BOROPROLINE COMPOUND AND CYTOKINE COMBINATION THERAPY

Related Applications

This application claims priority to U.S. Provisional Applications having Serial Nos. 60/735491, 60/73571 1 and 60/801903 and filed on November 10, 2005, November 11, 2005 and May 19, 2006, respectively, the entire contents of all of which are incorporated by reference herein.

Field of the Invention This invention relates to methods for the treatment of conditions characterized by abnormal cell proliferation using boroproline compounds together with cytokines or the screening of subjects likely to respond to boroproline compounds.

Background of the Invention Cancer is the second leading cause of death, resulting in one out of every four deaths, in the United States. In 1997, the estimated total number of new diagnoses for lung, breast, prostate, colorectal and ovarian cancer was approximately two million. Due to the ever increasing aging population in the United States, it is reasonable to expect that rates of cancer incidence will continue to grow. Cancer is currently treated using a variety of modalities including surgery, radiation therapy and chemotherapy. The choice of treatment modality will depend upon the type, location and dissemination of the cancer. One of the advantages of surgery and radiation therapy is the ability to control to some extent the impact of the therapy, and thus to limit the toxicity to normal tissues in the body. Chemotherapy is arguably the most appropriate treatment for disseminated cancers such as leukemia and lymphoma as well as metastases. Chemotherapy is generally administered systemically and thus toxicity to normal tissues is a major concern. Not all tumors, however, respond to chemotherapeutic agents and others, although initially responsive to chemotherapeutic agents, may develop resistance. As a result, the search for effective anti-cancer drugs has intensified in an effort to find even more effective agents with less non-specific toxicity.

In addition, the ability to identify patients that would respond to and thus benefit from a particular therapy would be beneficial. Such patients could be identified using for example biomarkers which include but are not limited to pre-treatment presence (and/or particular levels) of a biologic such as a protein, gene, carbohydrate, and the like.

Summary of the Invention

The invention relates to the use of cytokines in the treatment of cancer using boroproline compounds. The invention contemplates cytokines as therapeutic agents as well as biomarkers for the selection of patient populations that will respond to boroproline compound therapy. It was previously shown that certain boroproline compounds stimulate IL-I, thereby leading to a cascade of cytokine and chemokine induction. It has now been found that a treatment strategy that incorporates both boroproline compounds and certain cytokines is more effective in the treatment of cancer.

Thus, in one aspect the invention provides a method for treating a subject having a cancer using a non-immunoglobulin therapy comprising administering to a subject in need thereof at least one agent of Formula I and at least one cytokine, in an amount effective to treat the cancer, wherein the subject has normal hematopoietic cell levels.

In another aspect, the invention provides a method for treating a subject having a cancer using a non-immunoglobulin therapy comprising administering to a subject in need thereof at least one agent of Formula I and interferon, in an amount effective to treat the condition.

In yet another aspect, the invention provides a method for treating a subject having a cancer (e.g., a tumor) using a non-immunoglobulin therapy comprising administering to a subject in need thereof at least one agent of Formula I and at least one cytokine, in an amount effective to treat the cancer, wherein the subject has not been treated using chemotherapy or radiation.

In still another aspect, the invention provides a method for treating a subject having a cancer (e.g., a tumor) comprising administering to a subject in need thereof in an amount effective to treat the cancer, at least one cytokine, and then at least one agent of Formula I, wherein the cancer is non-immunogenic. In one embodiment, the at least one agent of Formula I is administered less than 4 months, 6 months, 8 months, or a year after the at least one cytokine. In another embodiment, the at least one agent of Formula I is administered

within 4 months, 3 months, 2 months, 1 month, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day of at least one cytokine,

In another aspect, the invention provides a method for treating a subject having melanoma or renal cell carcinoma using a non-immunoglobulin therapy comprising administering to a subject in need thereof at least one agent of Formula I and at least one cytokine, in an amount effective to treat the melanoma or renal cell carcinoma.

In still another aspect, the invention provides a method for treating a subject having a cancer comprising administering to a subject in need thereof at least one agent of Formula I and a heat shock protein, in an amount effective to treat the cancer. In some embodiments, the heat shock protein is administered to the subject prior to the agent of Formula I. In related embodiments, there is a delay between the time of administration of these agents, including but not limited to one to thirty days, one to four weeks, or one to twelve months. The heat shock protein may be gp96, but is not so limited. In some embodiments, the cancer is melanoma, including metastatic melanoma. In some embodiments, the method further comprises administering cisplatin to the subjects.

In yet another aspect, the invention provides a method for treating a subject having a cancer comprising administering to a subject in need thereof at least one agent of Formula I and a first and optionally a second anti-cancer agent, in an amount effective to treat the cancer. In some embodiments, the first anti-cancer agent is temozolomide/thalidomide protein. In some embodiments, the second anti-cancer agent is cisplatin. In some embodiments, the first anti-cancer agent is administered prior to the agent of Formula I, and optionally prior to the second anti-cancer agent. In related embodiments, there is a delay from the time of administration of these agents, including but not limited to one to thirty days, one to four weeks, or one to twelve months.

In one embodiment, the cancer is a tumor. In some embodiments, the cancer is melanoma (including metastatic melanoma) or renal cell carcinoma.

In one embodiment, the non-immunoglobulin therapy is a non-immunotherapy.

In one embodiment, the method further comprises administering a plurality of cytokines. In one embodiment, the at least one cytokine is IL-2, IFN, G-CSF or GM-CSF.

In various embodiments, the at least one cytokine is administered before the agent of Formula I. In related embodiments, the cytokine is administered substantially simultaneously with the agent of Formula I. In other embodiments, the at least one cytokine and the at least one agent of Formula I are both adminstered in the same (i.e., an identical) multi-day cycle

(e.g., the cytokine is adminsitered on day 1 of a multi-day cycle and the agent is administered on days 2-14 of the same cycle).

The agent of Formula I may be an agent of Formula II. The agent of Formula I may be an agent of Formula III. The agent of Formula I may be Val-boroPro or Glu-boroPro, although it is not so limited.

The agent of Formula I may be provided in a composition, including a mixture of agents. In such compositions or mixtures, the agent of Formula I may be at least 96%, at least 97%, at least 98%, or at least 99% pure L-isomer. The L-isomer refers to the stereochemistry at the pyrrolidine carbon to reactive moiety bond (e.g., the bond between the carbon in pyrrolidine to the boron in an organo boronate reactive group). This bond can also be referred to as being in the R configuration if the reactive moiety is an organo boronate and the carbon is bonded to the boron.

The agent of Formula I may be provided and/or present in a composition or mixture of linear and cyclic forms. The cyclic. form may represent (as a percentage of total agent present) at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more.

In one aspect, the invention provides a method for treating a subject having a cancer comprising administering to the subject an agent of Formula I in combination with cisplatin, wherein the subject has been previously administered or is currently being administered IFN, GM-CSF, a heat shock protein, or temozolomide. The cancer may be melanoma, including metastatic melanoma. The agent of Formula I is Val-boroPro in important embodiments.

In another aspect, the invention provides a method for treating a subject having melanoma, including metastatic melanoma, with an agent of Formula I, wherein the subject has been previously administered or is currently being administered IL-2 or IFN. The agent of Formula I is Val-boroPro in important embodiments. Such subjects may also be screened prior to the administration of the agent for responsiveness based on production of IL-I beta, IL-2 and/or GM-CSF after exposure to the agent, either in vivo or in vitro. The effective amount may be one that induces a partial or complete response in the subject.

In yet another aspect, the invention provides a method for identifying a subject having a cancer or a pre-cancerous condition and likely to respond to treatment with an agent of Formula I (e.g., a boroproline compound) comprising measuring a pre-treatment cytokine level in a subject, and comparing the pre-treatment cytokine level with a control, wherein a

pre-treatment cytokine level that is greater than the control is indicative of a subject likely to respond to treatment with the agent of Formula I. In one embodiment, the pre-treatment cytokine level is a pre-treatment level of IL-2 or IFN. The control may be a cytokine level in a normal subject that has not received any exogenous cytokines or chemokines or any agents of Formula I including but not limited to any boroproline compounds, and who does not have cancer. The control may be a population average wherein the population has the same condition as the subject. The control may be a cytokine level in the same subject taken prior to the current therapeutic regimen (e.g., a historical cytokine level measured months or years before the current measurement). The effective amount may be one that induces a partial or complete response in the subject.

In yet another aspect, the invention provides a method for identifying a subject having a cancer and likely to respond to treatment with an agent of Formula I (e.g., boroproline compound) comprising determining a cytokine response profile to an agent of Formula I of a subject having a cancer, and comparing the cytokine response profile to a control. A cytokine response profile comprising an increased level of one or more cytokines (such as but not limited to IL-lbeta, IL-lalpha, IL-2 and/or GM-CSF) is indicative of a subject who is likely to respond to treatment with the agent of Formula I. The control may be the cytokine level in the subject prior to contact with the agent of Formula I.

In one embodiment, the cytokine response profile is determined in vitro. Altered levels of cytokines can be assessed at the protein or mRNA level, and if at the protein level by analysis of secreted or cell bound cytokine.

In one embodiment, the agent of Formula I comprises a pyrrolidine ring bonded to an organo boronate reactive moiety and is referred to as a boroproline compound.

In one embodiment, the boroproline compound is an agent of Formula II which optionally may be an agent of Formula III. The boroproline compound may be Val-boroPro. The cancer may be a tumor. In one embodiment, the cancer is melanoma or renal cell carcinoma. The cancer may be metastatic.

The method may further comprise administering to an identified subject the boroproline compound in an effective amount to treat the cancer. These and other aspects of the invention will be described in greater detail below.

Throughout this disclosure, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains unless defined otherwise.

Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention.

Detailed Description of the Invention

The invention relates broadly to a particular therapy for cancer and the selection of subjects that are most likely to respond to such therapy. Cytokines are important to the invention both as therapeutic agents and as biomarkers for patient selection. The invention is premised in part on the finding that human subjects having melanoma and previously treated with certain cytokines were more responsive to treatment with a boroproline compound. The invention is further premised in part on the finding that responsive subjects also demonstrated increased levels of certain cytokines following treatment with the boroproline compound. Together these findings identify certain cytokines as therapeutic adjuncts to boroproline therapy and as biomarkers that can differentiate a patient population in terms of its expected level of response to boroproline therapy.

