Patent Application
20120282168
NOT APPLICABLE
NOT APPLICABLE
Proteasome inhibitors have been shown to induce cell killing alone and/or in combination with drugs in drug-resistant tumor cells. In 2003, the FDA approved the first proteasome inhibitor VELCADE, “bortezomib” for treating patients with multiple myeloma who relapsed after two therapies and are progressing on current treatments. Thus, proteasome inhibitors prove to be clinically effective. However, like many other drugs, resistance to bortezomib starts to emerge as well as bortezomib-induced tissue toxicity has been noted. The development of new proteasome inhibitors that can override bortezomib resistance and exhibiting less toxicity is highly desirable. The chemical compound Salinosporamide A (NPI-0052, Nereus Pharmaceuticals, San Diego) was discovered during the fermentation of Salinospora species, a new marine gram positive actinomycete. It is related to two less potent 20S proteasome inhibitors, structurally related to lactacystin, omuralide, and PS-519.
Several in vitro findings indicated that Salinosporamide A exhibited cytotoxicity against a variety of tumor cell lines (Feling, et al., Angew. Chem. Int. Ed., 2003, 42(3): 355-357) and can exert apoptosis and inhibition of NF-κB (Macherla, et al., Journal of Medicinal Chemistry, 2005, 48:3684). It has also been shown that Salinosporamide A is effective in bortezomib-resistant cell lines. In vivo, Salinosporamide A exerted anti-tumor effects whether administered orally or intravenously (Chuahan et al., Cancer Cell, Nov. 8, 407-419 (2005)). Salinosporamide A has been synthesized chemically. Studies on cytotoxicity with the NCI screening panels of 60 human tumor cell lines showed that Salinosporamide A affected many cancer cells, and had a mean growth inhibition of less than 10 nM. Other tumor cell lines examined showed significant cytotoxic activity. Noteworthy, Salinosporamide A was also cytotoxic to both drug sensitive HL60 and drug resistant HL60MX2 with equal doses.
Salinosporamide A also had the effect of inducing a range of direct apoptosis on different tumor cell lines. The effect of Salinosporamide A on the induction of apoptosis suggests that Salinosporamide A may be used as an agent to identity anti-apoptotic pathways that may serve as targets for cancer therapy by examining changes in the expression of nucleic acids and proteins upon the treatment of cancer cells with this compound.
In the present application, we have examined if Salinosporamide A can sensitize therapy sensitive and therapy-resistant B Non-Hodgkin's Lymphoma to therapy-induced apoptosis. We also investigated whether Salinosporamide A could act as a therapeutic agent and induce apoptosis after sensitization by another compound such as rituximab. Furthermore, we have also examined the effect of Salinosporamide A on the induction of Raf kinase inhibitor protein (RKIP), a metastasis tumor suppressor protein that potentiates anti-apoptotic pathways in cancer cells, and on the inhibition of expression of YY1, a transcriptional regulator protein overexpressed in cancer cells that regulates tumor cell resistance to both chemotherapy and immunotherapy
The present application demonstrates that Salinosporamide A, in combination with subtoxic therapeutically effective amounts of cancer therapeutic agents, sensitizes both resistant and sensitive cancer cells to therapy-induced cytotoxicity. The cancer cells can be either therapy-sensitive or therapy resistant. Furthermore, the present application demonstrates that Salinosporamide A acts as a therapeutic agent to induce apoptosis in cancer cells after sensitization of the cells by an antibody or by various chemo- and immuno sensitizing agents. The cancer cells can be either therapy-sensitive or therapy resistant. Additionally, the present application demonstrates that Salinosporamide A induces the expression of RKIP, thereby inhibiting survival anti-apoptotic signaling pathways and resulting in reducing the threshold of anti-apoptotic gene expression and when used alone, or in combination with other agents, results in apoptosis. Furthermore, induction of RKIP also exerts anti-angiogenic activity as well as prevents metastasis. Further Salinosporamide A treatment inhibits the transcription repressor YY1, resulting in the upregulation of death receptors and sensitization of tumor cells to cytotoxic immunotherapy. It also regulates death receptor expression in rituximab-resistant clones. Salinosporamide A-induces the expression of the AKT inhibitor PTEN resulting in downstream inhibition of the AKT anti-apoptotic and survival pathway and resulting in inhibition of anti-apoptotic gene products Salinosporamide A also inhibits the overexpression of pleiotrophin (PTN) a growth factor and resistance factor in tumor cells and circulating levels of PTN have been shown to have a prognostic importance.
In a first embodiment, the invention provides a method of treating, preventing or inhibiting a cancer by administering to a subject a therapeutically effective amount of a cancer therapy reagent and a sensitizingly effective amount of a compound of Formula I:
in which each of R1, R2 and R3 can independently be: H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; R4 is a 5-8 membered cycloalkyl optionally substituted with 1-8 R5 groups or a 5-8 membered cycloalkenyl optionally substituted with 1-8 R5 groups; each R5 can independently be: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, or substituted cycloalkyl; each of X1, X2, X3 and X4 can be independently: O, NR6 and S; and R6 is H or C1-C6 alkyl.
In some aspects of the first embodiment, each of R1 and R2 can independently be: H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, and substituted cycloalkyl; R3 can be alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; R4 is a 5-8 membered cycloalkenyl optionally substituted with 1-8 R5 groups; each of X1, X3 and X4 is O; and X2 is NH.
In yet another aspect of the first embodiment, each of R1 and R2 can independently be: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl; R3 can be alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, and amino; R4 is cyclohexenyl optionally substituted with 1-8 R5 groups; each of X1, X3 and X4 is O; and X2 is NH.
