METHODS FOR DIAGNOSING AND TREATING DISEASES BASED ON MODULATING DRUG EFFLUX BY BINDING TO CRYPTIC REGION OF CD44

Abstract

The present invention relates to methods for modulating efflux of drugs based on binding to a protein region of CD44 in part comprising the amino acid sequence specified in the provisional application referenced above, and incorporated in its entirety by reference herein (PROV). In some aspects, the present invention relates to methods for treatment of cancer by preventing the cellular efflux of therapeutic drugs; for example, preventing the cellular efflux of anti-cancer agents, for example a taxane, by contacting a CD44-modulating peptide, comprising the amino acid sequence specified in the provisional application referenced above, and incorporated in its entirety by reference herein (PROV).

Claims

1. A method for preventing efflux of a therapeutic agent that has been internalized into a cell, the method comprises contacting an anti-efflux agent to a region of a protein associated with the cell, wherein the region of the protein in part comprises amino acid sequence having a sequence specified in the PROV.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0028] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

[0029] FIG. 1A shows a semi-transparent molecular surface representation of the homology model of hCD44 built with SWISS-MODEL using the structure of mCD44 in complex with HA8 (PDB 2JCR) as template. The HA8 binding pocket is depicted in orange while in purple are highlighted the putative glycosylation sites. The most perturbed residues are labeled and localized in a putative back pocket opposite from the HA8 binding pocket. The red square indicates the portion of hCD44 with sequence homology with A6 peptide.

[0030] FIG. 1B shows the cryptic binding site, an unexpected binding site for a peptide on the back side of chain B, A6 binding site (yellow) in relation to the known HA binding site (orange). (HA position modelled by superposition of murine HABD structure 2JCR).

[0031] FIG. 2 illustrates that the A6 polypeptide of a sequence specified in the PROV shares sequence homology with a portion of the Link-Domain of CD44, the cryptic binding region, a sequence specified in the PROV.

[0032] FIG. 3 illustrates testing of A6 plus paclitaxel (PTX) in the B16F10-DsRed Cell Lung Metastasis Model. Tumor burden (number of tumor nodules) in the lungs was not significantly reduced by A6 in the presence or absence of paclitaxel.

[0033] FIG. 4 Illustrates testing of A6 plus paclitaxel (PTX) in HEY [cisplatin (DDP) sensitive] and HEY/C2 (DDP resistant) cells. A6 did not affect sensitivity of HEY or HEY/C2 cells to paclitaxel.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The terms polypeptide and protein are used interchangeably herein and refer to any molecule that includes at least 2 or more amino acids.

[0035] As used herein, administering and the like refer to the act physically delivering a composition or other therapy (e.g. a radiation therapy) described herein into a subject by such routes as oral, mucosal, topical, transdermal, suppository, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration. Parenteral administration includes intravenous, intramuscular, intra-arterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Radiation therapy can be administered using techniques described herein, including for example, external beam radiation or brachytherapy. When a disease, disorder or condition, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of disease, disorder or condition or symptoms thereof. When a disease, disorder or condition, or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease, disorder or condition or symptoms thereof.

[0036] The term coadministration refers to administration of two or more agents (e.g., a polypeptide described herein and another active agent such as an anti-cancer agent or other therapy (e.g. a radiation therapy) described herein). The timing of coadministration depends in part on the combination and compositions or other therapies administered and can include administration at the same time, just prior to, or just after the administration of one or more additional therapies, for example cancer therapies such as chemotherapy, hormonal therapy, radiotherapy, or immunotherapy. Coadministration is meant to include simultaneous or sequential administration of a composition or therapy individually or in combination (more than one polypeptide described herein or an anti-cancer agent described herein or radiation therapy as described herein). Coadministration can include administration of two or more agents where the agents are optionally combined with other active substances (e.g., to reduce metabolic degradation). The polypeptides, anti-cancer agents and radiation therapies described herein can be used in combination with one another, with other active agents known to be useful in treating a disease associated with cells expressing a particular kinase as described herein, or with adjunctive agents that cannot be effective alone, but can contribute to the efficacy of the active agent.

[0037] As used herein, the terms subject and patient are used interchangeably. As used herein, a subject can be a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkey and human). In specific embodiments, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) having a disease, disorder or condition described herein. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing a disease, disorder or condition described herein. In certain instances, the term patient refers to a human.