The term "cytokine" as used herein refers to a diverse group of soluble proteins and peptides which act as humoral regulators at nano- to picomolar concentrations and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. These proteins also mediate interactions between cells directly and regulate processes taking place in the extracellular environment. As used herein, cytokines embrace interleukins (IL) (i.e., those produced by and which act on white blood cells), monokines (i.e., those produced by monocytes), Iymphokines (i.e., those produced by lymphocytes), chemokines (i.e., those with chemotactic activity), as well as growth factors in general. Other subclasses of cytokines include but are not limited to Activins, Inhibins, Angiogenic Factors, Ephrin, Epidermal Growth Factors (EGF), NRG, Fibroblast Growth

Factors (FGF), Hematopoietic Cytokines, Hepatocyte Growth Factor/Scatter Factor, Insulin- like Growth Factors (IGF), Interferons (IFN), Neurotrophic Factors, Oncostatin M (OSM), Platelet Growth Factor (PDGF), Pleiotrophin, Transforming Growth Factor-b (TGF-b), Tumor Necrosis Factor (TNF), and Vascular Endothelial Growth Factor (VEGF). Thus, the broad class of cytokines as defined herein includes, but is not limited to, IL- lalpha and beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-Il, IL-12, IL-13, IL- 14, IL-15, IL- 16, IL-17, IL- 18, macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-

CSF), GRO, GRO-alpha, interferon-γ (IFN-γ), IFN-α, IFN-β, tumor necrosis factor (TNF), TGF-β, TGF-βl, EPO, MIP- lα, MIP- lβ, FLT-3 ligand, kit ligand (or stem cell factor), and CD40 ligand. The cytokine may act upon a hematopoietic cell, a non-hematopoietic cells, or both. Chemokines are a class of cytokine molecules that are involved in cell recruitment and activation in inflammation. These chemokines have been classified into four subgroups, depending on the nature of the spacing of two highly-conserved cysteine amino acids that are located near the amino terminus of the polypeptide. The first chemokine subgroup is referred to as "CXC"; the second subgroup is referred to as "CC"; the third chemokine subgroup is referred to as "CX3C"; and the fourth chemokine subgroup is referred to as "C". Within these subgroups, the chemokines are further divided into related families that are based upon amino acid sequence homology. The CXC chemokine families include the IP-IO and Mig family; the GROα, GROβ, and GROβ family; the interleukin-8 (IL-8) family; and the PF4 family. The CC chemokine families include the monocyte chemoattractant protein (MCP) family; the family including macrophage inhibitory protein- lα (MIP- lα), macrophage inhibitory protein- lβ (MIP- lβ), and regulated on activation normal T cell expressed (RANTES). The stromal cell-derived factor lα (SDF- lα) and stromal cell-derived factor lβ (SDF-I β) represent a chemokine family that is approximately equally related by amino acid sequence homology to the CXC and CC chemokine subgroups. The CX3C chemokine family includes fractalkine.

Particularly important cytokines include IL-I beta, IL-2, IFN, G-CSF and GM-CSF. These can be used individually, in any combination of two, three or four of each other, or in combination with one or more other cytokines.

The cytokines and/or chemokines can be administered directly (e.g., in a substantially pure form) or may be administered in the form of a nucleic acid vector that encodes the cytokine, such that the cytokine can be expressed in vivo. In one embodiment, the cytokine or chemokine is administered in the form of a plasmid expression vector. The cytokines may be harvested from naturally occurring sources (and substantially purified therefrom), or they may be synthesized in vitro such as for example recombinant versions. In addition, cytokine hybrids or chimerae can also be used.

Some aspects and embodiments of the invention relate to the use of heat shock proteins together with the agents of Formula I, and optionally also together with other anti-

cancer agents, such as but not limited to temozolomide/thalidomide and cisplatin. Heat shock proteins are proteins that are more highly expressed in stressed cells as compared to non- stressed cells. An example of a cell stress is a temperature above 37°C. Accordingly, they are also referred to as stress proteins. Heat shock proteins are thought to provide chaperoning functions that preserve the integrity of certain proteins or other molecules within the cell during times of stress. Examples include hsp70, hsp90, gp96 (ONCOPHAGE, vitespen, HSPPC-96, Antigenics, Inc.) and protein disulfide isomerase. Heat shock proteins are discussed in greater detail in U.S. Pat. Nos. 6410027 and 6410026.

The term "substantially purified" as used herein refers to a compound which is substantially free of other compounds such as proteins, lipids, carbohydrates or other materials with which it is naturally associated. The substantially pure polypeptide will often yield a single major band on a non-reducing polyacrylamide gel. In the case of partially glycosylated polypeptides or those that have several start codons, there may be several bands on a non-reducing polyacrylamide gel, but these will form a distinctive pattern for that polypeptide.

When used therapeutically, cytokines are used together with agents of Formula I which have the following structure:

Formula I PR wherein P is a targeting group which binds to the reactive site of a post proline- cleaving enzyme, and R is a reactive group that reacts with a functional group in the reactive site of a post proline-cleaving enzyme. Post proline-cleaving enzymes are enzymes which have a specificity for cleaving Xaa-Pro or Xaa-Ala dipeptides (where Xaa represents any amino acid) at the carboxy end of the dipeptide. Dipeptidyl peptidases (DPP) do so from the amino terminus of a peptide or protein. Examples of post-proline cleaving enzymes include, but are not limited to, CD26, dipeptidyl peptidase IV (DP IV or DPP IV) and fibroblast activation protein (FAP) all of which show DPP activity. Prolyl endopeptidase (PEP or POP) are endopeptidases with the same specificity. FAP demonstrates both exo- and endopeptidase activities. The targeting group (P) can be composed of single or multiple amino acid residues, whether naturally occurring or not, or of a peptide or a peptidomimetic. In certain embodiments, the portion of P that is involved in binding to the reactive site of a post proline- cleaving enzyme is formed of amino acid residues and the remaining portion of P is formed

of non-amino acid components. Therefore P can be composed wholly of amino acid residues, wholly of non-amino acid substituents, or a combination of both. P may be 100 or more residues in length including 30, 20, 10 or less than 10 residues in length. P or a portion(s) thereof may mimic a substrate of the protease. Targeting groups can be synthesized from other biomolecules including but not limited to saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs of the above, or combinations thereof and the like. Also envisioned in the invention is the use of targeting groups made from peptoids, random bio-oligomers (U.S. Patent 5,650,489), benzodiazepines, diversomeres such as dydantoins, benzodiazepines and dipeptides, nonpeptidyl peptidomimetics with a beta-D-glucose scaffolding, oligocarbamates or peptidyl phosphonates.

Many if not all of these compounds can be synthesized using recombinant or chemical library approaches. A vast array of candidate targeting groups can be generated from libraries of synthetic or natural compounds. The methods of the invention utilize this library technology to identify small molecules which bind to protease reactive sites. One advantage of using libraries for inhibitor identification is the facile manipulation of millions of different putative candidates of small size in small reaction volumes (i.e., in synthesis and screening reactions). Another advantage of libraries is the ability to synthesize targeting groups which might not otherwise be attainable using naturally occurring sources. The use of library technology, such as phage display, and combinatorial chemistry, such as compound array methods, in the synthesis and screening of protease inhibitors has been previously described in U.S. Patent Application entitled "Multivalent Compounds for Crosslinking Receptors and Uses Thereof" filed on April 12, 1999 and assigned U.S. S.N. 09/290,376, the contents of which are incorporated in their entirety by reference. Examples of parallel synthesis mixtures and parallel synthesis methods are provided in U. S. S.N.

08/177,497, filed January 5, 1994 and its corresponding PCT published patent application W095/18972, published July 13, 1995 and U.S. Patent No. 5,712,171 granted January 27, 1998 and its corresponding PCT published patent application W096/22529, which are hereby incorporated by reference. Libraries can be screened to identify naturally or non-naturally occurring putative targeting groups by assaying protease binding (and optionally cleavage activity) in the presence of the library molecule or member. The cleavage assay includes determining whether the library molecule inhibits cleavage by the protease of a known substrate or of a

substrate analog (e.g., a chromophoric substrate analog which is easily detectable in a spectrophotometric assay). Those library molecules which exhibit binding and optionally inhibition of a post-prolyl cleaving enzyme then can be covalently coupled to the reactive groups disclosed herein and again tested for binding and inhibition of the enzyme. In this manner, a simple, high-through-put screening assay is provided for identifying inhibitors.

In general, P is covalently coupled to R. In some embodiments, the covalent coupling occurs via a carboxyl group at the carboxyl terminus in P.

Examples of reactive groups useful in the invention include an organo boronate, an organo phosphonate, a fluoroalkylketone, an halomethyl ketone, a diazomethyl ketone, a dimethylsulphonium salt, an alphaketo carboxylic acid, an alphaketo ester, an alphaketo amide, an alpha-diketone, an acyloxymethyl ketone, an aldehyde, an epoxysuccinyl, an N- peptidyl-O-acylhydroxyl amine, an azapeptide, a fluoroolefin, a peptidyl (alpha-aminoalkyl) phosphonate ester, or a nitrile.

Agents of Formula I can be further defined by Formula II as follows:

Formula II

wherein m is an integer between 0 and 100 or more, inclusive; A and Ai are L- or D- naturally or non-natura!ly occurring amino acid residues or a peptide or a peptidomimetic such that when m >1 and A is an amino acid each A in A _m may be an amino acid residue different from another or all other A in A _m , and when A is a peptide or a peptidomimetic m is 0 or 1 ; the C bonded to B is in the L- or R- configuration; optionally the bonds between Ai and N and between A and Ai are peptide bonds; and each Xi and X ₂ is independently a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH.

The proline residue attached to the reactive group is referred to as a pyrrolidine ring. In some embodiments, the pyrrolidine may be replaced with an azetidine or a thiazolidine. In other embodiments, the pyrrolidine-T moiety is replaced with a 4-cyanothiazoIidine. In some

wherein m is an integer between 0 and 100 or more, inclusive; A and Ai are L- or D- naturally or non-naturally occurring amino acid residues such that when m >1 A in each repeating bracketed unit is independently selected; the C bonded to B is in the L- or R- configuration; and optionally the bonds between A and N, Ai and C, and Ai and N are peptide bonds. The agent may however also be an isostere in which case the bonds are non- peptide bonds. Each Xi and X ₂ is, independently, a hydroxyl group or a group capable of being hydrolyzed to a hydroxyl group in aqueous solution at physiological pH

The proline residue attached to the boronyl group is referred to as a pyrrolidine ring. The amino acid or peptide attached to the pyrrolidine ring may be in an R- or S- configuration.

