WO2012097351A1 - Matériels et procédés d'inhibition de cellules cancéreuses de myélome multiple - Google Patents

Matériels et procédés d'inhibition de cellules cancéreuses de myélome multiple Download PDF

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WO2012097351A1
WO2012097351A1 PCT/US2012/021393 US2012021393W WO2012097351A1 WO 2012097351 A1 WO2012097351 A1 WO 2012097351A1 US 2012021393 W US2012021393 W US 2012021393W WO 2012097351 A1 WO2012097351 A1 WO 2012097351A1
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lll12
cells
stat3
inhibition
multiple myeloma
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PCT/US2012/021393
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English (en)
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Pui-Kai Li
Chenglong Li
Jiayuh Lin
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The Ohio State University Research Foundation
Nationwide Children's Hospital, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/22—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound oxygen atoms
    • C07C311/29—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound oxygen atoms having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/22—Ortho- or ortho- and peri-condensed systems containing three rings containing only six-membered rings
    • C07C2603/24—Anthracenes; Hydrogenated anthracenes

Definitions

  • the present invention is based on the observation that a certain class of small molecules inhibits multiple myeloma cancer cells. Therefore, the present invention is in the fields of biochemistry, molecular biology and medicine.
  • MM Multiple Myeloma
  • STAT Signal Transducers and Activators of Transcription (STAT) proteins are transcription factors which normally mediate orderly and tightly regulated signaling processes initiated through extracellular cytokines and growth factors.
  • constitutive activation of STATs has been demonstrated to contribute to oncogenesis and STAT3 in particular, is considered to be an oncogene due to its ability to promote malignancy. STAT3 activation occurs through
  • STAT3 phosphorylation of the tyrosine 705 (Tyr 70 5) residue, leading to dimerization and translocation from the cytoplasm to the nucleus.
  • STAT3 binding to target genes induces the transcription and up regulation of proliferation and anti-apoptotic associated proteins.
  • Constitutively active STAT3 may be sufficient for inducing cellular transformation and resistance to transformation was observed in ST AT3 -deficient cells.
  • STAT3 is frequently activated in many types of human solid and blood cancer and contribute to cancer progression.
  • the STAT3 signaling pathway is especially important in the proliferation, chemoresistance, and survival of MM cells through constitutive phosphorylation of STAT3 or in response to interleukin (IL)-6 produced by cells in the bone marrow microenvironment or by MM cells, per se.
  • IL interleukin
  • STAT3 may be important for normal embryologic development, it appears to be less important for the function of differentiated tissues. For example, no discernable deleterious effects were observed when STAT3 antisense therapy was used to deplete protein from normal cells in mice. Furthermore, fibroblasts deficient in STAT3 exhibited similar proliferative capacities compared to their wild-type counterparts, similar survival in vitro, and responded appropriately to several growth factors.
  • LLL12 ( Figure 6).
  • the inventors herein have now shown, inter alia, that LLL12 and similar molecules inhibit multiple myeloma cancer cells.
  • This invention therefore contributes effective therapeutic, diagnostic and prophylactic agents having increased positive results and fewer side effects.
  • the invention also provides methods for making related compounds, formulations, compositions, kits, etc.
  • LLL12 characterize the effects of LLL12 on MM tumor cells.
  • the inventors demonstrate that LLL12 exhibits high specificity for inhibiting STAT3 phosphorylation and leads to down regulation of STAT3-modulated proliferation and survival genes.
  • LLL12 inhibits proliferation and induces apoptosis of primary, human MM cells in vitro, even in samples procured from patients with MM clinically resistant to lenalidomide and bortezomib.
  • LLL12 suppresses in vivo MM tumor growth in a mouse xenograft model.
  • Rl, R2 are independently hydrogen or alkyl M, wherein M is 1, 2, 3, 4, 5 or 6 carbons;
  • ii. R3, R5, R6, R8, R9 are independently hydrogen, alkyl, alkoxy, halogen, N0 2 , NH 2 , or hydroxyl; and
  • R4, R7 are independently alkyl, alkoxy, O-alkyl, N-alkyl, aromatic, heteroaromatic, cyclic, or heterocyclic.
  • LLL12 a compound of Formula I, wherein Rl, R2, R3, R4, R5, R7, R8 and R9 are each hydrogen and R6 is hydroxyl.
  • the present invention also includes methods to inhibit at least one multiple myeloma cancer cell, comprising introducing a compound of Formula I to at least one multiple myeloma cancer cell, and inhibiting at least one multiple myeloma cancer cell.
