阅读说明：本技术 靶向治疗剂 (Targeted therapeutic agents ) 是由 尼拉·贾殷 英伟文 D·U·基马纳马达 张俊怡 A·卡勒 于 2018-06-19 设计创作，主要内容包括：本发明提供了药理学化合物,其包括与结合部分缀合的效应物部分,该结合部分将效应物部分引导至受关注的生物学靶标。类似地,本发明提供了包括该化合物的组合物、试剂盒和方法(例如治疗、诊断和成像)。该化合物可以被描述为蛋白质相互作用结合部分-药物缀合物(SDC-TRAP)化合物,其包括蛋白质相互作用结合部分和效应物部分。例如,在针对治疗癌症的某些实施方案中,SDC-TRAP可包括与作为效应物部分的细胞毒性剂缀合的Hsp90抑制剂。(The present invention provides pharmacological compounds comprising an effector moiety conjugated to a binding moiety that directs the effector moiety to a biological target of interest. Similarly, the invention provides compositions, kits and methods (e.g., therapy, diagnosis and imaging) comprising the compounds. The compounds may be described as protein interaction binding moiety-drug conjugate (SDC-TRAP) compounds, which include a protein interaction binding moiety and an effector moiety. For example, in certain embodiments directed to treating cancer, a SDC-TRAP can comprise an Hsp90 inhibitor conjugated to a cytotoxic agent as an effector moiety.)

1. A pharmaceutical composition comprising a drug conjugate (SDC-TRAP) or a carboxylate derivative salt form thereof and at least one excipient, wherein the SDC-TRAP comprises a binding moiety and an effector moiety, wherein the binding moiety is bound to HSP90 and the effector moiety is a cytotoxic moiety.

2. The pharmaceutical composition of claim 1, wherein the binding moiety comprises an HSP90 inhibitor.

3. The pharmaceutical composition of claim 2, wherein the HSP90 inhibitor is ganetespib or a tautomer/derivative/analog thereof.

4. The pharmaceutical composition of claim 1, wherein the cytotoxic moiety is a small molecule.

5. The pharmaceutical composition of claim 4, wherein the effector moiety comprises SN-38 or irinotecan, or a fragment/derivative/analog thereof.

6. The pharmaceutical composition of claim 1, comprising SDC-TRAP-0063 sodium.

7. The pharmaceutical composition of claim 6, wherein the excipient is t-butanol.

8. The pharmaceutical composition of claim 6, further comprising water.

9. The pharmaceutical composition of claim 8, wherein the concentration of SDC-TRAP-0063 sodium is about 100mg/m L.

10. The pharmaceutical composition of claim 8, wherein the concentration of SDC-TRAP-0063 sodium is about 50mg/m L.

11. The pharmaceutical composition of claim 8, wherein the pH of the composition is at least 9.8.

12. The pharmaceutical composition of claim 8, further comprising sodium chloride.

13. The pharmaceutical composition of claim 12, wherein the concentration of SDC-TRAP-0063 sodium is from about 0.1 to about 2.0mg/m L.

14. The pharmaceutical composition of claim 13, wherein the pH of the composition is from about 8.1 to about 9.6.

15. The pharmaceutical composition of claim 14, wherein the pH of the composition is at least 9.3.

16. The pharmaceutical composition of claim 12 for intravenous administration by infusion.

17. A method for treating a tumor in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 1, thereby treating the subject.

18. The method of claim 17, wherein the tumor is colorectal cancer, breast cancer, small cell lung cancer, sarcoma, or pancreatic cancer.

19. A compound having the structure:

or a tautomer thereof.

20. A process for the preparation of SDC-TRAP-0063 comprising the steps of:

1) synthesizing STA-31-0229 from STA-31-0228;

2) synthesizing STA-31-0222 from SN-38;

3) synthesizing SDC-TRAP-0063 crude product from STA-31-0229 and STA-31-0222; and

4) and purifying the crude product of SDC-TRAP-0063.

21. A process for the preparation of SDC-TRAP-0063 sodium comprising the steps of:

1) dissolving SDC-TRAP-0063 in tert-butanol at 25-35 deg.C;

2) adding 0.3 equivalents of aqueous sodium hydroxide and water for injection to adjust the pH to above about 9.8;

3) filtering the mixture from step 2) with at least two 0.2 μm filters in succession; and

4) sterile vial filling and freeze drying were performed.

Brief description of the drawings

Figure 1 is a flow chart of the process for preparing SDC-TRAP-0063 sodium and process control.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Detailed Description

The present invention provides molecules comprising an effector moiety conjugated to a binding moiety that directs the effector moiety to a biological target of interest. The molecules of the invention allow for selective targeting of effector moieties by capturing the molecules of the invention in a desired cell, such as a cancer cell. Due to the selective binding of molecules to high concentrations of intracellular proteins, these molecules can be described as small molecule drug conjugates (SDC-TRAPs) that are trapped inside the cell. To capture the molecules of the invention within the cells of interest, the binding moiety, which is part of the SDC-TRAP molecule, interacts with proteins that are overexpressed in the target cells. In exemplary embodiments, the overexpressed protein is characteristic of a particular disease or disorder. Accordingly, the invention provides compositions, kits and methods (e.g., therapy, diagnosis and imaging) comprising the molecules of the invention.

In one embodiment of the invention, SDC-TRAP allows for delivery of effector molecules that would otherwise be unsuitable for administration alone due to toxicity and/or unwanted systemic effects. The use of targeted delivery molecules (SDC-TRAP) described herein allows effector moieties that are too toxic to be administered by current methods to be administered at lower levels, thereby targeting toxic effectors to specific diseased cells at sub-toxic levels.

In various exemplary aspects and embodiments, the present invention provides compounds for the treatment of cancer. For example, a SDC-TRAP can comprise an Hsp90 binding moiety (i.e., a targeted Hsp90 that is overexpressed in cancer cells compared to normal cells) and an effector moiety (e.g., an Hsp90 binding moiety can be an Hsp90 inhibitor conjugated to a cytotoxic agent). As noted above, the invention is exemplified herein by Hsp90 targeted binding moieties and cytotoxic agents. Other combinations of parts considered, mentioned or described herein are intended to be included within the scope of the invention.

In various aspects and embodiments, the invention provides a SDC-TRAP comprising a binding moiety and an effector moiety, wherein the SDC-TRAP molecule is capable of entering a cell by passive transport. The ability of the SDC-TRAP to enter cells through passive transport may be a result of one or more unique chemical properties of the SDC-TRAP (e.g., size, weight, charge, polarity, hydrophobicity, etc.), and may facilitate the delivery and/or action of the SDC-TRAP. The ability of SDC-TRAP to enter cells through passive transport is a functional property that, together with its physicochemical properties, distinguishes SDC-TRAP from other targeting molecules (e.g., antibody-drug conjugates).

In various aspects and embodiments, the invention provides a SDC-TRAP comprising a binding moiety and an effector moiety, wherein the SDC-TRAP molecule is capable of entering a cell by active transport. The ability of the SDC-TRAP to enter cells through active transport may be a result of one or more unique chemical properties of the SDC-TRAP, and may facilitate the delivery and/or action of the SDC-TRAP. Examples of active transport of SDC-TRAP may include, for example, endocytosis, phagocytosis, pinocytosis and exocytosis.

In various aspects and embodiments, the invention provides SDC-TRAPs having a molecular weight of less than about 5000 daltons (e.g., less than about 5000, 2500, 2000, 1600, 1550, 1500, 1450, 1400, 1350, 1300, 1250, 1200, 1150, 1100, 1050, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, etc.). Similarly, in various aspects and embodiments, the invention provides binding moieties having a molecular weight of less than about 2500 daltons (e.g., less than about 2500, 2000, 1600, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, etc.) and/or effector moieties having a molecular weight of less than about 2500 daltons (e.g., less than about 2500, 2000, 1600, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, etc.). The overall molecular weight of the SDC-TRAP, as well as the individual weights of the binding moiety, effector moiety, and any linking moiety, may affect the transport of the SDC-TRAP. In various instances, it has been observed that lower molecular weights may facilitate delivery and/or activity of SDC-TRAP.

In various aspects and embodiments, the invention provides SDC-TRAPs comprising an Hsp90 binding moiety and an effector moiety, wherein the Hsp90 binding moiety and the effector moiety are approximately equal in size (e.g., the Hsp90 binding moiety and the effector moiety have a difference in molecular weight of less than about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, etc. daltons). In various instances, it has been observed that lower molecular weight differences may facilitate delivery and/or activity of SDC-TRAP.

In various aspects and embodiments, the invention provides SDC-TRAPs comprising a binding moiety that interacts with a target protein. The binding moiety that interacts with the target protein may selectively interact with any one or more domains of the target protein. For example, when the target protein is Hsp90, the binding moiety may be an Hsp90 binding moiety that interacts with the N-terminal domain of Hsp90, the C-terminal domain of Hsp90, and/or the intermediate domain of Hsp 90. Selective interaction with any one or more domains of a target protein may advantageously increase specificity and/or increase the concentration of molecular targets in a target tissue and/or cell.

In various aspects and embodiments, the present invention provides compositions comprisingHaving high affinity for molecular targets (e.g., K)_d50, 100, 150, 200, 250, 300, 350, 400nM or higher) binding moiety. For example, where the binding moiety is an Hsp90 binding moiety, the K of the Hsp90 binding moiety_dCan be 50, 100, 150, 200, 250, 300, 350, 400nM or higher. Binding moieties with high affinity for the molecular target may advantageously improve targeting and/or increase the resonance time of the SDC-TRAP in target cells and/or tissues.

In various aspects and embodiments, the invention provides a SDC-TRAP comprising a binding moiety (e.g., an Hsp90 binding moiety) and an effector moiety, wherein the SDC-TRAP is present in tumor cells in a ratio of about 2:1 compared to plasma when administered to a subject. The ratio may be higher, for example, about 5:1, 10:1, 25:1, 50:1, 75:1, 100:1, 150:1, 200:1, 250:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1 or higher. In various aspects and embodiments, the ratio is 1,2, 3,4, 5, 6,7, 8, 12, 24, 48, 72 or more hours after administration. The effectiveness of targeting can be reflected in the ratio of SDC-TRAP to plasma in the target cells and/or tissues.

In various aspects and embodiments, the invention provides a SDC-TRAP comprising a binding moiety (e.g., an Hsp90 binding moiety) and an effector moiety, wherein the SDC-TRAP is present in target (e.g., cancer) cells for at least 24 hours. SDC-TRAP can be present in cancer cells for a longer period of time, e.g., at least 48, 72, 96, or 120 hours. It may be advantageous for the SDC-TRAP to be present in the target cells for a longer period of time to increase the therapeutic effect of a given dose of SDC-TRAP and/or to increase the interval between SDC-TRAP administrations.