Many of the agents of the invention and methods for their manufacture have been previously disclosed in U.S. Patent 4,935,493, the contents of which are incorporated by reference herein.

It is to be understood that each and every reactive group described herein can be substituted for the reactive group in Formulae II and III (e.g., boronyl group in the form of a boronic ester or a boronic acid). Thus, other inhibitors have an analogous structure to the agents of Formulae II or III but with the boronyl group replaced by, for example, an organo phosphonate, a fϊuoroalkylketone, an halomethyl ketone, a diazomethyl ketone, a dimethyl sulphonium salt, an alphaketo carboxylic acid, an alphaketo ester, an alphaketo amide, an alpha-diketone, an acyloxymethyl ketone, an aldehyde, an epoxysuccinyl, an N- peptidyl-O-acylhydroxylamine, an azapeptide, a fluoroolefm, a peptidyl (alpha-aminoalkyl) phosphonate ester, or a nitrile.

The proline residue attached to the reactive group is referred to as a pyrrolidine ring. In some embodiments, the pyrrolidine may be replaced with an azetidine or a thiazolidine. In other embodiments, the pyrrolidine-T moiety is replaced with a 4-cyanothiazolidine. In some embodiments, the amino acids are attached via non-peptide bonds or the peptide comprises non-peptide bonds, such as for example in an isostere moiety.

The amino acid residues may be naturally and non-naturally occurring amino acids. Examples of naturally occurring amino acids are glycine (GIy), and the L-forms of alanine (Ala), valine (VaI), leucine (Leu), isoleucine (lie), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), cysteine (Cys), methionine (Met), serine (Ser), threonine (Thr), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (GIu), asparagine (Asn), glutamine (GIn) and proline (Pro). Non-naturally occurring amino acids include the

D-forms of Ala, VaI, Leu, lie, Phe, Tyr, Trp, Cys, Met, Ser, Thr, Lys, Arg, His, Asp, GIu,

Asn, GIn, and Pro.

Other examples of non-naturally occurring amino acids include 2-azetidinecarboxylic acid or pipecolic acid (which have 6-membered, and 4-membered ring structures respectively), 4-hydroxy-proline (Hyp), 5-hydroxy-lysine, norleucine (NIe), 5- hydroxynorleucine (Hyn), 6-hydroxynorleucine, ornithine, cyclohexylglycine (Chg), N-

Methylglycine (N-MeGIy), N-Methylalanine (N-MeAIa), N-Methylvaline (N-MeVaI), N-

Methylleucine (N-MeLeu), N-Methylisoleucine (N-MeIIe), N-Methylnorleucine (N-MeNIe),

N-Methyl-2-aminobutyric acid (N-MeAbu) and N-Methyl-2-aminopentanoic acid (N- MeNva), methylthreonine, nitroglutamine, norleucine (NIe), norvaline, ornithine, phosphoserine, pipecolic acid, sarcosine, taurine, tert-leucine, thiazolidine carboxylic acid, thyroxine, trans-4-hydroxyproline, and trans-3-methylproline.

Non-naturally occurring amino acids also include beta-amino acids and alpha-amino acids with side chains replaced with synthetic derivatives. Representative side chains of naturally occurring and non-naturally occurring α-amino acids are shown below.

Non-naturally occurring amino acids also include D, L, and racemic configurations of hydrophobic amino acids. Hydrophobic amino acids include amino acid analogs having the formula -NH-CHR-CO-, wherein R is an aliphatic group, a substituted aliphatic group, a benzyl group, a substituted benzyl group, an aromatic group or a substituted aromatic group and wherein R does not correspond to the side chain of a naturally-occurring amino acid. As used herein, aliphatic groups include straight chained, branched or cyclic C1-C8 hydrocarbons which are completely saturated, which contain one or two heteroatoms such as nitrogen, oxygen or sulfur and/or which contain one or more units of desaturation. Aromatic groups include carbocyclic aromatic groups such as phenyl and naphthyl and heterocyclic aromatic groups such as imidazolyl, indolyl, thienyl, furanyl, pyridyl, pyranyl, oxazolyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl and acridintyl.

Suitable substituents on an aliphatic, aromatic or benzyl group include -OH, halogen (-Br, -Cl, -I and -F) -O (aliphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group), -CN, -NO ₂ , -COOH, -NH ₂ , -NH (aliphatic group, substituted aliphatic, benzyl, substituted benzyl,. aryl or substituted aryl group), -N (aliphatic group, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group) ₂ , -COO (aliphatic group, substituted aliphatic, benzyl, substituted benzyl, aryl or substituted aryl group), -CONH ₂ , -CONH (aliphatic, substituted aliphatic group, benzyl, substituted benzyl, aryl or substituted aryl group)), -SH, -S (aliphatic, substituted aliphatic, benzyl, substituted benzyl, aromatic or substituted aromatic group) and -NH-C(=NH)-NH ₂ . A substituted benzylic or aromatic group can also have an aliphatic or substituted aliphatic group as a substituent. A substituted aliphatic group can also have a benzyl, substituted benzyl, aryl or substituted aryl group as a substituent. A substituted aliphatic, substituted aromatic or substituted benzyl group can have one or more substituents. Modifying an amino acid substituent can increase, for example, the lypophilicity or hydrophobicity of natural amino acids which are hydrophilic.

A number of the suitable amino acids, amino acids analogs and salts thereof can be obtained commercially. Others can be synthesized by methods known in the art. Synthetic techniques are described, for example, in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991.

These agents of Formula I can be provided in linear or cyclic form or as mixtures thereof, as described in U.S. Patent No. 6,355,614, issued March 12, 2002. The proportion of cyclic forms (relative to the total amount of agent present) in these mixtures may vary (e.g.,

at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the agents in the mixture may be cyclic) depending on the formulation.

The agents of Formula I may be provided as A-boroPro containing compoxmds that are converted (via enzymatic, chemical, metabolic, or any other means, in vivo or ex vivo) to A-boroPro. A-boroPro containing compounds include prodrugs. A prodrug of for example A-boroPro, as used herein, is a compound that is metabolized in vivo to A-boroPro or that disintegrates (e.g., upon contact with stomach acid) to form A-boroPro. Some prodrugs are converted into A-boroPro via hydrolysis or oxidation in vivo. These include alcohol precursors of A-boroPro that are oxidized in vivo (e.g., in the liver) and a boroxine derivative of A-boroPro, as well as esters of Glu-boroPro and related compounds. Prodrugs of A- boroPro also include cyclized versions of the molecule, as discussed above.

Another category of prodrugs includes compounds that are converted to A-boroPro by enzymes. These enzymes may be post-prolyl cleaving enzymes (e.g., DPP-IV.) or non-post- prolyl cleaving enzymes. Examples of this class of prodrug moieties are disclosed in U.S. Patent Nos. 5,462,928 issued October 31, 1995; and 6,100,234 issued August 8, 2000; and published PCT applications WO 91/16339 published October 31, 1991; WO 93/08259 published April 29, 1993; and WO 03/092605, published November 13, 2003, among others. The length of such prodrug compounds may be 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 50, 100 or more residues in length (whereby the length includes A and proline residues). Multiples of 3 are also contemplated.

All amino acids, with the exception of glycine, contain an asymmetric or chiral carbon and may contain more than one chiral carbon atom. The asymmetric α carbon atom of the amino acid is referred to as a chiral center and can occur in two different isomeric forms. These forms are identical in all chemical and physical properties with one exception, the direction in which they can cause the rotation of plane-polarized light. These amino acids are referred to as being "optically active," i.e., the amino acids can rotate the plane-polarized light in one direction or the other.

The four different substituent groups attached to the α carbon can occupy two different arrangements in space. These arrangements are not superimposable mirror images of each other and are referred to as optical isomers, enantiomers, or stereo isomers. A solution of one stereo isomer of a given amino acid will rotate plane polarized light to the left and is called the levorotatory isomer [designated (-)]; the other stereo isomer for the amino

acid will rotate plane polarized light to the same extent but to the right and is called dextrorotatory isomer [designated (+)].

A more systematic method for classifying and naming stereo isomers is the absolute configuration of the four different substituents in the tetrahedron around the asymmetric carbon atom (e.g., the α carbon atom). To establish this system, a reference compound was selected (glyceraidehyde), which is the smallest sugar to have an asymmetric carbon atom. By convention in the art, the two stereo isomers of glyceraidehyde are designated L and D. Their absolute configurations have been established by x-ray analysis. The designations, L and D, also have been assigned to the amino acids by reference to the absolute configuration of glyceraidehyde. Thus, the stereo isomers of chiral compounds having a configuration related to that of L-glyceraldehyde are designed L, and the stereo isomers having a configuration related to D-glyceraldehyde are designated D, regardless of the direction in which they rotate the plane-polarized light. Thus, the symbols, L and D, refer to the absolute configuration of the four substituents around the chiral carbon. In general, naturally occurring compounds which contain a chiral center are only in one stereo isomeric form, either D or L. The naturally occurring amino acids are the L stereo isomers. As discussed herein, the invention embraces inhibitors comprising amino acid residues which can be in the D stereo isomer configuration also.

Most amino acids that are found in proteins can be unambiguously named using the D L system. However, compounds which have two or more chϊral centers may be in 2" possible stereo isomer configurations, where n is the number of chiral centers. These stereo isomers sometimes are designated using the RS system to more clearly specify the configurations of amino acids that contain two or more chiral centers. For example, compounds such as threonine and isoleucine contain two asymmetric carbon atoms and therefore have four stereo isomer configurations. The isomers of compounds having two chiral centers are known as diastereomers. A complete discussion of the RS system of designating optical isomers for amino acids is provided in Principles in Biochemistry, editor AX. Lehninger, page 99-100, supra. A brief summary of this system follows.

