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`THERAPEUTIC CANCER TREATMENTS
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`BACKGROUND
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`Hedgehogsignaling is essential in many stages of development, especially in
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`formation of left-right symmetry. Loss or reduction of hedgehog signaling leads to multiple
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`developmental deficits and malformations, one of the most striking of which is cyclopia.
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`Manytumors and proliferative conditions have been shown to depend on the
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`hedgehog pathway. The growth of such cells and survival can be affected by treatment with
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`the compounds disclosed herein. Recently, it has been reported that activating hedgehog
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`pathway mutations occur in sporadic basal cell carcinoma (Xie ef al. (1998) Nature 391: 90-
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`92) and primitive neuroectodermal tumorsof the central nervous system (Reifenbergeretal.
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`(1998) Cancer Res 58: 1798-803). Uncontrolled activation of the hedgehog pathwayhas also
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`been shown in numerous cancer types such as GItract cancers including pancreatic,
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`esophageal, gastric cancer (Bermanef al. (2003) Nature 425: 846-51, Thayer et al. (2003)
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`Nature 425: 851-56) lung cancer (Watkins et al. (2003) Nature 422: 313-317, prostate cancer
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`(Karhadkaret al (2004) Nature 431: 707-12, Sheng et al. (2004) Molecular Cancer 3: 29-42,
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`Fan et al. (2004) Endocrinology 145: 3961-70), breast cancer (Kuboet al. (2004) Cancer
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`Research 64: 6071-74, Lewis et al. (2004) Journal of Mammary Gland Biology and
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`Neoplasia 2: 165-181) and hepatocellular cancer (Sicklick et al. (2005) ASCO conference,
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`Mohiniet al. (2005) AACR conference).
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`For example, small molecule inhibition of the hedgehog pathway has been shown to
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`inhibit the growth of basal cell carcinoma (Williams, et al., 2003 PNAS 100: 4616-21),
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`medulloblastoma (Bermanet al., 2002 Science 297: 1559-61), pancreatic cancer (Berman et
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`al,, 2003 Nature 425: 846-51), gastrointestinal cancers (Berman et al., 2003 Nature 425:
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`846-51, published PCT application WO 05/013800), esophageal cancer (Bermanet al., 2003
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`Nature 425: 846-51), lung cancer (Watkins et al., 2003. Nature 422: 313-7), and prostate
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`cancer (Karhadkare¢ al., 2004. Nature 431: 707-12).
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`In addition, it has been shown that many cancer types have uncontrolled activation of
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`the hedgehog pathway, for example, breast cancer (Kuboet al., 2004. Cancer Research 64:
`6071-4), hepatocellular cancer(Patil et al., 2005. 96" Annual AACR conference, abstract
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`30
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`#2942 Sicklick e¢ al., 2005. ASCO annual meeting, abstract #9610), hematological
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`malignancies (Watkins and Matsui, unpublished results), basal cell carcinoma (Bale & Yu,
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`2001. Human Molec. Genet. 10:757-762 Xie et al., 1998 Nature 391: 90-92),
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`medulloblastoma (Pietsch et al., 1997. Cancer Res. 57: 2085-88), prostate cancer (Karhadkar
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`et al., 2003, Nature, 431:846-851), and gastric cancer (Maet al., 2005 Carcinogenesis May
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`19, 2005 (Epub)).
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`SUMMARY
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`The invention relates generally to methods of extending relapse free survival in a
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`cancerpatient who is undergoing treatment with a chemotherapeutic, by administering a
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`therapeutically effective amount of a hedgehog inhibitor to the patient. In some
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`embodiments, the hedgehog inhibitor is administered concurrently with the
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`chemotherapeutic. In instances of concurrent therapy, the hedgehog inhibitor may continue
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`to be administered after treatment with the chemotherapeutic has ceased.
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`In other
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`embodiments, the hedgehog inhibitor is administered after treatment with the
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`chemotherapeutic has ceased(1.e., sequential treatment with no period of concurrent
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`treatment with the chemotherapeutic).