In various aspects and embodiments, the invention provides SDC-TRAPs comprising a binding moiety (e.g., an Hsp90 binding moiety) and an effector moiety, wherein the effector moiety is released for a period of at least 6 hours. The effector moiety may be released for a longer period of time, for example at least 12, 24, 48, 72, 96 or 120 hours. Selective release can be used to control, delay, and/or prolong the time of release of the effector moiety, and thus increase the therapeutic effect of a given dose of SDC-TRAP, reduce undesirable side effects of a given dose of SDC-TRAP, and/or increase the interval between administrations of SDC-TRAP.

In various aspects and embodiments, the invention provides SDC-TRAPs comprising an Hsp90 binding moiety and an effector moiety, wherein the effector moiety is selectively released within target (e.g., cancer) cells. Selective release can be achieved, for example, by a cleavable linker (e.g., an enzymatically cleavable linker). Selective release can be used to reduce unwanted toxicity and/or unwanted side effects. For example, SDC-TRAPs can be designed in which such an effector moiety is inactive (or relatively inactive) in conjugated form, but is active (or more active) after it is selectively released into the interior of a target (e.g., cancer) cell.

In various aspects and embodiments, the invention provides SDC-TRAPs comprising a binding moiety (e.g., an Hsp90 binding moiety) and an effector moiety, wherein the SDC-TRAP permits the use of effector moieties that are otherwise toxic or otherwise unsuitable for administration to a subject. Due to undesirable toxicity, the effector moiety may not be suitable for administration to a subject. In this case, strategies such as selective release can be used to address the undesirable toxicity. The effector moiety may not be suitable for administration to a subject due to undesired targeting or lack of targeting. Targeting can address these issues, for example, by minimizing systemic toxicity while maximizing local toxicity at the target (e.g., tumor).

In various aspects and embodiments, the invention provides SDC-TRAPs comprising a binding moiety (e.g., an Hsp90 binding moiety) and an effector moiety, wherein the binding moiety is an inhibitor that is not effective as a therapeutic agent (e.g., an Hsp90 inhibitor) when administered alone. In such cases, the SDC-TRAP can promote additive or synergistic effects between the binding moiety and the effector moiety, thereby advantageously increasing the therapeutic effect and/or reducing side effects of the therapy.

In order that the invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter is recited, it is intended that values and ranges intermediate to the recited values also form part of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting.

Definition of

The articles "a," "an," and "the" are used herein to refer to one or more (i.e., to at least one) of the grammatical object of the article, unless otherwise clearly indicated by contrast. For example, "an element" means one element or more than one element.

The term "including" is used herein to mean, and is used interchangeably with, the phrase "including, but not limited to".

The term "or" is used herein to mean, and is used interchangeably with, the term "and/or," unless the context clearly dictates otherwise.

The term "for example" is used herein to mean, and is used interchangeably with, the phrase "for example, but not limited to".

Unless otherwise indicated or apparent from the context, as used herein, the term "about" is to be understood as being within the ordinary tolerance of the art, e.g., within 2 standard deviations of the mean. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise apparent from the context, all numbers provided herein may be modified by the term "about".

Ranges provided herein are to be understood as shorthand for all values within the range. For example, a range of 1 to 50 should be understood to include any number, combination of numbers, or subrange from 1,2, 3,4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 in the group.

Recitation of one or more chemical groups herein in any definition of a variable includes defining the variable as any single group or combination of groups recited. Recitation of embodiments of variables or aspects herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any of the compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

As used herein, the term "subject" refers to both human and non-human animals, including veterinary subjects. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. In a preferred embodiment, the subject is a human and may be referred to as a patient.

As used herein, the term "treating" preferably refers to an effect that achieves a beneficial or desired clinical result, including, but not limited to, alleviation or amelioration of one or more signs or symptoms of a disease or disorder, diminishment of extent of disease, stability (i.e., not worsening) state of disease, amelioration or palliation of the disease state, diminishment of rate or time to progression, and remission (whether partial or total), whether detectable or undetectable. "treatment" may also mean an increase in survival compared to the expected survival in the absence of treatment. The treatment is not necessarily curative.

A "therapeutically effective amount" is an amount sufficient to treat a disease in a subject. A therapeutically effective amount may be administered in one or more administrations.

As used herein, "diagnosis" or the like refers to the clinical or otherwise assessment of a subject's condition based on observations, tests or circumstances for identifying a subject suffering from a disease, disorder or condition based on the presence of at least one indicator (e.g., signs or symptoms of the disease, disorder or condition). In general, diagnosis using the methods of the invention includes observing a subject for multiple indicators of a disease, disorder, or condition in conjunction with the methods provided herein. The diagnostic method provides an indication of the presence or absence of a disease. A single diagnostic test typically does not provide a definitive conclusion about the disease state of the subject being tested.

The term "administering" includes any method of delivering a pharmaceutical composition or agent into the system of a subject or to a specific area within or on the subject. In certain embodiments of the invention, the agent is administered intravenously, intramuscularly, subcutaneously, intradermally, intranasally, orally, transdermally, or mucosally. In a preferred embodiment, the agent is administered intravenously. Administration of the medicament may be performed by a plurality of persons working in concert. Administering the agent includes, for example, prescribing to the subject the agent to be administered and/or providing instructions, either directly or by another person, to take the particular agent, the taking being by self-delivery (e.g., by oral delivery, subcutaneous delivery, intravenous delivery through a centerline, etc.); or by a trained professional, e.g., intravenous delivery, intramuscular delivery, intratumoral delivery, etc.

As used herein, the term "survival" refers to the continuation of life of a subject that has been treated for a disease or disorder, such as cancer. Survival time may be defined from any point, such as time to enter a clinical trial, time to begin with completion or failure or an earlier treatment regimen, time to begin with diagnosis, and the like.

As used herein, the term "recurrence" refers to the regrowth of tumor or cancer cells in a subject to whom a primary treatment for the tumor has been administered. Tumors may recur at the original site or another part of the body. In one embodiment, the recurrent tumor is of the same type as the original tumor of the subject being treated. For example, if a subject has an ovarian cancer tumor, is treated, and then develops another ovarian cancer tumor, the tumor has recurred. In addition, the cancer may recur or metastasize to an organ or tissue that is different from the organ or tissue that originally developed.

As used herein, the term "identify" or "select" refers to a selection that takes precedence over another selection. In other words, identifying a subject or selecting a subject is performing an active step of picking the particular subject from the group and confirming the identity of the subject by name or other distinguishing characteristic.

Similarly, if a subject exhibits a reduction in at least one sign or symptom of a disease or disorder (e.g., tumor shrinkage, reduction in tumor burden, inhibition or reduction of metastasis, improvement in quality of life ("QO L"), if time to progression ("TTP") is delayed, if overall survival ("OS") is increased, etc.), or if disease progression is slowed or halted (e.g., tumor growth or metastasis is halted or the rate of tumor growth or metastasis is slowed), they will benefit from treatment.

The term "cancer" or "tumor" is well known in the art and refers to, for example, the presence of cells in a subject that are characteristic of oncogenic cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, decreased cell death/apoptosis, and certain characteristic morphological features. Cancer cells are often in the form of solid tumors. However, cancer also includes non-solid tumors, such as hematological tumors, e.g., leukemia, in which the cancer cells are derived from bone marrow. As used herein, the term "cancer" includes pre-malignant as well as malignant cancers. Cancers include, but are not limited to, auditory neuroma, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia (monocytic, myeloblastic, adenocarcinoma, angiosarcoma, astrocytoma, myelomonocytic and promyelocytic), acute T-cell leukemia, basal cell carcinoma, cholangiocarcinoma, bladder carcinoma, brain carcinoma, breast carcinoma, bronchial carcinoma, cervical carcinoma, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myelogenous (myelogenous) leukemia, chronic myelogenous leukemia, colon carcinoma, colorectal carcinoma, craniopharyngioma, cystadenocarcinoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, proliferative abnormalities (dysplasia and metaplasia), embryonic carcinoma, endometrial carcinoma, endothelial sarcoma, ependymoma, epithelial carcinoma, erythroleukemia, Esophageal cancer, estrogen receptor positive breast cancer, primary polycythemia, ewing's tumor, fibrosarcoma, follicular lymphoma, germ cell testicular cancer, glioma, heavy chain disease, hemangioblastoma, liver cancer, hepatocellular carcinoma, hormone-insensitive prostate cancer, leiomyosarcoma, liposarcoma, lung cancer, lymphatic endothelial sarcoma, lymphatic sarcoma, lymphoblastic leukemia, lymphoma (hodgkin's and non-hodgkin's), bladder, breast, colon, lung, ovary, pancreas, prostate, malignant tumors and hyperproliferative diseases of the skin and uterus, lymphoid malignancies of T-cell or B-cell origin, leukemia, lymphoma, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, myelogenous leukemia, myeloma, myxosarcoma, neuroblastoma, non-small cell lung cancer, Oligodendroglioma, oral cancer, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinoma, papillary carcinoma, pinealoma, polycythemia vera, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, sebaceous adenocarcinoma, seminoma, skin cancer, small cell lung tumor, solid tumor (tumor and sarcoma), small cell lung cancer, gastric cancer, squamous cell carcinoma, synovioma, sweat gland carcinoma, thyroid cancer, vardendrin's macroglobulinemia, testicular tumor, uterine cancer, and wilms tumor. Other cancers include primary cancer, metastatic cancer, oropharyngeal cancer, hypopharynx cancer, liver cancer, gallbladder cancer, bile duct cancer, small intestine cancer, urinary tract cancer, kidney cancer, urothelial cancer, female genital tract cancer, uterine cancer, gestational trophoblastic disease, male genital tract cancer, seminal vesicle cancer, testicular cancer, germ cell tumor, endocrine gland tumor, thyroid cancer, adrenal cancer, pituitary adenocarcinoma, hemangioma, sarcoma caused by bone and soft tissue, kaposi's sarcoma, neural cancer, eye cancer, meningeal cancer, glioblastoma, neuroma, neuroblastoma, schwannoma, solid tumor caused by hematopoietic malignancy such as leukemia, metastatic melanoma, recurrent or persistent epithelial carcinoma, fallopian tube cancer, primary peritoneal cancer, gastrointestinal stromal tumor, colorectal cancer, gastric cancer, melanoma, glioblastoma multiforme, non-squamous non-small cell lung cancer, Malignant glioma, epithelial ovarian cancer, primary peritoneal serous carcinoma, metastatic liver cancer, neuroendocrine cancer, refractory malignancy, triple negative breast cancer, HER2 amplified breast cancer, nasopharyngeal cancer, oral cancer, biliary tract, hepatocellular carcinoma, squamous cell carcinoma of the head and neck (SCCHN), non-medullary thyroid cancer, recurrent glioblastoma multiforme, neurofibromatosis type 1, CNS cancer, liposarcoma, leiomyosarcoma, salivary gland carcinoma, mucosal melanoma, acromelanoma, paraganglioma, pheochromocytoma, advanced metastatic cancer, solid tumor, triple negative breast cancer, colorectal cancer, sarcoma, melanoma, renal cancer, endometrial cancer, thyroid cancer, rhabdomyosarcoma, multiple myeloma, ovarian cancer, glioblastoma, gastrointestinal stromal tumors, mantle cell lymphoma, and refractory malignancy.