  • cells are resistant to lenalidomide, and/or those wherein the cells are resistant to bortezomib.
  • LLL12 inhibition is measured by determining an IC50 value from between 0.26 ⁇ and 1.96 ⁇ . Also provided are methods herein wherein LLL12 inhibition is measured by reduced proliferation of cells. Also provided are methods herein wherein LLL12 inhibition is measured by reduced resistance to lenalidomide.
  • methods to treat multiple myeloma cancer in a patient in need of such treatment comprising administering a therapeutically-effective pharmaceutically-acceptable formulation of at least one compound of Formula I.
  • administering to the patient at least one additional chemotherapeutic drug is administering to the patient at least one additional chemotherapeutic drug.
  • additional chemotherapeutic drug is lenalidomide.
  • additional chemotherapeutic drug is bortezomib.
  • additional chemotherapeutic drugs are lenalidomide and bortezomib.
  • crystallographical) attributes via scientifically-reliable assay, including optional use of any scientifically-reliable assay(s) described herein, and optional use of computer generation and/or analysis of the results of any assay(s).
  • MM.1S and "MM1.S” are interchangeable names for a human-source multiple myeloma cell line known in the art, and available from ATCC, Lonza or other non-commercial sources.
  • the application contains one or more figures executed in color and/or one or more photographs. Copies of color figures(s) and/or photograph(s) will be provided upon request and payment of the necessary fee.
  • FIGS 1A-1E LLL12 inhibited constitutive and cytokine-induced STAT3 phosphorylation, reduced expression of downstream STAT3 targets, and exhibited high specificity for STAT3.
  • Figure 1A, Figure IB The human MM cell lines U266 (Figure 1A) and ARH-77
  • Figure IB exhibit constitutively phosphorylated STAT3.
  • LLL12 (2.5 ⁇ or 5 ⁇ ) inhibited STAT3 phosphorylation at Tyr705, resulting in induction of apoptosis as shown by cleaved PARP, caspase-3 and caspase 8.
  • Other signaling pathways were not affected by LLL12 (mTOR, Src, and ERK1/2).
  • GAPDH is shown as a loading control.
  • Figure IE LLL12 inhibited STAT3 but not STAT1 and STAT2 phosphorylation induced by interferon-a (25ng/ml) in the MM. IS cell line. Cells were pre-treated with LLL12 and/or Stattic for 2 hours, then were stimulated with IL-6 or interferon-a for 30-minutes in the presence or absence of LLL12.
  • Figures 2A-2D LLL12 inhibited STAT3 DNA binding activity and expression of downstream targets associated with proliferation and survival of MM tumor cells.
  • Figure 2A U266 (left) and ARH-77 (right) MM cells were treated in LLL12 (2.5 -
  • Figure 2B U266 (left) and ARH-77 (right) human MM cell lines were cultured in
  • Figure 2C U266 (left) and ARH-77 (right) - Downstream STAT3 target proteins
  • Cyclin Dl, Survivin, Bcl-2, and DNMT1 were downregulated by LLL12 as shown by western blot analysis.
  • FIG. 2D Downstream STAT3 target proteins, Survivin, Bcl-2 and Bcl-XL were downregulated by LLL12 as shown by western blot analysis.
  • FIGS 3A-3B LLL12 induced apoptosis in U266 and ARH-77 multiple myeloma cells.
  • Figure 3A Representative histograms of U266 and ARH-77 MM cells cultured for 24 hours in DMSO or LLL12 (2.5 ⁇ or 5 ⁇ ) following annexin V/ PI staining and flow cytometric analysis.
  • Figure 3B U266 (left) and ARH-77 (right) LLL12 led to dose-dependent, statistically significant (*) increases in apoptosis of MM cells, p ⁇ 0.05 in all instances, results are representative of at least 3 independent experiments.
  • FIGS 4A-4G LLL12 inhibited cell proliferation induced by IL-6 and blocked IL-6 mediated drug resistance in human MM cells.
  • FIG. 4A MM. IS cells were treated with IL-6 (25ng/ml) with or without LLL12. IL-
  • Figure 4B Treatments of MM. IS cells with lenalidomide caused a dose-dependent reduction of cell viability. IL-6 reversed lenalidomide induced inhibition of cell viability. Pre- treatment of MM. IS cells with LLL12, further reversed the rescue of lenalidomide-mediated inhibition of cell viability by IL-6.
  • Figure 4C U266 cells secreted higher levels of IL-6 than MM.1S cells.