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`In another embodiment, the invention relates to a method of extending relapse free
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`survival in a cancer patient who had previously been treated with a chemotherapeutic, by
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`administering a therapeutically effective amount of a hedgehog inhibitor to the patient after
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`treatment with the chemotherapeutic has ceased.
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`The cancer treated by the methods described herein can be selected from, for
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`example, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), bladder
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`cancer, Ovarian cancer, and colon cancer. For treatment of small cell lung cancer according
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`to the invention, the chemotherapeutic can be selected from etoposide, carboplatin, cisplatin,
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`irinotecan, topotecan, gemcitabine, radiation therapy, and combinations thereof. An
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`example of suitable chemotherapeutics for treatment of non-small cell lung cancer according
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`to the invention include vinorelbine; cisplatin; docetaxel; pemetrexed; etoposide;
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`gemcitabine; carboplatin; targeted therapies including bevacizumab, gefitinib, erlotinib, and
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`cetuximab; radiation therapy; and combinations thereof. For treatment of bladder cancer
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`according to the invention, suitable chemotherapeutics include gemcitabine, cisplatin,
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`methotrexate, vinblastin, doxorubicin, paclitaxel, docetaxel, pemetrexed, mitomycin C, 5-
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`fluorouracil, radiation therapy, and combinations thereof. Examples of suitable
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`chemotherapeutics for the treatment of ovarian cancer according to the invention include
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`paclitaxel; docetaxel; carboplatin; gemcitabine; doxorubicin; topotecan; cisplatin; irinotecan;
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`targeted therapies such as bevacizumab; radiation therapy; and combinations thereof. For
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`treatment of colon cancer according to the invention, examples of suitable
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`chemotherapeutics include paclitaxel; 5-fluorouracil; leucovorin; irinotecan; oxaliplatin;
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`capecitabine; targeted therapies including bevacizumab, cetuximab, and panitumumab;
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`radiation therapy; and combinationsthereof.
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`An example of a suitable hedgehog inhibitor is a compound of formulaI:
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`or a pharmaceutically acceptable salt thereof. An example of a pharmaceutically acceptable
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`salt of the compound of formula I is a hydrochloride salt.
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`In some embodiments, the hedgehog inhibitor is administered as a pharmaceutical
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`composition comprising the hedgehog inhibitor, or a pharmaceutically acceptable salt
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`thereof, and a pharmaceutically acceptable excipient.
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`In another embodiment, the invention relates to a methodof treating pancreatic
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`cancer, by administering to a patient in need thereof a therapeutically effective amountof a
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`compoundof formulaI:
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`or a pharmaceutically acceptable salt thereof. An example of a therapeutically acceptable
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`salt of the compound of formula I is a hydrochloride salt. The method can also include
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`administration of the compound of formula I, or a pharmaceutically acceptable salt thereof,
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`in combination with a chemotherapeutic (e.g., gemcitabine). Administration of the
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`compound of formula I can continue after treatment with the chemotherapeutic has ceased.
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`The compound of formula I can administered as a pharmaceutical composition comprising
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`the compound of formula I, or a pharmaceutically acceptable salt thereof, and a
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`pharmaceutically acceptable excipient.
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`DETAILED DESCRIPTION
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`The invention relates to methodsfor treating various conditions and disorders, by
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`administering hedgehog inhibitors. In some instances, the hedgehog inhibitoris
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`administered in combination with other therapies, including other chemotherapeutics. The
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`hedgehog inhibitor can be administered concurrently, sequentially, or a combination of
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`concurrent administration followed by monotherapy with the hedgehoginhibitor.