As used herein, "solid tumor" is understood to be any pathogenic tumor that can be detected as having three-dimensional abnormal growth by palpation or using imaging methods. Solid tumors are distinguished from hematological tumors (e.g., leukemia). However, the cells of hematological tumors are derived from bone marrow; thus, the tissue from which the cancer cells originate is a solid tissue that may be hypoxic.

By "tumor tissue" is understood cells, extracellular matrix and other naturally occurring components associated with solid tumors.

As used herein, the term "isolated" refers to a preparation that is substantially free (e.g., 50%, 60%, 70%, 80%, 90%, or more by weight) of other proteins, nucleic acids, or compounds associated with the tissue from which the preparation is obtained.

As used herein, the term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject. The term "sample" includes any bodily fluid (e.g., urine, serum, blood, lymph fluid, gynecological fluid, cystic fluid, ascites, ocular fluid, and fluid collected by bronchial lavage and/or peritoneal irrigation), ascites, tissue sample (e.g., tumor sample), or cells from a subject. Other subject samples include tear drops, serum, cerebrospinal fluid, feces, sputum, and cell extracts. In one embodiment, the sample is removed from the subject. In a particular embodiment, the sample is urine or serum. In another embodiment, the sample does not comprise ascites or is not an ascites sample. In another embodiment, the sample does not include or is not peritoneal fluid. In one embodiment, the sample comprises cells. In another embodiment, the sample does not comprise cells. The sample is typically removed from the subject prior to analysis. However, tumor samples can be analyzed in a subject, for example, using imaging or other detection methods.

As used herein, the term "control sample" refers to any clinically relevant control sample, including, for example, a sample from a healthy subject who does not have cancer, a sample from a subject who has cancer of lower severity or that has progressed more slowly than the subject to be assessed, a sample from a subject who has some other type of cancer or disease, a sample from a subject prior to treatment, a sample of non-diseased tissue (e.g., non-tumor tissue), a sample from the same source and proximal to the tumor site, and the like. The control sample may be a purified sample, protein and/or nucleic acid provided with the kit. Such control samples may be diluted, for example, in a dilution series to allow quantitative measurement of the analyte in the test sample. Control samples may include samples derived from one or more subjects. The control sample may also be a sample prepared from the subject to be assessed at an earlier time point. For example, a control sample can be a sample taken from a subject to be evaluated prior to the onset of cancer, an earlier stage of disease, or prior to administration of a treatment or partial treatment. The control sample may also be a sample from an animal model of cancer or from a tissue or cell line of an animal model. The level in a control sample consisting of a set of measurements may be determined, for example, based on any suitable statistical measure, such as a measure of central tendency including mean, median or modal values.

As used herein, the term "obtained" is understood herein to be manufactured, purchased, or otherwise owned.

As used herein, the term "identity" as used herein in relation to amino acid or nucleic acid sequences refers to any gene or protein sequence that is at least 30% identical, more preferably 40%, 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, and most preferably 95%, 96%, 97%, 98%, 99% or more identical to a known gene or protein sequence over the length of the compared sequences. Protein or nucleic acid sequences that have a high degree of identity throughout the sequence can be said to be homologous. A "homologous" protein can also have at least one biological activity of a comparative protein. Typically, for proteins, the length of the comparison sequences will be at least 10 amino acids, preferably 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 175, 200, 250 or at least 300 amino acids or more. For nucleic acids, the length of the comparison sequences is typically at least 25, 50, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, or at least 850 nucleotides or more.

As used herein, "detecting" or detection, etc. is to be understood as performing an assay to identify a particular analyte in a sample. The amount of analyte or activity detected in the sample may be none or below the detection level of the assay or method.

The term "modulation" or modulation "refers to up-regulation of levels (i.e., activation or stimulation), down-regulation (i.e., inhibition or depression), or a combination of both or both, respectively. A "modulator" is a compound or molecule that modulates and may be, for example, an agonist, antagonist, activator, stimulator, inhibitor or inhibitor.

The term "expression" as used herein refers to the process of producing a polypeptide from DNA. The process involves transcription of a gene into mRNA and translation of the mRNA into a polypeptide. Depending on the context of use, "expression" may refer to the production of RNA or protein or both.

The term "expression level of a gene" or "gene expression level" refers to the level of mRNA as well as pre-mRNA nascent transcripts, transcript processing intermediates, mature mRNA and degradation products, or the level of protein encoded by the gene in the cell.

As used herein, "level of activity" is understood to be the amount of protein activity (typically enzymatic activity) determined by a quantitative, semi-quantitative or qualitative assay. Activity is typically determined by monitoring the amount of product produced in the assay using a substrate that produces a readily detectable product (e.g., a colored product, a fluorescent product, or a radioactive product).

As used herein, a sample or subject that "changes as compared to a control" is understood to have a level of analyte or diagnostic or therapeutic indicator (e.g., marker) to be detected that is statistically different from that from a normal, untreated or control sample, including, for example, cells in culture, one or more laboratory test animals, or one or more human subjects.

Because the level of a marker present in a subject will be the result of a disease, and not the result of treatment, it is generally unlikely that a control sample obtained from a patient prior to the onset of the disease will be obtained.

A "normal" expression level of a marker is the level of expression of the marker in cells of a subject or patient not suffering from cancer. In one embodiment, a "normal" expression level refers to the expression level of a marker under normoxic conditions.

By "overexpression" or "high expression level" of a marker is meant that the level of expression in the test sample is greater than the standard error of the assay used to assess expression, and preferably at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,4, 5, 6,7, 8, 9, or 10 fold greater than the level of expression of the marker in a control sample (e.g., a sample from a healthy subject that does not have the disease, i.e., cancer, associated with the marker). In one embodiment, the expression of the marker is compared to the average expression level of the marker in several control samples.

By "low expression level" or "under-expression" of a marker is meant that the expression level in the test sample is at least 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 fold lower than the expression level of the marker in a control sample (e.g., a sample from a healthy subject not having the disease, i.e., cancer, associated with the marker). In one embodiment, the expression of the marker is compared to the average expression level of the marker in several control samples.

As used herein, "binding" is understood to mean having at least 10 for binding to a specific binding partner as compared to a non-specific binding partner²Or more, 10³Or more, preferably 10⁴Or more, preferably 10⁵Or more, preferably 10⁶Or more preference (e.g., binding an antigen to a sample known to contain cognate antibodies).

As used herein, "determining" is understood to mean making an assay or using a diagnostic method to determine the presence, absence, level or extent of a person or something, such as a condition, biomarker, disease state or physiological condition.

As used herein, "prescribing" should be understood to indicate one or more particular agents for administration to a subject.

As used herein, the term "response" is understood to have a positive response to treatment with a therapeutic agent, wherein a positive response is understood to have a reduction in at least one sign or symptom of a disease or disorder (e.g., tumor shrinkage, tumor burden reduction, inhibition or reduction of metastasis, improvement in quality of life ("QO L"), delay in time to progression ("TTP"), improvement in overall survival ("OS"), etc.) or slowing or halting of disease progression (e.g., stopping tumor growth or metastasis or slowing the rate of tumor growth or metastasis).

The term "administering" or "administration" may include any method of delivering a pharmaceutical composition or agent into the system of a subject or to a specific area within or on a subject. In certain embodiments of the invention, the Hsp90 inhibitor is administered intravenously, intramuscularly, subcutaneously, intradermally, intranasally, orally, transdermally, or mucosally. In a preferred embodiment, the agent is administered intravenously. Administration of the medicament may be performed by a plurality of persons working in concert. Administering the agent includes, for example, prescribing to the subject the agent to be administered and/or providing instructions, either directly or by another person, to take the particular agent, the taking being by self-delivery (e.g., by oral delivery, subcutaneous delivery, intravenous delivery through a centerline, etc.); or by a trained professional, e.g., intravenous delivery, intramuscular delivery, intratumoral delivery, etc.

As used herein, the term "high concentration" refers to the concentration of SDC-TRAP that accumulates in a target cell of the invention due to selective binding of the binding moiety of the SDC-TRAP to the target protein. In one embodiment, the concentration is higher than in a similar cell that does not overexpress the target protein (e.g., a lung cancer cell) as compared to a non-cancerous lung cell. In another embodiment, the concentration in the target cell is higher compared to a cell that does not express or overexpress the target protein. In exemplary embodiments, the high concentration is 1.5, 2,3, 4, 5, 10, 15, 20, 50, 100, 1000-fold or more of the cells not targeted by the SDC-TRAP molecules of the invention.

The term "moiety" generally refers to a portion of a molecule, which may be a functional group, a group of functional groups, and/or a specific atomic group within the molecule that is responsible for a characteristic chemical, biological, and/or medical property of the molecule.

The term "binding moiety" refers to a low molecular weight (e.g., less than about 2500, 200, 1600, 800, 700, 600, 500, 400, 300, 200, or 100, etc. daltons) organic compound that can be used as a therapeutic or modulator of a biological process. Binding moieties include molecules that can bind to a biopolymer such as a protein, nucleic acid, or polysaccharide and act as effectors to alter the activity or function of the biopolymer. Binding moieties may have a variety of biological functions, serve as cell signaling molecules, serve as tools in molecular biology, serve as pesticides in medicine, serve as pesticides in agriculture, and many other roles. These compounds may be natural (e.g., secondary metabolites) or artificial (e.g., antiviral drugs); they may have beneficial effects against disease (e.g. drugs) or may be harmful (e.g. teratogens and carcinogens). Biopolymers such as nucleic acids, proteins and polysaccharides (e.g. starch or cellulose) are not binding moieties, although their constituent monomers (ribonucleotides or deoxyribonucleotides, respectively, amino acids and monosaccharides) are generally considered binding moieties. Small oligomers are also commonly considered binding moieties such as dinucleotides, peptides (e.g. the antioxidant glutathione) and disaccharides (e.g. sucrose).