In further aspects of the first embodiment, R1 is an alkyl or substituted alkyl; R2 is alkyl; R3 is hydroxy; R4 is cyclohexenyl; and each of X1, X3 and X4 is O; and X2 is NH; the substituted alkyl of R1 can be a halogenated alkyl, which can be fluorinated, chlorinated, brominated in different aspects. In some aspects, the halogenated alkyl compound has the following structure:
In yet another aspect of the first embodiment, the halogenated alkyl compound has the following structure:
In further aspects of the first embodiment, the sensitizingly effective amount of the compound of Formula I is sufficient to induce expression of RKIP or PTEN, thereby inducing or facilitating apoptosis. The expression of RKIP or PTEN can be at least 1, 2, 10, or 100 fold higher than in the absence of the compound of Formula I.
In yet further aspects of the first embodiment, the sensitizingly effective amount of the compound of Formula I is sufficient to inhibit the expression of YY1, and PTN, thereby inducing apoptosis. The expression of YY1, PTEN, and PTN can be at least 1, 2, 10, or 100 fold lower than in the absence of the compound of Formula I.
In another aspect of the first embodiment, the cancer therapy reagent can be a chemotherapeutic reagent, an immunotherapeutic reagent, a radiotherapeutic reagent, a hormonal therapeutic reagent, or a pharmacologic inhibitor.
In other aspects of the first embodiment, the cancer can be non-Hodgkin's lymphoma, B-acute lymphoblastic lymphoma, prostate cancer, ovarian cancer, renal cancer, lung cancer, breast cancer, colon cancer, leukemia, multiple myeloma and hepatocarcinoma.
In another aspect of the first embodiment, the cancer therapy reagent induces or facilitates apoptosis and can be a chemotherapeutic reagent, an immunotherapeutic reagent, a radiotherapeutic reagent, a hormonal therapeutic reagent, or a pharmacologic inhibitor. In this aspect, the cancer therapy reagent can be rituximab immunotherapy.
In various aspects of the first embodiment, the cancer is therapy-resistant, including resistance to immunotherapy, chemotherapy, radiotherapy, or hormonal therapy. However, in other aspects, the cancer can be therapy-sensitive.
In further aspects of the first embodiment, the therapeutically effective amount of a cancer therapy reagent and the sensitizingly effective amount of a compound of Formula I are administered concurrently or sequentially, in which the cancer therapy reagent is bortezomib administration. In related aspects, the cancer therapy reagent can be a chemotherapeutic reagent, an immunotherapeutic reagent, a radiotherapeutic reagent, a hormonal therapeutic reagent, or a pharmacologic inhibitor.
In an alternative aspect of the first embodiment, the therapeutically effective amount of a cancer therapy reagent and the sensitizingly effective amount of a compound of Formula I are administered sequentially.
In an aspect of the first embodiment, the subject can be a human.
A second embodiment of this invention provides a method of treating, preventing or inhibiting lymphoma by administering to a subject a therapeutically effective amount of a cancer therapy reagent and a sensitizingly effective amount of a compound of Formula I:
in which each of R1, R2 and R3 can independently be: H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; R4 is a 5-8 membered cycloalkyl optionally substituted with 1-8 R5 groups or a 5-8 membered cycloalkenyl optionally substituted with 1-8 R5 groups; each R5 can independently be: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, and substituted cycloalkyl; each of X1, X2, X3 and X4 is independently selected from the group consisting of O, NR6 and S; and R6 is H or C1-C6 alkyl.
In an aspect of the second embodiment, the sensitizingly effective amount of the compound of Formula I is sufficient to induce expression of RKIP or PTEN, thereby inducing or potentiating apoptosis.
In another aspect of the second embodiment, the sensitizingly effective amount of the compound of Formula I is sufficient to inhibit the expression of YY1 or PTN thereby inducing or potentating apoptosis.
In further aspects of the second embodiment, the lymphoma is therapy resistant, which can include a lymphoma which is rituximab therapy resistant.
In a third embodiment, this invention provides a method of treating, preventing or inhibiting lymphoma by administering to a subject a therapeutically effective amount of rituximab and a sensitizingly effective amount of a compound of Formula I:
in which each of R1, R2 and R3 can independently be: H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; R4 is a 5-8 membered cycloalkyl optionally substituted with 1-8 R5 groups or a 5-8 membered cycloalkenyl optionally substituted with 1-8 R5 groups; each R5 can independently be: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, and substituted cycloalkyl; each of X1, X2, X3 and X4 can independently be O, NR6 and S; and R6 is H or C1-C6 alkyl.
In an aspect of the third embodiment, the sensitizingly effective amount of the compound of Formula I is sufficient to induce expression of RKIP or PTEN, thereby inducing apoptosis.
In another aspect of the third embodiment, the sensitizingly effective amount of the compound of Formula I is sufficient to inhibit expression of YY1 or PTN, thereby inducing apoptosis.
In a fourth embodiment, the invention provides a composition containing a therapeutically effective amount of rituximab and a sensitizingly effective amount of a compound of Formula I in a physiologically acceptable excipient.
In a fifth embodiment, the invention provides a kit comprising a therapeutically effective amount of rituximab and a sensitizingly effective amount of a compound of Formula I.
In a sixth embodiment, this invention provides a method of treating, preventing or inhibiting a cancer with proteasome inhibitor therapy by administering to a subject a sensitizingly effective amount of an antibody or chemosensitizing reagent and a therapeutically effective amount of a compound of Formula I:
in which each of R1, R2 and R3 can independently be: H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; R4 is a 5-8 membered cycloalkyl optionally substituted with 1-8 R5 groups or a 5-8 membered cycloalkenyl optionally substituted with 1-8 R5 groups; each R5 can independently be: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, and substituted cycloalkyl; each of X1, X2, X3 and X4 can independently be O, NR6 and S; and R6 is H or C1-C6
In an aspect of the sixth embodiment, the sensitizingly effective amount of the compound of Formula I is sufficient to induce expression of RKIP or PTEN, thereby inducing apoptosis or sensitizing cells to apoptosis by various sub-toxic concentrations on cytotoxic agents. In other aspects of this embodiment, the antibody is rituximab and the cancer is lymphoma.