  • Figure 4D U266 cells expressed higher levels of IL-6 than MM.1S cells. Both MM cell lines expressed similar levels of IL-6R and Gpl30.
  • Figure 4E U266 cells expressed higher levels of STAT3 phosphorylation than MM. IS cells.
  • Figure 4F U266 cells were more resistance to lenalidomide than MM. IS cells.
  • FIG. 4G LLL12 enhanced lenalidomide-mediated inhibition of cell proliferation in
  • FIGS 5A-5E LLL12 abrogated in vivo MM tumor cell growth.
  • ARH-77 MM flank tumors were established in 12 NOD/SCID mice, then randomized to receive intraperitoneal injection with 5mg/kg of LLL12 or DMSO. Tumor volume was measured every other day and tumor mass was determined after 26 days.
  • Figure 5A, Figure 5B, Figure 5C LLL12 statistically significantly (*, all comparisons shown p ⁇ 0.05) impaired MM tumor cell growth as assessed by serial volume measurements
  • Figure 5D Western blotting demonstrated inhibition of STAT3 phosphorylation in response to LLL12 therapy.
  • Figure 5E The bodyweights of LLL12-treated mice were not decreased and similar to that of the vehicle -treated mice over 15-day of treatments.
  • FIG. 7 LLL 12 (2 hours exposure at 10 ⁇ ) inhibited cytokine-induction of STAT3 phosphorylation in the IM-9 MM cell line (which does not express constitutive STAT3
  • LLL12 did not inhibit phosphorylation of ST ATI, STAT2, STAT4, and STAT6 induced by 30-min stimulation with 25ng/ml of interferon-a interferon- ⁇ , and IL-4.
  • FIG. 8 LLL 12 inhibited nuclear localization of STAT3. Left panels show nuclear localization of STAT3 (red) in DMSO treated U266 human MM cells whereas right panels show that LLL 12 treatment leaded to STAT3 being retained in cytoplasm. The nuclei were stained with DAPI (blue).
  • FIGS 9A-9C LLL 12 inhibited the cell viability of CD138 (+) primary cells, but had no effect on cell viability of peripheral blood mononuclear cell (PBMC) and CD 138 (-) marrow fraction.
  • PBMC peripheral blood mononuclear cell
  • Figure 9B Bone marrow aspirate was obtained from a patient with MM.
  • CD138 (+) cells (most of them are myeloma cells) were isolated by passive selection technique.
  • the CD 138 (-) marrow fraction and the CD 138 (+) cells were cultured in DMSO or LLL 12 (10 ⁇ ) for 24h and viability was measured by MTS assay (5 replicates per condition).
  • LLL12 suppressed viability of CD138 (+) cells by 44% (p ⁇ 0.01) but did not affect viability of CD138 (-) cells.
  • Figure 9C IL-6 independent MM cell line RPMI-8226 were treated in LLL 12 (2.5 ⁇ ) or DMSO for 24 hour. LLL12 did not induce cleaved caspase-3 and cleaved PARP in this cell line.
  • MM remains incurable despite the advent of therapies and, with the incidence of the disease rising, new treatments are urgently needed.
  • STAT3 plays a crucial role in MM, proliferation, resistance to apoptosis, and survival and represents an important molecular target for the development of therapies.
  • Previous methods aimed at blocking STAT3 have included the use of RNA interference, STAT3 antisense oligonucleotides, and dominant negative STAT3.
  • Rl, R2 are independently hydrogen or alkyl M, wherein M is 1, 2, 3, 4, 5 or 6 carbons;
  • ii. R3, R5, R6, R8, R9 are independently hydrogen, alkyl, alkoxy, halogen, N0 2 , NH 2 , or hydroxyl; and
  • R4, R7 are independently alkyl, alkoxy, O-alkyl, N-alkyl, aromatic, heteroaromatic, cyclic, or heterocyclic.
  • LLL12 is a compound of Formula I wherein Rl , R2, R3, R4, R5, R7, R8 and R9 are each hydrogen and R6 is hydroxyl. LLL12 specifically inhibited STAT3 phosphorylation, nuclear localization, DNA binding activity down-regulated STAT3 downstream genes, and induced apoptosis in MM cells.
  • MM cells which came from patients that were clinically resistant to lenalidomide and bortezomib.
  • LLL12 is a potent inhibitor of cell proliferation with IC 50 values ranging between
  • LLL12 also inhibited STAT3 phosphorylation induced by interleukin-6 (IL-6) and interferon-a, but not STAT1, STAT2, STAT4, and STAT6 phosphorylation induced by interferon-a, interferon- ⁇ , and interleukin-4 indicating the selectivity of LLL12 for STAT3.