As used herein, "protein interaction binding moiety" or "binding moiety" refers to a binding moiety or a portion thereof that interacts with a predetermined target. The interaction is achieved by a certain degree of specificity and/or affinity for the target. Specificity and affinity are generally desirable, although in some cases, higher specificity may compensate for lower affinity, and higher affinity may compensate for lower specificity. Affinity and specificity requirements will vary depending on a variety of factors including, but not limited to, the absolute concentration of the target, the relative concentration of the target (e.g., in cancer as compared to normal cells), potency and toxicity, route of administration, and/or diffusion or transport into the target cell. The target may be a molecule of interest and/or located in a region of interest. For example, the target may be a therapeutic target and/or a protein that is located in a targeted region for treatment (e.g., overexpressed in cancer cells compared to normal cells). In a particular example, the target may be a chaperone protein, such as Hsp90, and the binding moiety may be an Hsp90 binding moiety (e.g., a therapeutic, cytotoxic, or imaging moiety). Preferably, the binding moiety will enhance, be compatible with, or not substantially reduce passive transport of the conjugate comprising the binding moiety into a cell (e.g., a cell comprising the target protein).

The term "effector moiety" refers to a molecule or portion thereof that functions on and/or near a target. In various preferred embodiments, the effector moiety is a binding moiety or a portion thereof. Effects may include, but are not limited to, therapeutic effects, imaging effects, and/or cytotoxic effects. At the molecular or cellular level, the effect may include, but is not limited to, promotion or inhibition of target activity, labeling of the target, and/or cell death. Preferably, the effector moiety will enhance, be compatible with, or not substantially reduce passive transport of the conjugate comprising the effector moiety into the cell comprising the target. Different effector moieties may be used together, and a therapeutic agent according to the invention may comprise more than one effector moiety (e.g. two or more different (or the same) effector moieties in a single therapeutic agent according to the invention, two or more different therapeutic agents according to the invention comprising different effector moieties).

In some embodiments, the effector moiety is selected from a peptidyl-prolyl isomerase ligand; rapamycin, cyclosporin a; steroid hormone receptor ligands, antimitotic agents, actin binding agents, camptothecin, topotecan, combretastatin, capecitabine, gemcitabine, vinca alkaloids, platinum containing compounds, metformin, HDAC inhibitors, thymidylate synthase inhibitors; a nitrogen mustard; 5-fluorouracil (5-FU) and derivatives thereof, or combinations thereof.

In some embodiments, the effector moiety is selected from FK 506; rapamycin, cyclosporin a, estrogen, progesterone, testosterone, taxane, colchicine, nocodazole (nocadozole), vinblastine, vincristine, cytochalasin, lachrrine, phalloidin, lenalidomide, pomalidomide, SN-38, topotecan, combretastatin, capecitabine, gemcitabine, vinca alkaloid, metformin, suberoylanilide hydroxamic acid (SAHA), methotrexate, pemetrexed, raltitrexed, bendamustine, melphalan; 5-fluorouracil (5-FU), a vildagliptin and DM1, or a combination thereof.

The term "small molecule drug conjugate that is trapped intracellularly" or "binding moiety drug conjugate that is trapped intracellularly" or "SDC-TRAP" refers to a binding moiety and an effector moiety that bind to or function as if they bind to each other. The binding moiety and the effector moiety can be bound by essentially any chemical or physical force, either directly (e.g., the binding moiety and the effector moiety are considered to be two moieties on the same molecule, or a single moiety with two functions) or through an intermediate (e.g., a linker). For example, the binding moiety and the effector moiety may be linked by one or more covalent bonds, ionic bonds, hydrogen bonds, hydrophobic interactions, dipole-dipole forces, ionic-dipole forces, dipole-induced dipole forces, transient dipole-induced dipole forces, and/or combinations thereof. Preferably, the SDC-TRAP will be capable of passive and/or active transport into cells comprising the target. Furthermore, the SDC-TRAP molecules of the invention can comprise a plurality of effector molecules conjugated to a binding moiety.

As used herein in the context of a binding moiety, effector moiety, and/or SDC-TRAP, the term "linker" or "linking moiety" refers to a chemical moiety that links two other moieties (e.g., a binding moiety and an effector moiety). The linker may covalently bind the binding moiety and the effector moiety. The linker may comprise a cleavable linker, for example an enzymatically cleavable linker. The linking group may include a disulfide, carbamate, amide, ester, and/or ether linking group.

As used herein, a "ligand" is a substance (e.g., a binding moiety) that can form a complex with a biomolecule. The formation of the ligand and/or ligand-biomolecule complex may have a biological or chemical effect, such as a therapeutic effect, a cytotoxic effect and/or an imaging effect.

As used herein, a "prodrug" is a pharmacological substance that is administered in an inactive or less than fully active form and is subsequently converted to an active pharmacological agent (i.e., drug) by a metabolic process. The prodrugs can be used to improve the absorption, distribution, metabolism, and/or excretion pattern of the desired drug. Prodrugs can also be used to improve the selectivity of the interaction of a desired drug with cells or processes that are not their intended target (e.g., to reduce adverse or unintended effects of a desired drug, such as a chemotherapeutic drug).

The phrase "Hsp 90 ligand or prodrug thereof" generally refers to molecules that bind to and in some cases affect Hsp90 and inactive forms thereof (i.e., prodrugs). Hsp90 ligands may be "Hsp 90 inhibitors", which are understood to be therapeutic agents that reduce Hsp90 activity by direct interaction with Hsp90 or by, for example, preventing the formation of Hsp90/CDC37 complexes, thereby inhibiting the expression and proper folding of at least one client protein of Hsp 90. "Hsp 90" includes each member of the heat shock protein family having a mass of about 90-kilodaltons. For example, in humans, the highly conserved Hsp90 family includes cytoplasmic Hsp90^αAnd Hsp90^βSubtypes, and GRP94 found in endoplasmic reticulum and HSP75/TRAP1 found in mitochondrial matrix as used herein, Hsp90 inhibitors include, but are not limited to, ganetespib, geldanamycin (tanespimycin) such as IPI-493, macbecin, tripterine, tanespicin such as 17-AAG (apramycin), KF-55823, radicicol, KF-58333, KF-58332, 17-DMAG, IPI-504, BIIB-021, BIIB-028, PU-H64, PU-H71, PU-DZ8, PU-HZ151, SNVERX-2112, SNX-2321, SNX-5422, SNX-7081, SNX-8891, SNX-823, ABI-287, ABI-13387, MPC-113497, SNNV-897, SNX-5432, SNX-8289, SNNV-57, SNX-8289, SNH-33, SNNV-43, SNH-43, SNNV-8226, SNH-43, SNX-8226, SNNV-8226, SNH-43, SNH-H-43, SNH-D-43, SNH-43, SND-D-8226, SNH-3, SNH-D-3, SNH-D-3, SNE-D-3HSP990, SST-0201C L1, SST-0115AA1, SST-0221AA1, SST-0223AA1, novobiocin (C-terminal Hsp90i), herbinmycinA, radicicol, CCT018059, PU-H71 or celastrin.

The term "therapeutic moiety" refers to a molecule, compound, or fragment thereof (e.g., a medicament, drug, etc.) that is used to treat a disease or to improve the health of an organism or otherwise exhibit a healing capacity. The therapeutic moiety may be a chemical substance of natural or synthetic origin or a fragment thereof for its specific effect against a disease such as cancer. Therapeutic agents used to treat cancer may be referred to as chemotherapeutic agents. As described herein, the therapeutic moiety is preferably a small molecule. Exemplary small molecule therapeutic agents include those less than 800 daltons, 700 daltons, 600 daltons, 500 daltons, 400 daltons, or 300 daltons.

The term "cytotoxic moiety" refers to a molecule, compound, or fragment thereof that has a toxic or toxic effect on or kills a cell. Chemotherapy and radiation therapy are forms of cytotoxic therapy. Treatment of cells with cytotoxic moieties can produce a variety of results: cells may die, stop actively growing and dividing, or activate genetic programs of controlled cell death (i.e., apoptosis). Examples of cytotoxic moieties include, but are not limited to, SN-38, bendamustine, VDA, doxorubicin, pemetrexed, vorinostat, lenalidomide, irinotecan, ganetespib, docetaxel, 17-AAG, 5-FU, abiraterone, crizotinib, KW-2189, BUMB2, DC1, CC-1065, adolesin, or fragments thereof.

The term "imaging moiety" refers to a molecule, compound, or fragment thereof that facilitates a technique and/or process for creating an image or making measurements of a cell, tissue, and/or organism (or a portion or function thereof) for clinical and/or research purposes. The imaging portion may generate signals by, for example, emitting and/or interacting with electromagnetic, nuclear, and/or mechanical (e.g., acoustic in ultrasound) energy. The imaging section may be used in various radiological, nuclear medicine, endoscopic, thermal imaging, photographic, spectroscopic, and microscopy methods, for example.

"drug conjugate" refers to a non-naturally occurring molecule that includes a binding moiety (e.g., an Hsp90 targeting moiety) associated with an effector moiety, where the two components may also be covalently bonded to each other, either directly or through a linking group.

The term "drug" refers to any active agent that affects any biological process. For the purposes of this application, an active agent, which is considered to be a drug, is an agent that exhibits pharmacological activity. Examples of medicaments include active agents for the prevention, diagnosis, alleviation, treatment or cure of disease conditions.

"pharmacological activity" refers to an activity that modulates or alters a biological process resulting in a phenotypic change such as cell death, cell proliferation, and the like.

"pharmacokinetic properties" refer to parameters that describe the configuration of an active agent in an organism or host.

"half-life" refers to the time to eliminate half of the administered drug by biological processes (e.g., metabolism, excretion, etc.).

The term "efficacy" refers to the effectiveness of a particular active agent for its intended purpose, i.e., the ability of a given active agent to elicit its desired pharmacological effect.

Intracellular trapped binding moiety-effector moiety drug conjugates (SDC-TRAP)

The invention provides SDC-TRAP and SDC-TRAP compositions, kits, and methods of use thereof. The SDC-TRAP includes a binding moiety (e.g., a binding moiety such as a ligand) conjugated to an effector moiety (e.g., a pharmacological agent such as a drug or an imaging agent). The two moieties may be linked by a linker, such as a covalently bonded linker. SDC-TRAP can be used in a variety of therapeutic, imaging, diagnostic, and/or research applications. In one illustrative example of cancer treatment, a SDC-TRAP can be a drug conjugate of an Hsp90 binding moiety (e.g., an Hsp90 ligand or inhibitor) associated with an effector moiety (e.g., a therapeutic agent or cytotoxic agent).

