.42 mmol, 1.33 mL) and stirred at room temperature for 2 hours. The product was precipitated with MTBE (20 mL). The solvent was decanted, and the solid was placed under vacuum to remove residual solvent. 172.4 mg of Compound 34A (96%) was obtained as an off-white solid (2.46m, M+H=657).

Synthesis of Compound 35

35A (58.61 mg, 73.74 umol, 2TFA) and 35B (64 mg, 73.74 uL) were charged in a vial and dissolved in DMF (4 mL) and PBS (pH 7.4, 2 mL) The reaction stirred at room temperature for 2 hours. The product was isolated by preparative HPLC (15-55% B, MeCN/water, 0.1% trifluoroacetic acid). 65.9 mg of Compound 35 (57%) was obtained as a white lyophilized powder (6.99m, M+H=1211).

Synthesis of Compound 35A

T-1818 (250 mg, 360.96 umol, 2TFA) and 4-((4-methoxybenzyl)thio)-4-methylpentanal (172.06 mg, 721.91 umol) were charged in a vial and suspended in THF (5 mL) and acetic acid (440.17 mg, 7.33 mmol, 360.80 uL). The imine formation stirred at room temperature for 1 hour before the addition of Sodium Triacetonxyborohydride (382.51 mg, 1.80 mmol). The reaction was heated to 40° C. and stirred overnight. The reaction was neutralized with saturated sodium bicarbonate (20 mL) and extracted with MTBE (3×20 mL). The organic phases were combined and washed with brine and dried over anhydrous sodium sulfate before evaporation. The remaining residue was dissolved in methanol and precipitated with 2M HCl in diethyl ether (5 mL). The product was filtered and placed under vacuum to remove residual solvent. The solid was then dissolved in Trifluoroacetic Acid (5.10 mL) followed by the addition of thioanisole (292.23 mg, 2.35 mmol, 275.69 uL) and trifluoromethanesulfonic acid (407.87 mg, 2.72 mmol, 241.34 uL). The reaction stirred at room temperature for 15 minutes before the product was isolated by preparative (10-50% B, MeCN/water, 0.1% trifluoroacetic acid). Pure fractions were pooled and evaporated to give 312.3 mg of Compound 35A (85%) as an off-white solid (2.99m, M+H=567).

Synthesis of Compound 35B

Synthesis of Compound 36

Synthesis of Compound 37

Synthesis of Compound 38

Synthesis of Compound 39

Synthesis of Compound 40

Synthesis of Compound 41

A 50 mL round bottom flask was charged with 27B (165 mg, 284 umol), flask was set under nitrogen then 25 mL THF was added. Sodium carbonate (200 mg) was added to the suspension, followed by tert-butyl N-(3-hydroxypropyl)carbamate (149 mg, 851 umol) and triphenylphosphine (298 mg, 1.14 mmol). The solution was cooled to −40° C. via an acetonitrile/dry ice bath. After 15 minutes of cooling, Diisopropyl azodicarboxylate (172 mg, 851 umol, 167 uL) was added dropwise over the course of 15 minutes until the solution was a milky off white color. The reaction was pulled from the cold bath after addition and allowed to warm to room temperature where it continued to stir under inert atmosphere. After 90 mins, triphenylphosphine (297 mg, 1.14 mmol) was added, solution was cooled to −40° C., then Diisopropyl azodicarboxylate (172 mg, 851 umol, 167 uL) was added. The reaction was pulled from the cold bath after addition and allowed to warm to room temperature where it continued to stir under inert atmosphere. After 1 h, the reaction was diluted with water (40 mL) and a white precipitated had formed. The solid was collected by filtration and dissolved in Trifluoroacetic acid (1.49 g, 13.1 mmol, 1.00 mL). Solution was stirred at room temperature for 15 minutes. Crude was diluted with water and purified by reverse phase chromatography (10-50% MeCN/water, 0.1% trifluoroacetic acid). Pure fractions were pooled, frozen and lyophilized. 142 mg (79%) of Compound 41A was obtained as a white lyophilized powder (2.05 mins, M+H=411).