  • IL-6 interleukin-6
  • IL-6 promoted the cell proliferation and resistance to lenalidomide in MM cells.
  • LLL12 significantly blocked tumor growth of MM cells in mouse model.
  • LLL12 can be synthesized using more than one method.
  • LL12 can be synthesized by reacting an unsubstituted or substituted naphthalene sulfonyl chloride compound with a nitrogen containing compound to form an unsubstituted or substituted naphthalene sulfonyl amine; oxidizing the unsubstituted or substituted naphthalene sulfonyl amine of step i) to yield an unsubstituted or substituted naphthoquinone compound; and, catalyzing via a Diels-Alder reaction of 3-hydroxy-2-pyrone with the unsubstituted or substituted naphthoquinone compound of step ii) to yield a compound of Formula I.
  • the nitrogen containing compound of step i) comprises ammonium hydroxide and the naphthalene sulfonyl chloride is unsubstituted.
  • LLL12 is a small molecule inhibitor of STAT3 with high specificity to the Tyr 70 5 residue mediating activation of STAT3 signaling via phosphorylation.
  • the inventors present the data characterizing LLL12 as a therapy for MM.
  • LLL12 prevents phosphorylation of STAT3 and this effect is associated with caspase-mediated apoptosis in MM cells.
  • the data show that LLL12 prevents STAT3 activation induced through cytokine stimulation, including IFN-a and IL-6. These effects appear STAT3-specific, in that no inhibitory effects of LLL12 on other STAT-family members such as STAT1, STAT2, STAT4, and STAT6 upon IFN-a, IFN- ⁇ , and IL-4 stimulation.
  • LLL12 leads to down regulation of expression of downstream targets of STAT3 that are involved in proliferation and survival of MM cells, providing further mechanistic detail regarding the pro-apoptotic effects of LLL12 in MM. These effects culminate in statistically significant, deleterious effects on MM cell in vitro viability as well as impaired in vivo MM tumor cell growth in a murine model.
  • IL-6 is a key cytokine mainly produced by myeloma cells and a variety of other cells in the marrow microenvironment. A high IL-6 serum level is often associated to worse progression- free survival and overall survival in myeloma. IL-6 exerts its biological effects through binding to two signal transducing receptor subunits gpl30 and IL-6R. IL-6 binding results in gpl30 and IL-6R dimerization and in the subsequent activation of Janus kinases/STAT3 pathway.
  • IL-6 increases cell proliferation/viability in MM. IS cells and this is inhibited by small molecule, LLL12. This is consistent with the stimulation of STAT3 phosphorylation by IL-6 and inhibited by LLL12 in MM. IS cells.
  • U266 cells express higher levels of IL-6 and STAT3 phosphorylation compared to the MM. IS cells.
  • U266 cells are also more resistant to lenalidomide than MM. IS cells.
  • Treatment of LLL12 blocks lenalidomide-resistant in U266 cells showing that LLL12 is likely to enhance the effects of lenalidomide through the inhibition of IL- 6/P-STAT3 pathway in U266 cells. Therefore, these results show that IL-6 conferred resistance to lenalidomide and this promoting activity was blocked by STAT3 inhibitor LLL12.
  • STAT3 activity has also been implicated in chemoresistance against a number of effective anti-MM therapies. While not wishing to be bound by theory, it is also now believed that STAT3 is also implicated in emergent resistance to lenalidomide and bortezomib.
  • curcumin a natural product derived from Curcuma longa, may enhance the effects of anticancer agents against MM and other cancers through STAT3 inhibition.
  • curcumin also has a number of off-target effects in addition to STAT3 inhibition and doses associated with biologic activity are associated with clinical toxicities.
  • LLL12 is not a derivative from curcumin and the inhibitory effects of LLL12 appear STAT3-selective, in that no effects of LLL12 on twelve other human protein kinases were observed.
  • LLL12 The absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) of LLL12 were computed. Fifty "drug-likeness” parameters were evaluated, including molecular weight, polarity, solubility, cell permeability, blood brain barrier, HERG K+ blockage, HSA binding, metabolic stability, and more. LLL12 showed desirable "druglike" properties.
  • Competitive assays using the present compounds, tissue localization assays, toxicology screens, etc. using the presently- invented compounds, compositions, formulations, etc. are within the scope of the present invention.