In various embodiments, the SDC-TRAP may also be characterized by the binding moiety (e.g., targeting moiety) and the effector moiety being different, such that the drug conjugate may be viewed as a heterodimeric compound produced by binding two different moieties. Functionally, SDC-TRAP molecules have both targeting and effector functions (e.g., therapeutic, imaging, diagnostic). These functions are provided by corresponding chemical moieties that may be different (or in some cases the same). The SDC-TRAP can include any one or more binding moieties conjugated to any one or more effector moieties. In some embodiments, a composition or method may comprise a combination (e.g., combination therapy and/or multi-target therapy) of two or more binding moieties and/or two or more effector moieties administered with one or more different types of SDC-TRAPs.

In various embodiments, the SDC-TRAP is further characterized by its ability to passively diffuse and/or actively transport into target cells of interest. The diffusion and/or transport properties of the SDC-TRAP may be derived at least in part from the ionic, polar and/or hydrophobic properties of the SDC-TRAP. In preferred embodiments, the SDC-TRAP enters the cell primarily by passive diffusion. The diffusion and/or transport properties of the SDC-TRAP can be derived at least in part from the molecular weight of the SDC-TRAP, the binding moiety, the effector moiety, and/or the weight similarity between the binding moiety and the effector moiety. The SDC-TRAP is desirably small, e.g., compared to an antibody-drug conjugate ("ADC"). For example, the molecular weight of the SDC-TRAP can be less than about 5000, 2500, 2000, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, or 400 daltons. The binding moiety and the effector moiety can each be less than about 1000, 900, 800, 700, 600, 500, 400, 300, or 200 daltons. The binding moiety and the effector moiety can be approximately equal in size (e.g., a weight difference of less than 400, 350, 300, 250, 200, 150, 100, or 50 daltons).

Delivery of an effector molecule by SDC-TRAP may result in greater efficacy compared to administration of a non-targeted drug comprising the same effector moiety, e.g., because SDC-TRAP may be localized at a desired target for an extended period of time by association of the binding moiety with its target. Such positioning may result in the effector moiety being activated and/or released in the target cell and/or tissue for an extended period of time. The resonance time can be selected by careful design of the linker moiety. In contrast, due to the lack of intracellular "anchoring", in vivo administration of a drug may itself be more prone to have a shorter resonance time in a given target cell and/or tissue if it does cross into the cell.

SDC-TRAPs can be efficiently taken up or internalized by target cells, in part because they contain a targeting moiety and are relatively small in size. In contrast, uptake or internalization is relatively inefficient for ADCs, which must handle limited antigen expression and relatively inefficient mechanisms of internalization for the antibody portion of the molecule. Hsp90 provides a good illustrative example of the distinction between SDC-TRAP and conventional ADC. By comparison, the localization rate of radiolabeled monoclonal antibody in the patient's tumor is low, approximately 0.003-0.08% of the injected dose per gram of tumor. In contrast, higher rates of accumulation were measured for SDC-TRAP in mouse tumor xenografts (15-20% of injected dose per g tumor).

The SDC-TRAP drug conjugates according to the invention may show significant improvements over the prior art in targeting drugs. SDC-TRAP has a wide range of applications in many therapeutic, imaging, and diagnostic applications. As discussed above, SDC-TRAP is advantageously small compared to ADC, enabling better penetration of solid tumors and faster clearance from normal tissues (e.g., reduced toxicity). Given the simpler chemistry involved, the design of SDC-TRAPs (e.g., structure-property relationships) can be established using methods and principles that are well within the skill of one of ordinary skill in the art, and can also readily provide concomitant imaging diagnostics for targeted therapies.

The SDC-TRAP of the invention is characterized by selectively targeting the SDC-TRAP to target cells in which a target protein is overexpressed. This results in a high intracellular concentration of SDC-TRAP molecules in the target cells compared to non-targeted cells. Similarly, the SDC-TRAP of the invention is characterized by a low concentration of SDC-TRAP in non-targeted cells.

One illustrative embodiment relates to conjugates of an Hsp90 binding moiety linked to a chelator (i.e., an effector moiety, which conjugate may serve as an imaging agent for the cell/tissue to which the conjugate is targeted for metals such as In or Gd). Another illustrative embodiment relates to conjugates of Hsp90 binding moieties linked to chemotherapeutic agents (i.e., effector moieties such as SN-38). Alternatively, illustrative SDC-TRAPs are contemplated in which an Hsp90 targeting moiety with a radiolabeled halogen (e.g., iodine isotope) can be used to image the cells/tissues targeted by the conjugate, and the effector moiety can be a drug to treat the targeted cells/tissues. Thus, the progress of treatment can be determined by imaging the tissue being treated and examining the image for the presence of the labeled conjugate. Such embodiments are readily adaptable to essentially any cancer or other chemotherapeutic target. Molecular targets (e.g., interacting with a binding moiety) for targeting a particular cell or tissue can be selected based on their presence in the particular cell or tissue and/or their relative abundance in the target cell or tissue (e.g., cells associated with disease as compared to normal cells).

The SDC-TRAP molecules of the invention represent a new class of drugs. One particular advantage of SDC-TRAPs is that they can be designed to selectively deliver effector moieties (e.g., chemotherapeutic drugs) to target cells due to the relative overexpression or presence of the molecular targets of the binding moieties in the cells. After the binding moiety binds to the molecular target, the effector moiety can then be used to act on the cell (e.g., by cleaving a linker moiety that binds the binding moiety and the effector moiety together). Thus, SDC-TRAP employs a different mechanism than strategies currently used in the art (e.g., using HPMA copolymer-Hsp 90i conjugates, Hsp90i prodrugs, nanoparticle-Hsp 90i conjugates, or micellar methods to deliver Hsp90 inhibitors to cells).

The SDC-TRAP can also be described by the following formula:

binding moiety-L-E

Where the "binding moiety" is a protein-interacting binding moiety, L is a conjugating or linking moiety (e.g., a bond or linking group), E is an effector moiety these elements are discussed in other illustrative examples below.

Once the SDC-TRAP molecules of the invention enter the target cells, the effector molecules are released from the SDC-TRAP. In one embodiment, the effector molecule is not active until released from the SDC-TRAP. Thus, once the SDC-TRAP molecule enters the target cell, there is an equilibrium between free and bound SDC-TRAP molecules. In one embodiment, the effector moiety is released from the SDC-TRAP only when the SDC-TRAP is not associated with the target protein. For example, when the SDC-TRAP molecule is unbound, intracellular enzymes may access the linker region, thereby releasing the effector moiety. Alternatively, when the free SDC-TRAP molecule may be capable of releasing the effector molecule by, for example, hydrolysis of the bond or linker connecting the binding moiety and the effector moiety.

Thus, the rate of release of the effector molecules and the amount of released effector molecules can be controlled by using binding moieties that bind to the target protein with different affinities. For example, a binding moiety that binds with lower affinity to the target protein will be free, resulting in a higher concentration of unbound intracellular SDC-TRAP and hence a higher concentration of free effector molecules. Thus, in at least one embodiment, irreversibly bound binding moieties are incompatible with certain aspects of the invention (e.g., those embodiments in which effector molecule release is based on free intracellular SDC-TRAP molecules).

In one embodiment, for example, SDC-TRAP has advantageous safety when compared to, for example, a binding moiety or effector molecule alone. One of the reasons for the increased safety is the lack of rapid clearance of SDC-TRAP molecules into the target cells.

In the examples a number of exemplary SDC-TRAP molecules are listed. In particular, various Hsp 90-specific SDC-TRAP molecules are described and used to demonstrate the efficacy of SDC-TRAP molecules.

Binding moieties

The primary function of the binding moiety is to ensure that the SDC-TRAP delivers its payload (effector moiety) to its target by binding to a molecular target in or on the target cell or tissue. In this regard, the binding moiety also need not have an effect on the target (e.g., in the case of Hsp90 targeting moieties, Hsp90 is inhibited in the manner known for Hsp90i to act, i.e., to exhibit pharmacological activity or interfere with its function), but in certain embodiments the binding moiety does have an effect on the target. Thus, in various embodiments, the activity of SDC-TRAP is due only to the effector moiety exerting a pharmacological effect on the target cells, which has been better facilitated by the drug conjugate targeting the target cells. In other embodiments, the activity of the SDC-TRAP is due in part to the binding moiety, i.e., the binding moiety may have an effect beyond targeting.

The molecular target of the binding moiety may or may not be part of a complex or structure of a plurality of biomolecules (e.g., lipids, wherein the complex or structure may include lipoproteins, lipid bilayers, etc.). However, in many embodiments, the molecular target to which the binding moiety binds will be free (e.g., cytoplasmic globular proteins and/or not part of a macromolecular assembly or aggregate). The present invention can exploit the selective high presence of molecular targets at highly physiologically active sites, such as Hsp90 in oncology procedures. For example, where the drug target is an intracellular drug target, the corresponding molecular target (e.g., Hsp90) may be present in the cell. Similarly, where the drug target is an extracellular drug target, the corresponding molecular target (e.g., Hsp90) may be extracellular, near, or associated with an extracellular cell membrane of the target cell or tissue.

In various embodiments, the binding moiety may affect a target cell or tissue (e.g., where an Hsp90 targeting moiety that actually inhibits Hsp90 (e.g., Hsp90 i)). in such embodiments, the pharmacological activity of the binding moiety contributes to, supplements or enhances the pharmacological activity of the effector moiety. such embodiments overcome the advantages of combination therapy (e.g., cancer combination therapy of Hsp90i and a second drug such as ganetespib or crizotinib) by providing a therapy that can be performed by administration of a single SDC-TRAP that achieves both the benefits of combination therapy and targetingSirolimus (e.g., Renal Cell Carcinoma (RCC), colon, breast and/or NSC L C); P L X4032 (e.g., in melanoma); cisplatin (e.g., colon, breast cancer); AZD8055 (e.g., in NSC L C); and crizotinib (e.g., A L K)⁺NSC L C)).

A range of pharmaceutical activities can be achieved by judicious and judicious selection of binding and effector moieties. For example, in order to treat solid tumors such as colon cancer, it is often necessary to use high continuous doses of antimetabolites such as capecitabine or gemcitabine, in combination with other drugs. To meet this need, conjugates can be designed with Hsp90 targeting moieties that have a lower binding affinity or inhibitory activity for Hsp90, as determined, for example, by HER2 degradation assay. Such conjugates can include an effector moiety such as 5-FU (which is a potent antimetabolite) to provide high doses of the conjugate that can be administered relatively frequently. Due to the plasma stability of SDC-TRAP of the invention and the ability of Hsp90 targeting moieties to deliver antimetabolites to desired cells or tissues, this approach not only achieves the goal of providing high doses of antimetabolite fragments at the tumor site, but also reduces the toxicity of the drug administered alone.