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`In one aspect, the invention relates to a method of treating cancer. The method
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`includes administering to a patient a first therapeutic agent, and a second therapeutic agent,
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`wherein the second therapeutic agent is a hedgehog inhibitor. The two agents can be
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`administered concurrently (.e., essentially at the same time, or within the same treatment) or
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`sequentially (i.e., one immediately following the other, or alternatively, with a gap in
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`between administration of the two). In some embodiments, the hedgehoginhibitor is
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`administered after the first therapeutic. The first therapeutic agent can be a
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`chemotherapeutic agent, such as etoposide, carboplatin, or a combination thereof.
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`In another aspect, the invention relates to a methodof treating cancer. The method
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`includesthe steps of: administering to a patient a first therapeutic agent; then administering
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`the first therapeutic agent in combination with a second therapeutic agent, wherein said
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`second therapeutic agent is a hedgehog inhibitor. Specific examples of conditions that can
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`be treated include lung cancer (e.g., small cell lung cancer, non-small cell lung cancer).
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`In another aspect, the invention relates to a method of treating a condition mediated
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`by the hedgehog pathway. The method includes administering to a patient a first therapeutic
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`agent, and a second therapeutic agent, where said second therapeutic agent is a hedgehog
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`inhibitor. In some embodiments, the method includesthe steps of: (1) administering to a
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`patient a first therapeutic agent; then (2) administering the first therapeutic agent in
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`combination with a second therapeutic agent, where the second therapeutic agentis a
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`hedgehoginhibitor. The first agent can be a chemotherapeutic agent.
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`Suitable hedgehog inhibitors include, for example, those described and disclosed in
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`U.S. Patent 7,230,004 and U.S. Patent Application Publication 2008/0293754, the entire
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`disclosures of which are incorporated by reference herein.
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`For example, the hedgehog inhibitor can be a compound having the following
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`structure:
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`
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`or a pharmaceutically acceptable salt thereof; wherein
`R'is H, alkyl, -OR, amino, sulfonamido, sulfamido, -OC(O)R°, - N(R°)C(O)R®, ora
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`sugar;
`
`R? is H,alkyl, alkenyl, alkynyl, aryl, cycloalkyl, nitrile, or heterocycloalkyl;
`or R! and R? taken together form =O, =S, =N(OR), =N(R), =N(NR32), or =C(R)2;
`R?is H,alkyl, alkenyl, or alkynyl;
`R*is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl,
`heteroaralkyl, haloalkyl, -OR, -C(O)R’, -CO2R’, -SO2R”, -C(O)N(R°)(R°), -[C(R)2]q-R’,
`-[COW)-N(RICCO)]gR”, -L(W)-C(O)]qR”, -L(W)-C(O)O]qR”,-[(W)-OC(O)]qR°, -[(W)-SO2]qR’,
`-[C(W)-N(R*)SO2]qR”, -[CW)-C(O)N(R*)]JgR°, -[(W)-O]gR®, -[(W)-N(R)]gR°, -W-NR3*Xor
`-[(W)-S]QR°s
`each W is independently for each occurrencea diradical;
`
`each q is independently for each occurrence 1, 2, 3, 4, 5, or 6;
`
`xXis a halide;
`each R° is independently for each occurrence H,alkyl, alkenyl, alkynyl, aryl,
`cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl or -[C(R)2])-R°;
`or any two occurrences of R° on the samesubstituent can be taken together to form a
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`4-8 membered optionally substituted ring which contains 0-3 heteroatomsselected from N,
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`20
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`O, S, and P;
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`p is 0-6;
`each R°is independently hydroxyl, -N(R)COR,-N(R)C(O)OR,-N(R)SO2(R),
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`-C(O)N(R)2, -OC(O)N(R)(R), -SO2N(R)(R), -NCR)(R), -COOR, -C(O)N(OH)(R),
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`-OS(O)20R, -S(O)2OR, -OP(O)(OR)(OR), -NP(O)(OR)(OR), or -P(O)(OR)(OR);
`provided that when R’, R° are H and R* is hydroxyl; R' can not be hydroxyl;
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`Examples of compoundsinclude:
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`‘are H ; R' can not be hydroxyl; and
`provided that when R’, R’, and R*
`provided that when R?, R?, and R"
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`‘are H ; R' can not be sugar.