  • the methods can be practiced in vitro, it is contemplated that, in certain embodiments, the preferred embodiments for the methods comprise contacting the cells in vivo, i.e., by administering the compounds to a subject harboring cancer cells in need of treatment.
  • MM cell lines were maintained in RPMI1640 medium supplemented with 10% Fetal Bovine Serum (FBS), 4.5 g/L L-glutamine, sodium pyruvate, and 1% penicillin/streptomycin and maintained in a humidified 37°C incubator with 5% C0 2 .
  • FBS Fetal Bovine Serum
  • 4.5 g/L L-glutamine sodium pyruvate
  • penicillin/streptomycin maintained in a humidified 37°C incubator with 5% C0 2 .
  • MM tumor cells were obtained with written informed consent from patients with MM under an IRB -approved procurement protocol and isolated by positive selection utilizing EasySep CD138(+) magnetic nanoparticles per manufacturer's instructions (StemCell Technologies, Vancouver, BC).
  • LLL12, a STAT3 inhibitor , and WP1066, a JAK2 inhibitor were synthesized at The
  • Lenalidomide was purchased from LC Laboratories (Woburn, MA). Drugs were dissolved in sterile dimethyl sulfoxide (DMSO) to make 20mM stock solution, stored at -20 ° C until use.
  • DMSO sterile dimethyl sulfoxide
  • MM cells were treated with LLL12 (5 ⁇ or 10 ⁇ ) or DMSO for 24 hours before cells were collected for Western blot analysis.
  • IFN-a interferon-a
  • IFN- ⁇ interferon- ⁇
  • IL-4 interferon- ⁇
  • IL-6 IL-6 stimulation experiments
  • IM-9 and MM IS MM cells were serum-starved for 24 hours and left untreated or pre-treated with LLL12 (2.5-10 ⁇ ), Stattic (2.5-20 ⁇ ) or DMSO for 2 hours. Then, 25ng/ml IFN-a, IFN- ⁇ , or IL-4 was added, the cells were harvested for Western Blot analysis 30 minutes later.
  • MM cells were lysed in cold RIPA lysis buffer containing protease inhibitors and subjected to SDS-PAGE. Proteins were transferred on to PVDF membrane and probed with antibodies.
  • Antibodies Cell Signaling Technology, Beverly, MA
  • phospho-specific STAT1 Tyrosine 701
  • phospho-specific STAT2 Tyrosine 690
  • phospho-specific STAT3 Tyrosine 705)
  • phospho-specific STAT4 Tyrosine 693
  • phospho-specific STAT6 Tyrosine 641
  • phospho-specific ERK1/2 Threonine 202/Tyrosine 204
  • phospho-specific Src Tyrosine 416
  • phospho-specific mTOR Serine 2448
  • cleaved Poly ADP-ribose) polymerase (PARP)
  • phospho-independent STAT3 cyclin D Bcl-2, Twis
  • RNAs of ARH77, U266 and MM. IS cells were collected using RNeasy Kits (Qiagen, Valencia, CA). Primer sequences and source information of STAT3 downstream target genes can be found in Figure 10. PCR amplification was done under the following conditions: 5 min at 94°C followed by 25 cycles of 30 seconds at 94°C, 30 sec at 55°C, and 30 seconds at 72°C with a final extension of 5 min at 72°C. Primer sequences [SEQ ID NOs:l-12] are shown in Figure 10.
  • Annexin-V / propidium iodide (PI) double staining (BD Pharmingen, San Jose, CA). After treatment with LLL12 or DMSO for 24 hours, U266 and ARH-77 MM cells were harvested and washed with cold PBS. The cell pellet was re-suspended in lx binding buffer at a concentration of 1 x 106 cells/ml. 100 ⁇ of cell suspension was transferred into another tube. 5 ⁇ of Annexin V-FITC and 5 ⁇ of PI were added for 15 minutes at room temperature (RT) in darkness, and then analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ) within 1 hour.
  • RT room temperature
  • mice Bodyweights of mice were measured daily during 14 days period treatments.
  • LLL12 specifically inhibits STAT3 phosphorylation and induces apoptosis in MM cells
  • STAT3 phosphorylation LLL12 inhibited STAT3 phosphorylation at tyrosine residue 705 (Tyr 705) ( Figure 1A and Figure IB). Inhibition of STAT3 phosphorylation was associated with induction of apoptosis, as evidenced by PARP, caspase-3, and caspase-8 cleavage ( Figure 1A and Figure IB). LLL12 also inhibited STAT3 phosphorylation and induced apoptosis in primary, human MM cells from three different patients ( Figure 1C). Importantly, patients M.M.P. 2 and M.M.P. 3 were clinically resistant to prior therapy with lenalidomide and bortezomib. These primary MM cells were all found to have constitutively active STAT3. Thus, therapy targeting STAT3 inhibition is an effective treatment for patients with lenalidomide and bortezomib resistant disease.