In another aspect of the sixth embodiment, the sensitizingly effective amount of the compound of Formula I is sufficient to inhibit expression of YY1 or PTN, thereby inducing apoptosis. In other aspects of this embodiment, the antibody is rituximab and the cancer is lymphoma.
In a seventh embodiment, this invention provides a composition comprising a sensitizingly effective amount of rituximab and a therapeutically effective amount of a compound of Formula I in a physiologically acceptable excipient.
In an eighth embodiment, this invention provides a kit comprising a sensitizingly effective amount of rituximab and a therapeutically effective amount of a compound of Formula I.
In a ninth embodiment, this invention provides a method of treating, preventing or inhibiting a cancer, the method comprising the step of administering to a subject a therapeutically effective amount of a compound of Formula I:
in which each of R1, R2 and R3 can independently be: H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; R4 is a 5-8 membered cycloalkyl optionally substituted with 1-8 R5 groups or a 5-8 membered cycloalkenyl optionally substituted with 1-8 R5 groups; each R5 can independently be: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, or substituted cycloalkyl; each of X1, X2, X3 and X4 can independently be O, NR6 and S; R6 is H or C1-C6 alkyl, and in which the therapeutically effective amount is sufficient to induce the expression of RKIP or PTEN, thereby inducing apoptosis.
In various aspects of the ninth embodiment, the expression of RKIP or PTEN is at least about 1, 2, 10, or 100 fold higher than in the absence of the compound of Formula I.
In a tenth embodiment, this invention provides a method of treating, preventing or inhibiting a cancer, the method comprising the step of administering to a subject a therapeutically effective amount of a compound of Formula I:
in which each of R1, R2 and R3 can independently be: H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; R4 is a 5-8 membered cycloalkyl optionally substituted with 1-8 R5 groups or a 5-8 membered cycloalkenyl optionally substituted with 1-8 R5 groups; each R5 can independently be: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, or substituted cycloalkyl; each of X1, X2, X3 and X4 can independently be O, NR6 and S; R6 is H or C1-C6 alkyl, and in which the therapeutically effective amount is sufficient to inhibit the expression of YY1 or PTN, thereby inducing apoptosis or lowering the threshold of resistance to apoptosis by cytotoxic drugs.
In various aspects of the tenth embodiment, the expression of YY1 or PTN is at least about 1, 2, 10, or 100 fold lower than in the absence of the compound of Formula I.
In a eleventh embodiment, this invention provides a method of treating, preventing or inhibiting lymphoma by administering to a subject, optionally in combination with cytotoxic agents, a therapeutically effective amount of a compound of Formula I:
in which each of R1, R2 and R3 can be independently: H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; R4 is a 5-8 membered cycloalkyl optionally substituted with 1-8 R5 groups or a 5-8 membered cycloalkenyl optionally substituted with 1-8 R5 groups; each R5 can independently be: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, or substituted cycloalkyl; each of X1, X2, X3 and X4 can independently be O, NR6 and S; R6 is H or C1-C6 alkyl, and in which the therapeutically effective amount is sufficient to induce the expression of RKIP or PTEN, thereby inducing apoptosis.
In a twelfth embodiment, this invention provides a method of treating, preventing or inhibiting lymphoma by administering to a subject a therapeutically effective amount of a compound of Formula I:
in which each of R1, R2 and R3 can be independently: H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; R4 is a 5-8 membered cycloalkyl optionally substituted with 1-8 R5 groups or a 5-8 membered cycloalkenyl optionally substituted with 1-8 R5 groups; each R5 can independently be: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, or substituted cycloalkyl; each of X1, X2, X3 and X4 can independently be O, NR6 and S; R6 is H or C1-C6 alkyl, and in which the therapeutically effective amount is sufficient to inhibit the expression of YY1 or PTN, thereby inducing apoptosis.
In an thirteenth embodiment, this invention provides a method of treating, preventing or inhibiting a cancer with proteasome inhibitor therapy by administering to a subject a therapeutically effective amount of a compound of Formula I:
in which each of R1, R2 and R3 can independently be: H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; R4 is a 5-8 membered cycloalkyl optionally substituted with 1-8 R5 groups or a 5-8 membered cycloalkenyl optionally substituted with 1-8 R5 groups; each R5 can independently be: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, or substituted cycloalkyl; each of X1, X2, X3 and X4 is independently selected from the group consisting of O, NR6 and S; and R6 is H or C1-C6 alkyl, in which the therapeutically effective amount is sufficient to induce the expression of RKIP or PTEN, thereby inducing apoptosis.
In an fourteenth embodiment, this invention provides a method of treating, preventing or inhibiting a cancer with proteasome inhibitor therapy by administering to a subject a therapeutically effective amount of a compound of Formula I:
in which each of R1, R2 and R3 can independently be: H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; R4 is a 5-8 membered cycloalkyl optionally substituted with 1-8 R5 groups or a 5-8 membered cycloalkenyl optionally substituted with 1-8 R5 groups; each R5 can independently be: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, or substituted cycloalkyl; each of X1, X2, X3 and X4 is independently selected from the group consisting of O, NR6 and S; and R6 is H or C1-C6 alkyl, in which the therapeutically effective amount is sufficient to induce the expression of YY1 or PTN thereby inducing apoptosis.
In a fifteenth embodiment of this invention, this invention provides a method of treating a therapy resistant cancer by administering to a subject a therapeutically effective amount of a compound of Formula I:
in which each of R1, R2 and R3 can independently be: H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; R4 is a 5-8 membered cycloalkyl optionally substituted with 1-8 R5 groups or a 5-8 membered cycloalkenyl optionally substituted with 1-8 R5 groups; each R5 can independently be: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, or substituted cycloalkyl; each of X1, X2, X3 and X4 can independently be O, NR6 and S; R6 is H or C1-C6 alkyl, in which the therapeutically effective amount is sufficient to induce the expression of RKIP or PTEN, thereby inducing apoptosis.