Synthesis of Compound 42

A 50 mL round bottom flask was charged with 27B (165 mg, 284 umol), flask was set under nitrogen then 25 mL THF was added. Sodium carbonate (200 mg) was added to the suspension, followed by tert-butyl N-(3-hydroxypropyl)-N-isopropyl-carbamate (185 mg, 851 umol) and triphenylphosphine (298 mg, 1.14 mmol). The solution was cooled to −40° C. via an acetonitrile/dry ice bath. After 15 minutes of cooling, Diisopropyl azodicarboxylate (172 mg, 851 umol, 167 uL) was added dropwise over the course of 15 minutes until the solution was a milky off white color. The reaction was pulled from the cold bath after addition and allowed to warm to room temperature where it continued to stir under inert atmosphere. After 90 mins, triphenylphosphine (297 mg, 1.14 mmol) was added, solution was cooled to −40° C., then Diisopropyl azodicarboxylate (172 mg, 851 umol, 167 uL) was added. The reaction was pulled from the cold bath after addition and allowed to warm to room temperature where it continued to stir under inert atmosphere. After 1 h, the reaction was diluted with water (40 mL) and a white precipitated had formed. The solid was collected by filtration and dissolved in Trifluoroacetic acid (1.49 g, 13.1 mmol, 1.00 mL). Solution was stirred at room temperature for 15 minutes. Crude was diluted with water and purified by reverse phase chromatography (10-50% MeCN/water, 0.1% trifluoroacetic acid). Pure fractions were pooled, frozen and lyophilized. 86 mg (66%) of Compound 41A was obtained as a white lyophilized powder (2.39 mins, M+H=453).

Synthesis of Compound 43

Synthesis of 43B

A 50 mL round bottom flask was charged with 27B (800 mg, 1.38 mmol), flask was set under nitrogen then 25 mL THF was added. Sodium carbonate (200 mg) was added to the suspension, followed by N-(3-hydroxypropyl)-N-methyl-carbamate (781 mg, 4.13 mmol) and triphenylphosphine (1.44 g, 5.50 mmol). The solution was cooled to −40° C. via an acetonitrile/dry ice bath. After 15 minutes of cooling, Diisopropyl azodicarboxylate (835 mg, 4.13 mmol, 810 uL) was added dropwise over the course of 15 minutes until the solution was a milky off white color. The reaction was pulled from the cold bath after addition and allowed to warm to room temperature where it continued to stir under inert atmosphere. After 90 mins, triphenylphosphine (1.44 g, 5.50 mmol) was added, solution was cooled to −40° C., then Diisopropyl azodicarboxylate (835 mg, 4.13 mmol, 810 uL) was added. The reaction was pulled from the cold bath after addition and allowed to warm to room temperature where it continued to stir under inert atmosphere. After 1 h, the reaction was diluted with water (25 mL) and a white precipitated had formed. The solid was collected by filtration and dissolved in Trifluoroacetic acid (7.24 g, 63.5 mmol, 4.86 mL). Solution was stirred at room temperature for 15 minutes. Crude was diluted with water and purified by reverse phase chromatography (10-50% MeCN/water, 0.1% trifluoroacetic acid). Pure fractions were pooled, frozen and lyophilized. 663 mg (89%) of Compound 43B was obtained as a white lyophilized powder (1.77 mins, M+H=425).

Synthesis of Compound 44

Example 2: In Vitro Studies Using the Conjugates

BT474 (breast cancer) cells were plated at 12,000 cells per well and incubated for 20-24 hrs at 37° C. at 5% CO2. Post cell incubation, compounds were reconstituted in DMSO to a stock concentration of 5 mM. A compound plate was then prepared containing a 10 point dilution in DMSO. 2 uL of these dilutions were then added to the cells for a final working concentration of 5 uM to 0.0003 uM. Compounds and cells were incubated for 16 hrs. Media was then removed, cells washed, lysed, and analyzed for human total EbB2/Her2 levels by ELISA.

The bindings of the conjugates to HSP90 were studied with the HSP90α Assay Kit. The HSP90α Assay Kit is designed for identification of HSP90α inhibitors using fluorescence polarization. The assay is based on the competition of fluorescently labeled geldanamycin for binding to purified recombinant HSP90α. The key to the HSP90α Assay Kit is the fluorescently labeled geldanamycin. The fluorescently labeled geldanamycin is incubated with a sample containing HSP90α enzyme to produce a change in fluorescent polarization that can then be measured using a fluorescence reader.