[0038] The terms treating or treatment refer to any indicia of success or amelioration of the progression, severity, and/or duration of a disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a patient's physical or mental well-being.

[0039] The term cancer refers to any physiological condition in mammals characterized by unregulated cell growth. Cancers described herein include solid tumors and hematological (blood) cancers. A hematological cancer refers to any blood borne cancer and includes, for example, myelomas, lymphomas and leukemias. A solid tumor or tumor refers to a lesion and neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues resulting in abnormal tissue growth. Neoplastic, as used herein, refers to any form of dysregulated or unregulated cell growth, whether malignant or benign, resulting in abnormal tissue growth.

[0040] An improvement in the cancer or cancer-related disease can be characterized as a complete or partial response. Complete response refers to an absence of clinically detectable disease with normalization of any previously abnormal radiographic studies, bone marrow, and cerebrospinal fluid (CSF) or abnormal monoclonal protein measurements. Partial response refers to at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decrease in all measurable tumor burden (i.e., the number of malignant cells present in the subject, or the measured bulk of tumor masses or the quantity of abnormal monoclonal protein) in the absence of new lesions. The term treatment contemplates both a complete and a partial response.

[0041] A refractory, resistant, or persistent cancer refers to a circumstance where patients, even after intensive treatment, have residual cancer cells (e.g., leukemia cells, lymphoma cells, circulating tumor cells or cancer stem cells) in their lymphatic system, blood and/or blood forming tissues (e.g., marrow).

[0042] The terms manage, managing, and management refer to preventing or slowing the progression, spread or worsening of a disease or disorder, or of one or more symptoms thereof. In certain cases, the beneficial effects that a subject derives from a prophylactic or therapeutic agent do not result in a cure of the disease or disorder.

[0043] The term preventing refers to the treatment with or administration of a polypeptide or agent (e.g. anti-cancer agent described herein) provided herein, with or without other additional active agent (e.g. an anti-cancer agent), prior to the onset of symptoms, particularly to patients at risk of cancer and/or other disorders described herein. The term also refers to coadministration of a polypeptide with other therapies including radiation therapies as described herein. It should be understood that the polypeptides described herein can be co-administered with one or more anti-cancer agents and radiation therapies described herein. The term prevention includes the inhibition or reduction of a symptom of the particular disease, as well as a reduced incidence of a symptom of the particular disease (e.g. by comparison to historical data for a given subject, or population data for similar subjects). Patients with familial history of a disease in particular are candidates for preventive regimens in certain embodiments. In addition, patients who have a history of recurring symptoms are also potential candidates for the prevention. In this regard, the term prevention may be interchangeably used with the term prophylactic treatment.

[0044] A prophylactically effective amount of a polypeptide or agent (e.g. an anti-cancer agent described herein) means an amount of therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the inhibition or reduced incidence of a symptom of a disease or recurrence of a disease. The term also refers to coadministration of a polypeptide described herein with other therapies including radiation therapies as described herein. The term prophylactically effective amount can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

[0045] The term effective amount as used herein refers to the amount of a therapy (e.g., a composition or radiation therapy provided herein) which is sufficient to reduce and/or ameliorate the severity and/or duration of a given disease, disorder or condition and/or a symptom related thereto. This term also encompasses an amount necessary for the reduction or amelioration of the advancement or progression of a given disease, disorder or condition, reduction or amelioration of the recurrence, development or onset of a given disease, disorder or condition, and/or to improve or enhance the prophylactic or therapeutic effect(s) of another therapy. In some embodiments, effective amount as used herein also refers to the amount of therapy provided herein to achieve a specified result.

[0046] As used herein, and unless otherwise specified, the term therapeutically effective amount of a polypeptide described herein, an anti-cancer agent described herein, or a radiation therapy described herein is an amount sufficient to provide a therapeutic benefit in the treatment or management of a cancer, or to delay or minimize one or more symptoms associated with the presence of the cancer. A therapeutically effective amount of a polypeptide described herein, an anti-cancer agent described herein, or a radiation therapy described herein means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment or management of the cancer. The term therapeutically effective amount can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of cancer, or enhances the therapeutic efficacy of another therapeutic agent.

[0047] A therapy is any protocol, method and/or agent that can be used in the prevention, management, treatment and/or amelioration of a given disease, disorder or condition. In certain embodiments, the terms therapies and therapy refer to a drug therapy, biological therapy, supportive therapy, radiation therapy, and/or other therapies useful in the prevention, management, treatment and/or amelioration of a given disease, disorder or condition known to one of skill in the art such as medical personnel.

