WO2017184851A1

WO2017184851A1 - Compositions and methods for treating cancer

Info

Publication number: WO2017184851A1
Application number: PCT/US2017/028597
Authority: WO
Inventors: George Tidmarsh; James Rolke
Original assignee: La Jolla Pharmaceutical Company
Priority date: 2016-04-20
Filing date: 2017-04-20
Publication date: 2017-10-26

Abstract

The invention provides methods for the treatment of a cancer using a galectin-3 inhibitor, such as a modified pectin (e.g., GCS-lOO). Also described are methods for assessing and/or monitoring the effects of a galectin-3 inhibitor, e.g., to adapt the dosing regimen of the inhibitor during therapy.

Description

COMPOSITIONS AND METHODS

FOR TREATING CANCER

BACKGROUND OF THE INVENTION

Galectins comprise a family of proteins which are expressed by plant and animal cells and which bind β-galactoside sugars. These proteins can be found on cell surfaces, in cytoplasm, in the nucleus, and in extracellular fluids. The two most studied galectins, galectin-1 and galectin-3, have a molecular weight in the general range of 13-16 kDa and 29- 35 kD, respectively; they have an affinity for β-galactoside containing materials, and have been found to play a number of important roles in biological processes including cell migration, cell-cell adhesion, angiogenesis, cell fusion and other cell-cell interactions, as well as immune-based reactions and apoptosis. While there are a large number of galectins which manifest the foregoing activities, galectin-3 and galectin-1 have been strongly implicated in connection with cellular processes involving cancers.

Galectin-3 is a carbohydrate binding protein having a molecular weight of approximately 30,000. It is composed of two distinct structural motifs, an amino-terminal portion containing Gly-X-Y tandem repeats which are characteristic of collagens, and a carboxyl-terminal portion containing a carbohydrate binding site. Galectin-3 is found in almost all tumors, and has a binding affinity for β-galactoside-containing gly co-conjugates. Galectin-3 is believed to play a role in mediating cell-cell interactions and thereby fostering cell adhesion, cell migration and metastatic spread. It has been found that cells which have high expressions of galectin-3 are more prone to metastasis and are more resistant to apoptosis induced by chemotherapy or radiation. It has also been reported in the literature that galectin-3 plays a role in promoting angiogenesis. Multiple studies have established that elevated expression of galectin-3 correlates with aggressiveness and relapse in multiple human cancers, making it an attractive target for cancer therapy.

SUMMARY OF THE INVENTION

The invention described herein provides a safe and effective treatment of cancer using galectin-3 inhibitors, particularly modified pectins, such as GCS-100. The invention further provides combination therapies for treating cancer with a galectin-3 inhibitor or modified pectin conjointly with one or more additional therapeutic agents useful in the treatment of cancer. Compositions and articles of manufacture, including kits, relating to the methods for treating cancer are also contemplated as part of the invention.

In certain embodiments, the galectin-3 inhibitor is administered at a dose that preferentially affects galectin-3 levels and/or activity relative to other galectins, especially galectin-9, e.g., because the agent inhibits galectin-3 levels and/or activity to a greater extent than it inhibits galectin-9 levels and or activity. For example, the IC50 of the agent against galectin-9 may be at least 2, 3, 5, 10, 20, 50, 100, or even over 100 times greater than its IC50 against galectin-3. Without wishing to be bound by theory, inhibiting galectin-9 levels and/or activity may induce undesirable side effects, and so it may be desirable to inhibit galectin-3 levels and/or activity to a therapeutically effective extent without substantially inhibiting galectin-9 levels and/or activity. Accordingly, in some embodiments, the methods described herein include measuring galectin-9 levels in a patient treated with a galectin-3 inhibitor, to determine whether galectin-9 levels and/or activity have been affected to a clinically significant extent. If the measurement shows that galectin-9 levels and/or activity have been significantly affected, one or more subsequent doses of the galectin-3 inhibitor may be reduced relative to the dose administered prior to the measurement.

One aspect of the invention provides a method for treating cancer in a patient, comprising: administering to the patient at least one galectin-3 inhibitor.

In some embodiments, the cancer is selected from renal cell cancer, Kaposi's sarcoma, chronic leukemia, prostate cancer, breast cancer, endocrine cancer, sarcoma, pancreatic cancer, ovarian carcinoma, rectal cancer, throat cancer, melanoma, colon cancer, bladder cancer, mastocytoma, lung cancer, mammary adenocarcinoma, myeloma, lymphoma, pharyngeal squamous cell carcinoma, B cell malignancy, and gastrointestinal or stomach cancer. Preferably, the cancer which is treated in the present invention is melanoma, lung cancer, breast cancer, pancreatic cancer, prostate cancer, colon cancer, or ovarian cancer.

In some embodiments, the cancer is breast cancer, the breast cancer comprises basal- type breast cancer cells, triple-negative breast cancer cells, or claudin-low breast cancer cells.

In some embodiments wherein the cancer is endocrine cancer, the endocrine cancer is selected from adrenal cortex adenoma, adrenal cortex carcicnoma, adrenal gland

pheochromocytoma, or parathyroid gland adenoma. In some embodiments wherein the cancer is a B cell malignancy, the B cell malignancy is selected from multiple myeloma, leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, lymphoma, Burkitt's lymphoma, Diffuse large B cell lymphoma, follicular lymphoma, or Hodgkin's lymphoma.

In some embodiments, the galectin-3 inhibitor is a modified pectin.

In some embodiments, the backbone of the modified pectin comprises

homogalacturonan and/or rhamnogalacturonan I.

In some embodiments, the modified pectin is de-esterified and partially

depolymerized, so as to have a disrupted rhamnogalacturonan backbone.

In some embodiments, the modified pectin has an average molecular weight between

50 and 200 kDa, preferably between 80 and 150 kDa.

In some embodiments, the modified pectin is substantially free of modified pectins having molecular weights below 25 kDa.

In some embodiments, the modified pectin is GCS-100.

In some embodiments, the modified pectin is made by passing modified or unmodified pectin through a tangential flow filter.

In some embodiments, the method comprises administering the modified pectin at a dose of about 0.1 to 2 mg/m².

In some embodiments, the dose is about 1.5 mg/m².

In some embodiments, the dose is about 1-10 mg.

In some embodiments, the dose is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg, preferably 1, 3, or

9 mg.

In some embodiments, the galectin-3 inhibitor is administered weekly or biweekly.

In some embodiments, the galectin-3 inhibitor is administered weekly for an induction phase and then biweekly for a maintenance phase.

In some embodiments, the induction phase is 1-3 months, preferably 2 months.

In some embodiments, the maintenance phase is at least 1 month, preferably at least 3 months, or even six months or more.

In some embodiments, the at least one galectin-3 inhibitor is administered in an amount that reduces a level of galectin-3 in serum of the patient.

In some embodiments, the at least one galectin-3 inhibitor is administered in an amount that reduces an expression level of galectin 3 in the patient. In some embodiments,the at least one galectin-3 inhibitor is administered in an amount that reduces an activity of galectin-3 in the patient.

In some embodiments, the concentration, expression level, or activity of galectin-3 is reduced 0.5, 1, 2, 3, 4, or 5-fold relative to control.

In some embodiments, the method further comprises 1) measuring the concentration, level, or activity of galectin-3 before administering the galectin-3 inhibitor and 2) measuring the concentration, level, or activity of galectin-3 after administering the galectin-3 inhibitor.

In some embodiments, a decrease in the concentration, level, or activity of galectin-3 after administering the galectin-3 inhibitor indicates that the dose of galectin-3 inhibitor is an effective dose of galectin-3 inhibitor for the treatment of cancer in a patient.

In some embodiments, an increase in the concentration, level, or activity of galectin-3 after administering the galectin-3 inhibitor indicates that the dose of galectin-3 inhibitor is an ineffective dose of galectin-3 inhibitor for the treatment of cancer in a patient.

In some embodiments, the method further comprises administering to the patient a second dose of the galectin-3 inhibitor in a lower amount than in the prior administration.

In some embodiments, the method further comprises administering an additional therapeutic agent.

In some embodiments, the additional therapeutic agent is useful for the treatment of cancer.

In some embodiments, the method comprises administering the galectin-3 inhibitor concurrently with the therapeutic agent.

In some embodiments, the method comprises administering the galectin-3 inhibitor subsequent to administration of the therapeutic agent.

In some embodiments, the method comprises administering the therapeutic agent subsequent to administration of the galectin-3 inhibitor.

In some embodiments, the method comprises administering multiple doses of the galectin-3 inhibitor over a period of at least 8 weeks.

In some embodiments, the method comprises administering the galectin-3 inhibitor weekly.

In some embodiments, the galectin-3 inhibitor is administered by injection or intravenous infusion.

In some embodiments, the galectin-3 inhibitor is administered by intravenous infusion. It is contemplated that all embodiments described herein, including those described under different aspects of the invention, can be combined with one another where not specifically prohibited. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the family of known mammalian galectins.

Figure 2 schematically depicts the structure of GCS-100 unbound and bound to galectin-3.

Figure 3 shows the GCS-100 concentration versus baseline galectin-3 following a single 1.5 mg/m² dose in cancer patients.

Figure 4 shows the GCS-100 concentration versus baseline galectin-3 following a single 30 mg/m² dose in cancer patients.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are methods for treating cancer using galectin-3 inhibitors, particularly modified pectins, such as GCS-100. The invention further provides combination therapies for treating a cancer with a galectin-3 inhibitor or modified pectin conjointly with one or more additional therapeutic agents useful in the treatment of cancer. Also described are methods for assessing and/or monitoring the effects of a galectin-3 inhibitor, e.g., to adapt the dosing regimen of the inhibitor during therapy. Compositions and articles of manufacture, including kits, relating to the methods for treating cancer are also contemplated as part of the invention.

Various aspects of the invention are described in further detail herein.

I. Definitions

Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature and techniques relating to chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry, described herein, are those well known and commonly used in the art.

Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" may be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components). The singular forms "a," "an," and "the" include the plurals unless the context clearly dictates otherwise. The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

"About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20%, preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" may mean values that are within an order of magnitude, preferably within 5 -fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated.

The "baseline" is the last assessment taken prior to the first study drug

administration.

The "change from baseline" is the arithmetic difference between a post-baseline value and the baseline value: Change from Baseline = (Post-baseline Value - Baseline Value) Percentage Change from Baseline = [(Post-baseline Value - Baseline Value) / Baseline Value] x 100.

The "Body Surface Area," or BSA is defined by the formula:

A "clinical response" as used herein is refers to an indicator of therapeutic effectiveness of an agent. In certain embodiments, a clinical response is the measurement of the effect of a modified pectin relative to control on 1) circulating galectin-3 levels; 2) serum markers; and/or 3) markers of cancer.

The term "combination" as in the phrase "a first agent in combination with a second agent" includes co-administration of a first agent and a second agent, which for example may be dissolved or intermixed in the same pharmaceutically acceptable carrier, or administration of a first agent, followed by the second agent, or administration of the second agent, followed by the first agent. The present invention, therefore, includes methods of combination therapeutic treatment and combination pharmaceutical compositions.

The term "concomitant" as in the phrase "concomitant therapeutic treatment" includes administering an agent in the presence of a second agent. A concomitant therapeutic treatment method includes methods in which the first, second, third, or additional agents are co-administered. A concomitant therapeutic treatment method also includes methods in which the first or additional agents are administered in the presence of a second or additional agents, wherein the second or additional agents, for example, may have been previously administered. A concomitant therapeutic treatment method may be executed step-wise by different actors. For example, one actor may administer to a subject a first agent and a second actor may administer to the subject a second agent, and the administering steps may be executed at the same time, or nearly the same time, or at distant times, so long as the first agent (and additional agents) are after administration in the presence of the second agent (and additional agents). The actor and the subject may be the same entity (e.g., human).

The terms "conjoint therapy" and "combination therapy," as used herein, refer to the administration of two or more therapeutic substances, e.g., a galectin-3 inhibitor or modified pectin, and another drug used in the treatment of cancer. The other drug(s) may be administered concomitant with, prior to, or following the administration of a galectin-3 inhibitor or modified pectin.

The term "dose," as used herein, refers to an amount of a therapeutic agent, such as a galectin-3 inhibitor or modified pectin (e.g., GCS-lOO), which is administered to a subject.

The term "dosing," as used herein, refers to the administration of a therapeutic agent, such as galectin-3 inhibitor or modified pectin (e.g., GCS-lOO), to achieve a therapeutic objective (e.g., treatment of a cancer). The level of dosing could be based on the baseline level of galectin-3. One way of determining an appropriate dose would be to measure baseline galectin to determine a target dose, followed by additional measurements after administration to determine the dose's effect on galectin-3.

A "dosing regimen" describes a schedule for administering a therapeutic agent, such as a galectin-3 inhibitor or modified pectin (e.g., GCS-lOO), e.g., a treatment schedule over a prolonged period of time or throughout the course of treatment, e.g., administering a first dose of a galectin-3 inhibitor or modified pectin (e.g., GCS-lOO) at week 0 followed by a second dose of a galectin-3 inhibitor or modified pectin (e.g., GCS-lOO) on a weekly or biweekly dosing regimen.

The term "fixed dose" or "total body dose" refers to a dose which is a constant amount of a therapeutic agent delivered with each administration to the subject being treated. In certain embodiments, a galectin-3 inhibitor or modified pectin (e.g., GCS-lOO), is administered to the subject at a fixed dose ranging from 0.1 mg/m² to 30 mg/m². In certain embodiments, a modified pectin or galectin-3 inhibitor, is administered to the subject in a fixed dose of 0.1 mg/m², 0.5 mg/m², 1 mg/m², 3 mg/m², 6 mg/m², 9 mg/m², 12 mg/m², 15 mg/m², 18 mg/m², 21 mg/m², 24 mg/m², 27 mg/m², 30 mg/m², 35 mg/m², 40 mg/m², 50 mg/m², 60 mg/m², 70 mg/m², 80 mg/m², 90 mg/m², 100 mg/m², 110 mg/m², 120 mg/m², 130 mg/m², 140 mg/m², 150 mg/m², 160 mg/m², 170 mg/m², 180 mg/m², 190 mg/m², 200 mg/m², efc. Ranges of values between any of the aforementioned recited values are also intended to be included in the scope of the invention, e.g., 0.2 mg/m², 0.6 mg/m², 1.9 mg/m², 4 mg/m², 8 mg/m², 10 mg/m², 13 mg/m², 17 mg/m², 20 mg/m², 23 mg/m², 25 mg/m², 26 mg/m², 28 mg/m², 32 mg/m², 45 mg/m², 55 mg/m², 65 mg/m², 75 mg/m², 85 mg/m², 95 mg/m², 105 mg/m², 115 mg/m², 125 mg/m², 135 mg/m², 145 mg/m², 155 mg/m², 165 mg/m², 175 mg/m², 185 mg/m², 195 mg/m², 205 mg/m², as are ranges based on the forementioned doses, e.g., 0.1-5 mg/m², 5-10 mg/m², 10-15 mg/m², 15-20 mg/m², 20-25 mg/m², 25-30 mg/m², 30-80 mg/m², 80-120 mg/m², 120-150 mg/m², 150-175 mg/m², 175-200 mg/m²,. The total body dose should not exceed 1 g/m² weekly or 200 mg/m² daily times 5.

The term "induction dose" or "loading dose," used interchangeably herein, refers to the first dose(s) of a modified pectin or galectin-3 inhibitor (e.g., GCS-100) which is initially used to treat a cancer. The loading dose may, for example, be administered during an induction phase. The loading dose may be larger in comparison to the subsequent maintenance or treatment dose. The induction dose can be a single dose or, alternatively, a set of doses. For example, a 1.5 mg/m² dose may be administered as a single 1.5 mg/m² dose, as two doses of 0.75 mg/m² each, or four doses of 0.375 mg/m² each. In certain embodiments, an induction dose is subsequently followed by administration of smaller doses of a modified pectin or galectin-3 inhibitor (e.g., GCS-100), e.g., the treatment or maintenance dose(s). The induction dose is administered during the induction or loading phase of therapy. The induction phase may be followed by a maintenance phase.

Those "in need of treatment" include mammals, such as humans, already having cancer, including those in which the disease or disorder is to be prevented, e.g., those identified as being at risk of developing the disease or disorder.

The term "lectin" refers to a protein found in the body that specifically interacts with carbohydrate sugars located in, on the surface of, and in between cells. This interaction causes the cells to change behavior, including cell movement, proliferation, and other cellular functions. Interactions between lectins and their target carbohydrate sugars occur via a carbohydrate recognition domain (CRD) within the lectin. Galectins are a subfamily of lectins.

The term "galectins" are a subfamily of lectins that have a CRD that bind specifically to β-galactoside sugar molecules. Galectins have a broad range of functions, including mediation of cell survival and adhesion, promotion of cell-cell interactions, growth of blood vessels, and regulation of the immune system and inflammatory response (Leffler et. al, 2004). Currently, there are 15 known mammalian galectins, which can be divided into three subclasses: those with one CRD (galectins 1, 2, 5, 7, 10, 13, 14, and 15), those with two CRDs (galectins 4, 6, 8, 9, and 12), and those with one CRD and a second domain comprising an amino acid tail (galectin 3), as depicted in Figure 1. At low concentrations, galectins exist as monomers. However, at higher concentrations, they exist as dimers and oligomers (Figure 1) and, thus, form lattice-like networks with β-galactoside-containing receptors within a cell and between the cell and its environment (Figure 1). As such, at low concentrations, galectins may have a different biological function that changes upon upregulation and overexpression (Rabinovich et. al, 2007).