The RS system was invented to avoid ambiguities when a compound contains two or more chiral centers. In general, the system is designed to rank the four different substituent atoms around an asymmetric carbon atom in order of decreasing atomic number or in order of decreasing valance density when the smallest or lowest-rank group is pointing directly away from the viewer. The different rankings are well known in the art and are described on page

99 of Lehninger (supra). If the decreasing rank order is seen to be clock- wise, the configuration around the chiral center is referred to as R; if the decreasing rank order is counter-clockwise, the configuration is referred to as S. Each chiral center is named accordingly using this system. Applying this system to threonine, one skilled in the art would determine that the designation, L-threonine, refers to (2S, 3R)-threonine in the RS system. The more traditional designations of L-, D-, L-allo, and D-allo, for threonine have been in common use for some time and continue to be used by those of skill in this art. However, the R S system increasingly is used to designate the amino acids, particularly those which contain more than one chiral center.

The agents of the invention may be provided in a composition or as a mixture of agents. In such compositions or mixtures, the agents may be provided in a substantially optically pure form. That is, at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% of the agents comprise carbon atoms bearing reactive groups such as boron that are in the L configuration in some embodiments. In embodiments in which the agents comprise the carbon of the pyrrolidine ring bonded to the boron, this bond is in the R-configuration. The agent may be provided as a mixture of R- and S-enantiomers of boron substituted pyrrolidine. Therefore, the mixture of R- and S-enantiomers of boron substituted pyrrolidine may contain at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the R-enantiomer of boron substituted pyrrolidine. Methods for synthesizing optically pure isomers of Formula I agents are disclosed in published PCT application WO 93/08259.

The invention provides screening methods for identifying subjects for whom boroproline therapy will be more effective. Such subjects are referred to herein as being "likely to respond to treatment with a boroproline compound". Such screening methods include identifying subjects based on a pre-treatment level of one or more cytokines and comparing such levels to a control, wherein a pre-treatment cytokine level that is greater than the control is indicative of a subject for whom boroproline compound therapy will be more effective.

The cytokine level may be measured in the plasma, serum or blood or blood cells of the subject, but it is not so limited. The following is one representative assay for measuring cytokine levels in blood. Briefly, these samples are collected and optionally placed on ice. The samples are preferably centrifuged within 3 hours of sample harvest. The resultant plasma sample is then stored at -7O ⁰ C until assay. All samples were processed in the same manner. Cytokines may be measured using anti-cytokine antibodies in an ELISA. Such

antibodies and assay systems are commercially available from sources such as R&D Systems (Minneapolis, MN), Genzyme (Cambridge, MA), and Endogen (Woburn, MA). mRNA levels of cytokines and chemokines can be determined using for example nucleic acid chip- based assays such as those commercially available from vendors such as Affymetrix.

Examples of normal levels of cytokines include < 3.9 pg/mL for IL-I alpha, < 0.60 pg/mL for IL-I beta, < 1.60 pg/mL for IL-2, < 12 pg/mL (and in some cases 1 pg/mL) for IL- 6, < 7.5 pg/mL (and in some cases 1.2 pg/ml) for TNF alpha, about 260 pg/mL for VEGF, < 24.5 pg/mL (and in some cases about 3 pg/mL) for IL-10, < 15.6 pg/ml for IFN-gamma, 9- 41.4 pg/mL for G-CSF, < 7.8 pg/mL for GM-CSF, and 6-25.4 pg/mL (and in some cases about 8 pg/mL) for IL-8. Normal levels of other cytokines will be known in the art and reference can be made to any standard medical textbook.

In some important embodiments, the pre-treatment levels of IL-2 and/or IFN are used in the screening method.

To be indicative of a subject that will be more responsive to boroproline compound, the pre-treatment level of cytokine can be at least 10%, at-ieast 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, more than the control level or it may be at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold of the control level.

The invention further provides another screening method that comprises determining a cytokine response profile to a boroproline compound of a subject and comparing the cytokine response profile to a control. A cytokine response profile is at a minimum the level of at least one cytokine. Preferably, it contains levels of more than one cytokine. It may also contain time course data relating to the level of cytokine as a function of time after exposure or contact to a boroproline compound.

In one instance, a cytokine response profile comprising an increased level of one or more cytokines is indicative of a subject who is likely responsive to treatment with the boroproline compound. The increased levels of cytokine can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, more than the control level or it may be at least 2-fold, at least 3-fold, at least 4- fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 50-fold of the control level. Important cytokines are IL-I beta, IL-lapha, IL-2 and GM-CSF.

The cytokine response profile may be determined in vitro by harvesting cells from a subject (e.g., blood cells) and incubating them with a boroproline of interest for a period of

time. At the end of such incubation,, the cell sample is assayed for the presence of cytokines using ELISAs or other known cytokine assay systems. Secreted and cell membrane bound cytokines may be assayed in this manner.

Alternatively, the cytokine response profile may be determined by harvesting samples from subjects that are undergoing boroproline therapy. In such assays, the sample is directly assayed for the presence and level of one or more cytokines as described herein.

The control may be a cytokine level from a normal subject. The normal subject may be a subject who does not have cancer, and preferably who has not been exposed to a boroproline compound, or optionally other Formula I agents, and who has not been administered one or more cytokines or chemokines in the last year, or at all. The control may be harvested at the same or a different time as the test subject sample. The control may alternatively be the average level of cytokine from a number of normal samples, and thus may be presented as a previously determined value and/or range.

The control may alternatively be an average cytokine level from a population of subjects having the same condition (e.g., melanoma). Preferably, the population has not been previously administered any cytokine therapy, although the method is not always so limited.

The control in some aspects may be the cytokine level prior to administration of boroproline therapy. For example, if the method measures cytokine level after contact with a boroproline agent, then that cytokine level may be compared to a pre-contact (or pre- treatment or pre-administration) level.

Subjects may be identified using one or both of these methods, alone or together with other screening methods. Some aspects of the invention further include treating the identified subjects by administering a boroproline compound such as those of Formula II or III.

Some of the treatment methods provided herein are referred to as non-immunotherapy or non-immunoglobulin therapy. As used herein, non-immunotherapy means a therapy that does not include active administration of an antigen (active immunity) or an antibody or antibody fragment (passive immunity). As used herein, non-immunoglobulin therapy means a therapy that does not include active administration of an antibody or an antigen-binding antibody fragment. Some aspects of the invention may comprise treating the subject with surgery, radiation and/or chemotherapy, before, during and/or after the treatment regimen of the invention.

Treatment as used herein means to reduce or ameliorate a disorder and/or its associated symptoms, or prevent it from becoming worse. In some instances, the disorder

may be partially or completely eliminated. A complete response is defined as a complete disappearance of all target cancerous lesions. A partial response is defined as at least a 30% decrease in the total number of all target lesions. Stable disease is defined as the absence of decrease or increase in number or size of lesions. Subjects treated according to the invention may demonstrate stable disease or remission for the short term (e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9. 10, 11, 12 or more weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12 or more months) or the long term (e.g., for 1 , 2, 3, 4, 5 or more years). Symptoms include but are not limited to edema, fatigue, nausea and hypotension. Prevention of a disorder aims to reduce the risk of developing the disorder. The cytokines may be first administered to a subject followed by administration of the agent of Formula I. The cytokine may be administered minutes, hours, days, weeks, or months before the administration of the agent of Formula I, including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months before the administration of the agent of Formula I. The cytokines may be administered at the same time as the agent of Formula I, in addition to or instead of prior cytokine administration. In some important examples, IL-2 and/or IFN either alone, in combination with each other, or in combination with other cytokines are administered to a subject prior to the administration of an agent of Formula I.

The cytokine and agent of Formula I may be administered substantially simultaneously with each other. As used herein, the term "substantially simultaneously" means that the compounds are administered within minutes of each other (e.g., within 10 minutes of each other) and intends to embrace joint administration as well as consecutive administration, but if the administration is consecutive it is separated in time for only a short period (e.g., the lime it would take a medical practitioner to administer two compounds separately). As used herein, concurrent administration and substantially simultaneous administration are used interchangeably.

The compounds may be administered in multi-day cycles. Such cycles may be 2, 3, 4, 5, 6, 7, 14, 21 or 28 day cycles, or longer. The agent of Formula I may be administered once, twice, or more times per day. The agent of Formula I may be administered daily on a subset of days in every cycle, and such subset of days may or may not be continuous. For example, the agent of Formula I may be administered on each of days 1-I4 in a 21 day cycle. The cytokine may be administered with the same frequency. Alternatively, it may be administered throughout the cycle, or on select days of the cycle. For example, the cytokine

may be administered on day 1 of a cycle, with the agent of Formula I being administered on all days of the cycle, all remaining days of the cycle, or a subset of days in the cycle, optionally including day 1. The cycle may also comprise one or more days in which the subject receives neither the agent of Formula I nor any cytokine. This latter period is referred to herein as a rest period. For example, days 15-21 of a 21 day cycle may be a rest period. The cycle may be repeated once, twice, or as may times as is necessary (and medically acceptable) to observe a response.

The agents of Formula I and/or the cytokines may be administered on a routine schedule. As used herein, a "routine schedule" refers to a predetermined designated period of time. The routine schedule may encompass periods of time which are identical or which differ in length, as long as the schedule is predetermined. For instance, the routine schedule may involve administration on a daily basis, every two days, every three days, every four days, every five days, every six days, a weekly basis, a monthly basis or any set number of days or weeks therebetween, every two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, etc. Alternatively, the predetermined routine schedule may involve administration on a daily basis for the first week, followed by a monthly basis for several months, and then every three months after that, Any particular combination would be covered by the routine schedule as long as it is determined ahead of time that the appropriate schedule involves administration on a certain day.

As used herein, a subject means a human or animal including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent e.g., rats and mice, primate, e.g., monkey, and fish or aquaculture species such as fin fish (e.g., salmon) and shellfish (e.g., shrimp and scallops). Subjects include vertebrate and invertebrate species. Subjects can be house pets (e.g., dogs, cats, fish, etc.), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), zoo animals (e.g., lions, giraffes, etc.), but are not so limited. Although many of the embodiments described herein relate to human disorders, the invention is also useful for treating other nonhuman vertebrates. In preferred embodiments, the subject is a human. The subject may be one that has normal hematopoietic cell levels (and therefore is not in need of hematopoietic stimulation). Normal hematopoietic cell levels are known in the art. In general, the normal range for neutrophils is about 1800-7250 per μl (mean -3650); for basophils 0-150 per μl (mean -30); for eosinophils 0-700 per μl (mean -150); for

macrophages and monocytes 200-950 per μl (mean -430); for lymphocytes 1500-4000 per μl (mean -2500); for erythrocytes 4.2 x 10 ⁶ - 6.1 x 10 ⁶ per μl; and for platelets 133 x 10 ³ - 333 x 10 ³ per μl. The foregoing ranges are at the 95% confidence level.