[0048] A regimen is a protocol for dosing and timing the administration of one or more therapies (e.g., combinations described herein, another active agent such as for example an anti-cancer agent described herein, or a radiation therapy described herein) for treating a disease, disorder, or condition described herein. A regimen can include periods of active administration and periods of rest as known in the art. Active administration periods include administration of combinations and compositions described herein and the duration of time of efficacy of such combinations, compositions, and radiation therapies. Rest periods of regimens described herein include a period of time in which no agent (e.g., a polypeptide described herein or an anti-cancer agent described herein) is actively administered, and in certain instances, includes time periods where the efficacy of such agents can be minimal. Rest periods of regimens described herein can include a period of time in which no radiation therapy is actively administered. Combination of active administration and rest in regimens described herein can increase the efficacy and/or duration of administration of the combinations described herein.

[0049] The term pharmaceutically acceptable as used herein refers to physiologically acceptable compounds, agents, or ingredients recognized by a regulatory agency of the Federal or state government, or another governmental agency with authorization for such approval, or and an agent listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.

[0050] A pharmaceutically acceptable excipient, refers to a substance that aids the administration of an active agent to a subject by for example modifying the stability of an active agent or modifying the absorption by a subject upon administration. A pharmaceutically acceptable excipient typically has no significant adverse toxicological effect on the patient. Examples of pharmaceutically acceptable excipients include, for example, water, NaCl (including salt solutions), normal saline solutions, sucrose, glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, alcohols, oils, gelatins, carbohydrates such as amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. One of skill in the art will recognize that other pharmaceutical excipients known in the art are useful in the present invention and include those listed in for example the Handbook of Pharmaceutical Excipients, Rowe R. C., Shesky P. J., and Quinn M. E., 6th Ed., The Pharmaceutical Press, RPS Publishing (2009). The terms binder, filler, disintegrant, and lubricant are used in accordance with the plain and ordinary meaning within the art.

[0051] In certain embodiments, a pharmaceutically acceptable excipient may be incompatible (e.g., cross-reacts) with other excipients or active agents described herein. In some embodiments, magnesium stearate, croscarmellose sodium, lactose, excipients comprising Mg, Ca, K, Li, or nucleic acid, acesulfame potassium, ammonium alginate, calcium acetate, calcium alginate, calcium carbonate, calcium chloride, calcium lactate, calcium phosphate, calcium silicate, calcium stearate, calcium sulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, docusate sodium, glycine, kaolin, magnesium aluminum silicate, magnesium carbonate, magnesium oxide, magnesium silicate, magnesium trisilicate, polacrilin potassium, polymethacrylates, potassium alginate, potassium benzoate, potassium bicarbonate, potassium chloride, potassium citrate, sodium alginate, sodium benzoate, sodium chloride, sodium lauryl sulfate, sodium starch glycolate, sodium stearyl fumarate, sulfobutylether beta-cyclodextrin, sodium stearate, talc, or zinc stearate are incompatible in the dosage forms described herein.

[0052] The term anti-cancer agent is used in accordance with its plain ordinary meaning and refers to a composition having anti-neoplastic properties or the ability to inhibit the growth or proliferation of cells. In certain embodiments, an anti-cancer agent is a chemotherapeutic. In certain embodiments, an anti-cancer agent is an agent identified herein having utility in methods of treating cancer. In certain embodiments, an anti-cancer agent is an agent approved by the FDA or similar regulatory agency of a country other than the USA, for treating cancer.

[0053] The term chemotherapeutic or chemotherapeutic agent is used in accordance with its plain ordinary meaning and refers to a chemical composition or compound having anti-neoplastic properties or the ability to inhibit the growth or proliferation of cells. Chemotherapy or cancer therapy refers to a therapy or regimen that includes administration of a combination, chemotherapeutic, or anti-cancer agent described herein.

[0054] The term radiation therapy is used in accordance with its plain ordinary meaning and refers to the medical use of radiation in the treatment of cancer. Preferably, the medical use of radiation in the treatment of cancer results in the killing of cancer cells in the subject. A variety of radiation therapies as anti-cancer agents can be used in accordance with the present disclosure, examples of which are provided herein.