The term "maintenance therapy" or "maintenance dosing regimen" refers to a treatment schedule for a subject or patient diagnosed with a cancer, to enable them to maintain their health in a given state, e.g., reduced transformed phenotype, decreased cancer cell proliferation, or achieving a clinical response. For example, a maintenance therapy of the invention may enable a patient to maintain their health in a state which is completely or substantially free of symptoms. Alternatively, a maintenance therapy of the invention may enable a patient to maintain his health in a state where there is a significant reduction in symptoms associated with the disease relative to the patient's condition prior to receiving therapy.

The term "maintenance phase" or "treatment phase," as used herein, refers to a period of treatment comprising administration of a modified pectin or galectin-3 inhibitor (e.g., GCS-100) to a subject in order to maintain a desired therapeutic effect, e.g., improved symptoms associated with cancer. The maintainance phase may be preceded by an induction phase, which is typically a dose larger than a maintenance dose, e.g., with the aim of quickly raising a patient's plasma level of a therapeutic agent, such as a modified pectin, from a baseline level (e.g., 0) into a therapeutically effective window, which is then maintained by administration in the maintenance phase. The term "maintenance dose" or "treatment dose" is the amount of a modified pectin or galectin-3 inhibitor (e.g., GCS-100) taken by a subject to maintain or continue a desired therapeutic effect. A maintenance dose can be a single dose or, alternatively, a set of doses. A maintenance dose is administered during the treatment or maintenance phase of therapy. Typically, a maintenance dose(s) is smaller than the induction dose(s) and maintenance doses may be equal to each other when administered in succession.

The phrase "multiple-variable dose" includes different doses of a modified pectin or galectin-3 inhibitor (e.g., GCS-100) which are administered to a subject for therapeutic treatment. "Multiple-variable dose regimen" or "multiple-variable dose therapy" describes a treatment schedule which is based on administering different amounts of modified pectin or galectin-3 inhibitor (e.g., GCS-100) at various time points throughout the course of treatment.

The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount of the composition or therapeutic agent, such as a galectin-3 inhibitor, effective to treat cancer in a patient, e.g., effecting a beneficial and/or desirable alteration in the general health of a patient suffering from cancer. A "pharmaceutically effective amount" or "therapeutically effective amount" also refers to an amount that improves the clinical symptoms of a patient.

The phrase "pharmaceutically acceptable excipient" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, lubricant, binder, carrier, humectant, disintegrant, solvent or encapsulating material, that one skilled in the art would consider suitable for rendering a pharmaceutical formulation suitable for administration to a subject. Each excipient must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, as well as "pharmaceutically acceptable" as defined above. Examples of materials which can serve as pharmaceutically acceptable excipients include but are not limited to: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; silica, waxes; oils, such as com oil and sesame oil; glycols, such as propylene glycol and glycerin; polyols, such as sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents; alginic acid; pyrogen- free water; isotonic saline; Ringer's solution; and other non-toxic compatible substances routinely employed in pharmaceutical formulations. The term "preventing" is art-recognized, and when used in relation to a medical condition such as a cancer, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition.

As used herein, "proliferating" and "proliferation" refer to cells undergoing mitosis.

The term "prophylactic" or "therapeutic" treatment is art-recognized and refers to administration of a drug to a host. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate or maintain the existing unwanted condition or side effects thereof). Prophylatic and therapeutic treatment may be used in conjunction with known methods of.

The terms "subject" and "patient", as used herein, are used interchangeably. In certain embodiments, a subject refers to an individual who may be treated therapeutically with a modified pectin or galectin-3 inhibitor (e.g., GCS-100).

By "substantially free" of modified pectins having a certain molecular weight below a certain number, it is meant that the composition has less than 1%, preferably less than 0.5% or even less than 0.1%, of modified pectins having a molecular weight below that number.

A "therapeutically effective amount" of a compound, such as a modified pectin of the present invention, with respect to the subject method of treatment, refers to an amount of the compound(s) in a preparation which, when administered as part of a desired dosage regimen to a subject achieves a therapeutic objective (e.g., treatment of cancer). A therapeutically effective amount may be determined by measuring baseline galectin-3 levels to determine a target dose, followed by additional measurements after administration to determine the effect of the dose on galectin-3. In such embodiments, if the patient's galectin-3 level or activity is decreased, inhibited, or reduced, then the dose is a therapeutically effective amount.

As used herein, "transformed cells" refers to cells that have spontaneously converted to a state of unrestrained growth, i.e., they have acquired the ability to grow through an indefinite number of divisions in culture. Transformed cells may be characterized by such terms as neoplastic, anaplastic and/or hyperplastic, with respect to their loss of growth control. For purposes, of this invention, the terms "transformed phenotype of malignant mammalian cells" and "transformed phenotype" are intended to encompass, but not be limited to, any of the following phenotypic traits associated with cellular transformation of mammalian cells: immortalization, morphological or growth transformation, and

tumorigenicity, as detected by prolonged growth in cell culture, growth in semi-solid media, or tumorigenic growth in immuno-incompetent or syngeneic animals.

The term "treatment," as used within the context of the present invention, is meant to include therapeutic treatment, as well as prophylactic or suppressive measures.

As used herein, "unwanted proliferation" means cell division and growth that is not part of normal cellular turnover, metabolism, growth, or propagation of the whole organism. Unwanted proliferation of cells is seen in tumors and other pathological proliferation of cells, does not serve normal function, and for the most part will continue unbridled at a growth rate exceeding that of cells of a normal tissue in the absence of outside intervention.

III. Galectin-3 Inhibitors

In certain embodiments of the present invention, the galectin-3 inhibitor is an agent that binds to and inhibits galectin-3, e.g., by reducing its anti-apoptotic activity. Such agents can work, for example, by preventing intracellular signal transduction pathways and/or translocation of galectin-3. Merely to illustrate, the agent can be one which inhibits the multimerization of galectin-3 and/or its interaction of galectin-3 with an anti-apoptotic Bcl-2 protein, such as Bcl-2 or bcl-xL. It may also be an agent that inhibits phosphorylation of galectin-3, such as by inhibiting phosphorylation of galectin-3 at Ser-6. At a gross mechanistic level, the inhibitor can be an agent that inhibits translocation of galectin-3 between the nucleus and cytoplasm or inhibits galectin-3 translocation to the perinuclear membranes and inhibits cytochrome C release from mitochondria. The inhibitor can also be an agent that induces proliferation of fibroblasts, e.g., by binding to and inhibiting galectin-3.

One class of galectin-3 inhibitors contemplated by the present invention is polymers, particularly carbohydrate-containing polymers, that bind to galectin-3 and inhibit its anti- apoptotic activity. Materials useful in the present invention may generally comprise natural or synthetic polymers and oligomers. Preferably, such polymers are very low in toxicity.

A preferred class of polymers for the practice of the present invention is

carbohydrate-derived polymers that contain an active galectin-binding sugar site, but that have higher molecular weights than simple sugars, making them capable of sustained blocking, activation, suppression, or other interaction with the galectin protein. A preferred class of therapeutic materials comprises oligomeric or polymeric species of natural or synthetic origin, rich in galactose or arabinose, such as pectin. Such materials may preferably have a molecular weight in the range of up to 500,000 daltons and, more preferably, in the range of up to 100,000 daltons. One particular material comprises a substantially demethoxylated polygalacturonic acid backbone which may be interrupted by rhamnose with galactose-terminated side chains pendent therefrom. Another particular material comprises a homogalacturonan backbone with or without side chains pendent therefrom.

Pectin is a complex carbohydrate having a highly branched structure comprised of a polygalacturonic backbone with numerous branching side chains dependent therefrom. The branching creates regions which are characterized as being "smooth" and "hairy." It has been found that pectin can be modified by various chemical, enzymatic or physical treatments to break the molecule into smaller portions having a more linearized, substantially

demethoxylated, polygalacturonic backbone with pendent side chains of rhamnose residues having decreased branching. The resulting partially depolymerized pectin is known in the art as modified pectin.

In certain embodiments, the invention provides a modified pectin comprising rhamnogalacturonan and/or homogalacturonan backbone with neutral sugar side chains, and having a low degree of neutral sugar branching dependent from the backbone. In certain embodiments, the modified pectin is de-esterified and partially depolymerized, so as to have a disrupted rhamnogalacturonan backbone.

In certain embodiments, the modified pectin includes a copolymer of galacturonic acid and rhamnogalacturonan I in which at least some of the galactose- and arabinose- containing sidechains are still attached. In preferred embodiments, the modified pectin has an average molecular weight of 50-200 kD, preferably 70-200 kD, more preferably 70-150 kD as measured by Gel Permeation Chromatography (GPC) with Multi Angle Laser Light Scattering (MALLS) detection.

In certain embodiments, the modified pectin comprises a homogalacturonan backbone with small amounts of rhamnogalacturonan therein, wherein the backbone has neutral sugar side chains having a low degree of branching dependent from the backbone. In particular embodiments, the galacturonic acid subunits of the homogalacturonan backbone have been partially de-esterified.

In certain embodiments, the invention may be described by either or both of formulas I and II below, and it is to be understood that variants of these general formula may be prepared and utilized in accord with the principles described in U. S. Pat. No. 8,128,966. Homogalacturonan

- [a-Gal/?A-(l 4)- a-Gal ]n- (I)

Rhamnogalacturonan

Y, m

(Π)

[-[a-Gal/?A]_n- X- [ a-Gak7A]o-]p

In the formula above, m is > 0, n, o and p are > 1, X is a-Rhap; and Y_m represents a linear or branched chain of sugars (each Y in the chain Y_m can independently represent a different sugar within the chain). The sugar Y may be, but is not limited to, any of the following: a-Ga\p, fi-Ga\p, β-Αρί fi-Rhap, α-Rhap , a-Fuc/?, P-Glc/?A, a-Gal Ά, β-Gal^A, β-Dha^A, Kdop, β-Ace J a-Ara J β-Ara J and <x-Xy\p.

An exemplary polymer of this type is modified pectin, preferably water-soluble pH- modified citrus pectin. Suitable polymers of this type are disclosed in, for example U.S. Patents 5,834,442, 5,895,784, 6,274,566, 6,500,807, 7,491,708, and 8,128,966, U.S. Patent Publication 2002/0107222, and PCT Publications WO 96/01640 and WO 03/000118.

It may be understood that natural pectin does not possess a strictly regular repeating structure, and that additional random variations are likely to be introduced by partial hydrolysis of the pectin, so that the identity of Y_m and the values of "n" and "o" may vary from one iteration to the next of the p repeating units represented by Formula II above.

Abbreviated sugar monomer names used herein are defined as follows: GalA:

galacturonic acid; Rha: rhamnose; Gal: galactose; Api: erythro-apiose; Fuc: fucose; GlcA: glucuronic acid; DhaA: 3-deoxy-D-/yxo-heptulosaric acid; Kdo: 3-deoxy-D-maw?o-2- octulosonic acid; Ace: aceric acid (3-C-carboxy-5-deoxy-L-lyxose); Ara: arabinose. Italicized p indicates the pyranose form, and italicized /indicates a furanose ring.

U.S. Patent Nos. 5,895,784, 8,128,966, 8,658,224, 8,409,635, 8,420,133, and

8,187,642, the disclosures of which are incorporated herein by reference, describe modified pectin materials, techniques for their preparation, and use of the material as a treatment for various cancers, and these materials may also be used in the compositions and methods described herein. As described in the '784 patent, modified pectins prepared by a pH-based modification procedure in which the pectin is put into solution and exposed to a series of programmed changes in pH results in the breakdown of the molecule to yield therapeutically effective modified pectin. A preferred starting material is citrus pectin, although it is to be understood that modified pectins may be prepared from pectin obtained from other sources, such as apple pectin. Also, modification may be done by enzymatic treatment of the pectin, or by physical processes such as heating. Further disclosure of modified pectins and techniques for their preparation and use are also found in U.S. Patents 5,834,442 and 7,491,708, the disclosures of which are incorporated herein by reference. Modified pectins of this type generally have molecular weights in the range of less than 100 kilodaltons. A group of such materials has an average molecular weight of less than 3 kilodaltons. Another group has an average molecular weight in the range of 1-15 kilodaltons, with a specific group of materials having a molecular weight of about 10 kilodaltons. In certain

embodiments, modified pectin has the structure of a pectic acid polymer with some of the pectic side chains still present. In preferred embodiments, the modified pectin is a copolymer of homogalacturonic acid and rhamnogalacturonan I in which some of the galactose- and arabinose-containing sidechains are still attached. The modified pectin may have an average molecular weight of 1 to 500 kilodaltons (kD), preferably 10 to 250 kD, more preferably 50-200 kD or 80-150 kD, and most preferably 80 to 100 kD as measured by Gel Permeation Chromatography (GPC) with Multi Angle Laser Light Scattering (MALLS) detection. In certain embodiments, the modified pectin is a modified apple pectin having an average molecular weight in the range of 20-70 kD. In certain embodiments, the modified pectin may have a average molecular weight in the range of 1-15 kD, while in other embodiments, the modified pectin has an average molecular weight in the range of 15-60 kD. See Gunning, et al, The FASEB Journal, (2009) vol. 23, p. 416, incorporated herein by reference in its entirety, for its discussion of galactans that bind galectin-3. Such galactans may also be used in the compositions and methods described herein.

In certain embodiments, the modified pectin is substantially free of modified pectins having a molecular weight below 25 kDa. The modified pectin may be prepared by passing modified or unmodified pectin through a tangential flow filter.

Degree of esterification is another characteristic of modified pectins. In certain embodiments, the degree of esterification may be between 0 and 80%, between 10 and 60%, between 0 and 50%, or between 20 and 60%, such as 20-45%, or 30-40% esterification.

Saccharide content is another characteristic of modified pectins. In certain embodiments, the modified pectin is composed entirely of a single type of saccharide subunit. In other embodiments, the modified pectin comprises at least two, preferably at least three, and most preferably at least four types of saccharide subunits. For example, the modified pectin may be composed entirely of galacturonic acid subunits. Alternatively, the modified pectin may comprise a combination of galacturonic acid and rhamnose subunits. In yet another example, the modified pectin may comprise a combination of galacturonic acid, rhamnose, and galactose subunits. In yet another example, the modified pectin may comprise a combination of galacturonic acid, rhamnose, and arabinose subunits. In still yet another example, the modified pectin may comprise a combination of galacturonic acid, rhamnose, galactose, and arabinose subunits. In some embodiments, the galacturonic acid content of modified pectin is greater than 50%, preferably greater than 60% and most preferably greater than 80%. In some embodiments, the rhamnose content is less than 25%, preferably less than 15% and most preferably less than 10%; the galactose content is less than 50%, preferably less than 40% and most preferably less than 30%; and the arabinose content is less than 15%, preferably less than 10% and most preferably less than 5%. In certain embodiments, the modified pectin may contain other uronic acids, xylose, ribose, lyxose, glucose, allose, altrose, idose, talose, gluose, mannose, fructose, psicose, sorbose or talalose in addition to the saccharide units mentioned above.

Modified pectin suitable for use in the subject methods may also have any of a variety of linkages or a combination thereof. By linkages it is meant the sites at which the individual sugars in pectin are attached to one another. In some embodiments, the modified pectin comprises only a single type of linkage. In certain preferred embodiments, the modified pectin comprises at least two types of linkages, and most preferably at least 3 types of linkages. For example, the modified pectin may comprise only alpha- 1,4 linked galacturonic acid subunits. Alternatively, the modified pectin may comprise alpha- 1,4- linked galacturonic acid subunits and alpha- 1,2-rhamnose subunits. In another example, the modified pectin may be composed of alpha- 1,4-linked galacturonic acid subunits and alpha- 1,2-rhamnose subunits linked through the 4 position to arabinose subunits. In another example, the modified pectin may comprise alpha- 1,4-linked galacturonic acid subunits and alpha-l,2-rhamnose subunits linked through the 4 position to arabinose subunits with additional 3-linked arabinose subunits. In another example, the modified pectin may comprise alpha- 1,4-linked galacturonic acid subunits and alpha- 1,2-rhamnose subunits linked through the 4 position to arabinose subunits with additional 5-linked arabinose units. In another example, the modified pectin may comprise alpha- 1,4-linked galacturonic acid subunits and alpha- 1,2-rhamnose subunits linked through the 4 position to arabinose subunits with additional 3-linked and 5-linked arabinose subunits. In another example, the modified pectin may comprise alpha- 1,4-linked galacturonic acid subunits and alpha- 1 ,2-rhamnose subunits linked through the 4 position to arabinose subunits with additional 3-linked and 5- linked arabinose subunits with 3, 5 -linked arabinose branch points. In another example, the modified pectin may comprise alpha- 1,4-linked galacturonic acid subunits and alpha- 1 ,2- rhamnose subunits linked through the 4 position to galactose subunits. In another example, the modified pectin may comprise alpha- 1 ,4-linked galacturonic acid subunits and alpha- 1,2- rhamnose subunits linked through the 4 position to galactose subunits with additional 3- linked galactose subunits. In another example, the modified pectin may comprise alpha-1 ,4- linked galacturonic acid subunits and alpha- 1,2-rhamnose subunits linked through the 4 position to galactose subunits with additional 4-linked galactose subunits. In another example, the modified pectin may comprise alpha- 1,4-linked galacturonic acid subunits and alpha-l ,2-rhamnose subunits linked through the 4 position to galactose subunits with additional 3-linked galactose subunits with 3,6-linked branch points. In another example, the modified pectin may comprise alpha- 1,4-linked galacturonic acid subunits and alpha- 1 ,2- rhamnose subunits linked through the 4 position to galactose subunits with additional 4- linked galactose subunits with 4,6-linked branch points. In certain embodiments, the side chains of the modified pectin may comprise uronic acids, galacaturonic acid, glucuronic acid, rhamnose, xylose, ribose, lyxose, glucose, allose, altrose, idose, talose, gluose, mannose, fructose, psicose, sorbose or talalose in addition to the saccharide units described above.