In embodiments where solid tumors such as SC L C or colorectal cancer are treated with drugs such as topotecan or irinotecan, only low doses of the drug may be administered because the intrinsic activity of these drugs is very high, SDC-TRAP should be designed to provide low doses of such drugs at the target tissue in this case, for example, Hsp90 targeting moieties with higher binding affinity or inhibitory activity to Hsp90 (as determined, for example, by HER2 degradation assays) may sufficiently maintain the presence of the drug in the tissue at very high levels to ensure that sufficient drug reaches and is retained by the desired target tissue due to the low dose.

In various illustrative embodiments in which the molecular target of the binding moiety is Hsp90, the binding moiety may be an Hsp90 targeting moiety, such as a triazole/resorcinol-based compound that binds Hsp90, or a resorcinol amide-based compound that binds Hsp90, such as ganetespib or a tautomer/derivative/analog thereof, AUY-922 or a tautomer/derivative/analog thereof, or AT-13387 or a tautomer/derivative/analog thereof.

In another embodiment, the binding moiety may advantageously be an Hsp90 binding compound of formula (I):

wherein

R¹May be an alkyl, aryl, halide, carboxamide or sulfonamide; r²Can be alkyl, cycloalkyl, aryl or heteroaryl, wherein when R is²When it is 6-membered aryl or heteroaryl, R²Substituted at the 3-and 4-positions relative to the point of attachment on the triazole ring through which linker L is attached, R³Can be SH, OH, -CONHR⁴Aryl or heteroaryl, wherein when R is³When it is 6-membered aryl or heteroaryl, R³Substitution at position 3 or 4.

In another embodiment, the binding moiety may advantageously be an Hsp90 binding compound of formula (II):

wherein

R¹Can be alkyl, aryl, halogen, carboxamido, sulfonamido; r²May be an optionally substituted alkyl, cycloalkyl, aryl or heteroaryl group. 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.

In another embodiment, the binding moiety may advantageously be an Hsp90 binding compound of formula (III):

wherein

X, Y and Z may independently be CH, N,O or S (with appropriate substitution and satisfying the valency of the corresponding atom and the aromaticity of the ring); r¹May be an alkyl, aryl, halide, carboxamido or sulphonamido group; r²Which may be substituted alkyl, cycloalkyl, aryl or heteroaryl, wherein the linker L is attached directly or to an extended substituent on these rings, R³Can be SH, OH, NR⁴R⁵and-CONHR⁶To which effector moieties may be attached; r⁴And R⁵May independently be H, alkyl, aryl or heteroaryl; r⁶Can be an alkyl, aryl or heteroaryl group having at least one functional group to which an effector moiety can be attached. Examples of such compounds include AUY-922:

in another embodiment, the binding moiety may advantageously be an Hsp90 binding compound of formula (IV):

wherein

R¹May be alkyl, aryl, halogen, carboxamido or sulphonamido; r²And R³Independently is optionally substituted by hydroxy, halogen, C₁-C₂Alkoxy, amino, mono-and di-C₁-C₂C substituted by one or more of alkylamino groups₁-C₅A hydrocarbyl group; a 5 to 12 membered aryl or heteroaryl; or, R²And R³Together with the nitrogen atom to which they are attached form a 4-to 8-membered monocyclic heterocyclyl, wherein up to 5 ring members are selected from O, N and S. Examples of such compounds include AT-13387:

in certain embodiments, the binding moiety may be a prodrug of the Hsp90 binding compound in order to enhance the bioavailability or delivery of the drug conjugate.

Specific examples of suitable Hsp90 targeting moieties include geldanamycin, e.g., IPI-493Macbecin, tripterine, tanespimycins, e.g. 17-AAGKF-55823Radicicol, KF-58333KF-5833217-DMAGIPI-504BIIB-021BIIB-028、PU-H64PU-H71PU-DZ8PU-HZ151SNX-2112SNX-2321SNX-5422SNX-7081SNX-8891、SNX-0723SAR-567530、ABI-287、ABI-328、AT-13387NSC-113497PF-3823863PF-4470296EC-102、EC-154、ARQ-250-RP、BC-274VER-50589KW-2478BHI-001、AUY-922EMD-614684EMD-683671、XL-888、VER-51047KOS-2484、KOS-2539、CUDC-305MPC-3100CH-5164840PU-DZ13PU-HZ151PU-DZ13VER-82576VER-82160VER-82576VER-82160NXD-30001NVP-HSP990SST-0201CL1SST-0115AA1SST-0221AA1SST-0223AA1Novobiocin (C-terminal Hsp90i) or a tautomer/derivative/analog thereof. The selection of other Hsp90 targeting moieties will be within the purview of one of ordinary skill in the art. Similarly, the choice of binding moieties suitable for other molecular targets and/or other applications will be within the ability of one of ordinary skill in the art.

In addition, Hsp90 targeting moieties may be used to construct SDC-TRAP molecules for the treatment of inflammation. For example, a binding moiety of a compound shown in tables 5, 6, and 7, including U.S. patent publication 2010/0280032 (which is incorporated herein by reference in its entirety), or a compound of any formula therein, or a tautomer, pharmaceutically acceptable salt, solvate, clathrate, hydrate, polymorph, or prodrug thereof, inhibits the activity of Hsp90, thereby causing degradation of Hsp90 client protein. Any of these compounds may be conjugated to an effector molecule to form SDC-TRAP. The glucocorticoid receptor is a client protein of Hsp90 and binds to Hsp90 when it is in a conformation that enables binding to glucocorticoid ligands, such as cortisol. Once glucocorticoids bind to GR, the receptor dissociates from Hsp90 and translocates to the nucleus where it regulates gene expression to reduce inflammatory responses, such as the production of proinflammatory cytokines. Thus, glucocorticoids may be administered to patients in need of immunosuppression as well as to patients suffering from inflammatory and autoimmune diseases. Unfortunately, although glucocorticoids are effective in relieving inflammation, they have a number of serious side effects including osteoporosis, muscle atrophy, hypertension, insulin resistance, trunk obesity and fat redistribution and inhibition of wound repair. Inhibition of Hsp90 causes a change in GR activity, resulting in a reduction in inflammatory response similar to that seen with glucocorticoids. However, since the mechanism of reducing inflammation is different from that of glucocorticoids, it is expected that some or all of the side effects of glucocorticoid therapy may be reduced or eliminated.

Effector moiety

The effector moiety may be any therapeutic or imaging agent that can be conjugated to the binding moiety and delivered to the molecular target of the binding moiety in such a conjugated state. In some cases, the effector molecule may require a linking moiety for conjugation (e.g., not directly conjugated to a binding moiety). Similarly, in certain instances, the effector molecule may block or reduce the ability of the binding moiety and/or SDC-TRAP to reach the target, as long as the SDC-TRAP can still affect the target. However, in preferred embodiments, the effector moiety is readily conjugated and may facilitate delivery to and impact on the target.

In various embodiments, the SDC-TRAP may have other cell penetration patterns via the effector moiety in addition to simple passive diffusion. Examples of such are SDC-TRAP comprising as an effector moiety an antifolate or fragment thereof, e.g. temozolomide, mitozolamide, nitrogen mustard (nitrogen mustard), estramustine or nitrogen mustard (chloromethine). In this case, a conjugate of a binding moiety (e.g., an Hsp90 inhibitor) and pemetrexed (or a folate-recognizing fragment thereof) may undergo folate-receptor-mediated endocytosis rather than passive diffusion. Once inside the target cell, the SDC-TRAP can bind to a molecular target (e.g., Hsp90 protein) via its binding moiety (e.g., an Hsp90 inhibitor).

As described in more detail below, the effector moiety may comprise a region that may be modified and/or participate in covalent attachment to the binding moiety without substantially adversely affecting the ability of the binding moiety to bind to its target. The effector moiety may be a drug molecule or derivative thereof which substantially retains activity when conjugated to the binding moiety. It will be appreciated that drugs that otherwise have good and desirable activity may prove challenging to administer routinely (e.g., due to poor bioavailability or undesirable side effects in vivo before reaching their target) -these drugs may be "restarted" for use as the effector moiety in the SDC-TRAP of the present invention.

Examples of effector moieties include: peptidyl-prolyl isomerase ligands, such as FK 506; rapamycin, cyclosporin a, etc.; steroid hormone receptor ligands, such as naturally occurring steroid hormones, e.g., estrogens, progesterone, testosterone, and the like, as well as synthetic derivatives and mimetics thereof; binding moieties that bind to cytoskeletal proteins, such as antimitotic agents, e.g., taxanes, colchicine, nocodazole, vinblastine, and vincristine; actin binding agents such as cytochalasin, latrunculin, phalloidin, and the like; lenalidomide, pomalidomide; camptothecin, including SN-38

Topotecan, combretastatin, capecitabine, gemcitabine, vinca alkaloids, platinum containing compounds, metformin, HDAC inhibitors (e.g., suberoylanilide hydroxamic acid (SAHA)); thymidylate synthase inhibitors such as methotrexate, pemetrexed, and raltitrexed; nitrogen mustards, such as bendamustine and melphalan; 5-fluorouracil (5-FU) and derivatives thereof; and agents used in ADC drugs, such as vildagliptin and DM1, or tautomers/derivatives/analogs thereof.

The effector moiety may be derived from a library of naturally occurring or synthetic molecules, including libraries of compounds produced by combinatorial means, i.e. a diverse combinatorial library of compounds. When obtained from such a library, the effector moiety employed will exhibit some desired activity in an appropriate activity screening assay. It is contemplated that in other embodiments, the drug conjugate may include more than one effector moiety, thereby providing greater flexibility to the medicinal chemist. The number of effector moieties attached to a binding moiety (e.g., an Hsp90 targeting moiety) is generally limited only by: a binding moiety (e.g., an Hsp90 targeting moiety) and/or the number of any available sites for attachment to an effector moiety; steric considerations, such as the number of effector moieties that may actually be linked to a binding moiety (e.g., an Hsp90 targeting moiety); and the ability of the drug conjugate to bind to a molecular target (e.g., Hsp90 protein) is retained.