[0055] A CD44-modulating polypeptide refers to a polypeptide that binds to CD44 and modulates its activity (e.g., signaling activity). A CD44-modulating polypeptide can be a polypeptide sequence described herein or, in some embodiments, an antibody that specifically binds to CD44 and inhibits its downstream signaling activity. In one embodiment, a CD44-modulating polypeptide can be a polypeptide sequence described herein or, in some embodiments, an antibody that disrupts or inhibits signaling activity of a CD44 dependent co-receptor. In certain instances, the CD44 dependent co-receptor is a receptor tyrosine kinase (RTK) such as, for example, Met, Ran, or VEGFR. In still another embodiment a CD44-modulating polypeptide can be a polypeptide sequence described herein or, in some embodiments, an antibody that disrupts CD44 co-receptor function or association of a CD44 co-receptor with CD44 or another signaling protein. In one embodiment, a CD44-modulating polypeptide described herein binds to CD44 and inhibits CD44 signaling activity or association with one or more ABC transporters. The ABC transporter can be a multidrug resistant protein (e.g., MDR1). In certain embodiments, CD44 levels can be elevated upon radiation therapy. Exemplary CD44-modulating polypeptides include polypeptides having homology to the CD44-v6 region of human CD44. Such peptides can include substitution variants, addition variants, or chemical derivatives thereof including peptidomimetics.

[0056] Provided herein are methods for treating a resistant or refractory cancer, where the cancer can be resistant to at least one anti-cancer agent or radiation therapy by administering a CD44-modulating polypeptide provided herein in combination with an anti-cancer agent described herein. Also provided herein are methods for treating a resistant or refractory cancer, where the cancer can be resistant to at least one anti-cancer agent or radiation therapy by administering a CD44-modulating polypeptide provided herein in combination with radiation therapy described herein. Further provided herein are methods for treating a resistant or refractory cancer, where the cancer can be resistant to at least one anti-cancer agent or radiation therapy by administering a CD44-modulating polypeptide provided herein in combination with an anti-cancer agent described herein and radiation therapy described herein. It is well known in the art that many cancers are resistant or refractory to many anti-cancer agents or radiation therapy(ies) or over the course of treatment, become resistant or refractory to treatment. The methods described herein can restore activity of anti-cancer agents having reduced or eliminated activity against one or more cancers and permit additional treatment options for cancer patients. In another embodiment, the methods described herein can restore activity of radiation therapies described herein having reduced or eliminated activity against one or more cancers and permit additional treatment options for cancer patients.

[0057] The present invention includes embodiments where a CD44-modulating polypeptide described herein establishes, restores or enhances the anti-cancer activity of an anti-cancer agent in treating a cancer that is resistant or refractory to the treatment. In one example, a CD44-modulating polypeptide establishes anti-cancer activity (e.g., creates efficacy of an anti-cancer agent in treating a cancer) of an anti-cancer agent described herein in the treatment of cancer. In another example, a CD44-modulating polypeptide restores the anti-cancer activity of an anti-cancer agent described herein. In another example, a CD44-modulating polypeptide enhances the anti-cancer activity of an anti-cancer agent described herein. In still another example, a combination therapy of a CD44-modulating polypeptide described herein and an anti-cancer agent described herein establishes, restores, or enhances activity of a CD44-modulating polypeptide.

[0058] The present invention includes embodiments where a CD44-modulating polypeptide described herein establishes, restores or enhances the anti-cancer activity of a radiation therapy in treating a cancer that is resistant or refractory to the treatment. In one example, a CD44-modulating polypeptide establishes anti-cancer activity (e.g., creates efficacy of a radiation therapy) of a radiation therapy described herein in the treatment of cancer. In another example, a CD44-modulating polypeptide restores the anti-cancer activity of a radiation therapy described herein. In another example, a CD44-modulating polypeptide enhances the anti-cancer activity of a radiation therapy described herein. Further provided herein, a CD44-modulating polypeptide can establish, restore, or enhance the anti-cancer activity of one or more anti-cancer agents and radiation therapy. In still another example, a combination therapy of a CD44-modulating polypeptide described herein and a radiation therapy described herein establishes, restores, or enhances activity of a CD44-modulating polypeptide.