Modified pectins suitable for the compositions and methods described herein may have one or more of the characteristics described above.

Other carbohydrate materials including galactose residues capable of binding and inhibiting galectin-3 can also be employed in the compositions and methods disclosed herein. For example, mannan, dextrans, polygalacturonate, polyglucosamine and other water-soluble polysaccharides (see, for example, U. S. Patent Publication 2005/0043272 to Piatt, et al, incorporated herein by reference for the compositions disclosed therein) can be used as galectin-3 inhibitors. The inclusion of target specific carbohydrates, such as, galactose, rhamnose, mannose, or arabinose can be varied to target specific lectin-type receptors on tumor cells, e.g., to modulate relative inhibition of galectin-3 vs. galectin-9. One of skill in the art will recognize that there could be a heterogenous population of

carbohydrate residues on the polymer, as is true of some naturally occurring polymers, such as modified pectin and some galactans. Particular polysaccharides include galactomannans (e.g., from Cyamopsis tetragonolobus), arabinogalactan (e.g., from Larix occidentalis), rhamnogalacturonan (e.g., from potato), carrageenan (e.g., from Eucheuma seaweed), and the locust bean gum (e.g., from Ceratonia siliqua).

Alkyl-modified polysaccharides can originate from natural sources and/or be synthetically prepared from naturally occurring carbohydrate polymers. Microbial sources for alkylated polysaccharides are well known to those in the art, see, e.g., U.S. Pat. No. 5,997,881, the teachings of which are incorporated herein in their entirety by reference. Some of the microbial sources have been used in oil spill remediation operations (see Gutnick and Bach "Engineering bacterial biopolymers for the biosorption of heavy metals; Applied Microbiology and Biotechnology, 54 (4) pp 451-460, (2000); also see U.S. Pat. No. 4,395,354, Gutnick, et al. 1983, the entire teachings of which are hereby incorporated herein by reference). These microbes involved in oil spill remediation activities have been referred to as "Emulsans", in which some of their polysaccharides are O-acylated. Similar alkylated carbohydrates were also isolated from yeast fermentation and are known as sophorolipids.

Another example of suitable polysaccharides is a polysaccharide chain consisting essentially of 2-amino-2,6-dideoxyaldohexose sugar, glucosamine and one or more non- aminated sugars, wherein the amine groups of the aminated sugars are substantially all in acetylated form. The polysaccharide chain is linked with an ester bond to an alkyl moiety consisting of saturated and/or unsaturated chain of about 10 to about 18 carbon atoms of which 50-95% comprises dodecanoic acid and 3-hydroxy-dodecanoic acid. In one particular aspect, the dodecanoic acid is present in an amount greater than the 3-hydroxy-dodecanoic acid.

Optionally, the alkylated polysaccharide can comprise anionic groups, such as phosphate, sulfate, nitrate, carboxyl groups, and/or sulfate groups, while maintaining the hydrophobic moieties.

For example, a synthetic polysaccharide can be esterified with straight or branched alkyl groups of about 8 to about 40 carbon atoms. These alkyl groups may be aliphatic or unsaturated, and optionally may contain one or more aromatic groups. In certain

embodiments, the surface of the alkylated polysaccharides can be further derivatized using carbohydrate ligands, e.g., galactose, rhamnose, mannose or arabinose, to further enhance recognition sites by lectins. The polysaccharides of the present invention can be derivatized using alkyl, aryl or other chemical moieties. In particular embodiments, the polysaccharide can be a galactomannan, as described in U.S. Patent Publications 2003/0064957, 2005/0053664, 2011/0077217, and

2013/0302471, all of which are hereby incorporated by reference herein for the compositions disclosed therein. For example, the molecular weight of the galactomannan can have an average molecular weight in the range of 20-600 kD, for example the galactomannan has a molecular weight in the range of 90 to 415 kD or 40-200 kD, such as an average molecular weight of 83 kD or 215 kD. Suitable galactomannans may be isolated from Gleditsia triacanthos_L Ceratonia siliqua, Xanthomonas campestris, Trigonella foenum-graecum,

or Cyamopsis tetragonoloba or may be prepared from galactomannans isolated therefrom.

In certain such embodiments, the galactomannan may be -l→4-D-galactomannan and include a ratio of galactose to mannose where mannose is in the range of 1.0-3.0 and galactose is in the range of 0.5-1.5. Alternatively, the galactomannan may have a ratio of 2.6 mannose to 1.5 galactose.

In certain embodiments, the galactomannan has a ratio of 2.2 mannose to 0.9 galactose. Alternatively, the galactomannan may have a ratio of 1.13 mannose to 1 galactose. Alternatively, the galactomannan may have a ratio of 2.2 mannose to 1 galactose.

In certain embodiments, the polysaccharide can be -l,4-D-galactomannan and include a ratio of mannose to galactose of about 1.7. In certain embodiments, the molecular weight of the galactomannan polysaccharide is in the range of about 4 to about 200 kD. In certain particular embodiments, the galactomannan has an average weight of about 40 to 60 kD. In another aspect, the structure of the galactomannan is a ροΓν-β-1,4 mannan backbone, with side substituents affixed via α-Ι-6-glycoside linkages. In certain embodiments, the galactomannan polysaccharide can be -l,4-D-galactomannan. In certain particular embodiments, the polysaccharide is (((l,4)-linked -D-mannopyranose)17-((l,6)-linked- - D-galactopy ranose) 10) 12).

Suitable polysaccharides can have side branches of target specific carbohydrates, such as galactose, rhamnose, mannose, or arabinose, to impart recognition capabilities in targeting specific lectin-type receptors on the surface of cells, e.g., to modulate relative inhibition of galectin-3 vs. galectin-9. Branches can be a single unit or two or more units of oligosaccharide.

Yet another suitable polysaccharide is disclosed in U.S. Patent Publication

2005/0282773, hereby incorporated by reference herein for the compositions disclosed therein. Such polysaccharides mayb have a uronic acid saccharide backbone or uronic ester saccharide backbones having neutral monosaccharides connected to the backbone about every one-in-twenty to every one-in-twenty-five backbone units. The resulting

polysaccharides may have at least one side chain comprising mostly neutral saccharides and saccharide derivatives connected to the backbone via the about one-in-seven to twenty-five neutral monosaccharides. Some preferred polysaccharides may have at least one side chain of saccharides further having substantially no secondary saccharide branches, with a terminal saccharide comprising galactose, glucose, arabinose, or derivatives thereof. Other preferred polysaccharides may have at least one side chain of saccharides terminating with a saccharide modified by a feruloyl group.

Suitable polysaccharides may have an average molecular weight range of between about 40,000-400,000 dalton with multiple branches of saccharides, for example, branches comprised of glucose, arabinose, galactose, etc., and these branches may be connected to the backbone via neutral monosaccharides such as rhamnose. These molecules may further include a uronic acid saccharide backbone that may be esterified from as little as about 10% to as much as about 90% of uronic acid residues. The multiple branches themselves may have multiple branches of saccharides, the multiple branches optionally including neutral saccharides and neutral saccharide derivatives.

Such polysaccharides may be prepared by a chemical modification procedure that involves a pH-dependent depolymerization into smaller, de-branched polysaccharide molecules, using sequentially controlled pH, temperature and time, e.g., pH 10.0 at 37 °C for 30 minutes and than pH of about 3.5 at 25 °C for 12 hours (see Example 1). An optional alternative modification procedure is hydrolysis of the polysaccharide in an alkaline solution in the presence of a reducing agent such as a potassium borohydride to form fragments of a size corresponding to a repeating subunit (see, e.g., U.S. Pat. No. 5,554,386). The molecular weight range for the chemically modified polysaccharides is in the range of 5 to 60 kD, more specifically, in the range of about 15-40 kD, and more specifically, for example, about 20 kD.

Still other suitable polysaccharides are disclosed in U.S. Patent Publication

2008/0107622, hereby incorporated by reference herein for the compositions disclosed therein. One type of such polysaccharides include galacto-rhamnogalacturonate (GR), a branched heteropolymer of alternating 1,2-linked rhamnose and 1,4-linked Gala residues that carries neutral side-chains of predominantly l,4- -D-galactose and/or 1,5-a-L-arabinose residues attached to the rhamnose residues of the RGI backbone. GR side-chains may be decorated with arabinosyl residues (arabinogalactan I) or other sugars, including fucose, xylose, and mannose. These are also referred to in commercial use as pectic material.

Preparation of these polysaccharides may include modifying naturally occurring polymers to reduce the molecular weight for the desired range, adjusting the alkylated groups (demethoxylation or deacetylation), and adjusting side chain oligosaccharides for optimum efficacy. For example, natural polysaccharides may have a molecular weight range of between about 40,000-1,000,000 with multiple branches of saccharides, for example, branches comprised of 1 to 20 monosaccharides of glucose, arabinose, galactose, etc., and these branches may be connected to the backbone via neutral monosaccharides such as rhamnose. These molecules may further include a uronic acid saccharide backbone that may be esterified from as little as about 2% to as much as about 30%. The multiple branches themselves may have multiple branches of saccharides, the multiple branches optionally including neutral saccharides and neutral saccharide derivatives creating mainly hydrophobic entities.

In certain embodiments, a rhamnogalacturonate has a molecular weight range of 2,0 to 200 kD. In specific examples, the rhamnogalacturonate may have an average size molecular weight of about 34 kD or about 135 kD and is obtained through chemical, enzymatic, and/or physical treatments. Starting materials may be obtained via isolation and/or purification from pectic substance of citrus peels, apple pomace, soybean hull, or sugar beets, or other suitable materials, as will be apparent to the skilled artisan.

In certain embodiments, soluble chemically altered galacto-rhamnogalacturonates are prepared by modifying naturally occurring polymers to reduce the molecular weight for the desired range, reducing the alkylated group (de-methoxylation or de acetylation). Prior to chemical modification, the natural polysaccharides may have a molecular weight range of between about 40,000-1,000,000 with multiple branches of saccharides, for example, branches comprised of 1 to 20 monosaccharides of glucose, arabinose, galactose, etc., and these branches may be connected to the backbone via neutral monosaccharides, such as rhamnose. These molecules may further include a single or chain of uronic acid saccharide backbone that may be esterified from as little as about 2% to as much as about 30%. The multiple branches themselves may have multiple branches of saccharides, the multiple branches optionally including neutral saccharides and neutral saccharide derivatives creating mainly hydrophobic entities. Smaller saccharides can also be used. Suitable compounds include N- acetyllactosamine and its derivatives (see, for example, Sorme, et al, Chembiochem. 2002 Mar l ;3(2-3): 183-9, incorporated by reference herein in its entirety, which discloses a range of 3 '-amino-N-acetyllactosamine derivatives), as well as oligomeric and polymeric derivatives thereof, such as poly-N-acetyllactosamine.

Other classes of galectin-3 inhibitors that bind to galectin-3 include antibodies specific to galectin-3, peptides and polypeptides that bind to and interfere with galectin-3 activity, and small (preferably less than 2500 amu) organic molecules that bind to and inhibit galectin-3.

To further illustrate, in certain embodiments of the present invention, the subject methods can be carried out using an antibody or fragment thereof that is immunoreactive with galectin-3 and inhibitory for its anti-apoptotic activity.

An exemplary protein therapeutic is described in PCT publication WO 02/100343. That reference discloses certain N-Terminally truncated galectin-3 proteins that inhibit the binding of intact galectin-3 to carbohydrate ligands and thereby also inhibit the

multimerization and cross-linking activities of galectin-3 that may be required for its anti- apoptotic activity.

Exemplary small molecule inhibitors of galectin-3 include thiodigalactoside (such as described in Leffler et al., 1986, J. Biol. Chem. 261 : 10119) and agents described in PCT publication WO 02/057284, incorporated herein by reference for the inhibitors disclosed therein.

In certain preferred embodiments of galectin-3 inhibitors that bind to galectin-3, the inhibitor is selected to having a dissociation constant (Kd) for binding galectin-3 of 10^"6 M or less, and even more preferably less than 10^"7 M, 10^"8 M or even 10^"9 M.

Certain of the galectin-3 inhibitors useful in the present invention act by binding to galectin-3 and disrupting galectin-3's interactions with one or more anti-apoptotic Bcl-2 proteins. A galectin-3 inhibitor may bind directly to the Bcl-2 binding site thereby competitively inhibits Bcl-2 binding. However, galectin-3 inhibitors which bind to the Bcl-2 protein are also contemplated, and include galectin-3 inhibitors that bind to a Bcl-2 protein and either competitively or allosterically inhibit interaction with galectin-3.

As mentioned above, certain of the subject galectin-3 inhibitors exert their effect by inhibiting phosphorylation of galectin-3. The binding of a galectin-3 inhibitor may block the access of kinases responsible for galectin-3 phosphorylation, or, alternatively, may cause conformational change of galectin, concealing or exposing the phosphorylation sites.

However, the present invention also contemplates the use of kinase inhibitors which act directly on the kinase(s) that is responsible for phosphorylating galectin-3.

In still other embodiments, inhibition of galectin-3 activity is also achieved by inhibiting expression of galectin-3 protein. Such inhibition is achieved using an antisense or RNAi construct having a sequence corresponding to a portion of the mRNA sequence transcribed from the galectin-3 gene.

In certain embodiments, the galectin-3 inhibitors can be nucleic acids. In certain embodiments, the invention relates to the use of antisense nucleic acid that hybridizes to the galectin-3 mRNA and decreases expression of galectin-3. Such an antisense nucleic acid can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes galectin-3. Alternatively, the construct is an oligonucleotide which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences encoding galectin-3. Such oligonucleotides are optionally modified oligonucleotide which are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, and is therefore stable in vivo.

Exemplary nucleic acid molecules for use as antisense oligonucleotides are

phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U. S. Patent Nos. 5, 176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in nucleic acid therapy have been reviewed, for example, by van der Krol et al., (1988) Biotechniques 6:958-976; and Stein et al., 1988, Cancer Res. 48:2659-2668.

In other embodiments, the invention relates to the use of RNA interference (RNAi) to effect knockdown of expression of the galectin-3 gene. RNAi constructs comprise double stranded RNA that can specifically block expression of a target gene. "RNA interference" or "RNAi" is a term initially applied to a phenomenon observed in plants and worms where double-stranded RNA (dsRNA) blocks gene expression in a specific and post-transcriptional manner. RNAi provides a useful method of inhibiting gene expression in vitro or in vivo. As used herein, the term "RNAi construct" is a generic term including small interfering RNAs (siRNAs), hairpin RNAs, and other RNA species which can be cleaved in vivo to form siRNAs. RNAi constructs herein also include expression vectors (also referred to as RNAi expression vectors) capable of giving rise to transcripts which form dsRNAs or hairpin RNAs in cells, and/or transcripts which can produce siRNAs in vivo.

RNAi constructs can comprise either long stretches of dsRNA identical or substantially identical to the target nucleic acid sequence or short stretches of dsRNA identical to substantially identical to only a region of the target nucleic acid sequence.

Optionally, the RNAi constructs contain a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the mRNA transcript for the gene to be inhibited (i.e., the "target" gene). The double-stranded RNA need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi. Thus, the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism or evolutionary divergence. The number of tolerated nucleotide mismatches between the target sequence and the RNAi construct sequence is no more than 1 in 5 base pairs, or 1 in 10 base pairs, or 1 in 20 base pairs, or 1 in 50 base pairs. Mismatches in the center of the siRNA duplex are most critical and may essentially abolish cleavage of the target RNA. In contrast, nucleotides at the 3' end of the siRNA strand that is complementary to the target RNA do not significantly contribute to specificity of the target recognition. Sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene is preferred. Alternatively, the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 °C or 70 °C hybridization for 12-16 hours; followed by washing).

The double-stranded structure may be formed by a single self-complementary RNA strand or two complementary RNA strands. RNA duplex formation may be initiated either inside or outside the cell. The RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded material may yield more effective inhibition, while lower doses may also be useful for specific applications. Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition.

The subject RNAi constructs can be "small interfering RNAs" or "siRNAs." These nucleic acids are around 19-30 nucleotides in length, and even more preferably 21 -23 nucleotides in length. The siRNAs are understood to recruit nuclease complexes and guide the complexes to the target mRNA by pairing to the specific sequences. As a result, the target mRNA is degraded by the nucleases in the protein complex. In some embodiments, the 21 -23 nucleotides siRNA molecules comprise a 3' hydroxyl group. In certain embodiments, the siRNA constructs can be generated by processing of longer double- stranded RNAs, for example, in the presence of the enzyme dicer. For example, the

Drosophila in vitro system may be used. In this system, dsRNA is combined with a soluble extract derived from Drosophila embryo, thereby producing a combination. The

combination is maintained under conditions in which the dsRNA is processed to RNA molecules of about 21 to about 23 nucleotides. The siRNA molecules can be purified using a number of techniques known to those of skill in the art. For example, gel electrophoresis can be used to purify siRNAs. Alternatively, non-denaturing methods, such as non- denaturing column chromatography, can be used to purify the siRNA. In addition, chromatography (e.g., size exclusion chromatography), glycerol gradient centrifugation, affinity purification with antibody can be used to purify siRNAs.