  • the inventors also detected the effects of LLL12 on the phosphorylation of mTOR, Src and ERK(l/2). They were not reduced significantly ( Figure 1A and Figure IB) which showed the selective inhibition of LLL12 to STAT3. In order to characterize the specificity of these effects of LLL12 in MM cells further, the inventors examined whether LLL12 exhibited inhibitory effects on 21 other human protein kinase activities using a standardized, validated kinase profile.
  • LLL12 does not inhibit BMX, BRK, CSK, Fgr, Fyn, JAK3, LcK, Lyn, or ZAP-70 which contain an SH2 domain (Table 1) at achievable, relevant in vivo concentrations (IC 50 are at least 57.19 ⁇ to > 100 ⁇ .
  • LLL12 exhibits no relevant inhibition against other protein kinases, including AKT2, CDKl/cyclin B, FAK, FGFR2, ⁇ , JNK1, JNK2, PAK1, PAK2, PDGFR-a, PKC-a, and PKC- ⁇ (IC50 are 91.76 ⁇ to > 100 ⁇ , Table 1).
  • LLL12 inhibits STAT3 but not other STAT s phosphorylation induced by interleukin-6, interferon-a, interferon- ⁇ , and IL-4.
  • the relationship between the ambient microenvironment and MM tumor cells is important to maintaining and perpetuating the tumor cell clone via cytokine-mediated signaling in particular, IL-6.
  • the MM. IS human MM cell line, which expresses very lower constitutively phosphorylated STAT3, was utilized to determine whether LLL12 is capable of inhibiting cytokine - induced STAT3 phosphorylation.
  • Pretreatment of MM. IS multiple myeloma cells stimulated by IL- 6 and IFN-a respectively with LLL12 prevented phosphorylation of STAT3, but not STAT1 or STAT2 ( Figure ID and Figure IE).
  • LLL12 inhibits STAT3 nuclear localization, STAT3 DNA binding activity and the expression ofSTAT3 downstream target genes
  • LLL12 inhibits STAT3 nuclear localization, because the main function of STAT3 is to function as a transcription factor in nucleus.
  • STAT3 was mainly observed localized in nuclei (Left panels, Figure 8).
  • most STAT3 was retained in cytoplasm and absent in nuclei (Right panels, Figure 8).
  • the inventors also examined the effect of LLL12 on STAT3 DNA binding activity in U266 and ARH-77 MM cells.
  • LLL12 caused a statistically significant inhibition (approximately 30% reduction with 2.5 ⁇ and 55% reduction, p ⁇ 0.05) of STAT3 DNA binding activity in U266 and ARH-77 MM cells (Figure 2A). These findings demonstrate that inhibition of STAT3 phosphorylation by LLL12 consequently impairs STAT3 function in MM cells.
  • LLL12 impairs MM cell proliferation and viability
  • IL-6 promotes cell viability, confers resistance to growth inhibition induced by lenalidomide and is suppressed by LLL12 in MM. IS human MM cells.
  • MM cells were stimulated with IL-6 for 48 hours and cells viability was detected by MTT assay.
  • IL-6 increases cell proliferation/viability by two-fold which could be blocked by small molecular STAT3 inhibitor, LLL12 ( Figure 4A). This is consistent with the stimulation of STAT3 phosphorylation by IL-6 and inhibited by LLL12 in MM. IS cells (see Figure ID).
  • LLL12 enhanced the activity of lenalidomide in U266 human MM cells with higher levels of IL-6/P-STAT3
  • the inventors also examined the effects of lenalidomide on U266 human MM cells.
  • the inventors compared the level of IL-6 /P-STAT3 pathway in U266 and MM. IS cell lines.
  • ELISA assay showed that the level of IL-6 in the medium of U266 cells is higher than MM.
  • IS cells Figure 4C.
  • U266 cell line also has higher level of IL-6 gene expression as detected by RT-PCR (Figure 4D).
  • Western blot results also showed the phosphorylation of STAT3 (Tyr 705) in U266 is higher than MM.