[0059] The cancer can optionally be resistant or refractory to a plurality of anti-cancer agents (e.g. two or more anti-cancer agents) and/or a plurality of radiation therapies. In one example the cancer can also be resistant, refractory, or non-responsive to treatment with a CD44-modulating polypeptide described herein. In one embodiment of methods of treating described herein, a patient can be administered a combination of a CD44-modulating polypeptide described herein and an anti-cancer agent where the cancer treated is resistant, refractory, or non-responsive to one of or both the CD44-modulating polypeptide and the anti-cancer agent. In one example, the cancer can be resistant, refractory, or non-responsive to treatment with the anti-cancer agent. Administration of the combination of the CD44-modulating polypeptide and anti-cancer agent(s) surprisingly can restore or enhance the activity of the anti-cancer agent against the refractory, resistant, or non-responsive cancer. Administration of the combination of the CD44-modulating polypeptide and anti-cancer agent(s) surprisingly can restore or enhance the activity of the CD44-modulating polypeptide against the refractory, resistant, or non-responsive cancer. Administration of the combination of the CD44-modulating polypeptide and anti-cancer agent(s) surprisingly can restore or enhance the activity of the CD44-modulating polypeptide and the anti-cancer agent against the refractory, resistant, or non-responsive cancer.

[0060] In one embodiment of methods of treating described herein, a patient can be administered a combination of a CD44-modulating polypeptide described herein and a radiation therapy where the cancer treated is resistant, refractory, or non-responsive to one of or both the CD44-modulating polypeptide and the radiation therapy. In one example, the cancer can be resistant, refractory, or non-responsive to treatment with radiation therapy. Administration of the combination of the CD44-modulating polypeptide and anti-cancer agent(s) surprisingly can restore or enhance the activity of the radiation therapy against the refractory, resistant, or non-responsive cancer. Administration of the combination of the CD44-modulating polypeptide and anti-cancer agent(s) surprisingly can restore or enhance the activity of the CD44-modulating polypeptide against the refractory, resistant, or non-responsive cancer. Administration of the combination of the CD44-modulating polypeptide and radiation therapy surprisingly can restore or enhance the activity of the CD44-modulating polypeptide and the radiation therapy against the refractory, resistant, or non-responsive cancer.

[0061] In one example, a CD44-modulating polypeptide described herein does not have activity against a cancer described herein when administered alone. In one embodiment, where a CD44-modulating polypeptide described herein does not have activity against a cancer described herein when administered alone, its activity can be established or restored when administered in combination with an anti-cancer agent described herein, a radiation therapy described herein, or a combination thereof. In another example, a CD44-modulating polypeptide described herein has minimal activity against a cancer described herein (e.g., insufficient anti-cancer activity to treat a cancer described herein) when administered alone. In one embodiment, where a CD44-modulating polypeptide described herein has minimal activity against a cancer described herein, its activity can be enhanced when administered in combination with an anti-cancer agent described herein, a radiation therapy described herein, or a combination thereof.

[0062] In another example, an anti-cancer agent described herein or a radiation therapy described herein does not have activity against a cancer described herein when administered alone (or in combination with another anti-cancer agent). In one embodiment, where an anti-cancer agent described herein or a radiation therapy described herein does not have activity against a cancer described herein when administered alone, its activity can be restored when administered in combination with CD44-modulating polypeptide described herein. In another example, an anti-cancer agent described herein or a radiation therapy described herein has minimal activity against a cancer described herein (e.g., insufficient anti-cancer activity to treat a cancer described herein) when administered alone. In one embodiment, where an anti-cancer agent described herein or a radiation therapy described herein has minimal activity against a cancer described herein, its activity can be enhanced when administered in combination with a CD44-modulating polypeptide described herein. In another example, an anti-cancer agent or a radiation therapy can lose its anti-cancer activity over the course of treatment due to, for example, progression of resistance or refraction in the cancer treated. In one embodiment, the loss of anti-cancer agent or a radiation therapy activity can be slowed, stopped, or reversed (e.g. enhanced activity) when the patient is administered the anti-cancer agent or a radiation therapy in combination with a CD44-modulating polypeptide described herein.

[0063] Non-limiting examples of standard methods to measure efflux of drugs include a direct measurement of transport of a fluorescent compound, e.g., Fura-2 (1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N,N-tetraacetic acid, pentapotassium salt), use of DNA inter-chelating dyes (e.g., Hoeschst H33342, ethidium bromide), an indirect measurement of intracellular accumulation of a substrate, in vivo imaging, and radiolabeled anti-cancer agents. Other examples methods for measuring drug efflux may comprise single-cell or cell-free technologies and/or mass spectrometry.