In a sixteenth embodiment of this invention, this invention provides a method of treating a therapy resistant cancer by administering to a subject a therapeutically effective amount of a compound of Formula I:
in which each of R1, R2 and R3 can independently be: H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl; R4 is a 5-8 membered cycloalkyl optionally substituted with 1-8 R5 groups or a 5-8 membered cycloalkenyl optionally substituted with 1-8 R5 groups; each R5 can independently be: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, or substituted cycloalkyl; each of X1, X2, X3 and X4 can independently be O, NR6 and S; R6 is H or C1-C6 alkyl, in which the therapeutically effective amount is sufficient to inhibit the expression of YY1 or PTN, thereby inducing apoptosis.
Drug and Salinosporamide A resistanct Ramos cells (106 ml) were treated with various concentrations of Salinosporamide A for one hour and then treated with CDDP (15 μg/ml) for an additional 20 h. The cultures were then washed and the cells harvested and examined for apoptosis using the propidium iodide method, which measures DNA fragmentation, by flow cytometry. The percent apoptotic cells was recorded. Very long concentrations of Salinosporamide A (0.1 nM) were effective. The treatment were performed in duplicates.
“Cancer” refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin's lymphomas (e.g., Burkitt's, Small Cell, and Large Cell lymphomas) and Hodgkin's lymphoma, leukemia (including AML, ALL, and CML), multiple myeloma, mantle cell lymphoma, Waldenstrom's macrogobulinemia, and Philadelphia positive cancers.
“Therapy resistant” cancers, tumor cells, and tumors refers to cancers that have become resistant to both apoptosis-mediated (e.g., through death receptor cell signaling, for example, Fas ligand receptor, TRAIL receptors, TNF-R1), various conventionally used chemotherapeutic drugs, hormonal drugs, and radiation, and non-apoptosis mediated (e.g., antimetabolites, anti-angiogenic, etc.) cancer therapies. “Therapy sensitive” cancers are not resistant to therapy. One of skill in the art will appreciate that some cancers are therapy sensitive to particular agents but not to others. Cancer therapies include chemotherapy, hormonal therapy, radiotherapy, immunotherapy, and gene therapy.
“Therapy-mediated or induced cytotoxicity” refers to all mechanisms by which cancer therapies kill or inhibit cancer cells, including but not limited to inhibition of proliferation, inhibition of angiogenesis, and cell death due to, for example, activation of apoptosis pathways (e.g., death receptor cell signaling, for example, Fas ligand receptor, TRAIL receptors, TNF-R1). Cancer therapies include chemotherapy, immunotherapy, radiotherapy, and hormonal therapy.
“Therapeutic treatment” and “cancer therapies” and “cancer therapy reagents” refers to apoptosis-mediated and non-apoptosis mediated cancer therapies that treat, prevent, or inhibit cancers, including chemotherapy, hormonal therapy (e.g., androgens, estrogens, antiestrogens (tamoxifen), progestins, thyroid hormones and adrenal cortical compounds), radiotherapy, and immunotherapy (e.g., ZEVALIN, BEXXAR, RITUXAN (rituximab), HERCEPTIN). Cancer therapies can be enhanced by administration with a sensitizing agent, as described herein, either before or with the cancer therapy.
“Chemotherapeutic drugs” include conventional chemotherapeutic reagents such as alkylating agents, anti-metabolites, plant alkaloids, antibiotics, and miscellaneous compounds e.g., cis-platinum, CDDP, methotrexate, vincristine, adriamycin, bleomycin, and hydroxyurea. Chemotherapeutic drugs also include proteasome inhibitors such as salinosporamides (e.g., Salinosporamide A), bortezomib, PS-519, omuralide, PR-171 and its analogs, and Gleevec. The drugs can be administered alone or combination (“combination chemotherapy”).
By “sensitizingly effective amount or dose” or “sensitizingly sufficient amount or dose” herein is meant a dose that produces cancer cell sensitizing effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). Sensitized cancer cells respond better to cancer therapy (are inhibited or killed faster or more often) than non-sensitized cells, as follows: Control samples (untreated with sensitizing agents) are assigned a relative cancer therapy response value of 100%. Sensitization is achieved when the cancer therapy response value relative to the control is about 110% or 120%, preferably 200%, more preferably 500-1000% or more, i.e., at least about 10% more cells are killed or inhibited, or the cells are killed or inhibited at least about 10% faster. Cancer therapy response value refers to the amount of killing or inhibition of a cancer cell, or the speed of killing or inhibition of a cancer cell when it is treated with a cancer therapy. Some compounds are useful both as therapeutic reagents and as sensitizing reagents. Often, a lower dose (i.e., lower than the conventional therapeutic dose) or sub-toxic dose of such a reagent can be used to sensitize a cell. Often, when a cell is sensitized, a lower dose of the chemotherapeutic reagent can be used to achieve the same therapeutic effect as with a cell that has not been sensitized.
By “therapeutically effective amount or dose” or “therapeutically sufficient amount or dose” herein is meant a dose that produces therapeutic effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, the therapeutically effective dose can often be lower than the conventional therapeutically effective dose for non-sensitized cells.
Apoptosis refers to a process of programmed cell death that is different from the general cell death or necrosis that results from exposure of cells to non-specific toxic events such as metabolic poisons or ischemia in being an ordered molecular process by which unwanted cells undergo death. Cells undergoing apoptosis show characteristic morphological changes such as chromatin condensation and fragmentation and breakdown of the nuclear envelope in a process called pyknosis. As apoptosis proceeds, the plasma membrane is seen to form blebbings cells and the apoptotic cells are either phagocytosed or else break up into smaller vesicles which are then phagocytosed. Typical assays used to detect and measure apoptosis include microscopic examination of pyknotic bodies as well as enzymatic assays such as TUNEL labeling, caspase assay, annexin assay, and DNA laddering, among others. Apoptotic cells can be quantitated by FACS analysis of cells stained with propidium iodide for DNA hypoploidy.