  • IS cell line Figure 4E. Since U266 cells express higher levels of IL-6 and STAT3 phosphorylation than MM. IS cells, the inventors determined that U266 cells may be more resistant to certain drugs. The results demonstrated that U266 cells were indeed more resistant to lenalidomide compared to MM. IS cells ( Figure 4F).
  • LLL12 could increase the sensitive of U266 cells to lenalidomide treatment (Figure 4G).
  • the Combinatorial Index (CI) value for all the combinations of treatments were less than one (1), indicating synergism between LLL12 and lenalidomide.
  • CI Combinatorial Index
  • LLL12 suppresses MM tumor growth in vivo
  • LLL12 significantly suppressed tumor volume (Figure 5A and Figure 5C, p ⁇ 0.05 for all comparisons indicated), tumor weight ( Figure 5B, p ⁇ 0.05), and inhibited STAT3 phosphorylation (Figure 5D) compared with DMSO-treated controls.
  • the present invention provides options that are advantageous over previously-known compounds, compositions, formulations, research tools, diagnostics, and therapies.
  • therapeutic superiority because the present compounds are selective for STAT3 inhibition, the present compounds do not have the potential toxic side effects of previously-known treatment methods. In other words, in certain embodiments, the present compounds and methods have little or no impact non-cancerous cells.
  • selective nature and potentency of the present compounds allow synergy with conventional anti-cancer agents, thereby reducing the overall toxic load of any given treatment. In effect, the present compounds allow conventional anti-cancer treatments to exert greater effect at lower dosage.
  • an effective dose (ED50) for an anti-cancer agent or combination of conventional anti-cancer agents when used in combination with the present compounds can be less than the ED50 for the anti-cancer agent alone.
  • the therapeutic index (TI) for such anti-cancer agent or combination of such anti-cancer agent when used in combination with a compound herein is greater than the TI for conventional anti-cancer agent regimen alone.
  • the method combines the present compounds with other therapies such as chemotherapies and/or radiation therapies, including ionizing radiation, gamma radiation, or particle beams.
  • therapies such as chemotherapies and/or radiation therapies, including ionizing radiation, gamma radiation, or particle beams.
  • the dosage regimen can be selected in accordance with a variety of factors including type, species, age, weight, sex and the type of cancer being treated; the severity (i.e., stage) of the cancer to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed.
  • An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to treat, for example, to prevent, inhibit (fully or partially) or arrest the progress of the disease.
  • Non-limiting examples of suitable dosages can include total daily dosage of between about 25-4000 mg/m 2 administered orally once-daily, twice-daily or three times-daily, continuous (every day) or intermittently (e.g., 3-5 days a week).
  • the compositions can be administered in a total daily dose, or divided into multiple daily doses such as twice daily, and three times daily.
  • Other non-limiting examples of suitable dosages and methods of administration can include the intravenous administration directly to the tumor site via a catheter.
  • the administration can be continuous, i.e., every day, or intermittently.
  • intermittent administration may be administration one to six days per week or it may mean administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week) or it may mean administration on alternate days.
  • compositions may be administered according to any of prescribed schedules, consecutively for a few weeks, followed by a rest period.
  • the composition may be administered according to any one of the prescribed schedules from two to eight weeks, followed by a rest period of one week, or twice daily at a dose for three to five days a week.
  • compositions suitable for oral administration can be incorporated into pharmaceutical compositions suitable for oral administration, together with a pharmaceutically acceptable carrier or excipient.
  • Such compositions typically comprise a therapeutically effective amount of any of the compounds described herein, and a pharmaceutically acceptable carrier.
  • the effective amount is an amount effective to selectively induce terminal differentiation of suitable neoplastic cells and less than an amount which causes toxicity in a patient.
  • any inert excipient that is commonly used as a carrier or diluent may be used in the formulations of the present invention, such as for example, a gum, a starch, a sugar, a cellulosic material, an acrylate, or mixtures thereof.
  • compositions may further comprise a disintegrating agent (e.g., croscarmellose sodium) and a lubricant (e.g., magnesium stearate), and in addition may comprise one or more additives selected from a binder, a buffer, a protease inhibitor, a surfactant, a solubilizing agent, a plasticizer, an emulsifier, a stabilizing agent, a viscosity increasing agent, a sweetener, a film forming agent, or any combination thereof.
  • a disintegrating agent e.g., croscarmellose sodium
  • a lubricant e.g., magnesium stearate
  • additives selected from a binder, a buffer, a protease inhibitor, a surfactant, a solubilizing agent, a plasticizer, an emulsifier, a stabilizing agent, a viscosity increasing agent, a sweetener, a film forming agent, or any combination thereof.