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`and pharmaceutically acceptable salts thereof.
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`One example of a suitable hedgehog inhibitor for the methods of the current
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`invention is the compoundof formulaI:
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`
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`or a pharmaceutically acceptable salt thereof. An example of a pharmaceutically acceptable
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`salt is a hydrochloride salt of the compound of formulaI.
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`Proliferative disorders that can be treated using the methods disclosed herein include,
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`for example, lung cancer (including small cell lung cancer and non small cell lung cancer),
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`other cancers of the pulmonary system, medulloblastoma and other brain cancers, pancreatic
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`cancer, basal cell carcinoma, breast cancer, prostate cancer and other genitourinary cancers,
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`gastrointestinal stromal tumor (GIST) and other cancers of the gastrointestinal tract, colon
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`cancer, colorectal cancer, ovarian cancer, cancers of the hematopoietic system (including
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`multiple myeloma, acute lymphocytic leukemia, acute myelocytic leukemia, chronic
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`myelocytic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin
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`lymphoma, and myelodysplastic syndrome), polycythemia Vera, Waldenstrom's
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`macroglobulinemia, heavy chain disease, soft-tissue sarcomas, such as fibrosarcoma,
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`myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
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`endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
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`mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamouscell
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`carcinoma, basal cell carcinoma, melanoma, and other skin cancers, adenocarcinoma, sweat
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`gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary
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`adenocarcinomas, stadenocarcinoma, medullary carcinoma, bronchogenic carcinoma,renal
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`cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
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`carcinoma, Wilms’ tumor, cervical cancer, uterine cancer, testicular cancer, bladder
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`carcinoma, and other genitourinary cances, epithelial carcinoma, glioma, astrocytoma,
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`medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma,
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`acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma,
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`endometrial cancer, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell
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`lymphoma, hepatocellular carcinoma, thyroid cancer, gastric cancer, esophageal cancer,
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`head and neck cancer, small cell cancers, essential thrombocythemia, agnogenic myeloid
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`metaplasia, hypereosinophilic syndrome, systemic mastocytosis, familiar hypereosinophilia,
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`chronic eosinophilic leukemia, thyroid cancer, neuroendocrine cancers, and carcinoid
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`tumors.
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`Certain methods of the current invention may be especially effective in treating
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`cancers that respond well to existing chemotherapies, but suffer from a high relapserate.
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`Examples of such cancers include lung cancer (e.g., small cell lung cancer or non-small cell
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`lung cancer), bladder cancer, ovarian cancer, breast cancer, colon cancer, and multiple
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`myeloma.
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`Hedgehoginhibitors useful in the current invention may contain a basic functional
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`group, such as amino or alkylamino, and are thus capable of forming pharmaceutically-
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`acceptable salts with pharmaceutically-acceptable acids. The term "pharmaceutically-
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`acceptable salts" in this respect, refers to the relatively non-toxic, inorganic and organic acid
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`addition salts of compoundsof the present invention. These salts can be prepared in situ in
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`the administration vehicle or the dosage form manufacturing process, or by separately
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`treating the compoundin its free base form with a suitable organic or inorganic acid, and
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`isolating the salt thus formed during subsequentpurification. Representative salts include
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`the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate,
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`oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate,
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`fumarate, succinate, tartrate, napthylate, mesylate, besylate, glucoheptonate, lactobionate,
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`and laurylsulphonate salts and the like (see, for example, Berge et al. (1977)
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`"Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).
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`The pharmaceutically acceptable salts of the present invention include the
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`conventional nontoxic salts or quaternary ammoniumsalts of the compounds, e.g., from non-
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`toxic organic or inorganic acids. For example, such conventional nontoxic salts include
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`those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic,
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`phosphoric, nitric, and the like; and the salts prepared from organic acids such asacetic,
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`propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic,
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`hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic,
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`fumaric, toluenesulfonic, methanesulfonic, benzenesulfonic, ethane disulfonic, oxalic,
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`isothionic, and the like.