“Inducing apoptosis” refers to an agent or process which causes a cell to undergo the program of cell death described above for apoptosis.
“Salinosporamide” refers to proteasome inhibitor compounds produced by Salinospora sp., a marine gram positive actinomycete, e.g., Salinosporamide A (Salinosporamide A), B, C, etc, and analogs thereof. Salinosporamides can be made by isolating the products from fermentation of Salinospora (wild type and mutant strains) and genetically engineered microorganisms, by biosynthesis in vitro using whole cells, enzymes, and recombinant enzymes, and by synthetic chemistry techniques.
“Salinosporamide A” refers to proteasome inhibitor compounds produced by Salinospora sp., a marine gram positive actinomycete. This term also refers to analogs of Salinosporamide A. Salinosporamide A and analogs thereof have structures as disclosed herein, e.g., in Formula 1 and
“Antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.
Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)′2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)′2 dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990))
For preparation of antibodies, e.g., recombinant, monoclonal, or polyclonal antibodies, many technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity (see, e.g., Kuby, Immunology (3rd ed. 1997)). Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Pat. No. 4,946,778, U.S. Pat. No. 4,816,567) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized or human antibodies (see, e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can also be made bispecific, i.e., able to recognize two different antigens (see, e.g., WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Suresh et al., Methods in Enzymology 121:210 (1986)). Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or immunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO 92/200373; and EP 03089).
Methods for humanizing or primatizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
A “chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
As used herein, the term “alkyl” refers to a monovalent straight or branched chain hydrocarbon group having from one to about 12 carbon atoms, including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like.
As used herein, the term “substituted alkyl” refers to alkyl groups further bearing one or more substituents selected from hydroxy, alkoxy, mercapto, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, aryloxy, substituted aryloxy, halogen, cyano, nitro, amino, amido, —C(O)H, acyl, oxyacyl, carboxyl, sulfonyl, sulfonamide, sulfuryl, and the like.
As used herein, the term “lower alkyl” refers to alkyl groups having from 1 to about 6 carbon atoms.
As used herein, the term “alkenyl” refers to straight or branched chain hydrocarbyl groups having one or more carbon-carbon double bonds, and having in the range of about 2 up to 12 carbon atoms, and “substituted alkenyl” refers to alkenyl groups further bearing one or more substituents as set forth above. Alkenyl groups useful in the present invention include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, pentenyl, hexenyl, and the like.
As used herein, the term “alkynyl” refers to straight or branched chain hydrocarbyl groups having at least one carbon-carbon triple bond, and having in the range of about 2 up to 12 carbon atoms, and “substituted alkynyl” refers to alkynyl groups further bearing one or more substituents as set forth above. Alkynyl groups useful in the present invention include, but are not limited to, ethynyl, propynyl, butyryl, pentynyl, hexynyl, and the like.
As used herein, the term “aryl” refers to aromatic groups having in the range of 6 up to 14 carbon atoms and 1 to 3 rings, and “substituted aryl” refers to aryl groups further bearing one or more substituents as set forth above. Aryl groups useful in the present invention include, but are not limited to, phenyl, benzyl, naphthyl, biphenyl, phenanthrenyl, and anthrenyl.
As used herein, the term “heteroaryl” refers to aromatic rings containing one or more heteroatoms (e.g., N, O, S, or the like) as part of the ring structure, having in the range of 3 up to 14 carbon atoms and 1 to 3 rings. “Substituted heteroaryl” refers to heteroaryl groups further bearing one or more substituents as set forth above. Heteroaryl groups useful in the present invention include, but are not limited to, pyridyl, pyridyl N-oxide, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzopyranyl, benzopyranyl, benzothiopyranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, and thienyl.
As used herein, the term “alkoxy” refers to the moiety —O-alkyl-, wherein alkyl is as defined above, and “substituted alkoxy” refers to alkoxyl groups further bearing one or more substituents as set forth above.
As used herein, the term “thioalkyl” refers to the moiety —S-alkyl-, wherein alkyl is as defined above, and “substituted thioalkyl” refers to thioalkyl groups further bearing one or more substituents as set forth above.
As used herein, the term “cycloalkyl” refers to ring-containing alkyl groups containing in the range of about 3 up to 8 carbon atoms, and “substituted cycloalkyl” refers to cycloalkyl groups further bearing one or more substituents as set forth above. Cycloalkyl groups useful in the present invention include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane and cyclooctane.
As used herein, the term “cycloalkenyl” refers to a 3 to 8 membered cycloalkyl group having at least one carbon-carbon double bond (alkene) in the ring, and “substituted cycloalkenyl” refers to cycloalkenyl groups further bearing one or more substituents as set forth above. Cycloalkenyl rings useful in the present invention include, but are not limited to, 1-cyclopentenyl, 2-cyclopentenyl, 3-cyclopentenyl, 1-cyclohexenyl, 2-cyclohexenyl, 3-cyclohexenyl, as well as cyclopropenyl, cyclobutenyl, cycloheptenyl and cyclooctenyl. Cycloalkadienyls are also useful in the present invention and include, but are not limited to, cyclopentadienyl, cyclohexadienyl, cycloheptadienyl and cyclooctadienyl.
As used herein, the term “heterocyclic”, refers to cyclic (i.e., ring-containing) groups containing one or more heteroatoms (e.g., N, O, S, or the like) as part of the ring structure, having in the range of 3 up to 14 carbon atoms and 1 to 3 rings. “Substituted heterocyclic” refers to heterocyclic groups further bearing one or more substituents as set forth above. Heterocyclic groups useful in the present invention, include, but are not limited to, pyrrolidinyl, pyrrolinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidinyl, piperazinyl, indolinyl, quinuclidinyl, morpholinyl, tetrahydrofuranyl, tetrahydrothienyl and dioxane.