Production of RNAi constructs can be carried out by chemical synthetic methods or by recombinant nucleic acid techniques. Endogenous RNA polymerase of the treated cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vitro. The RNAi constructs may include modifications to either the phosphate-sugar backbone or the nucleoside, e.g., to reduce susceptibility to cellular nucleases, improve bioavailability, improve formulation characteristics, and/or change other pharmacokinetic properties. For example, the phosphodiester linkages of natural RNA may be modified to include at least one of an nitrogen or sulfur heteroatom. Modifications in RNA structure may be tailored to allow specific genetic inhibition while avoiding a general response to dsRNA. Likewise, bases may be modified to block the activity of adenosine deaminase. The RNAi construct may be produced enzymatically or by partial/total organic synthesis, any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis. Methods of chemically modifying RNA molecules can be adapted for modifying RNAi constructs (see, e.g., Heidenreich et al., 1997, Nucleic Acids Res. , 25:776-780; Wilson et al. , 1994, J. Mol. Recog. 7:89-98; Chen et al, 1995 , Nucleic Acids Res . 23:2661-2668;

Hirschbein et al , 1997 ^', Antisense Nucleic Acid Drug Dev. 7:55-61). Merely to illustrate, the backbone of an RNAi construct can be modified with phosphorothioates, phosphoramidate, phosphodithioates, chimeric methylphosphonate-phosphodiesters, peptide nucleic acids, 5- propynyl-pyrimidine containing oligomers or sugar modifications (e.g., 2' -substituted ribonucleosides, a-configuration).

In some cases, at least one strand of the siRNA molecules has a 3' overhang from about 1 to about 6 nucleotides in length, though may be from 2 to 4 nucleotides in length. More preferably, the 3' overhangs are 1-3 nucleotides in length. In certain embodiments, one strand having a 3' overhang and the other strand being blunt-ended or also having an overhang. The length of the overhangs may be the same or different for each strand. In order to further enhance the stability of the siRNA, the 3' overhangs can be stabilized against degradation. In some embodiments, the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine nucleotide 3' overhangs by 2'-deoxythyinidine is tolerated and does not affect the efficiency of RNAi. The absence of a 2' hydroxyl significantly enhances the nuclease resistance of the overhang in tissue culture medium and may be beneficial in vivo.

The RNAi construct can also be in the form of a long double-stranded RNA. In certain embodiments, the RNAi construct is at least 25, 50, 100, 200, 300 or 400 bases. In certain embodiments, the RNAi construct is 400-800 bases in length. The double-stranded RNAs are digested intracellularly, e.g., to produce siRNA sequences in the cell. However, use of long double-stranded RNAs in vivo is not always practical, presumably because of deleterious effects which may be caused by the sequence-independent dsRNA response. In such embodiments, the use of local delivery systems and/or agents which reduce the effects of interferon or PKR are preferred.

Alternatively, the RNAi construct is in the form of a hairpin structure (named as hairpin RNA). The hairpin RNAs can be synthesized exogenously or can be formed by transcribing from RNA polymerase III promoters in vivo. Examples of making and using such hairpin RNAs for gene silencing in mammalian cells are described in, for example, Paddison et al, Genes Dev ., 2002, 16:948-58; McCaffrey et al, Nature, 2002, 418:38-9; McManus et al, RNA, 2002, 8:842-50; Yu et al, Proc. Nat'lAcad. Sci. USA, 2002, 99:6047- 52). Preferably, such hairpin RNAs are engineered in cells or in an animal to ensure continuous and stable suppression of a desired gene. It is known in the art that siRNAs can be produced by processing a hairpin RNA in the cell.

In other embodiments, the invention relates to the use of ribozyme molecules designed to catalytically cleave galectin-3 mRNA transcripts to prevent translation of mRNA (see, e.g., PCT International Publication WO90/11364, published October 4, 1990; Sarver et al, 1990, Science 247: 1222-1225; and U.S. Patent No. 5,093,246). While ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy particular mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591. The ribozymes of the present invention also include RNA endoribonucleases ("Cech-type ribozymes") such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS or L-19 IVS RNA) and which has been extensively described (see, e.g., Zaug, et al, 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231 :470-475; Zaug, et al, 1986, Nature, 324:429-433; published

International patent application No. WO88/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216).

In further embodiments, the invention relates to the use of DNA enzymes to inhibit expression of the galectin-3 gene. DNA enzymes incorporate some of the mechanistic features of both antisense and ribozyme technologies. DNA enzymes are designed so that they recognize a particular target nucleic acid sequence, much like an antisense

oligonucleotide, however much like a ribozyme they are catalytic and specifically cleave the target nucleic acid. Briefly, to design an ideal DNA enzyme that specifically recognizes and cleaves a target nucleic acid, one of skill in the art must first identify the unique target sequence. Preferably, the unique or substantially sequence is a G/C rich of approximately 18 to 22 nucleotides. High G/C content helps insure a stronger interaction between the DNA enzyme and the target sequence. When synthesizing the DNA enzyme, the specific antisense recognition sequence that may target the enzyme to the message is divided so that it comprises the two arms of the DNA enzyme, and the DNA enzyme loop is placed between the two specific arms. Methods of making and administering DNA enzymes can be found, for example, in U.S. Patent No. 6,110,462. Other inhibitors may include monoclonal, polyclonal, humanized, and/or chimeric antibodies that bind to galectin-3. The term "antibody," as used herein, is intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Representative antibodies are described in further detail in U.S. Patent Nos. 6,090,382; 6,258,562; and 6,509,015.

The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., galectin-3). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full- length antibody. Binding fragments include Fab, Fab', F(ab')2, Fabc, Fv, single chains, and single-chain antibodies. Examples of binding fragments encompassed within the term

"antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al. (1989)

Nature 341 :544-546 ), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).

Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) . Such single chain antibodies are also intended to be encompassed within the term "antigen- binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al.

(1994) Structure 2: 1121-1123). The antibody portions of the invention are described in further detail in U.S. Patent Nos. 6,090,382, 6,258,562, 6,509,015, each of which is incorporated herein by reference in its entirety.

Still further, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule, formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S.M., et al. (1995) Human Antibodies and Hybridomas 6:93- 101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S.M., et al. (1994) Mol.

Immunol. 31 : 1047- 1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

"Chimeric antibodies" refers to antibodies wherein one portion of each of the amino acid sequences of heavy and light chains is homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class, while the remaining segment of the chains is homologous to corresponding sequences from another species. In certain embodiments, the invention features a chimeric antibody or antigen- binding fragment, in which the variable regions of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals, while the constant portions are homologous to the sequences in antibodies derived from another species. In certain embodiments, chimeric antibodies are made by grafting CDRs from a mouse antibody onto the framework regions of a human antibody.

"Humanized antibodies" refer to antibodies which comprise at least one chain comprising variable region framework residues substantially from a human antibody chain (referred to as the acceptor immunoglobulin or antibody) and at least one complementarity determining region (CDR) substantially from a non-human-antibody (e.g., mouse). In addition to the grafting of the CDRs, humanized antibodies typically undergo further alterations in order to improve affinity and/or immmunogenicity.

The term "multivalent antibody" refers to an antibody comprising more than one antigen recognition site. For example, a "bivalent" antibody has two antigen recognition sites, whereas a "tetravalent" antibody has four antigen recognition sites. The terms

"monospecific," "bispecific," "trispecific," "tetraspecific," etc. refer to the number of different antigen recognition site specificities (as opposed to the number of antigen recognition sites) present in a multivalent antibody. For example, a "monospecific" antibody's antigen recognition sites all bind the same epitope. A "bispecific" or "dual specific" antibody has at least one antigen recognition site that binds a first epitope and at least one antigen recognition site that binds a second epitope that is different from the first epitope. A "multivalent monospecific" antibody has multiple antigen recognition sites that all bind the same epitope. A "multivalent bispecific" antibody has multiple antigen recognition sites, some number of which bind a first epitope and some number of which bind a second epitope that is different from the first epitope.

The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term "recombinant human antibody," as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant, combinatorial human antibody library (described further below), monoclonal antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

IV. Serum Markers and Biomarkers

Serum markers may be measured in conjunction with galectin-3 to measure the effect of treatment with a galectin-3 inhibitor, such as a modified pectin (e.g., GCS-100). Whole blood samples may be drawn for determination of the levels of circulating galectin-3 and/or other serum markers. Assays for galectin-3 concentration and serum markers may be performed according to the methods described herein and known in the art.

In certain embodiments, the present methods reduce the galectin-3 levels by 0.1, 0.2,

0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 4, 6, 8, or even 10-fold in patients given a low dose of galectin-3 inhibitor (e.g., 1.5 mg/m² of modified pectin, such as GCS-100), e.g., relative to galectin-3 measured in an untreated patient or a patient treated with placebo.

V. Galectin-3 and biomarker protein detection techniques

Methods for the detection of protein, e.g., galectin-3 protein and biomarkers, are well known to those skilled in the art, and include ELISA (enzyme linked immunosorbent assay), RIA (radioimmunoassay), Western blotting, and immunohistochemistry. Immunoassays such as ELISA or RIA, which can be extremely rapid, are more generally preferred. These methods use antibodies, or antibody equivalents, to detect galectin-3 protein. Antibody arrays or protein chips can also be employed, see for example U.S. Patent Application Nos: 20030013208A1; 20020155493A1, 20030017515 and U.S. Pat. Nos: 6,329,209; 6,365,418, herein incorporated by reference in their entirety.

ELISA and RIA procedures may be conducted such that a galectin-3 standard is labeled (with a radioisotope such as ¹²⁵I or ⁵S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase), and, together with the unlabelled sample, brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first, and radioactivity or the immobilized enzyme assayed (competitive assay). Alternatively, galectin-3 in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme-labeled anti-galectin-3 antibody is allowed to react with the system, and radioactivity or the enzyme assayed (ELISA-sandwich assay). Other conventional methods may also be employed as suitable.

The above techniques may be conducted essentially as a "one-step" or "two-step" assay. A "one-step" assay involves contacting antigen with immobilized antibody and, without washing, contacting the mixture with labeled antibody. A "two-step" assay involves washing before contacting, the mixture with labeled antibody. Other conventional methods may also be employed as suitable.

In certain embodiments, a method for measuring galectin-3 levels comprises:

contacting a biological specimen with an antibody or variant (e.g., fragment) thereof which selectively binds galectin-3, and detecting whether said antibody or variant thereof is bound to said sample and thereby measuring the levels of galectin-3. A method may further comprise contacting the specimen with a second antibody, e.g., a labeled antibody. The method may further comprise one or more steps of washing, e.g., to remove one or more reagents.

Enzymatic and radiolabeling of galectin-3 and/or the antibodies may be effected by any suitable means. Such means may generally include covalent linking of the enzyme to the antigen or the antibody in question, such as by glutaraldehyde, specifically so as not to adversely affect the activity of the enzyme, by which is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be effected. Indeed, some techniques for binding enzyme are non-specific (such as using formaldehyde), and may only yield a proportion of active enzyme.

It may be desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed without laborious and time-consuming labor. It is possible for a second phase to be immobilized away from the first, but one phase is usually sufficient.

It is possible to immobilize the enzyme itself on a support, but if solid-phase enzyme is required, then this is generally best achieved by binding to antibody and affixing the antibody to a support, models and systems for which are well-known in the art. Simple polyethylene may provide a suitable support. Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availability and cheapness, as well as the ready availability of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art.

Other techniques may be used to detect galectin-3 according to a practitioner's preference based upon the present disclosure. One such technique is Western blotting (Towbin et al., Proc. Nat. Acad. Sci. 76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter. Anti-galectin-3 antibodies (unlabeled) are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or antiimmunoglobulin (suitable labels including ¹²⁵I, horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.

Immunohistochemistry may be used to detect expression of human galectin-3, e.g., in a biopsy sample. A suitable antibody is brought into contact with, for example, a thin layer of cells, washed, and then contacted with a second, labeled antibody. Labeling may be by fluorescent markers, enzymes, such as peroxidase, avidin, or radiolabelling. The assay is scored visually, using microscopy. The results may be quantitated, e.g., as described in the Examples.

Immunohistochemical analysis optionally coupled with quantification of the signal may be conducted as follows. Galectin-3 and biomarker expression may be directly evaluated in the tissue by preparing immunohistochemically stained slides with, e.g., an avidin-biotinylated peroxidase complex system.

Evaluation of the presence of stains, i.e., galectin-3 or biomarker, may also be done by quantitative immunohistochemical investigation, e.g., with a computerized image analyzer (e.g., Automated Cellular Imaging System, ACIS, ChromaVision Medical System Inc., San Juan Capistrano, CA) may be used for evaluation of the levels of galectin-3or biomarker expression in the immunostained tissue samples. Using ACIS, "cytoplasmic staining" may be chosen as program for galectin-3 or biomarker detection. Different areas of immunostained tumor samples may be analyzed with the ACIS system. An average of the ACIS values that is more or less than 1, e.g., about 1.1, 1.2, 1.3, 1.4, 1.5., 2, 2.5, 3, 5, 10, 30, 100 or more indicates an elevated or decreased galectin-3 or biomarker expression.

Other machine or autoimaging systems may also be used to measure immunostaining results for galectin-3. As used herein, "quantitative" immunohistochemistry refers to an automated method of scanning and scoring samples that have undergone

immunohistochemistry, to identify and quantitate the presence of a specified biomarker, such as an antigen or other protein. The score given to the sample is a numerical representation of the intensity of the immunohistochemical staining of the sample, and represents the amount of target biomarker present in the sample. As used herein, Optical Density (OD) is a numerical score that represents intensity of staining. As used herein, semi-quantitative immunohistochemistry refers to scoring of immunohistochemical results by human eye, where a trained operator ranks results numerically (e.g., as 1, 2 or 3).

Various automated sample processing, scanning and analysis systems suitable for use with immunohistochemistry are available in the art. Such systems may include automated staining (see, e.g, the Benchmark™ system, Ventana Medical Systems, Inc.) and

microscopic scanning, computerized image analysis, serial section comparison (to control for variation in the orientation and size of a sample), digital report generation, and archiving and tracking of samples (such as slides on which tissue sections are placed). Cellular imaging systems are commercially available that combine conventional light microscopes with digital image processing systems to perform quantitative analysis on cells and tissues, including immunostained samples. See, e.g., the CAS-200 system (Becton, Dickinson & Co.).

Another method that may be used for detecting and quantitating galectin-3 or biomarker protein levels is Western blotting, e.g., as described in the Examples. Tumor tissues may be frozen and homogenized in lysis buffer. Immunodetection can be performed with a galectin-3 antibody using the enhanced chemiluminescence system (e.g., from PerkinElmer Life Sciences, Boston, MA). The membrane may then be stripped and re- blotted with a control antibody, e.g., anti-actin (A-2066) polyclonal antibody from Sigma (St. Louis, MO). The intensity of the signal may be quantified by densitometry software (e.g., NIH Image 1.61). After quantification of the galectin-3, biomarker, and control signals (e.g., actin), the relative expression levels of galectin-3 or biomarker are normalized by amount of the actin in each lane, i.e., the value of the galectin-3 or biomarker signal is divided by the value of the control signal. Galectin-3 or biomarker protein expression is considered to be elevated when the relative level is more than 1, e.g., about 1.1, 1.2, 1.3, 1.4, 1.5., 2, 2.5, 3, 5, 10, 30, or even 100. Conversely, galectin-3 or biomarker protein expression is considered to be reduced when the relative level is less than 1, e.g., about 1.1, 1.2, 1.3, 1.4, 1.5., 2, 2.5, 3, 5, 10, 30, or even 100.

Anti-galectin-3 or biomarker antibodies may also be used for imaging purposes, for example, to detect the presence of galectin-3 or biomarkers in cells and tissues of a subject. Suitable labels include radioisotopes, iodine (¹²⁵1, ¹²¹I), carbon (¹⁴C), sulphur ( ⁵S), tritium (¾), indium (¹¹²In), and technetium (⁹⁹mTc), fluorescent labels, such as fluorescein and rhodamine, and biotin. Immunoenzymatic interactions can be visualized using different enzymes such as peroxidase, alkaline phosphatase, or different chromogens such as DAB, AEC or Fast Red.

For in vivo imaging purposes, antibodies are not intrinsically detectable from outside the body, and so must be labeled, or otherwise modified, to permit detection. Markers for this purpose may be any that do not substantially interfere with the antibody binding, but which allow external detection. Suitable markers may include those that may be detected by X-radiography, NMR or MRI. For X-radiographic techniques, suitable markers include any radioisotope that emits detectable radiation but that is not overtly harmful to the patient, such as barium or caesium, for example. Suitable markers for NMR and MRI generally include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by suitable labeling of nutrients for the relevant hybridoma, for example.

The size of the subject, and the imaging system used, may determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected may normally range from about 5 to 20 millicuries of technetium-99m. The labeled antibody or antibody fragment may then preferentially accumulate at the location of cells which contain galectin-3. The labeled antibody or variant thereof, e.g., antibody fragment, can then be detected using known techniques.