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`In other cases, the compoundsof the present invention may contain one or more
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`acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts
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`with pharmaceutically-acceptable bases. The term "pharmaceutically-acceptable salts” in
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`these instances refers to the relatively non-toxic, inorganic and organic base addition salts of
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`compoundsof the present invention. These salts can likewise be preparedin situ in the
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`administration vehicle or the dosage form manufacturing process, or by separately treating
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`the compoundin its free acid form with a suitable base, such as the hydroxide, carbonate or
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`bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a
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`pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative
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`alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium,
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`and aluminumsalts and the like. Representative organic amines useful for the formation of
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`base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine,
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`diethanolamine, piperazine and the like (see, for example, Berge et al., supra).
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`To practice the methods of the invention, the hedgehog inhibitor and/or the
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`chemotherapeutic agent may be delivered in the form of one or more pharmaceutically
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`acceptable compositions which comprise a therapeutically-effective amount of one or more
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`hedgehog inhibitors and/or one or more chemotherapeutic formulated together with one or
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`more pharmaceutically acceptable carriers (additives) and/or diluents. In some instances, the
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`hedgehog inhibitors described herein and the chemotherapeutic agent are not administered in
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`the same pharmaceutical composition, and may, because of different physical and chemical
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`characteristics, be administered by different routes (for example, one compound can be
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`administered orally, while the second therapeutic is administered intravenously). In other
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`instances, the hedgehog inhibitor and the chemotherapeutic are administered in the same
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`pharmaceutical composition.
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`Pharmaceutical compositions may be specially formulated for administration in solid
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`or liquid form, including those adapted for the following: oral administration, for example,
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`drenches (aqueous or non-aqueoussolutions or suspensions), tablets, e.g., those targeted for
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`buccal, sublingual, and systemic absorption, capsules, boluses, powders, granules, pastes for
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`application to the tongue; parenteral administration, for example, by subcutaneous,
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`intramuscular, intravenous or epidural injection as, for example, a sterile solution or
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`suspension, or sustained-release formulation; topical application, for example, as a cream,
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`ointment, or a controlled-release patch or spray applied to the skin; intravaginally or
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`intrarectally, for example, as a pessary, cream or foam; sublingually; ocularly; transdermally;
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`pulmonarily, or nasally.
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`Examples of suitable aqueous and nonaqueous carriers which may be employed in
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`pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene
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`glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such
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`as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be
`
`maintained, for example, by the use of coating materials, such as lecithin, by the
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`maintenance of the required particle size in the case of dispersions, and by the use of
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`surfactants.
`
`These compositions may also contain adjuvants such as preservatives, wetting
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`agents, emulsifying agents, dispersing agents, lubricants, and/or antioxidants. Prevention of
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`the action of microorganisms upon the compounds of the present invention may be ensured
`
`by the inclusion of various antibacterial and antifungal agents, for example, paraben,
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`chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic
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`agents, such as sugars, sodium chloride, and the like into the compositions. In addition,
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`prolonged absorption of the injectable pharmaceutical form may be brought about by the
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`inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
`
`Methodsof preparing these formulations or compositions include the step of bringing
`
`into association the hedgehog inhibitor and/or the chemotherapeutic with the carrier and,
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`optionally, one or more accessory ingredients.
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`In general, the formulations are prepared by
`
`uniformly and intimately bringing into association a compoundof the present invention with
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`liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the
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`product.
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`Whenthe hedgehog inhibitors of the present invention are administered to humans
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`and animals, they can be given per se or as a pharmaceutical composition containing, for
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`example, about 0.1 to 99%, or about 10 to 50%, or about 10 to 40%, or about 10 to 30, or
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`about 10 to 20%, or about 10 to 15% of active ingredient in combination with a
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`pharmaceutically acceptable carrier.