The compounds of the invention may be formulated into pharmaceutical compositions as natural or salt forms. Pharmaceutically acceptable non-toxic salts include the base addition salts (formed with free carboxyl or other anionic groups) which may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, procaine, and the like. Such salts may also be formed as acid addition salts with any free cationic groups and will generally be formed with inorganic acids such as, for example, hydrochloric, sulfuric, or phosphoric acids, or organic acids such as acetic, p-toluenesulfonic, methanesulfonic acid, oxalic, tartaric, mandelic, and the like. Salts of the invention include amine salts formed by the protonation of an amino group with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like. Salts of the invention also include amine salts formed by the protonation of an amino group with suitable organic acids, such as p-toluenesulfonic acid, acetic acid, and the like. Additional excipients which are contemplated for use in the practice of the present invention are those available to those of ordinary skill in the art, for example, those found in the United States Pharmacopeia Vol. XXII and National Formulary Vol. XVII, U.S. Pharmacopeia Convention, Inc., Rockville, Md. (1989), the relevant contents of which is incorporated herein by reference.
The compounds according to this invention may contain one or more asymmetric carbon atoms and thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. The term “stereoisomer” refers to chemical compounds which differ from each other only in the way that the different groups in the molecules are oriented in space. Stereoisomers have the same molecular weight, chemical composition, and constitution as another, but with the atoms grouped differently. That is, certain identical chemical moieties are at different orientations in space and, therefore, when pure, have the ability to rotate the plane of polarized light. However, some pure stereoisomers may have an optical rotation that is so slight that it is undetectable with present instrumentation. All such isomeric forms of these compounds are expressly included in the present invention.
Each stereogenic carbon may be of R or S configuration. Although the specific compounds exemplified in this application may be depicted in a particular configuration, compounds having either the opposite stereochemistry at any given chiral center or mixtures thereof are also envisioned. When chiral centers are found in the derivatives of this invention, it is to be understood that this invention encompasses all possible stereoisomers. The terms “optically pure compound” or “optically pure isomer” refers to a single stereoisomer of a chiral compound regardless of the configuration of the compound.
Compounds useful in the present invention include those of Formula I:
wherein each of R1, R2 and R3 are independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, and sulfuryl. R4 is a 5-8 membered cycloalkyl optionally substituted with 1-8 R5 groups or a 5-8 membered cycloalkenyl optionally substituted with 1-8 R5 groups. Each R5 is independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, and substituted cycloalkyl. Each of X1, X2, X3 and X4 is independently selected from the group consisting of O, NR6 and S. And R6 is H or C1-C6 alkyl.
Additional compounds useful in the present invention include the following:
Salinosporamides disclosed in J. Org. Chem., 70(16), 6196-6203, 2005 are incorporated herein by reference. Additional Salinsoporamides are described in US20050049294, herein incorporated by reference in its entirety.
The compounds of the present invention can be prepared by a variety of methods including fermentation, recombinant biosynthesis and via synthetic methodologies.
A. Fermentation
The compounds of the present invention can be prepared, for example, by bacterial fermentation, which generates the compounds in sufficient amounts for pharmaceutical drug development and for clinical trials. In some embodiments, invention compounds are produced by fermentation of the actinomycete strains CNB392 and CNB476 in A1Bfe+C or CKA-liquid media. Essential trace elements which are necessary for the growth and development of the culture should also be included in the culture medium. Such trace elements commonly occur as impurities in other constituents of the medium in amounts sufficient to meet the growth requirements of the organisms. It may be desirable to add small amounts (i.e. 0.2 mL/L) of an antifoam agent such as polypropylene glycol (M.W. about 2000) to large scale cultivation media if foaming becomes a problem. The organic metabolites are isolated by adsorption onto an amberlite XAD-16 resin. For example, Salinosporamide A is isolated by elution of the XAD-16 resin with methanol:dichlormethane 1:1, which affords about 105 mg crude extract per liter of culture. Salinosporamide A is then isolated from the crude extract by reversed-phase flash chromatography followed by reverse-phase HPLC and normal phase HPLC, which yields 6.7 mg of Salinosporamide A. FIG. 5 and Example 1 of US 2004/0259856 (incorporated herein by reference) set forth a fermentation procedure for the preparation of the compounds of the instant invention. US20050049294, herein incorporated by reference in its entirety, also provides methods of isolating the compounds from fermentation broth.
B. Recombinant Biosynthesis
Recombinant biosynthesis uses cells expressing cloned genes and optionally naturally occurring pathways to create biosynthetic pathways to produce natural and novel metabolites (see, e.g., Altreuter et al., Curr. Opin. Biotehcnol. 10:130-136 (1999); Reynolds, PNAS 95:12744-12746 (1998); and Cane et al., Science 282:63-68 (1998)). Several biosynthetic pathways are possible for the production of the compounds of the present invention, including a mixed polyketide-non-ribosomal peptide synthesis pathway. Polyketides and non-ribosomal peptides are synthesized from small chain carboxylic acid and amino acid monomers, respectively, by large multifunctional protein complexes called polyketide synthetases and nonribosomal peptide synthetases. US20050049294, herein incorporated by reference in its entirety, also provides information on recombinant biosynthesis.
C. Synthetic Procedure
The compounds of the present invention can also be prepared using standard organic synthesis procedures known in the art. An exemplary synthetic procedure can be found in US 2005/0228186 (incorporated herein by reference) for the synthesis of
One of skill in the art will recognize that additional pathways exist for the synthetic preparation of the compounds of the present invention. US20050049294, herein incorporated by reference in its entirety, also provides information on synthesis of the compounds.