NCI-H460 cells (non-small cell lung cancer) were plated at 500 cells per well and incubated for 20-24 hrs at 37° C. at 5% CO2. Post cell incubation, compounds were reconstituted in DMSO to a stock concentration of 200 uM. A compound plate was then prepared containing a 10 point dilution in RPMI+10% FBS+0.25% DMSO. 5 uL of the dilution was then added for a final working concentration range of 10 uM to 0.0005 uM. Compounds and cells were then incubated for 48 hrs. Cells were then analyzed by CellTiter-Glo for ATP levels and percent inhibition calculated.

Example 3: In Vivo Studies Using the Conjugates

Female athymic nude mice were implanted with 5×106H1975 cells (non-small cell lung cancer) per mouse in the right hind flank. Once tumors reached a volume of 50 mm3to 150 mm3they were randomized into two groups of ten to obtain a group average starting tumor volume of 114.4 mm3. Mice were then treated with either a vehicle control or 12.5 mg/kg of Conjugate 38. All dosing was carried out twice a week intravenously for three weeks, with five total doses being administered. Tumor volumes of the mice were measured. Final study measurements were taken on Day 19, at which point the study was terminated due to vehicle control tumor volumes approaching IACUC limits. As shown inFIG. 1, at the end of study, Conjugate 38 administered twice a week at 12.5 mg/kg, was able to achieve 70.8% tumor growth inhibition when compared to its vehicle control.

Conjugate and unconjugated payload accumulation at 96 and 144 h in H460 tumor-bearing mice. Conjugate 38 was dosed at 25 mg/kg intravenously.

Conjugate 38 accumulated and was retained in xenograph tumor tissue. It released its payload, Copanlisib, in its active form, which drives the efficacy superior to Copanlisib alone or ganetespib alone as shown inFIG. 2.

LS174t Colon Cancer Model

In another study using LS174t colon tumor xenograft model, significant tumor growth inhibition is observed with Conjugate 38. As shown inFIG. 3A, Conjugate 38 showed superior efficacy in contrast to the lack of efficacy for PI3K inhibitor (copanlisib) or HSP90 inhibitor (ganetespib) each alone. Surprisingly, Conjugate 38 also worked much better than a combination therapy comprising copanlisib and ganetespib.

Accumulation and retention of Conjugate 38 was tested in this tumor xenograft model. Conjugate 38 showed a strong and sustained pharmacodynamic response. As shown inFIG. 4, Conjugate 38 remained in the tumor xenograft and released the active PI3K inhibitor payload for >96 hours. Copanlisib itself has a much shorter tumor retention time. Inhibition of PI3K builds through the course of 48 h as shown by significant reduction of pAKT (S473) (FIG. 5).

Other In Vivo Models

PIK3A mutations occur in about 15% to 30% of breast, endometrial and colon cancers. In additional to LS174t model, other xenograft models harboring common PIK3CA mutations were chosen for testing. In one study, mice bearing SKOV3 (ovarian cancer) tumor xenograft were treated with IV dose of Conjugate 38 at 25 mg/kg and Copanlisib at 6 mg/kg. Doses were selected based on the maximum tolerated dose. Average tumor volume changes are shown inFIG. 3B. In another study, mice bearing BT474 (breast cancer) tumor xenograft were treated with IV dose of Conjugate 38 at 25 mg/kg and Copanlisib at 6 mg/kg. Average tumor volume changes are shown inFIG. 3C. In all xenograft models tested, a statistically significant increase in tumor growth inhibition was seen when comparing Conjugate 38 to Copanlisib.

Example 4: Masking Payload to Reduce Normal Tissue Toxicity

In this example, conjugates of the present disclosure derived from multiple payloads are found to be significantly less active in their respective in vitro functional assays while still retaining HSP90 targeting. The activity of the respective payload is blocked until the linker moiety gets cleaved in the tumor and releases active unblocked payload. Through the HSP90 platform, toxicity is mitigated by masking the payload's active site until it can be delivered to the tumor.