`
`Actual dosage levels of the active ingredients in the pharmaceutical compositions of
`
`the present invention may be varied so as to obtain an amountof the active ingredient which
`
`is effective to achieve the desired therapeutic response for a particular patient, composition,
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`and mode of administration, without being toxic to the patient.
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`The selected dosage level will depend upon a variety of factors including the activity
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`of the particular compoundof the present invention employed, or the ester, salt or amide
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`thereof, the route of administration, the time of administration, the rate of excretion or
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`metabolism of the particular compound being employed, the rate and extent of absorption,
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`the duration of the treatment, other drugs, compounds and/or materials used in combination
`
`with the particular compound employed, the age, sex, weight, condition, general health and
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`prior medical history of the patient being treated, and like factors well knownin the medical
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`arts.
`
`In general, a suitable daily dose of a hedgehog inhibitor and/or a chemotherapeutic
`
`will be that amount of the compound whichis the lowest dose effective to produce a
`
`therapeutic effect. Such an effective dose will generally depend upon the factors described
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`above. Generally, oral, intravenous and subcutaneous doses of the compounds of the present
`
`invention for a patient, when used forthe indicated effects, will range from about 0.0001 mg
`
`to about 100 mgperday, or about 0.001 mg to about 100 mg perday, or about 0.01 mg to
`
`about 100 mg per day, or about 0.1 mg to about 100 mgperday, or about 0.0001 mg to
`
`about 500 mg per day, or about 0.001 mg to about 500 mg per day, or about 0.01 mg to
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`about 500 mg per day, or about 0.1 mg to about 500 mgperday.
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`The subject receiving this treatment is any animal in need, including primates, in
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`particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry
`
`and pets in general.
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`The compounds can be administered daily, every other day, three times a week, twice
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`a week, weekly, or bi-weekly. The dosing schedule can include a “drug holiday,” i.e., the
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`drug can be administered for two weeks on, one week off, or three weeks on, one weekoff,
`
`or four weeks on, one weekoff, etc., or continuously, without a drug holiday. The
`
`compounds can be administered orally, intravenously, intraperitoneally, topically,
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`transdermally, intramuscularly, subcutaneously, intranasally, sublingually, or by any other
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`route.
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`The hedgehog inhibitors disclosed herein can be combined with other cancer
`
`treatments. For example, they can be combined with surgical treatments; radiation;
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`biotherapeutics (such as interferons, cytokines - e.g. Interferon ao, Interferon y, and tumor
`
`necrosis factor, hematopoietic growth factors, monoclonal serotherapy, vaccines and
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`immunostimulants); antibodies (e.g. Avastin, Erbitux, Rituxan, and Bexxar); endocrine
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`therapy (including peptide hormones, corticosteroids, estrogens, androgens and aromatase
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`inhibitors); anti-estrogens (e.g. Tamoxifen, Raloxifene, and Megestrol); LHRH agonists(e.g.
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`goscrclin and Leuprolide acetate); anti-androgens(e.g. flutamide and Bicalutamide); gene
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`therapy; bone marrow transplantation; photodynamic therapies (e.g. vertoporfin (BPD-MA),
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`Phthalocyanine, photosensitizer Pc4, and Demethoxy-hypocrellin A (2BA-2-DMHA)); and
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`chemotherapeutics.
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`Examples of chemotherapeutics include gemcitabine, methotrexate, taxol,
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`mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide,
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`nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, etoposides,
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`prednisolone, dexamethasone, cytarbine, campathecins, bleomycin, doxorubicin, idarubicin,
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`daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine,
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`vincristine, vinorelbine, . Additional agents include nitrogen mustards (e.g.
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`cyclophosphamide, Ifosfamide, Trofosfamide, Chlorambucil, Estramustine, and Melphalan),
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`nitrosoureas (e.g. carmustine (BCNU) and Lomustine (CCNU)), alkylsulphonates(e.g.