  • compositions can be administered orally, and are thus formulated in a form suitable for oral administration, i.e., as a solid or a liquid preparation.
  • suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like.
  • Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration, such as sterile pyrogen-free water. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • Non-limiting examples of solid carriers/diluents include, but are not limited to, a gum, a starch (e.g., corn starch, pregelatinized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
  • a gum e.g., corn starch, pregelatinized starch
  • a sugar e.g., lactose, mannitol, sucrose, dextrose
  • a cellulosic material e.g., microcrystalline cellulose
  • an acrylate e.g., polymethylacrylate
  • Non-limiting examples of liquid formulations, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, emulsions or oils.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil.
  • Solutions or suspensions can also include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • compositions may further comprise binders (e.g., acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g., cornstarch, potato starch, alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate, Primogel), buffers (e.g., tris-HCI, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol,
  • binders e
  • poloxamines e.g., ethyl cellulose, acrylates, polymethacrylates
  • coating and film forming agents e.g., ethyl cellulose, acrylates, polymethacrylates
  • adjuvants e.g., adjuvants.
  • the active compounds can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • the compounds may be administered intravenously on the first day of treatment, with oral administration on the second day and all consecutive days thereafter.
  • the compounds of the present invention may be administered for the purpose of preventing disease progression or stabilizing tumor growth.
  • compositions that contain an active component are well understood in the art, for example, by mixing, granulating, or tablet-forming processes.
  • the active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient.
  • the active agents are mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions and the like as detailed above.
  • the amount of the compound or formulation administered to the patient is less than an amount that would cause toxicity in the patient. In the certain embodiments, the amount of the compound that is administered to the patient is less than the amount that causes a concentration of the compound in the patient's plasma to equal or exceed the toxic level of the compound.
  • the concentration of the compound in the patient's plasma is maintained at about 10 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 25 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 50 nM.
  • the concentration of the compound in the patient's plasma is maintained at ranges between about 10 to about 50 nM.
  • the optimal amount of the compound that should be administered to the patient in the practice of the present invention will depend on the particular compound used and the type of cancer being treated.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des molécules dont l'utilisation a une incidence sur des cellules cancéreuses, ainsi que des procédés associés. Les présents composés, formulations, nécessaires et procédés sont utiles pour le diagnostic, la thérapie et la recherche. L'invention concerne des inhibiteurs de STAT3, en particulier LLL12. Les inhibiteurs de STAT3 sont utiles en général pour traiter le cancer du sein et en particulier les cellules à l'origine du cancer du sein.
PCT/US2012/021393 2011-01-14 2012-01-14 Matériels et procédés d'inhibition de cellules cancéreuses de myélome multiple WO2012097351A1 (fr)

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EP2884971A4 (fr) * 2012-08-16 2016-01-06 Ohio State Innovation Foundation Inhibiteurs de stat3 et leur utilisation anticancéreuse
US20200209247A1 (en) * 2017-09-15 2020-07-02 University Of Iowa Research Foundation Methods for identifying myeloma tumor-initiating cells and targeted therapy
EP3687514A4 (fr) * 2017-09-27 2021-06-30 Ohio State Innovation Foundation Méthodes et compositions pour l'inhibition de stat3
US11485750B1 (en) 2019-04-05 2022-11-01 Kymera Therapeutics, Inc. STAT degraders and uses thereof

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Cited By (7)

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Publication number Priority date Publication date Assignee Title
EP2884971A4 (fr) * 2012-08-16 2016-01-06 Ohio State Innovation Foundation Inhibiteurs de stat3 et leur utilisation anticancéreuse
US9783513B2 (en) 2012-08-16 2017-10-10 Ohio State Innovation Foundation STAT3 inhibitors and their anticancer use
US20200209247A1 (en) * 2017-09-15 2020-07-02 University Of Iowa Research Foundation Methods for identifying myeloma tumor-initiating cells and targeted therapy
EP3687514A4 (fr) * 2017-09-27 2021-06-30 Ohio State Innovation Foundation Méthodes et compositions pour l'inhibition de stat3
US11420946B2 (en) 2017-09-27 2022-08-23 Ohio State Innovation Foundation Methods and compositions for inhibition of STAT3
US11485750B1 (en) 2019-04-05 2022-11-01 Kymera Therapeutics, Inc. STAT degraders and uses thereof
US11746120B2 (en) 2019-04-05 2023-09-05 Kymera Therapeutics, Inc. Stat degraders and uses thereof

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