The invention aims to treat subjects having conditions characterized by abnormal cell proliferation. Abnormal cell proliferation is defined as cell proliferation that occurs at a rate that is greater than the rate of proliferation for normal cells of the same type, or of an equivalent type, or proliferation that is not responsive to stimuli that inhibit the proliferation of normal cells of the same or equivalent type. Such conditions include precancerous conditions as well as cancer. Precancerous conditions include abnormal epithelial cell proliferation conditions such as keloids, seborrheic keratosis, papilloma virus infection (e.g. producing verruca vulbaris, verruca plantaris, verruca plana, condylomata, etc.) and eczema; and benign and premalignant epithelial tumors, such as breast fibroadenoma and colon adenoma.

Thus in some aspects, the invention aims to treat subjects having cancer. A subject having a cancer is a subject that has detectable cancerous cells. Such subjects may also have a genetic abnormality that has been demonstrated to be associated with a higher likelihood of developing a cancer or a familial disposition to cancer, or may have been exposed to cancer causing agents (i.e., carcinogens) such as tobacco, asbestos, or other chemical toxins, or have been previously treated for cancer and in apparent remission. "Cancer" as used herein refers to an uncontrolled growth of cells which interferes . with the normal functioning, of the bodily organs and systems. Cancers which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. Hemopoietic- cancers, such as leukemia, are able to outcompete the normal hemopoietic compartments in a subject, thereby leading to hemopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death.

A metastasis is a region of cancer cells, distinct from the. primary tumor location resulting from the dissemination of cancer cells from the primary tumor to other parts of the body. At the time of diagnosis of the primary tumor mass, the subject may be monitored for. the presence of metastases. Metastases are most often detected through the sole or combined use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood and platelet counts, liver function studies, chest X-rays and bone scans in addition to the monitoπng of specific symptoms. ^' .

A cancer cell is a cell that divides and reproduces abnormally due to a loss of normal growth control. Cancer cells almost always arise from at least one genetic mutation. In some instances, it is possible to distinguish cancer cells from their normal counterparts based on profiles of expressed genes and proteins, as well as to the level of their expression. Genes commonly affected in cancer cells include oncogenes, such as ras, neu/HER2/erbB, myb, myc and abl, as well as tumor suppressor genes such as p53, Rb ₃ DCC, RET and WT. Cancer-related mutations in some of these genes leads to a decrease in their expression or a complete deletion. In others, mutations cause an increase in expression or the expression of an activated variant of the normal counterpart. The term "tumor" is usually equated with neoplasm, which literally means "new growth" and is used interchangeably with "cancer." A "neoplastic disorder" is any disorder associated with cell proliferation, specifically with a neoplasm. A "neoplasm" is an abnormal mass of tissue that persists and proliferates after withdrawal of the carcinogenic factor that initiated its appearance. There are two types of neoplasms, benign and malignant. Nearly all benign tumors are encapsulated and are noninvasive; in contrast, malignant tumors are almost never encapsulated but invade adjacent tissue by infiltrative destructive growth. This infiltrative growth can be followed by tumor cells implanting at sites discontinuous with the original tumor. The method of the invention can be used to treat neoplastic disorders in humans, including but not limited to sarcoma, carcinoma, fibroma, leukemia, lymphoma, melanoma including metastatic melanoma, myeloma, neuroblastoma, rhabdomyosarcoma, retinoblastoma, and glioma as well as each of the other tumors described herein.

Cancers include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; intra-epithelial neoplasm; kidney cancer; larynx cancer; leukemia including acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia; liver cancer; lung cancer (e.g., small cell lung cancer and non-small cell lung cancer); lymphoma including Hodgkin's and Non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; renal cell carcinoma; cancer of the respiratory system; sarcoma; skin cancer; stomach cancer; testicular cancer; thyroid cancer; uterine cancer; cancer of the urinary system, as well as other

carcinomas and sarcomas. Particularly important cancers include melanoma and renal cell carcinoma.

Sarcomas are rare mesenchymal neoplasms that arise in bone and soft tissues. Sarcomas include liposarcomas (including myxoid Iiposarcomas and pleomorphic liposarcomas), leiomyosarcomas, rhabdomyosarcomas, malignant peripheral nerve sheath tumors (also called malignant schwannomas, neurofibrosarcomas, or neurogenic sarcomas), Ewing's tumors including Ewing's sarcoma of bone, extraskeletal (i.e., non-bone) Ewing's sarcoma, and primitive neuroectodermal tumor (PNET), synovial sarcoma, angiosarcomas, hemangiosarcomas, lymphangiosarcomas, Kaposi's sarcoma, hemangioendothelioma, fibrosarcoma, desmoid tumor (also called aggressive fibromatosis), dermatoflbrosarcoma prσtuberans (DFSP), malignant fibrous histiocytoma (MFH), hemangiopericytoma, malignant mesenchymoma, alveolar soft-part sarcoma, epithelioid sarcoma, clear cell sarcoma, desmoplastic small cell tumor, gastrointestinal stromal tumor (GIST) (also known as GI stromal sarcoma), osteosarcoma (also known as osteogenic sarcoma)-skeletal and extraskeletal, and chondrosarcoma.

The cancers to be treated may be refractory cancers. A refractory cancer as used herein is a cancer that is resistant to the ordinary standard of care prescribed. These cancers may appear initially responsive to a treatment (and then recur), or they may be completely non-responsive to the treatment. The ordinary standard of care will vary depending upon the cancer type, and the degree of progression in the subject. It may be a chemotherapy, or surgery, or radiation, an immunotherapy or a combination thereof. Those of ordinary skill in the art are aware of such standards of care. Examples of ordinary standard of care for melanoma are provided in the Examples. Subjects being treated according to the invention for a refractory cancer therefore may have already been exposed to another treatment for their cancer. Alternatively, if the cancer is likely to be refractory (e.g., given an analysis of the cancer cells or history of the subject), then the subject may not have already been exposed to another treatment.

Examples of refractory cancers include but are not limited to leukemias, melanomas, renal cell carcinomas, colon cancer, liver (hepatic) cancers, pancreatic cancer, Non- Hodgkin's lymphoma, and lung cancer such as non-small cell lung cancer.

The invention contemplates the treatment of subjects that were previously administered a cytokine therapy but who failed to respond adequately to such therapy. Such subjects can be treated via the combined use of an agent of the invention and the prior

treatment or a new treatment. If the combination therapy includes the prior treatment, the invention contemplates that the agent leads to an enhanced response to the prior treatment. In some embodiments, the invention contemplates that the agent enhances the response of any therapy regardless of whether the subject has been pretreated. The invention can also be used to treat cancers that are immunogenic. Cancers that are immunogenic are cancers that are known to (or likely to) express cancer antigens on their surface or to release such antigens upon cell death and fragmentation. The release of these endogenous antigens can be exploited by the methods of the invention in order to treat the cancer. The cancer may be non-immunogenic. As used herein, a non-immunogenic cancer is one that is not known to express, either differentially or uniquely, any antigen which could be targeted for therapeutic effect. Such antigens could be used as targets for active or passive immunization strategies.

A cancer antigen encompasses antigens that are differentially expressed between cancer and normal cells. Due to this differential expression, these antigens can be targeted in anti-tumor therapies. Cancer antigens may be expressed in a regulated manner in normal cells. For example, they may be expressed only at certain stages of differentiation or at certain points in development of the organism or cell. Some are temporally expressed as embryonic and fetal antigens. Still others are never expressed in normal cells, or their expression in such cells is so low as to be undetectable. -

Other cancer antigens are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses. Cancer antigens can be classified in a variety of ways. Cancer antigens include antigens encoded by genes that have undergone chromosomal alteration. Many of these antigens are found in lymphoma and leukemia. Even within this classification, antigens can be characterized as those that involve activation of quiescent genes. These include BCL-I and IgH (Mantel cell lymphoma), BCL-2 and IgH (Follicular lymphoma), BCL-6 (Diffuse large B-cell lymphoma), TAL-I and TCRa or SIL (T-cell acute lymphoblastic leukemia), c- MYC and IgH or IgL (Burkitt lymphoma), MUN/IRF4 and IgH (Myeloma), PAX-5 (BSAP) (Immunocytoma).

Other cancer antigens that involve chromosomal alteration and thereby create a novel fusion gene and/or protein incmde RARa, PML, PLZF, NPM or NuMA (Acute promyelocyte leukemia), BCR and ABL (Chronic myeloid/acute lymphoblastic leukemia), MLL (HRX) (Acute leukemia), E2A and PBX or HLF (B-cell acute lymphoblastic leukemia), NPM, ALK (Anaplastic large cell leukemia), and NPM, MLF-I (Myelodysplastic syndrome/acute myeloid leukemia).

Other cancer antigens are specific to a tissue or cell lineage. These include cell surface proteins such as CD20, CD22 (Non-Hodgkin's lymphoma, B-cell lymphoma, Chronic lymphocytic leukemia (CLL)), CD52 (B-cell CLL), CD33 (Acute myelogenous leukemia (AML)). CDlO (gplOO) (Common (pre-B) acute lymphocytic leukemia and malignant melanoma), CD3/T-cell receptor (TCR) (T-cell lymphoma and leukemia), CD79/B-cell receptor (BCR) (B-cell lymphoma and leukemia), CD26 (Epithelial and lymphoid malignancies), Human leukocyte antigen (HLA)-DR, HLA-DP, and HLA-DQ (Lymphoid malignancies), RCASl (Gynecological carcinomas, bilary adenocarcinomas and ductal adenocarcinomas of the pancreas), and Prostate specific membrane antigen (Prostate cancer).

Tissue- or lineage- specific cancer antigens also include epidermal growth factor receptors (high expression) such as EGFR (HERl or erbB 1) and EGFRvIII (Brain, lung, breast, prostate and stomach cancer), erbB2 (HER2 or HER2/neu) (Breast cancer and gastric cancer), erbB3 (HER3) (Adenocarcinoma), and erbB4 (HER4) (Breast cancer).