As described herein, Salinosporamide A is useful for sensitizing both sensitive and resistant cancer cells to therapy based apoptosis when administered in combination with low dose or sub-toxic amounts of cancer therapeutic reagents. Salinosporamide A and the low dose or sub-toxic amount of a cancer therapeutic can be administered alone to sensitize cells for subsequent therapies or co-administered in combination with chemotherapy, radiotherapy, hormonal therapy, or immunotherapy. In another embodiment, Salinosporamide A is used as a chemotherapeutic agent after cellular sensitization using an antibody. Salinosporamide A as a therapeutic can be administered alone or co-administered in combination with chemotherapy, radiotherapy, hormonal therapy, or immunotherapy. Methods of using Salinosporamide A are also described in US patent application 20050239866 and 20050049294, herein incorporated by reference in their entirety.
Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 20th ed., 2003, supra).
Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the compound suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
The compound of choice, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be “nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
Suitable formulations for rectal administration include, for example, suppositories, which consist of the compound with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the compound of choice with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally. Parenteral administration, oral administration, and intravenous administration are the preferred methods of administration. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Cells transduced by nucleic acids for ex vivo therapy can also be administered intravenously or parenterally as described above.
The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The composition can, if desired, also contain other compatible therapeutic agents.
Preferred pharmaceutical preparations deliver one or the compounds of the invention, optionally in combination with one or more therapeutic agents, in a sustained release formulation. Typically, Salinosporamide A is administered therapeutically as a sensitizing agent that increases the susceptibility of tumor cells to other cytotoxic cancer therapies, including chemotherapy, radiation therapy, immunotherapy and hormonal therapy. In some embodiments, Salinosporamide A acts as a chemotherapeutic reagent after cellular sensitization using an antibody.
In therapeutic use for the treatment of cancer, the compounds utilized in the pharmaceutical method of the invention are administered at the initial dosage of about 0.001 mg/kg to about 1000 mg/kg daily. A daily dose range of about 0.01 mg/kg to about 500 mg/kg, or about 0.1 mg/kg to about 200 mg/kg, or about 1 mg/kg to about 100 mg/kg, or about 10 mg/kg to about 50 mg/kg, can be used. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. For example, dosages can be empirically determined considering the type and stage of cancer diagnosed in a particular patient. The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound in a particular patient. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired.
The pharmaceutical preparations are typically delivered to a mammal, including humans and non-human mammals. Non-human mammals treated using the present methods include domesticated animals (i.e., canine, feline, murine, rodentia, and lagomorpha) and agricultural animals (bovine, equine, ovine, porcine).
The following examples are offered to illustrate, but not to limit the claimed invention.
The CDDP resistant B-NHL Ramos cell line was treated with various concentrations of Salinosporamide A for one hour and then treated with predetermined nontoxic concentration of CDDP (15 μg/ml) for an additional 20 hours. The cells were then harvested and examined for apoptosis using the propidium iodide (PI) technique by flow cytometry examining DNA fragmentation.
Rituximab (chimeric anti-CD20 monoclonal antibody) has been used in the treatment of Non-Hodgkin's Lymphoma alone or in combination with chemotherapy. The clinical response has been very encouraging; however, some patients are initially unresponsive or develop resistance following treatment. In order to investigate the mechanism of rituximab resistance we have developed in our laboratory rituximab resistant clones from B-NHL cell lines. We have selected certain clones for further analysis of the underlying mechanism of resistance. In the present study we have examined the Daudi RR1 clone which is resistant to rituximab-induced signaling and unlike Daudi wild type, rituximab failed to sensitize Daudi RR1 to drug-induced apoptosis. In addition, we have found that Daudi RR1 also develops the highest degree of drug resistance compared to wild type. We investigated whether Salinosporamide A can sensitize Daudi RR1 to CDDP-induced apoptosis.
3) Comparison Between Salinosporamide A and Bortezomib in their Ability to Sensitize B-NHL Cell Lines to CDDP-Induced Apoptosis
a) Study with Daudi and Wild Type Cells
We examined the effect of Salinosporamide A and bortezomib in their ability to sensitize Daudi wild type cells to CDDP-induced apoptosis. The tumor cells were treated for one hour with various concentrations of Salinosporamide A or bortezomib (range 1-15 nM) and then treated with CDDP (10 μg/ml) for an additional 20 h. The cells were then examined for apoptosis as described above. The findings shown in
b) Study with Daudi RR1
We performed similar experiments as above with rituximab resistant Daudi RR1 cells and the findings are summarized in
The findings with both Daudi and Daudi RR1 cells demonstrate that Salinosporamide A and bortezomib showed similar findings under the conditions used and the model system utilized. Further analysis by changing the time of treatment and with other cell lines will determine if there were differences with respect to the concentrations and cytotoxicity when using Salinosporamide A or bortezomib in sensitization experiments.
4) Comparison Between Salinosporamide A and the NF-κB Inhibitor, DHMEQ in their Ability To Sensitize Drug Resistant Tumor Cells to CDDP-Induced Apoptosis.
a) Study with Daudi Wild Type Cells
DHMEQ is a NF-κB inhibitor that has been shown to be selective and preventing NF-κB translocation from the cytoplasm to the nucleus (Horiguchi, et al., Expert Rev. Anticancer Ther., 2003, 3(6): 793-8.). We have reported that DHMEQ can sensitize drug-resistant tumor cells to drug-induced apoptosis. We examined the differential effects of Salinosporamide A and DHMEQ in their ability to sensitize Daudi to CDDP-induced apoptosis. Tumor cells were treated with different concentrations of DHMEQ (range 1-65 μM) and Salinosporamide A (range 1-50 nM) for 1 h and then treated with CDDP (10 μg/ml) for an additional 20 h and the cells were then examined for apoptosis as described above. The findings in
b) Study with Daudi RR1 Cells
Similar studies were performed as above in a) with Daudi RR1 cells. The findings in
This finding demonstrates that Salinosporamide A is a superior inhibitor and sensitizing agent as compared to DHMEQ based on the concentration used. However, further studies are needed to demonstrate selectivity with other tumor cell lines.