Antibodies that may be used to detect galectin-3 include any antibody, whether natural or synthetic, full length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to the galectin-3 to be detected, e.g., human galectin-3. An antibody may have a Kd of at most about 10^"6M, 10^"7M, 10^"8M, 10^"9M, 10^"10M, 10^"nM, 10^"12M. The phrase "specifically binds" refers to binding of, for example, an antibody to an epitope or antigen or antigenic determinant in such a manner that binding can be displaced or competed with a second preparation of identical or similar epitope, antigen or antigenic determinant. An antibody may bind preferentially to galectin-3 relative to other proteins, such as related proteins, e.g., galectin 1-15.

Antibodies and derivatives thereof that may be used encompasses polyclonal or monoclonal antibodies, chimeric, human, humanized, primatized (CDR-grafted), veneered or single-chain antibodies, phase produced antibodies (e.g., from phage display libraries), as well as functional, i.e., galectin-3 binding fragments, of antibodies. For example, antibody fragments capable of binding to galectin-3 or portions thereof, including, but not limited to Fv, Fab, Fab' and F(ab')2 fragments can be used. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab') 2 fragments, respectively. Other proteases with the requisite substrate specificity can also be used to generate Fab or F(ab')2 fragments. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH, domain and hinge region of the heavy chain.

In some embodiments, agents that specifically bind to galectin-3 or other than antibodies are used, such as peptides. Peptides that specifically bind to galectin-3 can be identified by any means known in the art. For example, specific pepride binders of galectin- 3 can be screened for using peptide phage display libraries.

Generally, a reagent that is capable of detecting a galectin-3 or biomarker polypeptide, such that the presence of galectin-3 or other biomarker is detected and/or quantitated, may be used. As defined herein, a "reagent" refers to a substance that is cabable of identifying or detecting galectin-3 in a biological sample (e.g., identifies or detects galectin-3 or biomarker mRNA, DNA, and protein). In some embodiments, the reagent is a labeled or labelable antibody which specifically binds to galectin-3 or biomarker polypeptide. As used herein, the phrase "labeled or labelable" refers to the attaching or including of a label (e.g., a marker or indicator) or ability to attach or include a label (e.g., a marker or indicator). Markers or indicators include, but are not limited to, for example, radioactive molecules, colorimetric molecules, and enzymatic molecules which produce detectable changes in a substrate.

In addition, an galectin-3 or biomarker protein may be detected using Mass

Spectrometry such as MALDI/TOF (time-of-flight), SELDI/TOF, liquid chromatography- mass spectrometry (LC-MS), gas chromatography -mass spectrometry (GC-MS), high performance liquid chromatography -mass spectrometry (HPLC-MS), capillary

electrophoresis-mass spectrometry, nuclear magnetic resonance spectrometry, or tandem mass spectrometry (e.g., MS/MS, MS/MS/MS, ESI-MS/MS, etc.). See for example, U.S. Patent Application Nos: 20030199001, 20030134304, 20030077616, which are herein incorporated by reference.

Mass spectrometry methods are well known in the art and have been used to quantify and/or identify biomolecules, such as proteins (see, e.g., Li et al. (2000) Tibtech 18: 151-160; Rowley et al. (2000) Methods 20: 383-397; and Kuster and Mann (1998) Curr. Opin.

Structural Biol. 8: 393-400). Further, mass spectrometric techniques have been developed that permit at least partial de novo sequencing of isolated proteins. Chait et al, Science

262:89-92 (1993); Keough et al, Proc. Natl. Acad. Sci. USA. 96:7131-6 (1999); reviewed in Bergman, EXS 88: 133-44 (2000).

In certain embodiments, a gas phase ion spectrophotometer is used. In other embodiments, laser-desorption/ionization mass spectrometry is used to analyze the sample. Modem laser desorption/ionization mass spectrometry ("LDI-MS") can be practiced in two main variations: matrix assisted laser desorption/ionization ("MALDI") mass spectrometry and surface-enhanced laser desorption/ionization ("SELDI"). In MALDI, the analyte is mixed with a solution containing a matrix, and a drop of the liquid is placed on the surface of a substrate. The matrix solution then co-crystallizes with the biological molecules. The substrate is inserted into the mass spectrometer. Laser energy is directed to the substrate surface where it desorbs and ionizes the biological molecules without significantly fragmenting them. However, MALDI has limitations as an analytical tool. It does not provide means for fractionating the sample, and the matrix material can interfere with detection, especially for low molecular weight analytes. See, e.g., U.S. Pat. No. 5,118,937 (Hillenkamp et al), and U.S. Pat. No. 5,045,694 (Beavis & Chait).

In SELDI, the substrate surface is modified so that it is an active participant in the desorption process. In one variant, the surface is derivatized with adsorbent and/or capture reagents that selectively bind the protein of interest. In another variant, the surface is derivatized with energy absorbing molecules that are not desorbed when struck with the laser. In another variant, the surface is derivatized with molecules that bind the protein of interest and that contain a photolytic bond that is broken upon application of the laser. In each of these methods, the derivatizing agent generally is localized to a specific location on the substrate surface where the sample is applied. See, e.g., U.S. Pat. No. 5,719,060 (Hutchens & Yip) and WO 98/59361 (Hutchens & Yip). The two methods can be combined by, for example, using a SELDI affinity surface to capture an analyte and adding matrix- containing liquid to the captured analyte to provide the energy absorbing material.

For additional information regarding mass spectrometers, see, e.g., Principles of Instrumental Analysis, 3rd edition., Skoog, Saunders College Publishing, Philadelphia, 1985; and Kirk-Othmer Encyclopedia of Chemical Technology, 4 * ed. Vol. 15 (John Wiley & Sons, New York 1995), pp. 1071-1094.

Detection of the presence of a marker or other substances may typically involve detection of signal intensity. This, in turn, can reflect the quantity and character of a polypeptide bound to the substrate. For example, in certain embodiments, the signal strength of peak values from spectra of a first sample and a second sample can be compared (e.g., visually, by computer analysis etc.), to determine the relative amounts of particular biomolecules. Software programs such as the Biomarker Wizard program (Ciphergen Biosystems, Inc., Fremont, Calif.) can be used to aid in analyzing mass spectra. The mass spectrometers and their techniques are well known to those of skill in the art.

Any person skilled in the art understands, any of the components of a mass spectrometer (e.g., desorption source, mass analyzer, detect, etc.) and varied sample preparations can be combined with other suitable components or preparations described herein, or to those known in the art. For example, in some embodiments a control sample, a reference sample, and or one or more test samples may be distinguished by the presence of heavy atoms (e.g., ¹ C), optionally by using isotopically differentiated labels linked to the substrate to be detected in an array of samples, thereby permitting multiple samples to be combined and differentiated in the same mass spectrometry run.

In certain preferred embodiments, a laser desorption time-of-flight (TOF) mass spectrometer is used. In laser desorption mass spectrometry, a substrate with a bound marker is introduced into an inlet system. The marker is desorbed and ionized into the gas phase by laser from the ionization source. The ions generated are collected by an ion optic assembly, and then in a time-of-flight mass analyzer, ions are accelerated through a short high voltage field and let drift into a high vacuum chamber. At the far end of the high vacuum chamber, the accelerated ions strike a sensitive detector surface at a different time. Since the time-of- flight is a function of the mass of the ions, the elapsed time between ion formation and ion detector impact can be used to identify the presence or absence of molecules of specific mass to charge ratio. In some embodiments, the relative amounts of one or more biomolecules present in a first or second sample is determined, in part, by executing an algorithm with a programmable digital computer. The algorithm identifies at least one peak value in the first mass spectrum and the second mass spectrum. The algorithm then compares the signal strength of the peak value of the first mass spectrum to the signal strength of the peak value of the second mass spectrum of the mass spectrum. The relative signal strengths are an indication of the amount of the biomolecule that is present in the first and second samples. A standard containing a known amount of a biomolecule can be analyzed as the second sample to better quantify the amount of the biomolecule present in the first sample. In certain embodiments, the identity of the biomolecules in the first and second sample can also be determined.

VI. Galectin-3 and biomarker RNA detection techniques

Any method for qualitatively or quantitatively detecting galectin-3/biomarker RNA, e.g., mRNA, may be used.

Detection of RNA transcripts may be achieved by Northern blotting, for example, wherein a preparation of RNA is run on a denaturing agarose gel, and transferred to a suitable support, such as activated cellulose, nitrocellulose or glass or nylon membranes. Radiolabeled cDNA or RNA is then hybridized to the preparation, washed and analyzed by autoradiography.

Detection of RNA transcripts can further be accomplished using amplification methods. For example, it is within the scope of the present invention to reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Pat. No. 5,322,770, or reverse transcribe mRNA into cDNA followed by symmetric gap ligase chain reaction (RT-AGLCR) as described by R. L. Marshall, et al, PCR Methods and Applications 4: 80-84 (1994).

In certain embodiments, quantitative real-time polymerase chain reaction (qRT-PCR) is used to evaluate mRNA levels of galectin-3 (see Examples). Galectin-3/biomarker and a control mRNA, e.g., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels may be quantitated in cancer tissue and adjacent benign tissues. For this, frozen tissues may be cut into 5 micron sections and total RNA may be extracted, e.g., by Qiagen RNeasy Mini Kit (Qiagen, Inc., Valencia, CA). A certain amount of RNA, e.g., five hundred nanograms of total RNA, from each tissue may be reversely transcribed by using, e.g., Qiagen

Omniscript RT Kit. Two-step qRT-PCR may be performed, e.g., with the ABI TaqMan PCR reagent kit (ABI Inc, Foster City, CA), and galectin-3 primers and GAPDH primers, and the probes for both genes on ABI Prism 7700 system. Suitable primers that may be used are set forth in the Examples. The galectin-3/biomarker copy number may then be divided by the GAPDH copy number and multiplied by 1,000 to give a value for the particular subject. In other words, the amount of galectin-3/biomarker mRNA was normalized with the amount of GAPDH mRNA measured in the same RNA extraction to obtain a galectin-3/biomarker /GAPDH ratio. A ratio that is equal to or more than 1, e.g., about 1.1, 1.2, 1.3, 1.4, 1.5., 2, 2.5, 3, 5, 10, 30, or 100 may be considered as a high galectin-3/biomarker expression.

Other known amplification methods which can be utilized herein include but are not limited to the so-called "NASBA" or "3SR" technique described in PNAS USA 87: 1874- 1878 (1990) and also described in Nature 350 (No. 6313): 91-92 (1991); Q-beta

amplification as described in published European Patent Application (EPA) No. 4544610; strand displacement amplification (as described in G. T. Walker et al, Clin. Chem. 42: 9-13 (1996) and European Patent Application No. 684315; and target mediated amplification, as described by PCT Publication W09322461.

Primers that may be used for amplification of galectin-3 nucleic acid portions are set forth in the Examples.

In situ hybridization visualization may also be employed, wherein a radioactively labeled antisense RNA probe is hybridized with a thin section of a biopsy sample, washed, cleaved with RNase and exposed to a sensitive emulsion for autoradiography. The samples may be stained with haematoxylin to demonstrate the histological composition of the sample, and dark field imaging with a suitable light filter shows the developed emulsion. Nonradioactive labels such as digoxigenin may also be used.

Another method for evaluation of galectin-3/biomarker expression is to detect gene amplification by fluorescent in situ hybridization (FISH). FISH is a technique that can directly identify a specific region of DNA or RNA in a cell and therefore enables visual determination of the galectin-3/biomarker expression in tissue samples. The FISH method has the advantages of a more objective scoring system and the presence of a built-in internal control consisting of the galectin-3/biomarker gene signals present in all non-neoplastic cells in the same sample. Fluorescence in situ hybridization is a direct in situ technique that is relatively rapid and sensitive. FISH test also can be automated. Immunohistochemistry can be combined with a FISH method when the expression level of galectin-3/biomarker is difficult to determine by immunohistochemistry alone. Alternatively, mRNA expression can be detected on a DNA array, chip or a microarray. Oligonucleotides corresponding to the galectin-3/biomarker may be immobilized on a chip which is then hybridized with labeled nucleic acids of a test sample obtained from a patient. Positive hybridization signal can be obtained with the sample containing galectin- 3/biomarker transcripts. Methods of preparing DNA arrays and their use are well known in the art. (See, for example U.S. Pat. Nos: 6,618,6796; 6,379,897; 6,664,377; 6,451,536; 548,257; U.S. 20030157485 and Schena et al. 1995 Science 20:467-470; Gerhold et al. 1999 Trends in Biochem. Sci. 24, 168-173; and Lennon et al. 2000 Drug discovery Today 5: 59- 65, which are herein incorporated by reference in their entirety). Serial Analysis of Gene Expression (SAGE) can also be performed (See for example U.S. Patent Application 20030215858).

To monitor mRNA levels, for example, mRNA can be extracted from the biological sample to be tested, reverse transcribed, and fluorescent-labeled cDNA probes are generated. The microarrays capable of hybridizing to galectin-3/biomarker cDNA are then probed with the labeled cDNA probes, the slides scanned and fluorescence intensity measured. This intensity correlates with the hybridization intensity and expression levels.

Types of probes for detection of galectin-3/biomarker RNA include cDNA, riboprobes, synthetic oligonucleotides and genomic probes. The type of probe used may generally be dictated by the particular situation, such as riboprobes for in situ hybridization, and cDNA for Northern blotting, for example. Most preferably, the probe is directed to nucleotide regions unique to galectin-3/biomarker RNA. The probes may be as short as is required to differentially recognize galectin-3/biomarker mRNA transcripts, and may be as short as, for example, 15 bases; however, probes of at least 17 bases, more preferably 18 bases and still more preferably 20 bases are preferred. Preferably, the primers and probes hybridize specifically under stringent conditions to a DNA fragment having the nucleotide sequence corresponding to the galectin-3 gene. As herein used, the term "stringent conditions" means hybridization may occur only if there is at least 95% and preferably at least 97% identity between the sequences.

The form of labeling of the probes may be any that is appropriate, such as the use of radioisotopes, for example, ²P and ⁵S. Labeling with radioisotopes may be achieved, whether the probe is synthesized chemically or biologically, by the use of suitably labeled bases. VII. Methods for treating cancer with a galectin-3 inhibitor

The invention provides methods for treating cancer in patients with a galectin-3 inhibitor or modified pectin, e.g., GCS-100.

A. Dose and dose regimen

In some embodiments, the total amount of a therapeutically effective substance

(galectin-3 inhibitor or modified pectin, e.g., GCS-100) in a composition to be administered (e.g., injected or intravenously infused) to a patient is one that is suitable for that patient. One of skill in the art would appreciate that different individuals may require different total amounts of the galectin-3 inhibitor or modified pectin. In some embodiments, the amount of the galectin-3 inhibitor or modified pectin is a pharmaceutically effective amount. The skilled worker would be able to determine the amount of the galectin-3 inhibitor or modified pectin in a composition needed to treat a patient based on factors such as, for example, the age, weight, and physical condition of the patient. The concentration of the galectin-3 inhibitor or modified pectin depends in part on its solubility in the intravenous administration solution and the volume of fluid that can be administered.

In certain embodiments, a galectin-3 inhibitor or modified pectin (e.g., GCS-100), is administered to the subject at a fixed dose ranging from 0.1 mg/m² to 30 mg/m². For example, a modified pectin or galectin-3 inhibitor may be administered to the subject in a fixed dose of 0.1 mg/m², 0.5 mg/m², 1 mg/m², 3 mg/m², 6 mg/m², 9 mg/m², 12 mg/m², 15 mg/m², 18 mg/m², 21 mg/m², 24 mg/m², 27 mg/m², 30 mg/m², 35 mg/m², 40 mg/m², 50 mg/m², 60 mg/m², 70 mg/m², 80 mg/m², 90 mg/m², 100 mg/m², 110 mg/m², 120 mg/m², 130 mg/m², 140 mg/m², 150 mg/m², 160 mg/m², 170 mg/m², 180 mg/m², 190 mg/m², 200 mg/m², etc. Ranges of values between any of the aforementioned recited values are also intended to be included in the scope of the invention, e.g., 0.2 mg/m², 0.6 mg/m², 1.5 mg/m², 2 mg/m², 4 mg/m², 8 mg/m², 10 mg/m², 13 mg/m², 17 mg/m², 20 mg/m², 23 mg/m², 25 mg/m², 26 mg/m², 28 mg/m², 32 mg/m², 45 mg/m², 55 mg/m², 65 mg/m², 75 mg/m², 85 mg/m², 95 mg/m², 105 mg/m², 115 mg/m², 125 mg/m², 135 mg/m², 145 mg/m², 155 mg/m², 165 mg/m², 175 mg/m², 185 mg/m², 195 mg/m², 205 mg/m², as are ranges based on the forementioned doses, e.g., 0.1-5 mg/m², 5-10 mg/m², 10-15 mg/m², 15-20 mg/m², 20-25 mg/m², 25-30 mg/m², 30-80 mg/m², 80-120 mg/m², 120-150 mg/m², 150-175 mg/m², 175-200 mg/m²,. The total body dose should not exceed 1 g/m² weekly or 200 mg/m² daily times 5.

In certain embodiments, a galectin-3 inhibitor or modified pectin (e.g., GCS-100), is administered to the subject at a fixed dose ranging from 1-10 mg, e.g., weekly. For example, the fixed dose may be 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg, e.g., weekly in each case. In certain such embodiments, a modified pectin, preferably GCS- 100, is administered weekly for an initial period (e.g., an induction phase, such as 1-3 months, preferably 2 months) followed by biweekly administration (e.g., a maintenance or treatment phase, such as 1-6 months, or even indefinitely) thereafter. In certain such embodiments, the fixed dose is the same throughout both phases, with only the frequency of administration varying between the two phases.