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`busulfan and Treosulfan), triazenes (e.g. Dacarbazine and Temozolomide), platinum
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`containing compounds(e.g. Cisplatin, Carboplatin, and oxaliplatin), vinca alkaloids (e.g.
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`vincristine, Vinblastine, Vindesine, and Vinorelbine), taxoids (e.g. paclitaxel and
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`Docetaxol), epipodophyllins (e.g. etoposide, Teniposide, Topotecan, 9-Aminocamptothecin,
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`Camptoirinotecan, Crisnatol, Mytomycin C, and Mytomycin C), anti-metabolites, DHFR
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`inhibitors (e.g. methotrexate and Trimetrexate), IMP dehydrogenaseInhibitors (e.g.
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`mycophenolic acid, Tiazofurin, Ribavirin, and EICAR), ribonuclotide reductase Inhibitors
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`(e.g. hydroxyurea and Deferoxamine), uracil analogs (e.g. Fluorouracil, Floxuridine,
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`Doxifluridine, Ratitrexed, and Capecitabine), cytosine analogs (e.g. cytarabine (ara C),
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`Cytosine arabinoside, and Fludarabine), purine analogs (e.g. mercaptopurine and
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`Thioguanine), Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH 1060), isoprenylation
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`inhibitors (e.g. Lovastatin), dopaminergic neurotoxins (e.g. 1-methyl-4-phenylpyridinium
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`ion), cell cycle inhibitors (e.g. staurosporine), actinomycins(e.g. Actinomycin D and
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`Dactinomycin), bleomycins (e.g. bleomycin A2, Bleomycin B2, and Peplomycin),
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`anthracyclines (e.g. daunorubicin, Doxorubicin (adriamycin), Idarubicin, Epirubicin,
`Pirarubicin, Zorubicin, and Mitoxantrone), MDRinhibitors (e.g. verapamil), Ca** ATPase
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`inhibitors (e.g. thapsigargin), imatinib, thalidomide, lenalidomide, erlotinib, gefitinib,
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`sorafenib, sunitinib, and proteasome inhibitors such as bortezomib.
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`Whenthe hedgehog inhibitors disclosed herein are administered in combination with
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`other treatments (such as additional therapeutics or with radiation or surgery) the doses of
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`P A T EN T
`Docket 578133000901
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`each agent or therapy may be lowerthan the corresponding dose forsingle-agent therapy.
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`The dose for single-agent therapy ranges from about 0.0001 to about 200 mg,or about 0.001
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`to about 100 mg, or about 0.01 to about 100 mg, or about 0.1 to about 100 mgper, or about 1
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`to about 50 mg per kilogram of body weight per day. The determination of the mode of
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`administration and the advisability of administration, where possible, in the same
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`pharmaceutical composition, is well within the knowledge of the skilled clinician.
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`EXAMPLES
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`The invention now being generally described, it will be more readily understood by
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`reference to the following examples, which are included merely for purposesofillustration
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`of certain aspects and embodiments of the present invention, and are not intended to limit the
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`invention.
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`Example 1: Activity in the Hedgehog Pathway
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`Hedgehog pathwayspecific cancer cell killing effects may be ascertained using the
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`following assay. C3H10T1/2 cells differentiate into osteoblasts when contacted with the
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`sonic hedgehog peptide (Shh-N). Upondifferentiation, these osteoblasts produce high levels
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`of alkaline phosphatase (AP) which can be measured in an enzymatic assay (Nakamura et
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`al., 1997 BBRC 237: 465). Compoundsthat block the differentiation of C3H10T1/2 into
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`osteoblasts (a Shh dependent event) can therefore be identified by a reduction in AP
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`production (van der Horstet al., 2003 Bone 33: 899). The assay details are described below.
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`Cell Culture
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`Mouse embryonic mesoderm fibroblasts C3H10T1/2 cells (obtained from ATCC)
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`were cultured in Basal MEM Media (Gibco/Invitrogen) supplemented with 10% heat
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`inactivated FBS (Hyclone), 50 units/ml penicillin and 50ug/ml streptomycin
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`(Gibco/Invitrogen) at 37 °C with 5% CO,in air atmosphere.