The above findings have demonstrated the following:
1) Salinosporamide A at very low concentrations (0.1-10 nM) sensitizes both rituximab sensitive and rituximab resistant B-NHL tumor cells to drug-induced apoptosis.
2) Comparing the effectiveness of Salinosporamide A and bortezomib in the model system used, revealed that both agents at similar concentrations sensitize B-NHL cells to drug-induced apoptosis.
3) Comparing Salinosporamide A and the specific NF-κB inhibitor DHMEQ revealed that both agents sensitized tumor cells to drug-induced apoptosis; however, sensitization by DHMEQ required a 4,000 fold increase in the concentration as compared to Salinosporamide A.
Our published work with B-NHL cells revealed that rituximab sensitized drug resistant tumor cells to drug induced apoptosis. Sensitization was the result of inhibition of survival pathways such as the Raf-Mek-Erk and NF-κB pathways. These pathways resulted to down regulation of the anti-apoptotic gene product, selectively Bclx1 (Jazirehi and Bonavida, 2005). Since Salinosporamide A was shown to be cytotoxic in sensitive tumor cells, we considered that it might behave like a chemotherapeutic drug and thus we examined whether rituximab can sensitize tumor cells to Salinosporamide A induced apoptosis. We have reported that rituximab treatment of B-NHL cell lines sensitized the drug-resistant cells to drug-induced apoptosis. One of the mechanisms by which rituximab sensitizes the tumor cells to drug-induced apoptosis has been shown to be mediated via inhibition of the NF-κB pathway and downstream the selective inhibition of the anti-apoptotic product BclXL expression. Inhibitors of this pathway mimicked rituximab in sensitizing the cells to drug-induced apoptosis (Jazirehi, et al, 2005, Cancer Research, 65(1):264-76). We hypothesized that proteasome inhibitors that inhibit NF-κB activity and downstream anti-apoptotic gene products may sensitize tumor cells to drug-induced apoptosis. The new proteasome inhibitor Salinosporamide A (Nereus Pharmaceuticals), which inhibits NF-κB activity, has been shown to sensitize B-NHL cells to drug (CDDP, adriamycin)-induced apoptosis. Salinosporamide A has also been shown to directly kill sensitive tumor cells by apoptosis. Also, Salinosporamide A induces apoptosis in multiple myeloma cells resistant to conventional and bortezomib therapies (Chauhan et al., Cancer Cell 2005 In Press).
We hypothesized that Salinosporamide A may behave like a chemotherapeutic drug and rituximab may therefore sensitize the tumor cells to Salinosporamide A-induced apoptosis. Ramos cells were treated with rituximab (20 ug/ml) (12 h to 18 h) and the cells were treated with various concentrations of Salinosporamide A (1-10 nM) for an additional 20 h and the cells were examined for apoptosis using the PI method detecting DNA fragmentation by flow cytometry. The combination treatment resulted in significant apoptosis. The synergistic activity was detected with very low concentrations of Salinosporamide A>=1 nM. By comparison, several thousand fold higher concentrations of chemotherapeutic drugs (e.g. CDDP, adriamycin) were used for rituximab-mediated chemosensitization of Ramos cells. We also examined the Salinosporamide A resistant Daudi cells following treatment with rituximab for 1 h and Salinosporamide A for an additional 20 h and apoptosis was measured as before. The findings in
We further found that rituximab sensitizes cells to Salinosporamide A-induced apoptosis to a higher level than does adriamycin (ADR). As shown in
These results demonstrate that rituximab sensitizes the tumor cells to the proteasome inhibitor Salinosporamide A-mediated apoptosis. In addition, the findings suggest that rituximab (or chemo sensitizing agents) used in combination with Salinosporamide A may result in synergistic activity and can reverse drug and/or rituximab resistance of B-NHL.
The acquisition of resistance to conventional therapies such as chemotherapy, radiation, and immunotherapy remains a major obstacle in the successful treatment of cancer. Among the mechanisms of resistance is the acquisition of resistance to apoptotic stimuli by tumor cells. Hence, tumor cells develop mechanisms to resist apoptosis and exhibit constitutive hyperactivation of survival and anti-apoptotic signaling pathways. Tumor suppressors exist in normal cells that negatively regulate cell survival and enhance response to apoptotic stimuli. The dysregulation of such controls that regulate cell survival and proliferation leads to neoplastic transformation. Thus, most tumor cells have dysregulated expression or function of functional tumor suppressors through deletion or mutation or low expression. Therefore, agents that can upregulate the expression of functional tumor suppressors would be useful to counteract the survival and anti-apoptotic pathways in tumor cells. Such agents would be expected to inhibit tumor cell proliferation or survival and/or sensitize cells to the cytotoxic effect of conventional cytotoxic therapies.
As shown in
It has been shown that RKIP inhibits the Raf/MEK/ERK 1/2 and the NF-κB survival signaling pathways, and consequently, the expression of several anti-apoptotic gene products that are regulated by these pathways. Furthermore, expression of RKIP has been shown to reverse the resistance of drug-resistant cancer cells to drug-induced apoptosis. In addition, RKIP expression has been found to be depressed in primary tumors as compared to normal tissues and has been found to be lost following malignancy and metastasis in tumors. Thus, the ability of Salinosporamide A to induce the expression of RKIP in tumor cells provides a novel therapeutic target for avoiding or reversing therapy resistance of cancer cells and may be especially useful in treating metastases.
As shown in
Analogs of Salinosporamide A, as shown in Formula I and in US 20050049294 are tested for activity as sensitizing agents and as chemotherapeutic agents as described above in Examples I, II, and III.
The findings in
The findings in
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
This application claims priority to U.S. Ser. No. 60/733,965, filed on Nov. 4, 2005, and U.S. Ser. No. 60/840,811, filed Aug. 28, 2006, the teachings of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60733965 | Nov 2005 | US | |
| 60840811 | Aug 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12282343 | Feb 2009 | US |
| Child | 13457293 | US |