The concentration of the galectin-3 inhibitor or modified pectin in the composition administered can be at least 16 ug/ml. In some embodiments, the concentration of the galectin-3 inhibitor or modified pectin may be about 1.0 ug/ml, about 2.0 ug/ml, about 3.0 ug/ml, about 4.0 ug/ml, about 5.0 ug/ml, about 6.0 ug/ml, about 7.0 ug/ml, about 8.0 ug/ml, about 9.0 ug/ml, about 10.0 ug/ml, about 11.0 ug/ml, about 12.0 ug/ml, about 13.0 ug/ml, about 14.0 ug/ml, about 15.0 ug/ml, etc. The composition including the galectin-3 inhibitor or modified pectin can be administered at a rate sufficient to achieve an increase in apopotosis, reduction in transformed phenotype, or reduction in cellular proliferation, etc., or in the levels of one or more biomarkers, as discussed herein. A patient may be coupled to a monitor that provides continuous, periodic, or occasional measurements during some or all of the course of treatment. The rate of administration may be modulated manually (e.g., by a physician or nurse) or automatically (e.g., by a medical device capable of modulating delivery of the composition in response to physiological parameters received from the monitor) to maintain the patient's physiological and/or biomarker parameters within a desired range or above or below a desired threshold, or example, the rate of administration of the galectin-3 inhibitor or modified pectin may be from about 0.032 ng/kg/min to about 100 ug/kg/min in the injectable composition. In some embodiments, the rate of administration of the galectin-3 inhibitor or modified pectin may be from about 0.4 to about 45 ug/min, from about 0.12 to about 19 ug/min, from about 3.8 to about 33.8 ug/min, from about 0.16 to about 2.6 ug/min, etc. In particular embodiments, the rate of administration of the galectin-3 inhibitor or modified pectin may be about 0.032 ng/kg/min, about 0.1 ng/kg/min, about 0.32 ng/kg/min, about 1 ng/kg/min, about 1.6 ng/kg/min, about 2 ng/kg/min, about 3 ng/kg/min, about 4 ng/kg/min, about 5 ng/kg/min, about 6 ng/kg/min, about 7 ng/kg/min, about 8 ng/kg/min, about 9 ng/kg/min, about 10 ng/kg/min, about 15 ng/kg/min, about 20 ng/kg/min, about 25 ng/kg/min, about 30 ng/kg/min, about 40 ng/kg/min, about 50 ng/kg/min, about 60 ng/kg/min, about 70 ng/kg/min, about 80 ng/kg/min, about 90 ng/kg/min, about 100 ng/kg/min, about 200 ng/kg/min, about 300 ng/kg/min, about 400 ng/kg/min, about 500 ng/kg/min, about 600 ng/kg/min, about 700 ng/kg/min, about 800 ng/kg/min, about 900 ng/kg/min, about 1 ug/kg/min, about 1.1 ug/kg/min, about 1.2 ug/kg/min, about 1.3 ug/kg/min, about 1.4 ug/kg/min, about 1.5 ug/kg/min, about 1.5 ug/kg/min, about 1.6 ug/kg/min, about 1.7 ug/kg/min, about 1.8 ug/kg/min, about 1.9 ug/kg/min, about 2 ug/kg/min, about 2.1 ug/kg/min, about 2.2 ug/kg/min, about 2.3 ug/kg/min, about 2.4 ug/kg/min, about 2.5 ug/kg/min, about 2.6 ug/kg/min, about 2.7 ug/kg/min, about 2.8 ug/kg/min, about 2.9 ug/kg/min, about 3.0 ug/kg/min, about 3.1 ug/kg/min, about 3.2 ug/kg/min, about 3.3 ug/kg/min, about 3.4 ug/kg/min, about 3.5 ug/kg/min, about 3.6 ug/kg/min, about 3.7 ug/kg/min, about 3.8 ug/kg/min, about 3.9 ug/kg/min, about 4.0 ug/kg/min, about 4.1 ug/kg/min, about 4.2 ug/kg/min, about 4.3 ug/kg/min, about 4.4 ug/kg/min, about 4.5 ug/kg/min, about 4.6 ug/kg/min, about 4.7 ug/kg/min, about 4.8 ug/kg/min, about 4.9 ug/kg/min, about 5.0 ug/kg/min, about 6 ug/kg/min, about 7 ug/kg/min, about 8 ug/kg/min, about 9 ug/kg/min, about 10 ug/kg/min, about 11 ug/kg/min, about 12 ug/kg/min, about 13 ug/kg/min, about 14 ug/kg/min, about 15 ug/kg/min, about 16 ug/kg/min, about 17 ug/kg/min, about 18 ug/kg/min, about 19 ug/kg/min, about 20 ug/kg/min, about 25 ug/kg/min, about 30 ug/kg/min, about 31 ug/kg/min, about 32 ug/kg/min, about 33 ug/kg/min, about 33.8 ug/kg/min, about 34 ug/kg/min, about 35 ug/kg/min, about 40 ug/kg/min, about 45 ug/kg/min, about 50 ug/kg/min, about 55 ug/kg/min, about 60 ug/kg/min, about 65 ug/kg/min, about 70 ug/kg/min, about 75 ug/kg/min, about 80 ug/kg/min, about 85 ug/kg/min, about 90 ug/kg/min, about 95 ug/kg/min, about 100 ug/kg/min, etc.

The composition may be administered over a period of time selected from at least 8 hours; at least 24 hours; and from 8 hours to 24 hours. The composition may be

administered continuously for at least 2-6 days, such as 2-11 days, continuously for 2-6 days, for 8 hours a day over a period of at least 2-6 days, such as 2-11 days. A weaning period (from several hours to several days) may be beneficial after prolonged infusion. In certain embodiments, the duration of treatment may last up to 8 consecutive weeks of dosing or until the development of dose-limiting toxicity.

B. Pharmaceutical formulations

The compositions of the invention can be administered through any suitable route. In some embodiments, the compositions of the invention are suitable for parenteral

administration. These compositions may be administered, for example, intraperitoneally, intravenously, intrarenally, or intrathecally. In some embodiments, the compositions of the invention are injected intravenously. One of skill in the art would appreciate that a method of administering a therapeutically effective substance formulation or composition of the invention would depend on factors such as the age, weight, and physical condition of the patient being treated, and the disease or condition being treated. The skilled worker would, thus, be able to select a method of administration optimal for a patient on a case-by-case basis.

The compositions may be solutions containing at least 0.5%, 1%, 5% or 10% by weight of the galectin-3 inhibitor or modified pectin, e.g., up to about 10% or 15% by weight. In certain embodiments, the modified pectin is provided as a colloidal solution in water. The size of the colloidal particles may be less than 1 μιτι in diameter, preferably less than about 0.65 μιτι, and most preferably less than about 0.2 μιτι.

The formulation may comprise suitable excipients including pharmaceutically acceptable buffers, stabilizers, local anesthetics, and the like that are well known in the art. For parenteral administration, an exemplary formulation may be a sterile solution or suspension; for oral dosage, a syrup, tablet or palatable solution; for topical application, a lotion, cream, spray or ointment; for intravaginal or intrarectal administration, pessaries, suppositories, creams or foams. Preferably, the route of administration is parenteral, more preferably intravenous.

In alternative embodiments, a pharmaceutical composition of the invention may be in a form adapted for oral dosage, such as for example a syrup or palatable solution; a form adapted for topical application, such as for example a cream or ointment; or a form adapted for administration by inhalation, such as for example a microcrystalline powder or a solution suitable for nebulization. Methods and means for formulating pharmaceutical ingredients for alternative routes of administration are well-known in the art, and it is to be expected that those skilled in the relevant arts can adapt these known methods to the galectin-3 inhibitors of the invention.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the modified therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The galectin-3 inhibitor can also be in microencapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the galectin-3 inhibitors of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the galectin-3 inhibitor, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Administration of medicament may be indicated for the treatment of mild, moderate or severe acute or chronic symptoms or for prophylactic treatment. It may be appreciated that the precise dose administered may depend on the age and condition of the patient, the particular particulate medicament used and the frequency of administration and may ultimately be at the discretion of the attendant physician. Typically, administration may occur weekly, though may occur at a regular or irregular frequency, such as daily or monthly or a combination thereof (e.g., daily for five days once a month). Pharmaceutical compositions of this invention suitable for parenteral administration comprise a galectin-3 inhibitor of the invention in combination with one or more

pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions.

Examples of pharmaceutically acceptable antioxidants include but are not limited to ascorbic acid, cysteine hydrochloride, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxy toluene (BHT), propyl gallate, alpha-tocopherol, and chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include

poly(orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The galectin-3 inhibitor may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

A pH-adjusting agent may be beneficial to adjust the pH of the compositions by including a pH-adjusting agent in the compositions of the invention. Modifying the pH of a formulation or composition may have beneficial effects on, for example, the stability or solubility of a therapeutically effective substance, or may be useful in making a formulation or composition suitable for parenteral administration. pH-adjusting agents are well known in the art. Accordingly, the pH-adjusting agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary pH-adjusting agents that may be used in the compositions of the invention. pH-adjusting agents may include, for example, acids and bases. In some embodiments, a pH-adjusting agent includes, but is not limited to, acetic acid, hydrochloric acid, phosphoric acid, sodium hydroxide, sodium carbonate, and combinations thereof. The pH of the compositions of the invention may be any pH that provides desirable properties for the formulation or composition. Desirable properties may include, for example, therapeutically effective substance stability, increased therapeutically effective substance retention as compared to compositions at other pHs, and improved filtration efficiency. In some embodiments, the pH of the compositions of the invention may be from about 3.0 to about 9.0, e.g., from about 5.0 to about 7.0. In particular embodiments, the pH of the compositions of the invention may be 5.5±0.1, 5.6±0.1 , 5.7±0.1 , 5.8±0.1 , 5.9±0.1, 6.0±0.1, 6.1±0.1, 6.2±0.1 , 6.3±0.1 , 6.4±0.1 , or 6.5±0.1.

In certain embodiments, the galectin-3 inhibitor is a modified pectin which is prepared substantially ethanol-free and suitable for parenteral administration. By substantially free of ethanol, it is meant that the compositions of the invention contain less than 5% ethanol by weight. In preferred embodiments the compositions contain less than 2%, and more preferably less than 0.5% ethanol by weight. In certain embodiments, the compositions further comprise one or more pharmaceutically acceptable excipients. Such compositions include aqueous solutions of the galectin-3 inhibitor of the invention. In certain embodiments of such aqueous solutions, the pectin modification occurs at a concentration of at least 7 mg/mL, at least 10, or 15 or more mg/ml. Any of such compositions are also substantially free of organic solvents other than ethanol.

A buffer may be used to resuspend the compound in solution. In certain

embodiments, a buffer may have a pKa of, for example, about 5.5, about 6.0, or about 6.5. One of skill in the art would appreciate that an appropriate buffer may be chosen for inclusion in compositions of the invention based on its pKa and other properties. Buffers are well known in the art. Accordingly, the buffers described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary buffers that may be used in the compositions of the invention. In certain embodiments, a buffer may include one or more of the following: Tris, Tris HC1, potassium phosphate, sodium phosphate, sodium citrate, sodium ascorbate, combinations of sodium and potassium phosphate, Tris/Tris HC1, sodium bicarbonate, arginine phosphate, arginine hydrochloride, histidine hydrochloride, cacodylate, succinate, 2-(N-moipholino)ethanesulfonic acid (MES), maleate, bis-tris, phosphate, carbonate, and any pharmaceutically acceptable salts and/or combinations thereof.

A solubilizing agent may be added to increase the solubility of a drug or compound.

In some embodiments, it may be beneficial to include a solubilizing agent to the galectin-3 inhibitor or modified pectin. Solubilizing agents may be useful for increasing the solubility of any of the components of the formulation or composition, including a therapeutically effective substance galectin-3 inhibitor or an excipient. The solubilizing agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary solubilizing agents that may be used in the compositions of the invention. In certain embodiments, solubilizing agents include, but are not limited to, ethyl alcohol, tert-butyl alcohol, polyethylene glycol, glycerol, methylparaben, propylparaben, polyethylene glycol, polyvinyl pyrrolidone, and any pharmaceutically acceptable salts and/or combinations thereof.

A stabilizing agent may help to increase the stability of a therapeutically effective substance in compositions of the invention. This may occur by, for example, reducing degradation or preventing aggregation of a therapeutically effective substance. Without wishing to be bound by theory, mechanisms for enhancing stability may include sequestration of the therapeutically effective substance from a solvent or inhibiting free radical oxidation of the anthracycline compound. Stabilizing agents are well known in the art. Accordingly, the stabilizing agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary stabilizing agents that may be used in the compositions of the invention. Stabilizing agents may include, but are not limited to, emulsifiers and surfactants.

A surfactant may be added to reduce the surface tension of a liquid composition. This may provide beneficial properties such as improved ease of filtration. Surfactants also may act as emulsifying agents and/or solubilizing agents. Surfactants are well known in the art. Accordingly, the surfactants described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary surfactants that may be used in the compositions of the invention. Surfactants that may be included include, but are not limited to, sorbitan esters such as polysorbates (e.g., polysorbate 20 and polysorbate 80), lipopolysaccharides, polyethylene glycols (e.g., PEG 400 and PEG 3000), poloxamers (i.e., pluronics), ethylene oxides and polyethylene oxides (e.g., Triton X-100), saponins, phospholipids (e.g., lecithin), and combinations thereof.

A tonicity-adjusting reagent may be used to help make a formulation or composition suitable for administration. The tonicity of a liquid composition is an important

consideration when administering the composition to a patient, for example, by parenteral administration. Tonicity-adjusting agents are well known in the art. Accordingly, the tonicity-adjusting agents described herein are not intended to constitute an exhaustive list, but are provided merely as exemplary tonicity-adjusting agents that may be used in the compositions of the invention. Tonicity-adjusting agents may be ionic or non-ionic and include, but are not limited to, inorganic salts, amino acids, carbohydrates, sugars, sugar alcohols, and carbohydrates. Exemplary inorganic salts may include sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate. An exemplary amino acid is glycine. Exemplary sugars may include sugar alcohols such as glycerol, propylene glycol, glucose, sucrose, lactose, and mannitol.

B. Articles of Manufacture and Kits

The invention also provides a packaged pharmaceutical composition wherein the galectin-3 inhibitor or modified pectin, e.g., GCS-100, is packaged within a kit or an article of manufacture. The kit or article of manufacture of the invention may contain materials useful for the treatment, including the improvement, and/or remission, prevention and/or diagnosis or monitoring of cancer. The kit or article of manufacture may comprise a container and a label or package insert or printed material on or associated with the container which provides information regarding use of the galectin-3 inhibitor or modified pectin for the treatment of cancer.

In certain embodiments, the invention provides an article of manufacture comprising a galectin-3 inhibitor and a package insert.

The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

In certain embodiments, the article of manufacture of the invention comprises (a) a first container holding a composition comprising a galectin-3 inhibitor or modified pectin; and (b) a package insert indicating how the galectin-3 inhibitor or modified pectin may be administered to a patient, as discussed herein. In preferred embodiments, the label or package insert indicates that the galectin-3 inhibitor or modified pectin (e.g. GCS-100), is used for treating a cancer. In certain embodiments, the invention features a kit comprising a sufficient number of containers to provide both loading and maintenance doses of the galectin-3 inhibitor or modified pectin. For example, the kit may contain containers containing about 1.5 and 30 mg/m², or amounts ranging from 0.1-5 mg/m², 5-10 mg/m², 10- 15 mg/m², 15-20 mg/m², 20-25 mg/m², 25-30 mg/m², 30-80 mg/m², 80-120 mg/m², 120-150 mg/m², 150-175 mg/m², 175-200 mg/m², of modified pectin for intravenous injection. The containers each containing the galectin-3 inhibitor or modified pectin (e.g. GCS-100) could, for example, provide enough modified pectin to be administered intravenously once weekly for up to 8 consecutive weeks, or at another suitable frequency such as daily or monthly.

Suitable containers for the galectin-3 inhibitor or modified pectin (e.g. GCS-100), include, for example, bottles, vials, syringes, including preloaded/pre-filled syringes, pens, including autoinjector pens, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or when combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port.

In certain embodiments, the pharmaceutical compositions and associated articles of manufacture are useful in treating certain patient populations who may respond favorably to the modified pectin. For example, the modified pectin, e.g., GCS-100, may be used to treat cancer in patients who have been unresponsive or intolerant to oral antibiotics or medication for treatment for their cancer.

In certain embodiments, the pharmaceutical compositions and/or associated articles of manufacture may provide a dose suitable for administration of the therapeutic agent for the treatment of a cancer. In certain embodiments, the article includes a loading dose of about 1.5 mg/m² to be administered at the outset of therapy. In certain embodiments, the article includes a maintenance dose of about 0.5 mg/m², e.g., for a number of weeks thereafter, such as starting from week 4. For example, a kit of the invention may include a loading dose and one or more maintenance doses.

In other embodiments, the article provides a galectin-3 inhibitor or modified pectin (e.g. GCS-100) suitable for subcutaneous injection.

In certain embodiments of the invention, the kit comprises a galectin-3 inhibitor or modified pectin, a second pharmaceutical composition comprising an additional therapeutic agent, and optionally instructions for administration of both agents for the treatment of cancer. The instructions may describe how, e.g., subcutaneously or intravenously, and when, e.g., at week 0, week 2, and weekly or biweekly thereafter, doses of modified pectin and/or the additional therapeutic agent shall be administered to a subject for treatment.