A known and potentially dose limiting side effect of inhibitors targeting PI3K pathways is hyperglycemia. In this study, Conjugate 38 was tested to determine whether the side effect of its PI3K inhibitor payload can be reduced.

In a cell free PI3K enzyme assay, Conjugate 38 was much less active than its PI3K enzyme inhibitor payload as shown inFIG. 6. Therefore, conjugating a PI3K enzyme inhibitor payload to a HSP90 targeting ligand masked the PI3K enzyme inhibition.

In an in vivo study in mice, glucose levels were monitored post PI3K inhibitor (Copanlisib) and Conjugate 38 dosing. As shown inFIG. 7, mice treated with Conjugate 38 had lower glucose concentrations than mice treated with the PI3K inhibitor payload. Therefore, conjugating a PI3K enzyme inhibitor payload to a HSP90 targeting ligand reduced the normal tissue activity of glucose increase in mice. Conjugate 38 was able to mitigate the increase in glucose levels observed post dosing with the PI3K inhibitor alone, demonstrating that selective delivery may be able to increase the therapeutic window in comparison to PI3K inhibitors alone.

These data demonstrate that by leveraging the preferential accumulation of HSP90-targeting ligands in tumors, PI3K inhibitors can be selectively delivered to achieve deep pathway inhibition leading to efficacy in multiple tumor models without hyperglycemia induction in mice.

In some further studies, conjugates comprising an HSP90 binding ligand and other payloads were tested. Conjugate 45 comprises a ganetespib derivative as a targeting moiety and talazoparib (PARP inhibitor) as a payload. Conjugate 46 comprises a ganetespib derivative as a targeting moiety and ulixertinib (ERK1/2 inhibitor) as a payload. Conjugate 47 comprises a ganetespib derivative as a targeting moiety and TAK-733 (MEK inhibitor) as a payload.

FIG. 8Acompares the activity of Conjugate 45 and its payload (talazoparib).FIG. 8Bcompares the activity of Conjugate 46 and its payload (ulixertinib).FIG. 8Ccompares the activity of Conjugate 47 and its payload TAK-733. The data further support that attaching a payload to a HSP90 binding ligand blocks the target activity of the payload. The HSP90 bindings of the HSP90 ligands are not affected. The conjugates retain high affinity for HSP90 as shown in the table below:

In one further study, DNA damage caused by Conjugate 48 (a conjugate comprising a ganetespib derivative and SN-38) was compared with the DNA damage caused by SN-38, the payload of Conjugate 48. SN-38 and its pre-drug, irinotecan (an analog of camptothecin), are inhibitors of topoisomerase-I and potently cause DNA damage that results in nicks in the DNA. As shown inFIG. 9, irinotecan showed a moderate level of DNA damaging activity and ganetespib showed none as expected. Conjugate 48 showed negligible levels of DNA damage up to 100 uM, while SN-38 showed DNA damage even at 1 uM. The conjugation of SN-38 to the HSP90 ligand results in the masking of the activity of SN-38 until it can be selectively delivered to tumors where linker cleavage occurs.

Therefore, conjugates of the present disclosure masks a wide range of payloads to reduce normal tissue toxicity while releasing the potent payload upon linker cleavage.

Example 5: Determining the Permeability of Payloads and Conjugates

In order to test the ability of the payloads and/or conjugates to enter cells, a cell monolayer assay was utilized employing Caco-2 cells (a human epithelial colorectal adenocarcinoma cell line).

Experimental Procedure: Caco-2 cells grown in tissue culture flasks are trypsinized, suspended in medium, and the suspensions were applied to wells of a Millipore 96 well Caco-2 plate. The cells are allowed to grow and differentiate for three weeks, feeding at 2-day intervals. For Apical to Basolateral (A→B) permeability, the test agent is added to the apical (A) side and amount of permeation is determined on the basolateral (B) side; for Basolateral to Apical (B→A) permeability, the test agent is added to the B side and the amount of permeation is determine on the A side.

Compound 27A was tested in the Caco-2 permeability assay and was shown to have very low permeability. The mean permeability (Papp) in the A to B direction was found to be 0.00320×10−6cm/s and in the B to A direction was 0.0162×10−6cm/s.