Tissue- or lineage- specific cancer antigens also include cell-associated proteins such as Tyrosinase, Melan-A/MART-1, tyrosinase related protein (TRP)- l/gp75 (Malignant melanoma), Polymorphic epithelial mucin (PEM) (Breast tumors), and Human epithelial mucin (MUCl) (Breast, ovarian, colon and lung cancers).

Tissue- or lineage- specific cancer antigens also include secreted proteins such as Monoclonal immunoglobulin (Multiple myeloma and plasmacytoma), Immunoglobulin light chains (Multiple Myeloma), α-fetoprotein (Liver carcinoma), Kallikreins 6 and 10 (Ovarian cancer), Gastrin-releasing peptide/bombesin (Lung carcinoma), and Prostate specific antigen (Prostate cancer).

Still other cancer antigens are cancer testis (CT) antigens that are expressed in some normal tissues such as testis and in some cases placenta. Their expression is common in tumors of diverse lineages and as a group the antigens form targets for immunotherapy. Examples of tumor expression of CT antigens include MAGE-Al, -A3, -A6, -Al 2, BAGE,

GAGE, HAGE, LAGE-1, NY-ESO-1, RAGE, SSX-1, -2, -3, -4, -5, -6, -7, -8, -9, HOM-TES- 14/SCP-l, HOM-TES-85 and PRAME. Still other examples of CT antigens and the cancers in which they are expressed include SSX-2, and -4 (Neuroblastoma), SSX-2 (HOM-MEL- 40), MAGE, GAGE, BAGE and PRAME (Malignant melanoma), HOM-TES- 14/SCP-l (Meningioma), SSX-4 (Oligodendroglioma), HOM-TES-14/SCP-l , MAGE-3 and SSX-4 (Astrocytoma), SSX member (Head and neck cancer, ovarian cancer, lymphoid tumors, colorectal cancer and breast cancer), RAGE-I, -2, -4, GAGE-I, -2, -3, -4, -5, -6, -7 and -8 (Head and neck squamous cell carcinoma (HNSCC)), HOM-TES 14/SCP-l, PRAME, SSX-I and CT-7 (Non-Hodgkin's lymphoma), and PRAME (Acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML) and chronic lymphocytic leukemia (CLL)).

Other cancer antigens are not specific to a particular tissue or cell lineage. These include members of the carcinoembryonic antigen (CEA) family: CD66a, CD66b, CD66c, CD66d and CD66e. These antigens can be expressed in many different malignant tumors and can be targeted by immunotherapy. Still other cancer antigens are viral proteins and these include Human papilloma virus protein (cervical cancer), and EBV-encoded nuclear antigen (EBNA)-I (lymphomas of the neck and oral cancer).

Still other cancer antigens are mutated or aberrantly expressed molecules such as but not limited to CDK4 and beta-catenin (melanoma). The cancer antigen may be selected from the group consisting of MART-I /Mel an- A, gplOO, adenosine deaminase-binding protein (AD Abp), cyclophilin b, colorectal associated antigen (CRC)-COl 7- 1 A/GA733, carcinoembryonic antigen (CEA), CAP-I, CAP-2, etv6, AMLl, prostate specific antigen (PSA), PSA-I, PSA-2, PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain, and CD20. The cancer antigen may also be selected from the group consisting of MAGE-A 1 , MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-AlO, MAGE-Al 1, MAGE-A 12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE- B4), MAGE-Cl, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5). In still another embodiment, the cancer antigen is selected from the group consisting of GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9. And in yet a further embodiment, the cancer antigen is selected from the group consisting of BAGE, RAGE, LAGE-I, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCASl, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin, p!20ctn, gplOO ^Pme1117 ,

PRAME, NY-ESO-I , cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, pi 5, gp75, GM2 ganglioside, GD2 ganglioside, human papilloma virus proteins, Smad family of tumor antigens, lmp-1, PlA, EBV-encoded nuclear antigen (EBNA)-I, brain glycogen phosphorylase, SSX-I, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-I and CT-7, and c-erbB-2.

Examples of cancer antigens include HER 2 (pl85), CD20, CD33, GD3 ganglioside, GD2 ganglioside, carcinoembryonic antigen (CEA), CD22, milk mucin core protein, TAG- 72, Lewis A antigen, ovarian associated antigens such as O V-TL3 and MOv 18, high Mr melanoma antigens recognized by antibody 9.2.27, HMFG-2, SM-3, B72.3, PR5C5, PR4D2, and the like. Other cancer antigens are described in U.S. Pat. No. 5,776,427. Still other cancer antigens are listed below.

These antigens can be classified as indicated below.

Adapted from Falini B. and Mason, D.Y. (2002) Blood 99: 409-426

Cancer testis (CT anti ens*

Proteins specific to a tissue or cell lineage

Proteins not-specific to a tissue or cell lineage*

Carcmoembryonic antigen (CEA) family: CD66a, CD66b, CD66c, CD66d and CD66e. These antigens can be expressed in many different malignant tumors and can be targeted by immunotherapy.

Viral proteins

Human papilloma virus protein (cervical cancer)

EBV-encoded nuclear antigen (EBNA)-I (lymphomas of neck and oral cancer)

Mutated or aberrantly expressed molecules

I CDK4 and beta-catenin in melanoma

Cancer or tumor antigens can also be classified according to the cancer or tumor they are associated with (i.e., expressed by). Cancers or tumors associated with tumor antigens include acute lymphoblastic leukemia (etvβ; amll; cyclophilin b), B cell lymphoma (Ig- idiotype); Burkitt's (Non-Hodgkin's) lymphoma (CD20); glioma (E-cadherin; α-catenin; β- catenin; γ-catenin; pl20ctn), bladder cancer (p21ras), biliary cancer (p21ras), breast cancer (MUC family; HER2/neu; c-erbB-2), cervical carcinoma (p53; p21ras), colon carcinoma (p21ras; HER2/neu; c-erbB-2; MUC family), colorectal cancer (Colorectal associated antigen (CRC)--C017-l A/GA733; APC), choriocarcinoma (CEA), epithelial cell-cancer (cyclophilin b), gastric cancer (HER2/neu; c-erbB-2; ga733 glycoprotein), hepatocellular cancer (α- fetoproteϊn), Hodgkin's lymphoma (lmp-1; EBNA-I), lung cancer (CEA; MAGE-3; NY- ESO-I), lymphoid cell-derived leukemia (cyclophilin b), melanoma (pi 5 protein, gp75, oncofetal antigen, GM2 and GD2 gangliosides), myeloma (MUC family; p21ras), non-small cell lung carcinoma (HER2/neu; c-erbB-2), nasopharyngeal cancer (lmp-1 ; EBNA-I), ovarian cancer (MUC family; HER2/neu; c-erbB-2), prostate cancer (Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-I, PSA-2, and PSA-3; PSMA; HER2/neu; c-erbB-2), pancreatic cancer (p21ras; MUC family; HER2/neu; c-erbB-2; ga733 glycoprotein), renal (HER2/neu; c-erbB-2), squamous cell cancers of cervix and esophagus (viral products such as human papilloma virus proteins and non-infectious particles), testicular cancer (NY-ESO-I), T cell leukemia (HTLV-I epitopes), and melanoma (Melan- A/MART-1; cdc27; MAGE-3; p21ras; gpl00 ^Pmc " ¹⁷ ).

Still more antigens are described in PCT Application PCT/US98/18601.

In some aspects of the invention the subject may be administered an anti-cancer agent in the form of a non-immunoglobulin chemotherapeutic agent. Several chemotherapeutic

agents can be categorized as DNA damaging agents and these include topoisomerase inhibitors (e.g., etoposide, ramptothecin, topoteςan, teniposide, mitoxantrone), anti- microtubule agents (e.g., vincristine, vinblastine), anti-metabolite agents (e.g., cytarabine, methotrexate, hydroxyurea, 5-fluorouracii, floxuridine, 6-thioguanine, 6-mercaptop urine, fludarabine, pentostatin, chlorodeoxyadenosine), DNA alkylating agents (e.g., cisplatin, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chorambucil, busulfan, thiotepa, carmustine, lomustine, carboplatin, dacarbazine, procarbazine, temozolomide (TEMODAR)), DNA strand break inducing agents (e.g., bleomycin, doxorubicin, daunombicin, idarubicin, mitomycin C).

In some embodiments, the chemotherapeutic agent may be docetaxel (TAXOTERE) (e.g., in non-small cell lung carcinoma), cisplatin (e.g., in lymphoma and melanoma), gemcitabine, pemetrexed (ALIMTA), erlotinib (TARCEVA), gefitinib (IRESSA), temozolomide (TEMODAR), carboplatin, cyclophosphamide, 5-fluorouracil, dacarbazine, paclitaxel, thalidomide, or doxorubicin. In some important embodiments, the chemotherapeutic agent is cisplatin or temozolomide/thalidornide.

Other chemotherapeutic agents include those selected from the group consisting of annonaceous acetogenins; asimicin; rolliniastatin; guanacone, squamocin, bullatacin; squamotacin; taxanes such as paclitaxel and docetaxel; gemcitabine; methotrexate FR- 900482; FK-973: FR-66979; FK-317; 5-FU; FUDR; FdUMP; discodermolide; epothilones; vinorelbine; meta-pac; irinotecan; SN-38; 10-OH campto; flavopiridol; mithramycin; capecitabine; cytarabine; 2-Cl-2'deoxyadenosine; Fludarabine-Pθ4; mitozolomide; Pentostatin; Tomudex; pemetrexed; erlotinib; adriamycin; aldesleukin, asparaginase, bleomycin; bleomycin sulfate, carboplatin; chlorambucil, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunoαibicin hydrochloride, docetaxel, doxorubicin, doxorubicin hydrochloride, epirubicin hydrochloride, etoposide, etoposide phosphate, floxuridine, fludarabine, fluorouracil, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride,. ifosfamide, interferons, interferon-α2a, interferon-α2b, intevferon- αn3, interferon-αlb, interleukins, irinotecan, mechlorethamine hydrochloride, melphalan, mercatopurine, methotrexate, methotrexate sodium, mitomycin, mitomycin C; mitoxantrone, paclitaxel, pegaspargase, pentostatin, prednisone, profimer sodium, procabazine hydrochloride, taxol, taxotere, teniposide, topotecan; topotecan hydrochloride, vinblastine; vinblastine sulfate, vincristine; vincristine sulfate and vinorelbine tartrate.