In certain embodiments, the kits contain a pharmaceutical composition comprising a galectin-3 inhibitor or modified pectin and a pharmaceutically acceptable carrier and one or more additional pharmaceutical compositions each comprising a drug useful for treating a cancer or a symptom thereof and a pharmaceutically acceptable carrier. Altematively, the kit comprises a single pharmaceutical composition comprising a galectin-3 inhibitor (such as a modified pectin), one or more drugs useful for treating a cancer, and a pharmaceutically acceptable carrier.

In another aspect, the invention provides a pharmaceutical package, comprising a vial or ampoule containing a galectin-3 inhibitor according to the invention in the form of a reconstitutable powder or a solution suitable for injection or infusion, optionally together with instructions for administering the composition to a patient suffering from

nephrotoxicity. Instructions include but are not limited to written and/or pictorial descriptions of: the active ingredient, directions for diluting the composition to a

concentration suitable for administration, suitable indications, suitable dosage regimens, contraindications, drug interactions, and any adverse side-effects noted in the course of clinical trials.

In alternative embodiments, the pharmaceutical package may comprise a plastic bag containing from 100 mL to 2 L of a pharmaceutical composition of the invention, in the form of a solution suitable for intravenous administration, optionally together with instructions as described above.

C. Additional therapeutic agents

Galectin-3 inhibitors or modified pectins, including GCS-100, may be used in the methods of the invention either alone or in combination with an additional therapeutic agent, said additional agent being selected by the skilled artisan for its intended purpose. For example, the additional agent can be a therapeutic agent art recognized as being useful to treat the disease or condition being treated by the galectin-3 inhibitor or modified pectins.

It should further be understood that the combinations which are to be included within this invention are those combinations useful for their intended purpose. The therapeutic agents set forth below are illustrative for purposes and not intended to be limited. The combinations, which are part of this invention, can be the galectin-3 inhibitor or modified pectin and at least one additional agent selected from the lists below. The combination can also include more than one additional agent, e.g., two or three additional therapeutic agents if the combination is such that the formed composition can perform its intended function. Modified pectins or galectin-3 inhibitors described herein may be used in combination with additional therapeutic agents for the treatment of cancer, which may act parallel to, dependent on or in concert with modified pectin function.

1. Conjoint Administration

In some embodiments, the method of treating or preventing cancer may comprise administering the modified pections or galectin-3 inhibitors of the invention conjointly with one or more other chemotherapeutic agent(s). Chemotherapeutic agents that may be conjointly administered with compounds of the invention include: ABT-263,

aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, bortezomib, buserelin, busulfan, campothecin, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, chloroquine, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, demethoxyviridin, dexamethasone, dichloroacetate, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, everolimus, exemestane, filgrastim, fludarabine, fludrocortisone, f uorouracil and 5-f uorouracil, f uoxymesterone, f utamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, lenalidomide, letrozole, leucovorin, leuprolide, levamisole, lomustine, lonidamine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, metformin, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, perifosine, PF-04691502, plicamycin, pomalidomide, porfimer, procarbazine, raltitrexed, rituximab, romidepsin, sorafenib, streptozocin, sunitinib, suramin, tamoxifen, temozolomide, temsirolimus, teniposide, testosterone, thalidomide, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, vinorelbine, and vorinostat (SAHA). For example, chemotherapeutic agents that may be conjointly administered with compounds of the invention include: aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, bortezomib, buserelin, busulfan, campothecin, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, chloroquine, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, demethoxyviridin, dichloroacetate, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, everolimus, exemestane, filgrastim, fiudarabine, fludrocortisone, fluorouracil,

fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, lenalidomide, letrozole, leucovorin, leuprolide, levamisole, lomustine, lonidamine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, metformin, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, perifosine, plicamycin, pomalidomide, porfimer, procarbazine, raltitrexed, rituximab, sorafenib, streptozocin, sunitinib, suramin, tamoxifen, temozolomide, temsirolimus, teniposide, testosterone, thalidomide, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine. In other embodiments, chemotherapeutic agents that may be conjointly administered with compounds of the invention include: ABT-263, dexamethasone, 5- fluorouracil, PF-04691502, romidepsin, and vorinostat (SAHA). In certain embodiments of the methods of the invention described herein, the chemotherapeutic agent conjointly administered with compounds of the invention is a taxane chemotherapeutic agent, such as paclitaxel or docetaxel. In certain embodiments of the methods of the invention described herein, the chemotherapeutic agent conjointly administered with compounds of the invention is doxorubicin. In certain embodiments of the methods of the invention described herein, a compound of the invention is administered conjointly with a taxane chemotherapeutic agent (e.g., paclitaxel) and doxorubicin.

Many combination therapies have been developed for the treatment of cancer.

In certain embodiments, the galectin-3 inhibitors of the invention may be conjointly administered with a combination therapy. Examples of combination therapies with which compounds of the invention may be conjointly administered are included in Table 1.

Table 1 : Exemplary combinatorial therapies for the treatment of cancer.

ABV Doxorubicin, Bleomycin, Vinblastine

ABVD Doxorubicin, Bleomycin, Vinblastine, Dacarbazine

AC (Breast) Doxorubicin, Cyclophosphamide

AC (Sarcoma) Doxorubicin, Cisplatin

AC (Neuroblastoma) Cyclophosphamide, Doxorubicin ACE Cyclophosphamide, Doxorubicin, Etoposide

ACe Cyclophosphamide, Doxorubicin

AD Doxorubicin, Dacarbazine

AP Doxorubicin, Cisplatin

ARAC-DNR Cytarabine, Daunorubicin

B-CAVe Bleomycin, Lomustine, Doxorubicin, Vinblastine

BCVPP Carmustine, Cyclophosphamide, Vinblastine, Procarbazine,

Prednisone

BEACOPP Bleomycin, Etoposide, Doxorubicin, Cyclophosphamide, Vincristine,

Procarbazine, Prednisone, Filgrastim

BEP Bleomycin, Etoposide, Cisplatin

BOMP Bleomycin, Vincristine, Cisplatin, Mitomycin

BIP Bleomycin, Cisplatin, Ifosfamide, Mesna

CA Cytarabine, Asparaginase

CABO Cisplatin, Methotrexate, Bleomycin, Vincristine

CAF Cyclophosphamide, Doxorubicin, Fluorouracil

CAL-G Cyclophosphamide, Daunorubicin, Vincristine, Prednisone,

Asparaginase

CAMP Cyclophosphamide, Doxorubicin, Methotrexate, Procarbazine

CAP Cyclophosphamide, Doxorubicin, Cisplatin

CaT Carboplatin, Paclitaxel

CAV Cyclophosphamide, Doxorubicin, Vincristine

CAVE ADD CA V and Etoposide

CA-VP16 Cyclophosphamide, Doxorubicin, Etoposide

CC Cyclophosphamide, Carboplatin

CDDPNP-16 Cisplatin, Etoposide

CEF Cyclophosphamide, Epirubicin, Fluorouracil

CEPP(B) Cyclophosphamide, Etoposide, Prednisone, with or without/

Bleomycin

CEV Cyclophosphamide, Etoposide, Vincristine CF Cisplatin, Fluorouracil or Carboplatin Fluorouracil

CHAP Cyclophosphamide or Cyclophosphamide, Altretamine, Doxorubicin,

Cisplatin

ChlVPP Chlorambucil, Vinblastine, Procarbazine, Prednisone

CHOP Cyclophosphamide, Doxorubicin, Vincristine, Prednisone

CHOP-BLEO Add Bleomycin to CHOP

CISCA Cyclophosphamide, Doxorubicin, Cisplatin

CLD-BOMP Bleomycin, Cisplatin, Vincristine, Mitomycin

CMF Methotrexate, Fluorouracil, Cyclophosphamide

CMFP Cyclophosphamide, Methotrexate, Fluorouracil, Prednisone

CMFVP Cyclophosphamide, Methotrexate, Fluorouracil, Vincristine,

Prednisone

CMV Cisplatin, Methotrexate, Vinblastine

CNF Cyclophosphamide, Mitoxantrone, Fluorouracil

CNOP Cyclophosphamide, Mitoxantrone, Vincristine, Prednisone

COB Cisplatin, Vincristine, Bleomycin

CODE Cisplatin, Vincristine, Doxorubicin, Etoposide

COMLA Cyclophosphamide, Vincristine, Methotrexate, Leucovorin,

Cytarabine

COMP Cyclophosphamide, Vincristine, Methotrexate, Prednisone

Cooper Regimen Cyclophosphamide, Methotrexate, Fluorouracil, Vincristine,

Prednisone

COP Cyclophosphamide, Vincristine, Prednisone

COPE Cyclophosphamide, Vincristine, Cisplatin, Etoposide

COPP Cyclophosphamide, Vincristine, Procarbazine, Prednisone

CP (Chronic Chlorambucil, Prednisone lymphocytic leukemia)

CP (Ovarian Cancer) Cyclophosphamide, Cisplatin

CT Cisplatin, Paclitaxel

CVD Cisplatin, Vinblastine, Dacarbazine

CVI Carboplatin, Etoposide, Ifosfamide, Mesna CVP Cyclophosphamide, Vincristine, Prednisome

CVPP Lomustine, Procarbazine, Prednisone

CYVADIC Cyclophosphamide, Vincristine, Doxorubicin, Dacarbazine

DA Daunorubicin, Cytarabine

DAT Daunorubicin, Cytarabine, Thioguanine

DAV Daunorubicin, Cytarabine, Etoposide

DCT Daunorubicin, Cytarabine, Thioguanine

DHAP Cisplatin, Cytarabine, Dexamethasone

DI Doxorubicin, Ifosfamide

DTIC/Tamoxifen Dacarbazine, Tamoxifen

DVP Daunorubicin, Vincristine, Prednisone

EAP Etoposide, Doxorubicin, Cisplatin

EC Etoposide, Carboplatin

EFP Etoposie, Fluorouracil, Cisplatin

ELF Etoposide, Leucovorin, Fluorouracil

EMA86 Mitoxantrone, Etoposide, Cytarabine

EP Etoposide, Cisplatin

EVA Etoposide, Vinblastine

FAC Fluorouracil, Doxorubicin, Cyclophosphamide

FAM Fluorouracil, Doxorubicin, Mitomycin

FAMTX Methotrexate, Leucovorin, Doxorubicin

FAP Fluorouracil, Doxorubicin, Cisplatin

F-CL Fluorouracil, Leucovorin

FEC Fluorouracil, Cyclophosphamide, Epirubicin

FED Fluorouracil, Etoposide, Cisplatin

FL Flutamide, Leuprolide

FZ Flutamide, Goserelin acetate implant

HDMTX Methotrexate, Leucovorin

Hexa-CAF Altretamine, Cyclophosphamide, Methotrexate, Fluorouracil ICE-T Ifosfamide, Carboplatin, Etoposide, Paclitaxel, Mesna

IDMTX/6-MP Methotrexate, Mercaptopurine, Leucovorin

IE Ifosfamide, Etoposie, Mesna

IfoVP Ifosfamide, Etoposide, Mesna

IPA Ifosfamide, Cisplatin, Doxorubicin

M-2 Vincristine, Carmustine, Cyclophosphamide, Prednisone, Melphalan

MAC-III Methotrexate, Leucovorin, Dactinomycin, Cyclophosphamide

MACC Methotrexate, Doxorubicin, Cyclophosphamide, Lomustine

MACOP-B Methotrexate, Leucovorin, Doxorubicin, Cyclophosphamide,

Vincristine, Bleomycin, Prednisone

MAID Mesna, Doxorubicin, Ifosfamide, Dacarbazine

m-BACOD Bleomycin, Doxorubicin, Cyclophosphamide, Vincristine,

Dexamethasone, Methotrexate, Leucovorin

MBC Methotrexate, Bleomycin, Cisplatin

MC Mitoxantrone, Cytarabine

MF Methotrexate, Fluorouracil, Leucovorin

MICE Ifosfamide, Carboplatin, Etoposide, Mesna

MINE Mesna, Ifosfamide, Mitoxantrone, Etoposide

mini-BEAM Carmustine, Etoposide, Cytarabine, Melphalan

MOBP Bleomycin, Vincristine, Cisplatin, Mitomycin

MOP Mechlorethamine, Vincristine, Procarbazine

MOPP Mechlorethamine, Vincristine, Procarbazine, Prednisone

MOPP/ABV Mechlorethamine, Vincristine, Procarbazine, Prednisone,

Doxorubicin, Bleomycin, Vinblastine

MP (multiple Melphalan, Prednisone myeloma)

MP (prostate cancer) Mitoxantrone, Prednisone

MTX/6-MO Methotrexate, Mercaptopurine

MTX/6-MPNP Methotrexate, Mercaptopurine, Vincristine, Prednisone

MTX-CDDPAdr Methotrexate, Leucovorin, Cisplatin, Doxorubicin

MV (breast cancer) Mitomycin, Vinblastine MV (acute myelocytic leukemia) Mitoxantrone, Etoposide

M-VAC Methotrexate Vinblastine, Doxorubicin, Cisplatin

MVP Mitomycin Vinblastine, Cisplatin

MVPP Mechlorethamine, Vinblastine, Procarbazine, Prednisone

NFL Mitoxantrone, Fluorouracil, Leucovorin

NOVP Mitoxantrone, Vinblastine, Vincristine

OPA Vincristine, Prednisone, Doxorubicin

OPPA Add Procarbazine to OP A.

PAC Cisplatin, Doxorubicin

PAC-I Cisplatin, Doxorubicin, Cyclophosphamide

PA-CI Cisplatin, Doxorubicin

PC Paclitaxel, Carboplatin or Paclitaxel, Cisplatin

PCV Lomustine, Procarbazine, Vincristine

PE Paclitaxel, Estramustine

PFL Cisplatin, Fluorouracil, Leucovorin

POC Prednisone, Vincristine, Lomustine

Pro MACE Prednisone, Methotrexate, Leucovorin,

Doxorubicin,Cyclophosphamide, Etoposide

ProMACE/cytaBOM Prednisone, Doxorubicin, Cyclophosphamide, Etoposide,

Cytarabine, Bleomycin, Vincristine, Methotrexate, Leucovorin, Cotrimoxazole

PRoMACE/MOPP Prednisone, Doxorubicin, Cyclophosphamide, Etoposide,

Mechlorethamine, Vincristine, Procarbazine, Methotrexate, Leucovorin

Pt/VM Cisplatin, Teniposide

PVA Prednisone, Vincristine, Asparaginase

PVB Cisplatin, Vinblastine, Bleomycin

PVDA Prednisone, Vincristine, Daunorubicin, Asparaginase

SMF Streptozocin, Mitomycin, Fluorouracil TAD Mechlorethamine, Doxorubicin, Vinblastine, Vincristine, Bleomycin, Etoposide, Prednisone

TCF Paclitaxel, Cisplatin, Fluorouracil

TIP Paclitaxel, Ifosfamide, Mesna, Cisplatin

TTT Methotrexate, Cytarabine, Hydrocortisone

Topo/CTX Cyclophosphamide, Topotecan, Mesna

VAB-6 Cyclophosphamide, Dactinomycin, Vinblastine, Cisplatin, Bleomycin

VAC Vincristine, Dactinomycin, Cyclophosphamide

VACAdr Vincristine, Cyclophosphamide, Doxorubicin, Dactinomycin,

Vincristine

VAD Vincristine, Doxorubicin, Dexamethasone

VATH Vinblastine, Doxorubicin, Thiotepa, Flouxymesterone

VBAP Vincristine, Carmustine, Doxorubicin, Prednisone

VBCMP Vincristine, Carmustine, Melphalan, Cyclophosphamide, Prednisone

VC Vinorelbine, Cisplatin

VCAP Vincristine, Cyclophosphamide, Doxorubicin, Prednisone

VD Vinorelbine, Doxorubicin

YelP Vinblastine, Cisplatin, Ifosfamide, Mesna

VIP Etoposide, Cisplatin, Ifosfamide, Mesna

VM Mitomycin, Vinblastine

VMCP Vincristine, Melphalan, Cyclophosphamide, Prednisone

VP Etoposide, Cisplatin

V-TAD Etoposide, Thioguanine, Daunorubicin, Cytarabine

5+2 Cytarabine, Daunorubicin, Mitoxantrone

7+3 Cytarabine with/, Daunorubicin or Idarubicin or Mitoxantrone

"8 in 1" Methylprednisolone, Vincristine, Lomustine, Procarbazine,

Hydroxyurea, Cisplatin, Cytarabine, Dacarbazine

In some embodiments, a compound of the invention may be conjointly administered with non-chemical methods of cancer treatment. In some embodiments, a compound of the invention may be conjointly administered with radiation therapy. In some embodiments, a compound of the invention may be conjointly administered with surgery, with

hermoablation, with focused ultrasound therapy, with cryotherapy, or with any combination of these.

In some embodiments, different compounds of the invention may be conjointly administered with one or more other compounds of the invention.

Moreover, such combinations may be conjointly administered with other therapeutic agents, such as other agents suitable for the treatment of cancer, immunological or neurological diseases, such as the agents identified above. In some embodiments, conjointly administering one or more additional chemotherapeutic agents with a compound of the invention provides a synergistic effect. In some embodiments, conj ointly administering one or more additional chemotherapeutics agents provides an additive effect.