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`Alkaline Phosphatase Assay
`C3H10T1/2 cells were plated in 96 wells with a density of 8x10° cells/well. Cells
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`were grown to confluence (72 hrs.). After sonic hedgehog (250ng/ml) and/or compound
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`treatment, the cells were lysed in 110 uL of lysis buffer (50 mM Tris pH 7.4, 0.1%
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`TritonX 100), plates were sonicated and lysates spun through 0.2 um PVDFplates (Corning).
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`40 uL of lysates was assayed for AP activity in alkaline buffer solution (Sigma) containing
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`P A T EN T
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`1lmg/ml p-Nitrophenyl Phosphate. After incubating for 30 min at 37 °C, the plates were read
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`on an Envision plate reader at 405 nm. Total protein was quantified with a BCAprotein
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`assay kit from Pierce according to manufacturer’s instructions. AP activity was normalized
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`against total protein. Using the above-described assay, Compound 42 was shownto be an
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`antagonist of the hedgehog pathway with an ICsless than 20 nM.
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`
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`Compound 42
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`Example 2: Pancreatic Cancer Monotherapy Model
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`The activity of Compound 42 was tested in a human pancreatic model. BxPC-3 cells
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`were implanted subcutaneously into the flanks of the right legs of mice. On day 42 post-
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`tumor implant, the mice were randomizedinto two groups to receive either Vehicle (30%
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`HPBCD) or Compound 42. Compound 42 was dosed at 40mg/kg/day. After receiving 25
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`daily doses, Compound42statistically reduced tumor volume growth by about 40% when
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`compared to the vehicle control (p=0.0309) (see Figure 1).
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`Atthe end of the study, the tumors were harvested 4 hours post the last dose to
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`evaluate an on target response by q-RT-PCR analysis of the Hedgehog pathway genes. As
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`shown in Figure 2A, Human Gli-1 was not modulated in either the vehicle or the treated
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`group. However, murine Gli-1 mRNAlevels were significantly down-regulated in the
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`Compound 42 treated group when comparedto the vehicle treated group (see Figure 2B).
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`Example 3: Pancreatic Cancer Concurrent Combination Therapy Model
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`Animals bearing BxPC-3 pancreatic cancer xenografts were treated with the
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`chemotherapeutic drug gemcitabine in concurrent combination with Compound 42.
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`Gemcitabine was administered at a dose of 100 mg/kg twice weekly by intraperitoneal
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`injection while Compound 42 was administered at a dose of 40 mg/kg daily by oral gavage.
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`As shownin Figure 3, under these conditions the tumors showed a 33% response to
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`P A T EN T
`Docket 578133000901
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`gemcitabine alone, a 55% response to Compound 42 alone, and a 67% response to the
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`combination of Compound 42 and gemcitabine.
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`In another model, Animals bearing MiaPaCa pancreatic cancer xenografts were
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`treated with the chemotherapeutic drug gemcitabine in concurrent combination with
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`Compound 42. Gemcitabine was administered at a dose of 100 mg/kg once weekly by
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`intraperitoneal injection while Compound 42 was administered at a dose of 40 mg/kg daily
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`by oral gavage. As shownin Figure 4, under these conditions the tumors showed a 52%
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`response to gemcitabine alone, a 50% response to Compound 42 alone, and a 70% response
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`to the combination of Compound 42 and gemcitabine.
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`Example 4: Lung Cancer Concurrent Combination Therapy Model
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`To test the activity of Compound 42 in a human small cell lung cancer tumor model,
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`LX22 cells were implanted subcutaneously into the flank of the right leg of male Ncr nude
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`mice. LX22 1s primary xenograft model of SCLC derived from chemo-naive patients, which
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`has been maintained by mouse to mou
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