Other cancer therapies include hormonal manipulation, particularly for breast and gynecological cancers. Formula I compounds are also useful in combination with tamoxifen or aromatase inhibitor arimidex (i.e., anastrozole), or simply for disorders responsive to either (e.g., breast cancer). The term "effective amount" refers to the amount necessary or sufficient to realize a desired therapeutic or prophylactic effect. Svich effect may be the amelioration and or absolute elimination of symptoms resulting from the disorder being treated. Such effect may also be the complete abrogation of the disorder, for example as evidenced by the absence of a tumor or a biopsy or blood smear which is free of cancer cells. The effective amount may vary depending upon the particular agent of Formula I and the particular cytokine used. It may also vary depending on the nature or severity of the condition being treated and the size and profile of the subject. One of ordinary skill in the art can empirically determine the effective amount of a particular combination without undue experimentation, as this is routinely accomplished in the medical arts. Effective amounts have been described in references such as Remington's Pharmaceutical Sciences, 18th ed., 1990, or the Physician Desktop Reference, as well as many other medical references relied upon by the medical profession as guidance. Combined with the teachings provided herein, and by weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject.

For agents of Formula I effective amounts for treating (e.g., inhibiting) cancer cells can be initially determined from cell culture assays. Effective amounts can also be determined in animal studies. For instance, the effective amount of an agent of Formula I compound and a cytokine to treat a cancer (e.g., a tumor) can be determined using in vivo assays of tumor regression and/or prevention of tumor formation. Relevant animal models include assays in which malignant cells are injected into the animal subjects, usually in a defined site. Generally, a range of Formula I compound doses are administered into the animal along with a range of cytokine doses. Inhibition of the growth of a tumor following the injection of the malignant cells is indicative of the ability to reduce the risk of developing a cancer. Inhibition of further growth (or reduction in size) of a pre-existing tumor is indicative of the ability to treat the cancer. These animal models include mice which have

been modified to have human immune system elements as recipients of human cancer cell lines.

Subject doses of the compounds described herein typically range from about 0.1 μg to

10,000 mg per day, more typically from about 1 μg to 8000 mg per day, even more typically from about 10 μg to 5 mg per day, and most typically from about 100 μg to 1 mg per day.

Stated in terms of subject body weight, typical dosages range from about 0.001 to 20 mg/kg per day, more typically from about 0.005 to 5 mg/kg per day, and most typically from about

0.005 to 1 mg/kg per day.

In some important embodiments, the agents of Formula I are administered in doses of 100 μg, 200 μg, 300 μg, 400 μg or 500 μg (or any dose in between as if explicitly recited herein) once or twice a day (resulting in total daily doses of 200 μg, 400 μg, 600 μg, 800 μg or 1000 μg, or any dose in between as if explicitly recited herein).

In particularly important embodiments, the agent is administered in amounts equal to or less than 1.0 mg/kg per day (e.g., equal to or less than 0.9, 0-8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1 mg/kg per day). The agents may be administered in amounts of equal to or less than 0.1 mg/kg per day (e.g., equal to or less than 0.09, 0.08, 0.07, 0.06, 0.5, 0.04, 0.03, 0.02 or 0.01 mg/kg per day).

In some instances, a sub-therapeutic dosage of the agent Formula I compound and/or the cytokine(s) is used in the treatment of a subject. As used herein, a "sub-therapeutic dose" refers to a dose which is less than the dose which would produce a therapeutic result if the compound was administered in the absence of the other compound in the contemplated combination therapy. Thus, the sub-therapeutic dose of the agent of Formula I is one which would not produce the desired therapeutic result in the subject in the absence of the administration of the cytokine(s). As similar meaning is ascribed to a sub-therapeutic dose of a cytokine.

Accordingly, the agents and cytokines may be administered in reduced doses relative to therapeutically appropriate doses when agents are administered alone. For example, the unit dosage of one or both agents may be reduced by a factor of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,

20, 25, 50, 100 or more relative to the unit dosage required when a single agent is administered.

The agents of Formula I and the cytokines may be administered as a synergistic combination. As used herein, the term "synergistic" describes an effect resulting from the

combination of at least two compounds which is greater than the additive effect of each of the individual compounds when used alone. A synergistic response therefore includes an effect that is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% greater than the additive effect of the compounds alone. Alternatively, a synergistic response may be an effect that is at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50- fold, or at least 100-fold greater than the additive effect of the compounds alone.

The invention further provides compositions that comprise an agent of Formula I and at least one cytokine. Such compositions are preferably pharmaceutical preparations indicating that they are sterile, pharmaceutically acceptable, formulated for in vivo administration, prepared according to GMP (for example), and dosed in effective amounts. Such preparations may comprise a pharmaceutically acceptable carrier.

The invention further provides kits that comprise the compounds of the invention and ^' optionally instructions of use thereof. The compounds may be present in oral forms such as ■ tablets, pills, capsules, caplets and the like, or liquid injectable forms provided in ampoules or vials. Certain of the compounds may be provided in a one a day dispensing unitsuch as a blister pack or dial pack type dispenser, preferably with days of the week or day of the month (e.g., 1, 2, 3, 4, etc.) (and doses per day, where applicable) printed on the dispenser. For example, if the compounds are to be administered every other day or twice (or more) a day, the dispensing unit can be modified accordingly, with no more than routine reconfiguration, known in the art. The kit may further contain a second compound such as a second anticancer agent. The unit dosages provided in each form (e.g., tablet, pill, capsule, etc.) will depend upon the particular therapy and desired result. The kit may optionally comprise a housing such as a box or bag. Instructions for use may be supplied separately from the dispensing unit or housing or they may be imprinted on one or both.

The compounds of the invention may be administered neat, or in the context of a vector or delivery system. An example of a chemical/physical vector of the invention is a colloidal dispersion system. Colloidal dispersion systems include lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system of the invention is a liposome. Liposomes are artificial membrane vessels which are useful as a delivery vector in vivo or in vitro. It has been shown that large unilamellar vessels (LUV), which range in size from 0.2 - 4.0 μm can encapsulate large

macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. SW., (1981 ) 6:77).

Liposomes may be targeted to a particular tissue by coupling the liposome to a specific ligand such as a sugar, glycolipid, or protein. Ligands which may be useful for targeting a liposome to an cancer cell include, but are not limited to intact or fragments of molecules which interact with cell surface cancer antigens. The liposome may be targeted to the cancer by coupling it to an antibody specific for a cancer antigen. Additionally, the vector may be coupled to a nuclear targeting peptide, which will direct the vector to the nucleus of the host cell.

Lipid formulations for transfection are commercially available from QIAGEN, for example, as EFFECTENE™ (a non-liposomal lipid with a special DNA condensing enhancer) and SUPERFECT™ (a novel acting dendrimeric technology).

Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTIN™ and LIPOFECTACE™, which are formed of cationic lipids such as N-[l-(2, 3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammom ^' um bromide (DDAB). Methods for making liposomes are well known in the art and have been described in many publications. Liposomes also have been reviewed by Gregoriadis, G. in Trends in Biotechnology, (1985) 3:235-241. In another embodiment the chemical/physical vector is a biocompatible microsphere that is suitable for delivery, such as oral or mucosal delivery. Such microspheres are disclosed in Chickering et al., Biotech. AndBioeng., (1996) 52:96-101 and Mathiowitz et al., Nature, (1997) 386:.410-414 and PCT Patent Application WO97/03702.

Both non-biodegradable and biodegradable polymeric matrices can be used to deliver compounds to a subject. Biodegradable matrices are preferred. Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable. The polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multivalent ions or other polymers.

The polymeric matrix preferably is in the form of a microparticle such as a microsphere (wherein the compounds are dispersed throughout a solid polymeric matrix) or a

microcapsule (wherein the compounds are stored in the core of a polymeric shell). Other forms of the polymeric matrix for containing the agents include films, coatings, gels, implants, and stents. The size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix is introduced. The size of the polymeric, matrix further is selected according to the method of delivery which is to be used, typically injection into a tissue or administration of a suspension by aerosol into the nasal and/or pulmonary areas. Preferably when an aerosol route is used the polymeric matrix and the compounds are encompassed in a surfactant vehicle. The polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the matrix is administered to a nasal and/or pulmonary surface that has sustained an injury. The matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time.

Bioadhesive- polymers of particular interest include bioerodible hydrogels described by H.S. Sawhney, CP. Pathak and J.A. Hubell in Macromolecuϊes, (1993) 26:581-587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(bιιtylmethacrylate), poly(isobutyl methacrylate), . . . poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurel methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate). . . ^• . .

Other delivery vehicles can be used and these include cochleates (Gould-Eogerite et al., 1994, 1996); Emulsomes (Vancott et al., 1998, Lowell et al., 1997); ISCOMs (Mowat et al., 1993, Carlsson et al., 1991, Hu et., 1998, Morein et al., 1999); liposomes (Childers et ah, 1999, Michalek et al., 1989, 1992, de Haan' 1995a, 1995b); live bacterial vectors (e.g., Salmonella, Escherichia coli, Bacillus calmatte-guerin, Shigella, Lactobacillus) (Hone et al., 1996, Pouwels et al., 1998, Chatfield et a]., 1993, Stover et al., 1991, Nugent et al., 1998); live viral vectors (e.g., Vaccinia, adenovirus, Herpes Simplex) (Gallichan et al., 1993, 1995, Moss et al., 1996, Nugent et al., 1998, Flexner et al., 1988, Morrow et al., 1999); . microspheres (Gupta et al., 1998, Jones et al., 1996, Maloy et al., 1994, Moore et al., 1995, . O'Hagan et al., 1994, Eldridge et al., 1989); nucleic acid vaccines (Fynan et al., 1993, Kuklin et al., 1997, Sasaki et al., 1998, Okada et.al., 1997, Ishii et al., 1997); polymers (e.g. carboxymethylcellulose, chitosan) (Hamajima et al., 1998, Jabbal-Gill et al., 1998); polymer

rings (Wyatt et al., 1998); proteosomes (Vancott et al., 1998, Lowell et al., 1988, 1996, 1997); sodium fluoride (Hashi et al., 1998); transgenic plants (Tacket et al., 1998, Mason et al., 1998, Haq et al., 1995); virosomes (Gluck et al., 1992, Mengiardi et al., 1995, Cryz et al., 1998); and, virus-like particles (Jiang et al., 1999, Leibl et al., 1998).

The term "pharmaceutically-acceptable carrier" means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical preparations also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.