In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent. Any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in the patient, which may include synergistic effects of the two compounds). For example, the modified pectins can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. In certain embodiments, the different therapeutic compounds can be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or a week of one another. Thus, an individual who receives such treatment can benefit from a combined effect of different therapeutic compounds.

The conjoint administration of the galectin-3 inhibitor and an additional therapeutic agent may involve concurrent administration. In particular embodiments, conjoint administration involves administration of the two agents within about 10 min, about 20 min, or about 30 minutes of each other. In exemplary embodiments, the galectin-3 inhibitor is administered in an overlapping fashion with the additional therapeutic, e.g., the additional therapeutic is administered intravenously and the galectin-3 inhibitor is administered orally during the course of the intravenous dosing.

The galectin-3 inhibitor may be administered subsequent to administration of the additional therapeutic agent. The galectin-3 inhibitor may be administered immediately after the additional therapeutic agent or within, for example, 1 hour, 2 hours, 4 hours, 6 hours or 12 hours. In other embodiments, the additional therapeutic agent may be administered subsequent to the galectin-3 inhibitor. The additional therapeutic agent may be administered immediately after the galectin-3 inhibitor or within, for example, 1 hour, 2 hours, 4 hours, 6 hours or 12 hours.

The galectin-3 inhibtor may be administered by any suitable manner in order to contact the tumor and accumulate sufficient quantities to prevent or treat cancer. A galectin- 3 inhibitor or combination therapeutics containing a galectin-3 inhibitor may be

administered orally, parenterally by intravenous injection, transdermally, by pulmonary inhalation, by intravaginal or intrarectal insertion, by subcutaneous implantation, intramuscular injection or by injection directly into an affected tissue, as for example by injection into a tumor site. In some instances the materials may be applied topically at the time surgery is carried out.

The materials are formulated to suit the desired route of administration. The galectin- 3 inhibitor and any additional therapeutic agent may each be formulated in ways to facilitate administration. For example, the combination therapy may be formulated for intravenous administration while the galectin-3 inhibitor may be formulated for nebulization. The following discussion of formulation may be applied to the individual formulation of the combination therapy or galectin-3 inhibitor or combination of the two.

The galectin-3 inhibitor need not be administered in the same manner as the other combination therapy. For example, the galectin-3 inhibitor may be administered orally while the additional therapeutic agent is administered intravenously. In addition, the galectin-3 inhibitor may be administered, before, during or after the administration of the combination therapy, such as before the administration of the combination therapy. In preferred embodiments, the galectin-3 inhibitor is administered in a manner to accumulate an effective concentration of the galectin-3 inhibitor in the tumor cells. Any one or more of the above- mentioned therapeutic agents, alone or in combination, can be administered to a subject suffering from cancer, in combination with the galectin-3 inhibitors or modified pectins, e.g., using a multiple variable dose treatment regimen.

The method of treating a cancer may further comprise hydrating the patient with saline before, during, and/or after conjoint administration of the additional therapeutic agent and galectin-3 inhibitor.

In some embodiments, any one of the above-mentioned therapeutic agents, alone or in combination therewith, can be administered to a subject suffering from cancer in addition to a therapeutic agent used to treat cancer, cardiovascular disease, inflammation, etc. It should be understood that the additional therapeutic agents can be used in combination therapy as described above, but also may be used in other indications described herein wherein a beneficial effect is desired. The combination of agents used in the methods and pharmaceutical compositions described herein may have a therapeutic additive or synergistic effect on the condition(s) or disease(s) targeted for treatment. The combination of agents used within the methods or pharmaceutical compositions described herein also may reduce a detrimental effect associated with at least one of the agents when administered alone or without the other agent(s) of the particular pharmaceutical composition. For example, the toxicity of side effects of one agent may be attenuated by another agent of the composition, thus allowing a higher dosage, improving patient compliance, and/or improving therapeutic outcome. The additive or synergistic effects, benefits, and advantages of the compositions apply to classes of therapeutic agents, either structural or functional classes, or to individual compounds themselves.

VIII. Efficacy of galectin inhibitors and modified pectins

The invention also provides methods for assessing the effects of a galectin-3 inhibitor or modified pectin in a subject. Such methods may be used to determine the efficacy of a galectin-3 inhibitor or modified pectin, or to adjust a patient's dosage in response to the measured effects. Using the methods described herein, the effects of a galectin-3 inhibitor or modified pectin may be determined or confirmed, and, optionally, used in the method of treating cancer.

In certain embodiments, the invention provides a method for determining the efficacy of a galectin-3 inhibitor or modified pectin, including a GCS-lOO, for treating cancer in a subject, using the change in cancer cell proliferation, transformed phenotype, or apoptosis to determine efficacy. In certain embodiments, the efficacy of a galectin-3 inhibitor or modified pectin, including GCS-lOO, for treating cancer in a subject is assessed by detecting a change in galectin-3 levels and/or activity, with a reduction in the level of galectin-3 being indicative of a desirable result. Other suitable markers include cancer biomarkers.

In certain embodiments, the invention provides a method of treating cancer in a subject, comprising administering a galectin-3 inhibitor or modified pectin, e.g., GCS-lOO, to the subject such that cancer is treated, e.g., wherein the galectin-3 inhibitor or modified pectin achieves a statistically significant clinical response within a patient or patient population. In certain embodiments, the methods of the invention are used to determine whether a dose of galectin-3 inhibitor or modified pectin is an effective dose of galectin-3 inhibitor modified pectin with respect to a patient who has been treated with the galectin-3 or modified pectin.

In certain embodiments, the methods of the invention comprise administering the galectin-3 inhibitor or modified pectin to a patient and determining the efficacy of the modified pectin by determining changes, improvements, measurements, etc., in galectin-3 or biomarker serum levels of the patient (e.g., relative to a pretreatment condition of the patient, to a predetermined desired condition or standard, or to a condition of an untreated patient or a patient treated with placebo).

A method for determining efficacy may comprise assessing the effect on a subject who has cancer of a dosage regimen comprising a galectin-3 inhibitor or modified pectin in order to determine whether the galectin-3 inhibitor or modified pectin is an effective therapy or whether a change in dosage would be desirable.

The Examples and discoveries described herein are representative of a modified pectin, GCS-lOO, which is effective for treating cancer. As such, the studies and results described in the Examples section herein may be used as a guideline for using a galectin-3 inhibitor or modified pectin for the treatment of cancer.

Other embodiments of the present invention are described in the following Examples. The present invention is further illustrated by the following examples which should not be construed as limiting in any way.

EXEMPLIFICATIONS

Galectin-3 inhibitor. GCS-lOO is a complex polysaccharide that has the ability to bind to and potentially block the effects of galectin-3. GCS-lOO is a derivative of pectin, a naturally occurring polysaccharide found in the structure of various plants, including the pulp and peel of citrus fruits. Pectin is composed of several types of sugars arranged in a complex polymeric configuration with multiple side branches. In particular, pectins have multiple side-branches containing the sugar β-galactose which is recognized by the carbohydrate binding domain of galectin-3. Thus, GCS-lOO is able to bind to and sequester multiple molecules of extracellular (circulating) galectin-3 (Figure 2). Additionally, because of its high average molecular weight, GCS-lOO resides in the body for an extended period (half- life of approximately 30 hours), increasing the time to interact with and sequester circulating galectin-3.

Example 1: Summary of GCS-lOO pharmacology studies

GCS-lOO has been studied in a fibrotic, pro-inflammatory mouse model of fatty liver disease known as non-alcoholic steatohepatitis (NASH). In this model, NASH is established in mice by a single subcutaneous injection of streptozotocin after birth, followed by the feeding of a high-fat diet ad libitum after 4 weeks of age. NASH develops at about Week 7 with evidence of fibrosis at Week 9 and liver nodule formation at Week 11 - 12. In the present study, mice were randomized at 9 weeks of age into three groups treated

intravenously with inactive placebo (control), 1 mg/kg GCS-lOO, or 25 mg/kg GCS-lOO. All animals received their respective administrations three times per week during Weeks 9 - 12. At the end of Week 12, blood and tissue samples were collected and analyzed for liver enzymes, non-alcoholic fatty liver disease (NAFLD) activity score, and fibrosis.

Overall, treatment with GCS-lOO in NASH mice was well tolerated and resulted in decreased plasma ALT. No effect was observed on blood glucose levels. Histological analysis showed a significant improvement in NAFLD score with decreased micro- and macro-vesicular fat deposition, hepatocyte ballooning and inflammatory cell infiltration. A decrease in hydroxyproline was observed and a significant decrease in fibrosis, as measured by Sirius red staining, was also observed. This study demonstrates GCS-lOO is effective at reducing fibrosis.

Example 2: Use of GCS-lOO in Treatment of Patients with Chronic Kidney Disease

In order to achieve the desired therapeutic effect, it is helpful to achieve a circulating

GCS-lOO concentration sufficient to bind to and neutralize plasma galectin-3 at an effective level over an extended period. The average concentration of circulating galectin-3 in ESRD patients is about 64 ng/mL, which is equal to 2.21 x 10^"6 μιτιοΐ galectin-3/mL plasma

(de Boer et. al., 2011).

Based on human pharmacokinetic data, a single 1.5 mg/m² dose of GCS-lOO is expected to result in a starting plasma concentration in excess of the expected galectin-3 concentration. At this dose on a molar basis, GCS-lOO is about 6-fold more concentrated than circulating galectin-3 at the Cmax for GCS-lOO. The approximate average half-life of

GCS-lOO in plasma is 30 hours, thus the level of GCS-lOO would fall below this baseline prior to the next treatment (Figure 3). Similarly, a single 30 mg/m² dose of GCS-lOO is expected to result in a starting plasma concentration in excess of the expected galectin-3 concentration. At this dose on a molar basis, GCS-lOO is about 160-fold more concentrated than circulating galectin-3 at the Cmax for GCS-lOO and the plasma concentration of GCS-lOO may not fall below this baseline prior to the next treatment.

Based on the preceding rationale, the dose groups and dosing schedule used for this study was expected to allow for effective galectin-3 inhibition, while being well tolerated.

Study drug administration and dosage schedule

Patients assigned to treatment groups received placebo or GCS-lOO on Days 1, 8, 15, 22, 29, 36, 43, and 50. The amount (in mg) of GCS-lOO to be administered was determined based on body surface area, calculated based on body weight and height using Formula III or IV below.

Htiinches) x Wt(lbs)

BSA Formula III

3131 or

Ht(cm) x Wt(kg)

BSA Formula IV

3600

Dosing Regimen

Patients were dosed once weekly for 8 weeks. The study drug dose for each patient was calculated on Day 1.

Treatment

Patients were randomly assigned to receive placebo (0.9% Sodium Chloride

Injection, USP), 1.5 mg/m² GCS-lOO, or 30 mg/m² GCS-lOO. Placebo and GCS-lOO were administered as IV infusions once weekly for 8 weeks.

Table 8 shows the change galectin-3 in patients administered with 1.5 mg/m² of GCS-lOO, and 30 mg/m² of GCS-lOO.

Table 8: Change in baseline galectin-3 (ng/mL) in patients administered with placebo, 1.5 mg/m² of GCS-lOO, and 30 mg/m² of GCS-lOO.

I High I 1.29 I

Example 3: Phase 2 study of GCS-100 in chronic kidney disease (CKD) patients

A multicenter, randomized, blinded, placebo-controlled, Phase 2 study in 121 advanced CKD patients was performed. The phase 2 study met its primary efficacy endpoint of a statistically significant improvement in kidney function. Specifically, GCS-100, at a dose of 1.5 mg/m², led to a statistically significant (p=0.045) improvement in estimated glomerular filtration rate (eGFR) versus placebo after 8 weeks of dosing. This improvement, compared to placebo, was maintained 5 weeks following the completion of dosing (p=0.07). No statistically significant improvement in eGFR was observed in the 30 mg/m² dose group.

GCS-100 was well-tolerated. There were no serious adverse events, no Grade 3/4 adverse events and no early study discontinuations in the 1.5 mg/m² group. There was no observed effect on blood pressure in any dose group.

An extension study was conducted in which patients from the Phase 2 study were re- randomized to receive either 1.5 or 30 mg/m² of GCS-100 (complete data available through week 16). Of the 93 patients enrolled in total, 33 patients had previously received placebo in the Phase 2 study before being treated with GCS-100 in the extension study. This group, which represents a set of patients receiving GCS-100 for the first time, was analyzed for efficacy. Consistent with the blinded Phase 2 results, the 1.5 mg/m² group experienced a significant improvement in eGFR. This was observed when comparing these patients' responses to both: (1) the response in the parallel randomized group receiving 30 mg/m² (p=0.04); and (2) the previous response to placebo in the blinded Phase 2 study for placebo- treated patients enrolled in the extension study (p=0.02).

REFERENCES

All publications and patents mentioned herein, including those references listed below, are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually incorporated by reference. In case of conflict, the present application, including any definitions herein, may control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention may become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS What is claimed is:

1. A method for treating a cancer in a patient, comprising: administering to the patient at least one galectin-3 inhibitor.

2. The method of claim 1, wherein the cancer is selected from renal cell cancer, Kaposi's sarcoma, chronic leukemia, prostate cancer, breast cancer, sarcoma, pancreatic cancer, ovarian carcinoma, rectal cancer, throat cancer, melanoma, colon cancer, bladder cancer, mastocytoma, lung cancer, mammary adenocarcinoma, myeloma, lymphoma, pharyngeal squamous cell carcinoma, gastrointestinal cancer, stomach cancer, melanoma, lung cancer, breast cancer, pancreatic cancer, prostate cancer, colon cancer, or ovarian cancer.

3. The method of claim 1, wherein the galectin-3 inhibitor is a modified pectin.

4. The method of claim 3, wherein the backbone of the modified pectin comprises homogalacturonan and/or rhamnogalacturonan I.

5. The method of claim 3, wherein the modified pectin is de-esterified and partially depolymerized, so as to have a disrupted rhamnogalacturonan backbone.

6. The method of any of claims 3-5, wherein the modified pectin has an average molecular weight between 50 and 200 kDa, preferably between 80 and 150 kDa.

7. The method of any of claims 3-6, wherein the modified pectin is substantially free of modified pectins having molecular weights below 25 kDa.

8. The method of claim 3, wherein the modified pectin is GCS-100.

9. The method of any of claims 3-8, wherein the modified pectin is made by passing modified or unmodified pectin through a tangential flow filter.

10. The method of any one of claims 3-9, comprising administering the modified pectin at a dose of about 0.1 to 2 mg/m².

11. The method of claim 10, wherein the dose is about 1.5 mg/m².

12. The method of any one of claims 3-11, comprising administering the modified pectin at a dose of about 1-10 mg.

13. The method of claim 12, wherein the dose is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg, preferably 1, 3, or 9 mg.

14. The method of any preceding claim, wherein the galectin-3 inhibitor is administered weekly or biweekly.

15. The method of claim 14, wherein the galectin-3 inhibitor is administered weekly for an induction phase and then biweekly for a maintenance phase.

16. The method of claim 15, wherein the induction phase is 1-3 months, preferably 2 months.

17. The method of claim 15 or 16, wherein the maintenance phase is at least 1 month, preferably at least 3 months, or even six months or more.

18. The method of any of preceding claim, wherein the at least one galectin-3 inhibitor is administered in an amount that reduces a level of galectin-3 in serum of the patient.

19. The method of any of preceding claim, wherein the at least one galectin-3 inhibitor is administered in an amount that reduces an expression level of galectin 3 in the patient.

20. The method of any of preceding claim, wherein the at least one galectin-3 inhibitor is administered in an amount that reduces an activity of galectin-3 in the patient.

21. The method of any preceding claim, whereby the concentration, expression level, or activity of galectin-3 is reduced 0.5, 1, 2, 3, 4, or 5-fold relative to a control.

22. The method of any preceding claim, further comprising 1) measuring the concentration, level, or activity of galectin-3 in the patient before administering the galectin- 3 inhibitor and 2) measuring the concentration, level, or activity of galectin-3 after administering the galectin-3 inhibitor.

23. The method of claim 22, wherein a decrease in the concentration, level, or activity of galectin-3 after administering the galectin-3 inhibitor indicates that the dose of galectin-3 inhibitor is an effective dose of galectin-3 inhibitor for the treatment of cancer in a patient.

24. The method of claim 23, wherein an increase in the concentration, level, or activity of galectin-3 after administering the galectin-3 inhibitor indicates that the dose of galectin-3 inhibitor is an ineffective dose of galectin-3 inhibitor for the treatment of cancer in a patient.

25. The method of claim 24, further comprising administering to the patient a second dose of the galectin-3 inhibitor in a lower amount than in the prior administration.

26. The method of any preceding claim, further comprising administering an additional therapeutic agent.

27. The method of any preceding claim, wherein the additional therapeutic agent is useful for the treatment of cancer.

28. The method of any preceding claim, comprising administering the galectin-3 inhibitor concurrently with the therapeutic agent.

29. The method of any one of claims 1-27, comprising administering the galectin-3 inhibitor subsequent to administration of the therapeutic agent.

30. The method of any one of claims 1-27, comprising administering the therapeutic agent subsequent to administration of the galectin-3 inhibitor.

31. The method of any preceding claim, comprising administering multiple doses of the galectin-3 inhibitor over a period of at least 8 weeks.

32. The method of any preceding claim, comprising administering the galectin-3 inhibitor weekly.

33. The method of any preceding claim, wherein the galectin-3 inhibitor is administered by injection or intravenous infusion.

34. The method of claim 33, wherein the galectin-3 inhibitor is administered by intravenous infusion.