Specific drugs from which effector moieties may be derived include: psychopharmacological agents, e.g. inInhibitors of the central nervous system, e.g. general anesthetics (barbiturates, benzodiazepines)Steroids, cyclohexanone derivatives and other drugs), sedative-hypnotics (benzodiazepines)Barbiturates, piperidinediones and triones, quinazoline derivatives, carbamates, aldehydes and derivatives, amides, acyclic ureides, benzazepinesAnd related drugs, phenothiazines, etc.), central voluntary muscle tone modulators (anticonvulsants such as hydantoin, barbiturate, oxazolidinedione, succinimide, acylureide, glutarimide, benzodiazepineSecondary and tertiary alcohols, dibenzoazepinesDerivatives, valproic acid and derivatives, GABA analogs, etc.), analgesics (morphine and derivatives, oripavine derivatives, morphinan derivatives, phenylpiperidine, 2, 6-methane-3-benzazocine (2, 6-methane-3-benzazocine) derivatives, diphenylpropylamine and isostere, salicylates, p-aminophenol derivatives, 5-pyrazolone derivatives, arylacetic acid derivatives, fenamate and isostere, etc.), and antiemetics (anticholinergics, antihistamines, anti-dopaminergic agents, etc.); central nervous system stimulants such as stimulants (respiratory stimulants, convulsions stimulants, psychomotor stimulants), narcotic antagonists (morphine derivatives, oripavine derivatives, 2, 6-methane-3-benzoxazine (2,6-methane-3-benzoxacine) derivatives, morphinan derivatives), nootropic agents; psychopharmacologic/psychotropic agents, e.g. anxiolytic sedatives (benzodiazepines)Propylene glycol carbamate), antipsychotics (phenothiazine derivatives, thioxanthine derivatives, other tricyclic compounds, propylphenone derivatives and isosteres, diphenylbutylamine derivatives, substituted benzamides, arylpiperazine derivatives, indole derivatives, etc.), antidepressants (tricyclic compounds, MAO inhibitors, etc.); respiratory drugs, such as central antitussives (opioid alkaloids and derivatives thereof); an immunosuppressant; pharmacodynamic agents, such as peripheral nervous system drugs, e.g. local anesthetics (ester derivatives, amide derivatives); drugs acting on synapses or neuroeffector junction sites, such as cholinergic agents, cholinergic blockers, neuromuscular blockers, adrenergic agents, anti-adrenergic agents; smooth muscle active agents, such as spasmolytics (anticholinergics, muscular spasmolytics), vasodilators, smooth muscle stimulators; histamine and antihistamines, e.g. histamine and derivatives thereof (betazoles), antihistamines (H)₁-antagonists, H₂Antagonists), histamine-metabolizing drugs, cardiovascular drugs, such as cardiotonics (plant extracts, butenolide, pentadienolide, alkaloids from the species of the genus Gracilaria, ionophores, adrenergic receptor stimulators, etc.), antiarrhythmic drugs, antihypertensives, hypolipidemic drugs (Chlorofloxacin derivatives, nicotinic acid derivatives, hormones and analogs, antibiotics, salicylic acid and derivatives), anti-varicose drugs, hemostatic drugs, chemotherapeutic agents, such as anti-infectives, such as ectoparasiticides (chlorinated hydrocarbons, pyrethrins, sulfurized compounds), anthelmintics, antiprotozoals, antimalarials, anti-amebic agents, anti-leishmaniasis drugs, anti-trichomonas agents, anti-trypanosomiasis agents, sulfonamides, anti-mycobacterial drugs, chemotherapeutic antiviral agents, etc., and cytostatics, i.e., antitumor agents or cytotoxic drugs, such as alkylating agents, such as dichloromethyldiethylamine hydrochloride (nitrogen mustard, Murgen, HN2), Cyxanthane (cyclophosphamide), chlorambucil (Alphan-L), Alphan, Alphaninozide, Alphan-L), Alphane, 3, Alphanthin (E, AlphanThiophosphamide), carmustine (BiCNU, BCNU), lomustine (CEeNU, CCNU), streptozotocin (Zanosar) and the like, plant alkaloids such as vincristine (Oncovin), vinblastine (Velban, Velbe), Taxol (Taxol) and the like, antimetabolites such as Methotrexate (MTX), mercaptopurine (Purinethol, 6-MP), thioguanine (6-TG), fluorouracil (5-FU), cytarabine (Cytosar-U, Ara-C), azacitidine (Mylosar, 5-AZA) and the like, antibiotics such as Actinomycin (Actinomycin D, Cosmegen), doxorubicin (Adriamycin), daunorubicin (Duanomycin, Cerubicin), idarubicin (Idamycin), bleomycin (bleomycin), streptomycin (gentamycin, streptomycin (gentamycin, streptomycin, streptomyc,Nystatin, trichostatin, hamycin, lucinomycin, mepartricin, natamycin, nystatin, pecilostacin, epimycin; synthetic antifungal agents, such as allylamines, e.g., butenafine, naftifine, terbinafine; imidazoles such as bifonazole, butoconazole, chlordantoin, clomiprazole, and the like; thiocarbamates such as tosiramate; triazoles such as fluconazole, itraconazole, terconazole; anthelmintic agents, for example: arecoline, aspidin, aspidinol, dichlorophen, nobiletin, bitter octyl, naphthalene, niclosamide, punicine, quinacrine, alantolactone, amoxapine, nitrothiocyanamide, ascaridole, benomyl, disulfotobenzene, carbon tetrachloride, carvacrol, cyclobendazole, diethylcarbamazine, and the like; antimalarial drugs, for example: acephatane, amonoquine, arteether, artemether, artemisinin, artesunate, atovaquone, biaccordingly, berberine, swertia, proguanil, chloroquine, cinchrogaunil, cinchona, cinchonidine, cinchonine, cyclochloroguanidine, gentiopicroside, halofantrine, hydroxychloroquine, mefloquine hydrochloride, 3-methyl p-acetamidophenylarsonic acid, pamaquine, methoxamine, primaquine, pyrimethamine, quinacrine, quinidine, quinuclidine, quinoline, disodium hydrogen arsenate; and antiprotozoal agents, such as: chloromethoxyazine, tinidazole, isonitrodazole, ethylidene 30535, amine, pentamidine, arsanilide, acetaminidazole, anisomycin, nifuratel, tinidazole, benznidazole, suramin, etc.

Conjugation and linking moieties

The binding and effector moieties of the invention may be conjugated, for example, via a linker or linking moiety L, wherein L may be a bond or linking group.

The affinity, specificity and/or selectivity assays described herein can be used to identify appropriate linking moieties. The connecting portion may be selected based on, for example, size to provide a SDC-TRAP having size characteristics as described above. In various embodiments, the linking moiety may be selected from or derived from known chemical linking groups. The linking moiety may comprise a spacer having a reactive functional group at either end capable of covalently bonding to the drug or ligand moiety. Spacers of interest include aliphatic and unsaturated hydrocarbon chains, spacers containing heteroatoms such as oxygen (ethers, e.g. polyethylene glycol) or nitrogen (polyamines), peptides, carbohydrates, cyclic or acyclic systems which may contain heteroatoms. The spacer group may also consist of a ligand that binds to the metal such that the presence of the metal ion coordinates two or more ligands to form a complex. Specific spacing elements include: 1, 4-diaminohexane, xylylenediamine, terephthalic acid, 3, 6-dioxaoctanedioic acid, ethylenediamine-N, N-diacetic acid, 1 '-ethylenebis (5-oxo-3-pyrrolidinecarboxylic acid), 4' -ethylenedipiperidine. Potential reactive functional groups include nucleophilic functional groups (amines, alcohols, thiols, hydrazides), electrophilic functional groups (aldehydes, esters, vinyl ketones, epoxides, isocyanates, maleimides), functional groups capable of undergoing cycloaddition reactions, forming disulfide bonds or binding to metals. Specific examples include primary and secondary amines, hydroxamic acids, N-hydroxysuccinimidyl esters, N-hydroxysuccinimidyl carbonates, oxycarbonylimidazoles, nitrophenyl esters, trifluoroethyl esters, glycidyl ethers, vinyl sulfones, and maleimides. Specific linking moieties that may be used in SDC-TRAP include disulfide and stable thioether moieties.

In various embodiments, the linking moiety is cleavable, e.g., enzymatically cleavable. Once the SDC-TRAP is internalized, the cleavable linker can be used to release the effector moiety within the target cell. The sensitivity of the linking moiety to cleavage can be used to control the delivery of the effector molecule. For example, the linking moiety may be selected to provide extended or prolonged release of the effector moiety in the target cell over time (e.g., the carbamate linking moiety may undergo enzymatic cleavage by a carboxylesterase enzyme via the same cellular process used to cleave other carbamate prodrugs such as capecitabine or irinotecan). In these and various other embodiments, the linking moiety may exhibit sufficient stability to ensure good target specificity and low systemic toxicity, but not so high as to result in reduced potency and efficacy of the SDC-TRAP.

Exemplary linkers are described in U.S. Pat. No. 6,214,345(Bristol-Myers Squibb), U.S. patent application 2003/0096743 and U.S. patent application 2003/0130189 (both Seattles genetics), de Groot et al, J.Med.Chem.42,5277(1999), de Groot et al J.org.Chem.43,3093(2000), de Groot et al J.Med.Chem.66,8815, (2001), WO 02/083180(Syntarga), Carl et al J.Med.Chem. L ett.24,479, (1981), Dubowchik et al, Bioorg & Med.Chem. L ett.8,3347(1998) and Doronina et al BioConjug Chem.2006, Doronina et al Nat Biotech 2003.

In one embodiment, the SDC-TRAP comprises ganetespib or a tautomer or analog thereof as a binding moiety and SN-38 or a fragment/derivative/analog thereof as an effector moiety. One non-limiting example is SDC-TRAP-0063. The term SDC-TRAP-0063 includes compounds having the structure:

or a tautomer thereof:

identification and selection of targets and corresponding SDC-TRAPs

The present invention provides a broad spectrum of pharmacological compounds comprising an effector moiety conjugated to a binding moiety that directs the effector moiety to a biological target of interest. Although the use of Hsp90 inhibitor binding moieties conjugated to cytotoxic agent effector moieties to treat cancer is an illustrative example of the invention, SDC-TRAP is fundamentally broader in its composition and use.

In various embodiments, a broad class of SDC-TRAP pharmacological compounds directed against a biological target have the following properties:

a biological target (a cell and/or tissue target of interest, e.g., a tumor) should be accessible to the effector moiety, and the effector moiety should be known or developed against the biological target (e.g., a chemotherapeutic agent for the tumor); the biological target should be associated with a molecular target (e.g., a biomolecule capable of being specifically bound, which is uniquely represented in the biological target) that specifically interacts with a binding moiety, and the binding moiety should be known or developed for the molecular target (e.g., a ligand for the biomolecule); and the effector and binding moieties should be suitable for conjugation and should substantially retain their respective activities after conjugation. In addition, the conjugate should be able to reach and interact with the molecular target and should be suitable for administration to a subject in clinical applications (e.g., the subject may tolerate a therapeutically effective dose).

Examples of therapeutic molecular targets (i.e., binding moiety binding partners) for various conditions/disease states are given in the table below. Suitable binding moieties may be selected based on a given molecular target and/or suitable effector moieties may be selected based on a given condition/disease. In some cases, an FDA-approved therapeutic agent may be used as an effector moiety (i.e., where the FDA-approved therapeutic agent is an effector moiety described herein, e.g., a binding moiety rather than an antibody).

The following table lists examples of imaging/diagnostic molecular targets (i.e., binding moiety binding partners) for various conditions/disease states. Suitable binding moieties may be selected based on a given molecular target and/or suitable effector moieties may be selected based on a given condition/disease. In some cases, an FDA-approved imaging/diagnostic agent may be used as an effector moiety (i.e., where the FDA-approved imaging/diagnostic agent is an effector moiety described herein, e.g., a binding moiety rather than an antibody).

Imaging moieties and diagnostic agents and research applications

In various embodiments, the effector moiety is an imaging moiety, i.e., a molecule, compound, or fragment thereof that facilitates a technique and/or process for creating an image or making measurements of a cell, tissue, and/or organism (or a portion or function thereof) for clinical and/or research purposes. The imaging portion may generate signals by, for example, emitting and/or interacting with electromagnetic, nuclear, and/or mechanical (e.g., acoustic in ultrasound) energy. The imaging section may be used in various radiological, nuclear medicine, endoscopic, thermal imaging, photographic, spectroscopic, and microscopy methods, for example.

Imaging studies can be used, for example, in a clinical or research setting to diagnose a subject, select a subject for treatment, select a subject for participation in a clinical trial, monitor the progression of a disease, monitor the effect of a treatment to determine whether a subject should discontinue or continue treatment, determine whether a subject has reached a clinical endpoint, and determine the recurrence of a disease. Imaging studies can be used, for example, to conduct studies to identify effective interacting moieties and/or effector moieties and/or combinations thereof, to identify effective dosages and dosing schedules, to identify effective routes of administration, and to identify suitable targets (e.g., diseases susceptible to particular treatments).

Process for preparing drug conjugates

Any convenient method may be used to prepare the drug conjugates of the invention, i.e., SDC-TRAP. In a rational approach, drug conjugates are constructed from their individual component binding moieties, in some cases linkers, and effector moieties. As is known in the art, the components may be covalently bonded to each other through functional groups, wherein these functional groups may be present on the components or introduced onto the components using one or more steps such as oxidation reactions, reduction reactions, cleavage reactions, and the like. Functional groups that can be used to covalently bond components together to produce a drug conjugate include: hydroxyl, mercapto, amino, and the like. The particular moiety of the different components that is modified to provide covalent attachment will be selected so as not to substantially adversely interfere with the desired binding activity of that component, e.g., for the effector moiety, the region that will not affect the target binding activity will be modified so that a sufficient amount of the desired pharmaceutical activity is retained. As is known in the art, protecting Groups may be used to protect certain moieties on the components as necessary and/or desired, see, for example, Green & Wuts, Protective Groups in Organic Synthesis (John Wiley & Sons) (1991).

Alternatively, drug conjugates can be prepared using known combinatorial methods to generate a large library of potential drug conjugates, which can then be screened to identify bifunctional molecules with pharmacokinetic properties. Alternatively, drug conjugates can be prepared using pharmaceutical chemistry and known structure-activity relationships for targeting moieties and drugs. In particular, the method will provide insight as to where the two moieties are attached to the linker.

A number of exemplary methods for preparing SDC-TRAP molecules are given in the examples. As will be appreciated by those skilled in the art, the exemplary methods given in the examples can be modified to prepare other SDC-TRAP molecules.

Methods of use, pharmaceutical formulations and kits

The drug conjugates are useful for treating host conditions, such as disease conditions. In these methods, an effective amount of the drug conjugate is administered to the host, where an "effective amount" refers to a dose sufficient to produce a desired result, such as an improvement in a disease condition or a symptom associated therewith. In many embodiments, the amount of drug in the form of a drug conjugate that needs to be administered to a host in order to be an effective amount will be different from the amount that must be administered in free drug form. The difference in amount may vary, and in many embodiments may range from two to ten times. In certain embodiments, for example, where the resulting one or more modulated pharmacokinetic properties result in enhanced activity as compared to a free drug control, the amount of the effective amount of the drug is less than the amount of the corresponding free drug that needs to be administered, wherein the amount is less than two times, typically about four times, more typically about ten times, the amount of the free drug administered.

The drug conjugate may be administered to the host using any convenient means capable of producing the desired result. Thus, the drug conjugates can be incorporated into a variety of formulations for therapeutic administration. More particularly, the drug conjugate of the present invention can be formulated into a pharmaceutical composition by combining with an appropriate pharmaceutically acceptable carrier or diluent, and can be formulated into preparations in solid, semi-solid, liquid or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. As such, administration of the drug conjugate can be accomplished in a variety of ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, and the like. In pharmaceutical dosage forms, the drug conjugates can be administered alone or in combination with other pharmaceutically active compounds.

The methods of the invention can be used to treat a variety of different disease conditions. Of particular interest in certain embodiments is the use of the methods of the invention in disease conditions in which an active agent or drug having a desired activity has been previously identified, but which does not bind to its target with a desired affinity and/or specificity. For such active agents or drugs, the methods of the invention can be used to enhance the binding affinity and/or specificity of the agent to its target.

The specific disease conditions that can be treated with the bifunctional compounds of the present invention vary with the type of drug moiety that can be present in the drug conjugate. Thus, disease conditions include cell proliferative disorders, such as neoplastic disorders, autoimmune disorders, central nervous system or neurodegenerative disorders, cardiovascular disorders, hormonal abnormalities, infectious diseases, and the like.

Treatment refers to at least alleviation of symptoms associated with a disease condition afflicting the host, where alleviation, in a broad sense, refers to at least diminishment of the magnitude of a parameter (e.g., symptoms) associated with the pathological condition being treated (e.g., inflammation and pain associated therewith). Thus, treatment also includes the case: wherein the pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g., prevented from occurring) or halted (e.g., terminated), such that the host no longer suffers from the pathological condition, or at least symptoms that are characteristic of the pathological condition.

The use of the method of the present invention is beyond the scope of strict treatment of the disease. For example, the invention includes use in a clinical or research setting for diagnosing a subject, selecting a subject for treatment, selecting a subject for participation in a clinical trial, monitoring the progression of a disease, monitoring the effectiveness of a treatment, to determine whether a subject should discontinue or continue treatment, to determine whether a subject has reached a clinical endpoint, and to determine the recurrence of a disease. The invention also includes uses in conducting research for identifying effective interacting moieties and/or effector moieties and/or combinations thereof, identifying effective dosages and dosing schedules, identifying effective routes of administration, and identifying suitable targets (e.g., diseases susceptible to particular treatments).

A variety of hosts can be treated according to the methods of the invention. Typically, such hosts are "mammals" or "mammalian species," where these terms are used broadly to describe organisms belonging to the mammalian species, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the host will be a human.

The invention provides a kit for treating a subject in need thereof comprising at least one SDC-TRAP and instructions for administering a therapeutically effective amount of the at least one SDC-TRAP to the subject, thereby treating the subject. The invention also provides a kit for imaging, diagnosing and/or selecting a subject comprising at least one SDC-TRAP and instructions for administering an effective amount of the at least one SDC-TRAP to a subject, thereby imaging, diagnosing and/or selecting the subject.

Kits are provided having unit doses of drug conjugates, typically in oral or injectable dosages, and typically in storage stable formulations. In such kits, in addition to the containers containing the unit doses, an informational package insert will be included which describes the use and attendant benefits of the drug in treating the pathological condition of interest. Preferred compounds and unit doses are those described above.

The invention also provides methods for treating a disease or disorder, wherein a subject to be treated is selected based on the presence or overexpression of a particular protein. For example, a subject may be selected for treatment of cancer based on the presence of higher than normal levels of Hsp 90. In this case, the subject will be administered a SDC-TRAP comprising a binding moiety that selectively binds Hsp 90.

The present invention provides a method of treating or preventing an inflammatory disease in a subject comprising administering to the subject an effective amount of a compound represented by any one of formulas (I) to (L XXII), or any embodiment thereof, or a compound shown in tables 5, 6 or 7 disclosed in U.S. patent publication 2010/0280032 in one embodiment, the compound or binding moiety or SDC-TRAP may be administered to a human to treat or prevent the inflammatory disease in another embodiment, the inflammatory disease is selected from transplant rejection, skin transplant rejection, arthritis, rheumatoid arthritis, osteoarthritis and bone diseases associated with increased bone resorption, inflammatory bowel disease, ileitis, ulcerative colitis, Barrett ' S syndrome, Crohn ' S disease, asthma, adult respiratory distress syndrome, chronic obstructive airway disease, corneal dystrophy, trachoma, strongyloides coccosis, uveitis, sympathetic opthalmitis, gingivitis, periodontitis, tuberculosis, uremia syndrome, glomerulonephritis, psoriasis, neuroleptic sclerosis, psoriasis, autoimmune encephalomyelitis, lupus erythematosus, autoimmune encephalomyelitis, sjogren ' S, autoimmune encephalomyelitis, sjogrens, autoimmune encephalomyelitis, sjogrens, autoimmune encephalomyelitis, and other embodiments disclosed in another embodiment 2010/0280032, and AIDS 2010/0280032, and autoimmune encephalomyelitis may be administered in another embodiment, and.

In one embodiment, SDC-TRAP administered to a subject but not entering target cells is cleared rapidly from the body. In this embodiment, SDC-TRAP that does not enter the target cells is rapidly cleared to reduce toxicity due to components of the SDC-TRAP, degradation products of the SDC-TRAP, or SDC-TRAP molecules. Clearance can be determined by measuring the plasma concentration of SDC-TRAP molecules over time.

Similarly, SDC-TRAP molecules that enter non-target cells by passive diffusion either rapidly leave the non-target cells or tissues and are cleared from the subject or enter and are retained by the target cells or tissues. For example, SDC-TRAP intended to treat tumor cells and target tumor cells that overexpress, e.g., Hsp90, will selectively accumulate in tumor cells that overexpress Hsp 90. Thus, very low levels of this exemplary SDC-TRAP will be present in non-tumor tissues such as normal lung tissue, heart, kidney, etc. In one embodiment, the safety of the SDC-TRAP molecules of the invention can be determined by their lack of accumulation in non-target tissues. In contrast, the safety of SDC-TRAP molecules of the invention can be determined by their selective accumulation in target cells and/or tissues.