The agents may be administered pj≥r se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic • solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the

preparation of highly concentrated solutions. Another suitable compound for sustained release delivery is GELFOAM, a commercially available product consisting of modified collagen fibers.

Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The pharmaceutical preparations also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

For use in therapy, an effective amount of the Formula I compound can be administered to a subject by any mode that delivers the compound to the affected organ or tissue, or alternatively to the immune system. "Administering" the pharmaceutical preparation of the present invention may be accomplished by any means known to the skilled artisan. Preferred routes of administration include but are not limited to oral, parenteral, intramuscular, intranasal, intratracheal, inhalation, ocular, vaginal, and rectal. Systemic routes include oral and parenteral routes.

The compounds may be administered in the same route, and in the same formulation, or they may be administered in a different route, different formulation, and even on a different schedule. In an important embodiment, the agent of Formula I is administered orally, and cytokine is administered parenterally, preferably by intravenous, intramuscular or stibcutaneous injection.

For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or

alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any carriers.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compounds may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g.. dichlorodifluoromethane, trichlorofiuoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. Techniques for preparing aerosol delivery systems are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the therapeutic (see, for example, Sciarra and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712; incorporated by reference). Those of skill in the art can readily determine the various parameters and conditions for producing aerosols without resort to undue experimentation.

Inhaled medications are preferred in some embodiments because of the direct delivery to the lung, particularly in lung cancer patients. Several types of metered dose inhalers are regularly used for administration by inhalation. These types of devices include metered dose inhalers (MDI), breath-actuated MDI, dry powder inhaler (DPI), spacer/holding chambers in combination with MDI, and nebulizers.

The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g. , in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical preparations also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical preparations are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533. 1990, which is incorporated herein by reference.

Examples Example 1: Introduction

Talabostat is an oral inhibitor of dipeptidyl peptidases such as fibroblast activation protein found on the stroma of tumors, draining lymph nodes, and in melanomas. Administration of talabostat results in upregulation of cytokines and chemokines in humans and animals, leading to specific T-cell immunity and T-cell independent activity. In xenograft models of melanoma (A375, A2058), talabostat significantly reduced tumor size >60% in immunodeficient mice.

Study Design

• Treatment regimen o Talabostat 300 μg administered orally BID (twice a day) on days 1-34 of each

3-week cycle o Dose-escalation of talabostat to 400 μg BID allowed in cycle 2 or subsequent cycles depending on tolerability o Treatment could continue until disease progression or unacceptable toxicity

• Outcome measures: o Primary: Disease response using RECIST o Secondary: Survival, PFS, duration of response, and cytokine expression in serum

• CT scans after cycles 2, 4, 6 and every 2 months

• Patients followed for 12 months for survival and disease progression (PD)

Methods

Key Eligibility Criteria

• Men or women age >18 years with measurable disease

• Histologically or cytologically confirmed metastatic melanoma (unresectable Stage IV per AJCC 2002)

• No more then 1 prior chemotherapy or biotherapy regimen for Stage IV melanoma •No active CNS metastases

• ECOG Performance Status of O,I, or 2 and life expectancy >12 weeks

• Baseline laboratory results within the following parameters: o Platelets >100,000/μL, granulocytes >1500/μL o ALT or AST < 3 x upper limit of normal (ULN) o LDH < 3 x ULN o Total bilirubin < 1.5 institutional ULN (unless secondary to Gilbert's) o Serum creatinine < 2.0mg/dL

• No chemotherapy, radiation therapy or immunotherapy within 30 days of study day 1

Patients were seen in clinic after every 3-week cycle. CT scans (or MRI, X-ray, photograph as appropriate) were obtained at baseline and every 6 weeks for 18 weeks and then every 2 months thereafter. Adverse events, clinical laboratories, and vitals were monitored throughout the study.

Blood samples for serum cytokines and chemokines (IL- lα, IL- lβ, IL-2, IL-6, IL-8, IL-I O, TNF-α, IFN-γ, G-CSF, GM-CSF) were obtained at baseline and on study day 14 immediately prior to the morning talabostat dose and at 1 , 2, 4, and 6 hours post-dose.

Results

Patient Population

Forty-two patients were enrolled (27 men and 15 women) median age 51 years (range 34 to 84 years). The majority of patients (71%) had visceral metastases, 19% had lung metastases, and 10% had distant skin, subcutaneous, or nodal metastases. Most patients (64%) had received prior biotherapy or chemotherapy for Stage IV disease.

Thirty-one patients were evaluable for response (evaluability defined as completion of at least 2 cycles of treatment with a post-baseline disease assessment). Patients are still being followed for survival.

Clinical Disease Assessments

Two responses were observed in 31 evaluable patients: a CR in a-patient with in transit metastases and a PR in a patient with metastasis to a right hilar node (both patients had prior cytokine treatment).

The Kaplan-Meier estimate for median PFS in all 42 patients was 1.5 months (95% CI 1.3, 2.1 months). Median overall survival is currently estimated at 7.1 months (95% CI 5.4, 8.9 months).

Kaplan-Meier Estimation for Overall Survival

(N=42)

Cytokines Median pre-dose levels ofIL-6, IL-8, TNF-alpha, and G-CSF at Day 14 pre-dose were slightly higher than baseline for most patients, and the 2 responders were among the patients who also had increased serum levels of IL-I beta, ϊL-2, and GM-CSF post-dose at

Day 14.

Mean TNF-alpha levels at 4 and 6 hours post dose were approximately 20% higher in patients reporting edema or peripheral swelling than those who did not. In patients reporting

hypotension, mean IL-8 levels were approximately 2-fold higher at 4 and 6 hours post dose compared to those who did not report hypotension, and IL-8 levels were also higher in patients reporting dehydration or hypovolemia.

Conclusions

Talabostat is an orally administered agent that shows activity in patients with Stage IV melanoma. Two objective responses, including one CR, were reported. Median survival is currently estimated at 7.1 months. The most common AEs seem to be associated with up- regulation in TNF-alpha and IL-8.

Example 2: Introduction

Talabostat is an oral inhibitor of dipeptidyl peptidases such as fibroblast activation protein found on the stroma of tumors, draining lymph nodes, and in melanomas. Talabostat up-regulates cytokines and chemokines, leading to specific T-cell immunity and T-cell independent activity. Talabostat significantly enhances the activity of cisplatin in mice and reduces tumor size > 60% in melanoma xenografts (A375, A2058).

Study Design • • .

• Treatment regimen (21 -day cycles for up to 4 cycles) • ^• o Cisplatin 75mg/m ² intravenously day 1 of cycles 1 through 4 • o Talabostal 300 μg tablets BID orally on days 2-15. Dose-escalation to talabostat 400μg BID allowed depending on tolerability. o Single-agent talabostat continued beyond 4 cycles until disease progression or unacceptable toxicity.

• Key Outcome measures: •

^• o Primary: Disease response (per RECIST) o .Secondary: Progression-free survival (PFS), duration of response, survival

• Adverse events graded according to NCI-CTCAE ^'

• Patients followed for 12 months for survival and disease progression (PD)

Patients were seen in clinic after every 3 -week cycle. CT scans (or MRI, X-ray, photograph) were obtained at baseline and every 6 weeks for 12 weeks, then every 2 months thereafter. Adverse events, clinical laboratories, and vital signs were monitored throughout the study. •

Methods

Key Eligibility Criteria

• Men or women age >18 years

• Histologically or cytologically confirmed metastatic melanoma (unresectable Stage IV per AJCC 2002)

• ^" No more than 1 prior chemotherapy or biotherapy regimen for Stage IV melanoma

• No active CNS metastases

• ECOG Performance Status 0, 1 , or 2

• Baseline laboratory results within the following parameters: . o . Platelets >100,000/μL, granulocytes >1500/μL o LDH <3 x upper limit of normal (ULN) o ALT or AST <3 x ULN o Total bilirubin < 1.5 x ULN o Serum creatinine <2.0mg/dL and/or creatinine clearance >60mL.min ^• • No chemotherapy, immunotherapy, radiation, or surgery within 30 days of day 1

Results

Enrollment has concluded into this study, and data are being verified and summarized.

Patient Population

Patients were enrolled between August 2004 and July 2005. To be considered evaluable for response, patients had to complete 2 cycles of study including taking 75% of the protocol-specified dose of talabostat with no more than 5 days of nausea/vomiting, and have a post-baseline response assessment. Of the 74 patients who entered the study (50 men, 24 women), the median age was 58 years (range 27 to 79); 93.2% were Caucasian. Most patients (55.8%) were ECOG 1, and 43.2% were ECOG 0. The majority of patients (68.9%) were classified as MIc at original diagnosis.

Patient Demographics

(N=74)

Most patients (64.9%) were previously treated for Stage IV disease. Of these patients, 64.5% had received prior treatment with cytokines. In the 48 patients who had received prior treatment, 5 patients had a partial response (PR), 18 had stable disease (SD), and 24 patients had PD reported as the best response to treatment. The median duration of response of PR or SD was 4.0 months (range 1 to 7 months).

Prior Treatment for Stage JV Melanoma.

(N=74) ^{■ • '}

*Of the 48 patients who were previously treated for Stage IV melanoma

Clinical Disease Assessments

The following table lists the patients who showed a response to treatment. A PR was reported in 6/43 (13.9%) patients evaluable for response. Stable disease (>4 cycles) was reported in an additional 20/43 patients (46.5%).

Objective Response in Evaluable Patients

(N=43)

N/A = Not Applicable

The median Kaplan-Meier estimate of PFS in all patients (intent-to-treat [ITT]) is currently estimated at 2.8 months (95% CI 1.9, 3.4).

Kaplan-Meier Estimation for Progression-Free Survival

(N=74)

The median Kaplan-Meier estimate for overall survival in the ITT population is 8.5 months (95% C[ 5.0, infinity).

Kaplan-Meier Estimation for Overall Survival

(N=74»

Conclusions

• The combination of talabostat tablets and cisplatin showed activity in patients with Stage IV melanoma

• Six partial responses were observed in 43 evaluable patients • The estimate of median PFS and survival in all 74 patients was 2.8 and 8.5 months, respectively

• The most frequent AEs were nausea, fatigue, vomiting, edema, and constipation

Equivalents The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

All references, patents and patent publications that are recited in this application are incorporated in their entirety herein by reference.

What is claimed is: