Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
  • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
  • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
  • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
  • C07D239/32—One oxygen, sulfur or nitrogen atom
  • C07D239/42—One nitrogen atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

• the present disclosure relates to methods of treating chronic lymphocytic leukemia with HDAC inhibitors alone or in combination with BTK inhibitors.
• HDACs histone deactylases
• valproic acid (VPA-pan-HDAC inhibitor) (Stamatopoulos B, et al, Leukemia 2009; 23(12): 2281-9) and MGCD0103 (class I) (Blum KA, et al, Br J Haematol 2009; 147(4): 507-14) induced antileukemic activity in CLL, however undesirable side-effects due to non- selectivity remains to be a significant concern.
• HDACs role in BCR signaling pathways is still under investigation.
• This acknowledged disparity can be attributed, at least in part, to (1) the pan- inhibitory effect of HDAC inhibitors targeting all 11 zinc-dependent HDACs, and (2) the intrinsic differences in the expression profile of these enzymes between different cell types in both physiological and pathological conditions. Therefore, a desirable focus is on
• Ricolinostat is a well-tolerated HDAC6 inhibitor which is now in clinical trials alone or in combination therapy for relapsed-and refractory myeloma (Raje N, et a/., ASH Annual Meeting Abstracts. 2012; 120:4061; Vogl D, et al., Blood, December 3, 2015 126 (23)) as well as relapsed-refractory lymphoma (Amengual JE, et a/, Poster ASH Annual Meeting 2015).
• BTK inhibition is accompanied by a blockade in proliferation and an increase in apoptosis.
• most patients only reach partial responses with ibrutinib, and some of them still relapse, requiring additional therapies.
• CLL chronic lymphocytic leukemia
• BTK Bruton's tyrosine kinase
• various methods for achieving specific cellular responses in subjects who have CLL such as decreasing CLL cell viability, altering expression of inhibitory checkpoint molecules in a T- and/or B- cell compartment, reducing CLL cell expression of IL-10, and decreasing CLL cell proliferation.
• a method for treating chronic lymphocytic leukemia in a subject in need thereof comprising administering to the subject a
• HDAC6 selective inhibitor is a compound of Formula I:
• ring B is aryl or heteroaryl
• Ri is an aryl or heteroaryl, each of which may be optionally substituted by OH, halo, or Ci -6- alkyl;
• R is H or Ci-6-alkyl.
• the compound of Formula I is compound A:
• the compound of Formula I is compound B:
• the HDAC6 selective inhibitor is a compound of Formula
• R x and R y together with the carbon to which each is attached, form a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl;
• each R A is independently Ci -6- alkyl, Ci -6- alkoxy, halo, OH, -N0 2 , -CN, or - H 2 ; and m is 0, 1, or 2.
• th is compound C:
• the compound of Formula II is compound D:
• the method further comprises administering to the subject a therapeutically effective amount of a Bruton's tyrosine kinase (BTK) inhibitor, such as ibrutinib, or a pharmaceutically acceptable salt thereof.
• BTK Bruton's tyrosine kinase
• the HDAC6 selective inhibitor can be administered at a sub-therapeutic dose.
• the HDAC6 selective inhibitor induces apoptosis of chronic lymphocytic leukemia cells.
• a pharmaceutical combination for treating chronic lymphocytic leukemia comprising a therapeutically effective amount of a histone deacetylase 6 (HDAC6) selective inhibitor or a pharmaceutically acceptable salt thereof, and a Bruton's tyrosine kinase (BTK) inhibitor or a pharmaceutically acceptable salt thereof.
• HDAC6 selective inhibitor is a compound of Formula I:
• ring B is aryl or heteroaryl
• Ri is an aryl or heteroaryl, each of which may be optionally substituted by OH, halo, or Ci -6- alkyl;
• R is H or Ci-6-alkyl.
• the compound of Formula I is compound A:
• the compound of Formula I is compound B:
• the HDAC6 selective inhibitor is a compound of F
• R x and R y together with the carbon to which each is attached, form a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl;
• each R A is independently Ci -6- alkyl, Ci -6- alkoxy, halo, OH, -N0 2 , -CN, or - H 2 ; and m is 0, 1, or 2.
• the compound of Formula II is compound C:
• the compound of Formula II is compound D:
• the BTK inhibitor is ibrutinib or a pharmaceutically acceptable salt thereof.
• the combination can further comprise a pharmaceutically acceptable carrier.
• the pharmaceutical combination comprises Compound A:
• the pharmaceutical combination comprises Compound B:
• the pharmaceutical combination comprises Compound D:
• a pharmaceutical composition for treating chronic lymphocytic leukemia in a subject in need thereof comprising a therapeutically effective amount of a histone deacetylase 6 (HDAC6) selective inhibitor or a
• the HDAC6 selective inhibitor is a compound of Formula I:
• ring B is aryl or heteroaryl
• Ri is an aryl or heteroaryl, each of which may be optionally substituted by OH, halo, or Ci-6-alkyl;
• R is H or Ci -6- alkyl.
• the compound of Formula I is Compound A:
• the compound of Formula I is Compound B:
• the HDAC6 selective inhibitor is a compound of Formula II:
• R X and R Y together with the carbon to which each is attached, form a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl;
• each RA is independently Ci- 6 -alkyl, Ci- 6 -alkoxy, halo, OH, -N0 2 , -CN, or - H 2 ; and m is 0, 1, or 2.
• the compound of Formula II is Compound C:
• the compound of Formula II is Compound D:
• the BTK inhibitor is ibrutinib or a pharmaceutically acceptable salt thereof.
• the composition can further comprise a pharmaceutically acceptable carrier.
• a method for decreasing cell viability of chronic lymphocytic leukemia cells in a subject with chronic lymphocytic leukemia in need thereof by administering a histone deacetylase (HDAC) inhibitor, or a combination comprising a HDAC inhibitor and a Bruton's tyrosine kinase (BTK) inhibitor, to the subject.
• HDAC histone deacetylase
• BTK Bruton's tyrosine kinase
• a method for synergistically increasing apoptosis of chronic lymphocytic leukemia cells in a subject with chronic lymphocytic leukemia in need thereof by administering a combination comprising a histone deacetylase (HDAC) inhibitor and a Bruton's tyrosine kinase (BTK) inhibitor.
• HDAC histone deacetylase
• BTK Bruton's tyrosine kinase
• a method for altering expression of an inhibitory checkpoint molecule in a T- and/or B-cell compartment in a subject with chronic lymphocytic leukemia in need thereof by administering to the subject a therapeutically effective amount of a histone deacetylase (HDAC) inhibitor, or a combination comprising a HDAC inhibitor and a Bruton's tyrosine kinase (BTK) inhibitor.
• HDAC histone deacetylase
• BTK Bruton's tyrosine kinase
• the inhibitory checkpoint molecule is selected from the group consisting of: CD274 (PDL-1), CD273 (PDL-2), CD80 (B7-1), CD86 (B7-2), CD152 (CTLA4), CD275 (B7RP1), CD276 (B7-H3), B7-H4
• VTCNl CD270 (HVEM), BLTA, GAL9, CD366 ( ⁇ 3), A2aR, CD279 (PD-1), KIR, and CD223 (LAG3), and wherein the expression is reduced.
• CD274 (PDL-1) and CD273 (PDL-2) are reduced.
• the checkpoint molecule is reduced on effector T lymphocytes (CD4+ or CD8+).
• a method for altering expression of antigen presenting complexes in a subject with chronic lymphocytic leukemia in need thereof by administering to the subject a therapeutically effective amount of a histone deacetylase (HDAC) inhibitor, or a combination comprising a HDAC inhibitor and a Bruton's tyrosine kinase (BTK) inhibitor.
• HDAC histone deacetylase
• BTK Bruton's tyrosine kinase
• the presenting protein is MHC I or MHC II, and wherein the expression is increased.
• MHC II expression is increased.
• a method for reducing circulating regulatory T lymphocytes (Tregs) in a subject with chronic lymphocytic leukemia in need thereof by administering to the subject a therapeutically effective amount of a histone deacetylase (HDAC) inhibitor, or a combination comprising a HDAC inhibitor and a Bruton's tyrosine kinase (BTK) inhibitor.
• HDAC histone deacetylase
• BTK Bruton's tyrosine kinase
• a method for reducing IL-10 expression in chronic lymphocytic leukemia cells in a subject with chronic lymphocytic leukemia in need thereof by administering to the subject a therapeutically effective amount of a histone deacetylase (HDAC) inhibitor, or a combination comprising a HDAC inhibitor and a
• HDAC histone deacetylase
• BTK Bruton's tyrosine kinase
• a ninth aspect disclosed herein is a method for decreasing cell proliferation of chronic lymphocytic leukemia cells in a subject with chronic lymphocytic leukemia in need thereof by administering to the subject a therapeutically effective amount of a histone deacetylase (HDAC) inhibitor, or a combination comprising a HDAC inhibitor and a
• HDAC histone deacetylase
• BTK Bruton's tyrosine kinase
• lymphocytes in a subject with chronic lymphocytic leukemia (CLL) in need thereof by administering to the subject a therapeutically effective amount of a histone deacetylase (HDAC) inhibitor, or a combination comprising a HDAC inhibitor and a Bruton's tyrosine kinase (BTK) inhibitor.
• HDAC histone deacetylase
• BTK Bruton's tyrosine kinase
• a method for treating chronic lymphocytic leukemia (CLL) in a subject in need thereof, wherein the subject was previously ineffectively treated for CLL with a histone deacetylase 6 (HDAC6) selective inhibitor comprising administering to the subject a therapeutically effective amount of a HDAC6 selective inhibitor and a therapeutically effective amount of a Bruton's tyrosine kinase (BTK) inhibitor.
• CLL chronic lymphocytic leukemia
• HDAC6 histone deacetylase 6
• a method for treating chronic lymphocytic leukemia (CLL) in a subject in need thereof, wherein the subject was previously ineffectively treated for CLL with a Bruton's tyrosine kinase (BTK) inhibitor comprising administering to the subject a therapeutically effective amount of a histone deacetylase 6 (HDAC6) selective inhibitor and a therapeutically effective amount of a BTK inhibitor.
• CLL chronic lymphocytic leukemia
• BTK Bruton's tyrosine kinase
• the HDAC inhibitor is a HDAC6 selective inhibitor.
• the HDAC6 selective inhibitor is a compound of Formula I:
• ring B is aryl or heteroaryl
• Ri is an aryl or heteroaryl, each of which may be optionally substituted by OH, halo, or Ci -6- alkyl;
• R is H or Ci-6-alkyl.
• the compound of Formula I is Compound A:
• the compound of Formula I is Compound B:
• the HDAC6 selective inhibitor is a compound of Formula
• R x and R y together with the carbon to which each is attached, form a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl;
• each R A is independently Ci -6- alkyl, Ci -6- alkoxy, halo, OH, -N0 2 , -CN, or - H 2 ; and m is 0, 1, or 2.
• the compound of Formula II is Compound C:
• the compound of Formula II is Compound D:
• the method further comprises administering to the subject a therapeutically effective amount of a Bruton's tyrosine kinase (BTK) inhibitor, such as ibrutinib or a pharmaceutically acceptable salt thereof.
• BTK Bruton's tyrosine kinase
• the HDAC6 selective inhibitor can be administered at a sub -therapeutic dose.
• FIGS 1A-1B show HDAC6 overexpression in human primary B-CLL and altered cell viability in CLL cell lines with modulation of HDAC6 expression.
• Fig. 1A shows results of qRT-PCR analysis of HDAC6 expression in twenty B-CLL with Rai stages ranging from 0-4.
• Fig. IB shows viability of CLL cell line Mecl in this experiment as determined using MTS CellTiter 96® (Promega) analysis.
• Left side of Fig. IB Viability compared to non- targeted control counterpart of two polyclonal HDAC6 knock-down (HDAC6KD) cell lines.
• Right side of Fig. IB viability of Mecl cells transfected with increasing concentrations of HDAC6 over-expressing (H60E) plasmid, and viability of Mecl cells treated with two HDAC6 inhibitors at various doses.
• H60E HDAC6 over-expressing
• Figures 2A-2B show cell death measured using cellTox assay SyTox® Green (Promega) of OSU-CLL cells treated with Compound A (Fig. 2A) or Compound D (Fig. 2B) for 48 hours.
• Fig. 2C shows production of IL-10 by Mecl cells treated with Compound A for 24 hours and stimulated with LPS.
• Figures 3A-3C show synergy plots of cell viability determined using the CellTiter- Blue (Promega) assay in Mec2 cells (Fig. 3A) and OSU-CLL (Fig. 3B) cells after a 72 hour treatment with Compound A and ibrutinib. The results were analyzed for synergism using the combination index (CI) method developed by Chou & Talalay (Chou TC, Cancer research 2010; 70(2): 440-6).
• Fig. 3C shows Western blot results of PARP and pERK in Mecl cells treated with Compound A at varying doses (0.5 & ⁇ ⁇ ) alone or in combination with ibrutinib (0.2 & ⁇ ⁇ ) for 24hrs.
• Figures 4A-4E show disease burden and survival advantage with biological or pharmacological manipulation of HDAC6.
• Fig. 4A shows percent survival results from eight mice from eu-TCLl and euTCLl/HDAC6KO groups that were aged up to 300 days (the day last euTCLl expired). euTCLl mice succumb to disease starting day 200 while all eu- TCL1/HDAC6KO mice survived. Spleens from these mice were measured post-mortem and compared, (p value ⁇ 0.0005***).
• Fig. 4A shows percent survival results from eight mice from eu-TCLl and euTCLl/HDAC6KO groups that were aged up to 300 days (the day last euTCLl expired). euTCLl mice succumb to disease starting day 200 while all eu- TCL1/HDAC6KO mice survived. Spleens from these mice were measured post-mortem and compared, (p value ⁇ 0.0005***).
• Fig. 4A shows percent
• FIG. 4B shows the disease burden survival advantage of eight C57BL/6 mice adoptively transplanted with 5 x 10 6 splenocytes euTCLl (or euTCLl- HDAC6KO mice) over euTCLl splenocyte injected cohort.
• Fig. 4C shows disease burden in mice treated with Compound D in an aging model of CLL.
• Fig. 4D shows overall survival (Top) and disease burden (Bottom) of mice receiving Compound D treatment in adoptive transfer experiment model of CLL.
• Fig. 4E shows overall survival of mice treated with Compound D in an aging model of CLL.
• Figures 5A-5G show that inhibition of HDAC6 in vivo and in vitro alters the phenotypic profile of B- and T- cell compartment in CLL.
• Fig. 5A shows expression of PDL-1 and PDL-2 was reduced and the expression of MHC II was increased in HDAC6KD Mecl cells when compared to non-target control counterpart.
• Fig. 5B shows flow cytometry analysis demonstrating normalization of PD-1 and LAG3 on CD4+ T-cells in mice receiving Compound D treatment in the chow (week 10 post adoptive transfer).
• Fig. 5C shows flow cytometry analysis demonstrating number of T-regs in these mice.
• Fig. 5A shows expression of PDL-1 and PDL-2 was reduced and the expression of MHC II was increased in HDAC6KD Mecl cells when compared to non-target control counterpart.
• Fig. 5B shows flow cytometry analysis demonstrating normalization of PD-1 and LAG3 on CD4+ T-cells in mice receiving Com
• FIG. 5D shows the expression of PD-1 and LAG3 molecules on T-regs in mice under the various treatments.
• Fig. 5E shows a decrease in expression of PD-L1 on the B cells isolated from these treated mice with Compound D when compared to the none-treated animals.
• Fig. 5F shows mice adoptively transferred with euTCLl splenocytes and treated with Compound D (Feed) and Compound A (injection) exhibit lower expression of coinhibitory molecules PD-1 and LAG- 3 (10 weeks post adoptive transfer).
• Fig. 5G shows mice treated with Compound D feed daily in an aging model of CLL exhibit lower PD-1 and LAG-3 expression on T-regs.
• Figures 6A-6B show disease burden and survival advantage of mice treated with a combination of Compound D and Ibrutinib.
• Fig. 6A shows mice adoptively transferred with euTCLl splenocytes and treated with Compound D (Feed) and/or Ibrutinib (Drinking water) as well as combination treatment with both reagents. The data shows a significant decrease in the number of malignant cells in the combinatorial cohort when compared to mice treated with Compound D alone.
• Fig. 6B shows mice adoptively transferred with euTCLl splenocytes and treated with Compound D (Feed) and/or Ibrutinib (Drinking water) as well as combination treatment with both reagents.
• HDAC inhibitors are directed, generally, to HDAC inhibitors, combinations comprising a histone deacetylase (HDAC) inhibitor and a Bruton's tyrosine kinase (BTK) inhibitor, and methods for the treatment of chronic lymphocytic leukemia.
• HDAC histone deacetylase
• BTK Bruton's tyrosine kinase
• HDAC6 HDAC6-oxide-semiconductor
• selective HDAC6 inhibitors modify the expression of immunomodulatory molecules, which can ultimately lead to increases in the immunogenicity of CLL.
• CLL cells treated with HDAC6 inhibitors show 1) a dose-dependent cell kill, 2) a reduction of IL-10 - an important cytokine in the regulation of cell proliferation in CLL, and 3) synergistic reduction of CLL cell viability when combined with the BTK inhibitor ibrutinib.
• MEC2-HDAC6KD cells exhibit an increase in MHCII and a decrease in PD-Ll expression.
• alkyl refers to saturated, straight- or branched-chain hydrocarbon moieties containing, in certain embodiments, between one and six, or one and eight carbon atoms, respectively.
• Examples of Ci- 6 -alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, «-butyl, tert-butyl, neopentyl, n-hexyl moieties; and examples of Ci- 8 -alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, «-butyl, tert-butyl, neopentyl, n-hexyl, heptyl, and octyl moieties.
• the number of carbon atoms in an alkyl substituent can be indicated by the prefix
• C x - y where x is the minimum and y is the maximum number of carbon atoms in the substituent.
• a C x chain means an alkyl chain containing x carbon atoms.
• alkoxy refers to an -O-alkyl moiety.
• cycloalkyl or "cycloalkylene” denote a monovalent group derived from a monocyclic or polycyclic saturated or partially unsaturated carbocyclic ring compound.
• C 3 - 8 -cycloalkyl examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl; and examples of C 3 -Ci 2 -cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [2.2.1] heptyl, and bicyclo [2.2.2] octyl.
• monovalent groups derived from a monocyclic or polycyclic carbocyclic ring compound having at least one carbon- carbon double bond by the removal of a single hydrogen atom include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like.
• aryl refers to a mono- or poly-cyclic carbocyclic ring system having one or more aromatic rings, fused or non-fused, including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl, and the like.
• aryl groups have 6 carbon atoms.
• aryl groups have from six to ten carbon atoms.
• aryl groups have from six to sixteen carbon atoms.
• heteroaryl refers to a mono- or poly-cyclic (e.g., bi-, or tri-cyclic or more) fused or non-fused moiety or ring system having at least one aromatic ring, where one or more of the ring-forming atoms is a heteroatom such as oxygen, sulfur, or nitrogen.
• the heteroaryl group has from about one to six carbon atoms, and in further embodiments from one to fifteen carbon atoms.
• the heteroaryl group contains five to sixteen ring atoms of which one ring atom is selected from oxygen, sulfur, and nitrogen; zero, one, two, or three ring atoms are additional heteroatoms independently selected from oxygen, sulfur, and nitrogen; and the remaining ring atoms are carbon.
• Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, acridinyl, and the like.
• halo refers to a halogen, such as fluorine, chlorine, bromine, and iodine.
• combination refers to two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such combination of therapeutic agents may be in the form of a single pill, capsule, or intravenous solution. However, the term “combination” also encompasses the situation when the two or more therapeutic agents are in separate pills, capsules, or intravenous solutions. Likewise, the term “combination therapy” refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure.
• Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple, or in separate containers (e.g., capsules) for each active ingredient.
• administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein.
• composition refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier.
• the pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
• pharmaceutically acceptable carrier means a
• composition or carrier such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function.
• a pharmaceutically acceptable material, composition or carrier such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function.
• a pharmaceutically acceptable material, composition or carrier such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function.
• Such constructs are carried or transported from one
• materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil;
• glycols such as propylene glycol
• polyols such as glycerin, sorbitol, mannitol and polyethylene glycol
• esters such as ethyl oleate and ethyl laurate
• agar buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen- free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.
• pharmaceutically acceptable carrier also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions.
• pharmaceutically acceptable carrier may further include a
• HDAC histone deacetylases
• HDAC2 histone deacetylases
• HDAC3 histone deacetylase 8
• HDAC4 histone deacetylase 8
• HDAC5 histone deacetylase 8
• HDAC9 histone deacetylase 9
• HDAC 10 histone deacetylase 10
• HDAC6 selective means that the compound binds to HDAC6 to a substantially greater extent, such as, e.g., 5X, 10X, 15X, 20X greater or more, than to any other type of HDAC enzyme, such as HDACl or HDAC2. That is, the compound is selective for HDAC6 over any other type of HDAC enzyme.
• a compound that binds to HDAC 6 with an IC 50 of 10 nM and to HDACl with an IC 50 of 50 nM is HDAC 6 specific.
• a compound that binds to HDAC6 with an IC 50 of 50 nM and to HDAC1 with an IC 50 of 60 nM is not HDAC6 specific
• inhibitor is synonymous with the term antagonist.
• HDAC Histone deacetylase
• kits and pharmaceutical combinations for the treatment of chronic lymphocytic leukemia in a subject in need thereof are also provided herein. Also provided herein are methods for treating chronic lymphocytic leukemia in a subject in need thereof.
• the compounds, combinations, and methods disclosed herein comprise a histone deacetylase (HDAC) inhibitor.
• the HDAC inhibitor may be any HDAC inhibitor.
• the HDAC inhibitor may be selective or non-selective to a particular type of histone deacetylase enzyme.
• the HDAC inhibitor is a selective HDAC inhibitor. More preferably, the HDAC inhibitor is an HDAC6 selective inhibitor.
• the HDAC6 selective inhibitor is a compound of Formula I:
• ring B is aryl or heteroaryl
• Ri is an aryl or heteroaryl, each of which may be optionally substituted by OH, halo, or Ci -6- alkyl;
• R is H or Ci-6-alkyl.
• Representative compounds of Formula I include, but are not limited to Compounds A (ricolinostat) and B, or pharmaceutically acceptable salts thereof.
• the HDAC6 selective inhibitor is a compound of Formula II:
• R X and R Y together with the carbon to which each is attached, form a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl;
• each RA is independently Ci- 6 -alkyl, Ci- 6 -alkoxy, halo, OH, -N0 2 , -CN, or -NH 2 ; and m is 0, 1, or 2.
• the compounds described herein are unsolvated. In other embodiments, one or more of the compounds are in solvated form.
• the solvate can be any of pharmaceutically acceptable solvent, such as water, ethanol, and the like.
• “Pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form.
• pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
• Pharmaceutically acceptable salts include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
• salts can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.
• BTK Bruton 's tyrosine kinase
• Some embodiments comprise the use of a BTK inhibitor. Some embodiments of the methods also comprise a BTK inhibitor.
• the BTK inhibitor may be any BTK inhibitor.
• the BTK inhibitor is ibrutinib.
• Bruton' s tyrosine kinase inhibitor and “BTK inhibitor” refer to any compound that reduces a catalytic activity of Bruton' s tyrosine kinase (BTK), or homolog thereof, and thereby reduces BTK-mediated signaling.
• BTK Bruton's tyrosine kinase
• BTK Bruton's tyrosine kinase
• Bruton's tyrosine kinase homolog refers to orthologs of Bruton's tyrosine kinase, e.g., the orthologs from mouse (GenBank Accession No. AAB47246), dog (GenBank Accession No. XP-549139.), rat (GenBank Accession No. NP-001007799), chicken
• BTK-mediated signaling means any of the biological activities that are dependent on, either directly or indirectly, the activity of BTK.
• Examples of BTK-mediated signaling are signals that lead to proliferation and survival of BTK-expressing cells, and stimulation of one or more BTK-signaling pathways within BTK-expressing cells.
• a BTK "signaling pathway” or “signal transduction pathway” is intended to mean at least one biochemical reaction, or a group of biochemical reactions, that results from the activity of BTK, and which generates a signal that, when transmitted through the signal pathway, leads to activation of one or more downstream molecules in the signaling cascade.
• Signal transduction pathways involve a number of signal transduction molecules that lead to transmission of a signal from the cell-surface across the plasma membrane of a cell, and through one or more in a series of signal transduction molecules, through the cytoplasm of the cell, and in some instances, into the cell's nucleus.
• BTK signal transduction pathways ultimately regulate (either enhance or inhibit) the activation of NF- ⁇ via the NF-KB signaling pathway.
• a BTK inhibitor can be an antagonist anti-BTK antibody.
• the antagonist anti-BTK antibody is free of significant agonist activity in one cellular response.
• the antagonist anti-BTK antibody is free of significant agonist activity in assays of more than one cellular response ⁇ e.g., proliferation and differentiation, or proliferation, differentiation, and, for B cells, antibody production).
• the BTK inhibitor can be either a reversible or irreversible small molecule inhibitor (recently reviewed by D'Cruz et al., OncoTargets and Therapy 2013 :6 161-176).
• irreversible BTK inhibitor refers to an inhibitor of BTK that can form a covalent bond with an amino acid residue of BTK that results in a reduction of BTK signaling activity.
• the irreversible inhibitor of BTK can form a covalent bond with a Cys residue of BTK; in particular embodiments, the irreversible inhibitor can form a covalent bond with a Cys 481 residue (or a homolog thereof) of BTK.
• irreversible BTK inhibitors include, but are not limited to, for example, ibrutinib/PCI-32765 (see structure below and U.S. Patent No. 8,088,781), CNX-774, CC-292, AVL-101, and AVL-291/292.
• reversible BTK inhibitor refers to an inhibitor of BTK that reversibly binds to BTK to reduce BTK signaling activity.
• reversible BTK inhibitors include, but are not limited to Dasatinib (Sprycel/BMS-354825, Bristol-Myers Squibb) [N-(2- chloro-6-methylphenyl)-2-(6-(4-(2-hydroxy ethyl) piperazin- 1 -yl)-2-methylpyrimidin-4- lamino)thiazole5-carboxamide LFM-A13, ONO-WG-307, RN-486, and GDC-0834.
• the compounds described herein are unsolvated. In other embodiments, one or more of the compounds are in solvated form.
• the solvate can be any of pharmaceutically acceptable solvent, such as water, ethanol, and the like.
• compositions and combinations for the treatment of chronic lymphocytic leukemia in a subject in need thereof are provided herein.
• HDAC inhibitors or combinations comprising a histone deacetylase (HDAC) inhibitor and a Bruton's tyrosine kinase (BTK ) inhibitor for the treatment of chronic lymphocytic leukemia in a subject in need thereof.
• HDAC histone deacetylase
• BTK Bruton's tyrosine kinase
• HDAC inhibitors or combinations comprising a histone deacetylase (HDAC) inhibitor and a Bruton's tyrosine kinase (BTK) inhibitor for the treatment of chronic lymphocytic leukemia in a subject in need thereof, wherein the combination is administered at dosages that would not be effective when one or both of the compounds are administered alone, but which amounts are effective in combination.
• HDAC histone deacetylase
• BTK Bruton's tyrosine kinase
• the HDAC inhibitor is an HDAC6 selective inhibitor.
• the HDAC6 selective inhibitor is a compound of Formula I:
• the compound of Formula I is Compound B:
• the HDAC6 selective inhibitor is a compound of
• the compound of Formula II is Compound D:
• the Bruton's tyrosine kinase (BTK) inhibitor is ibrutinib:
• a combination therapy comprising an HDAC6 selective inhibitor and a Bruton's tyrosine kinase (BTK) inhibitor, wherein the HDAC6 selective inhibitor is a compound of Formula I:
• ring B is aryl or heteroaryl
• Ri is an aryl or heteroaryl, each of which may be optionally substituted by
• R is H or Ci -6- alkyl
• the BTK inhibitor is any BTK inhibitor.
• the HDAC6 selective inhibitor is:
• the BTK inhibitor is ibrutinib or a pharmaceutically acceptable salt thereof.
• the combination comprises Compound A:
• the BTK inhibitor is ibrutinib or a pharmaceutically acceptable salt thereof.
• the combination comprises Compound B:
• the BTK inhibitor is ibrutinib or a pharmaceutically acceptable salt thereof.
• a combination therapy comprising an HDAC6 selective inhibitor and a Bruton's tyrosine kinase (BTK) inhibitor, wherein the HDAC6 selective inhibitor is a compound of Formula II:
• R x and R y together with the carbon to which each is attached, form a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl; each R A is independently Ci -6- alkyl, Ci -6- alkoxy, halo, OH, -N0 2 , -CN, or - H 2 ; and
• n 0, 1, or 2;
• the BTK inhibitor any BTK inhibitor.
• the HDAC6 selective inhibitor is:
• the BTK inhibitor is ibrutinib or a pharmaceutically acceptable salt thereof, embodiment, the combination comprises Compound D:
• the BTK inhibitor is ibrutinib or a pharmaceutically acceptable salt thereof.
• the pharmaceutical combination comprises at least one compound of Formula I and a BTK inhibitor.
• the pharmaceutical combination comprises at least one compound of Formula II and a BTK inhibitor.
• the pharmaceutical combination comprises at least Compound A and a BTK inhibitor.
• the pharmaceutical combination comprises at least Compound B and a BTK inhibitor.
• the pharmaceutical combination comprises at least Compound C and a BTK inhibitor.
• the pharmaceutical combination comprises at least Compound D and a BTK inhibitor.
• the pharmaceutical combination comprises at least one compound of Formula I and ibrutinib.
• the pharmaceutical combination comprises at least one compound of Formula II and ibrutinib.
• the pharmaceutical combination comprises at least Compound A and ibrutinib.
• the pharmaceutical combination comprises at least Compound B and ibrutinib.
• the pharmaceutical combination comprises at least Compound C and ibrutinib.
• the pharmaceutical combination comprises at least Compound D and ibrutinib.
• the pharmaceutical composition comprises at least one compound of Formula I and a BTK inhibitor.
• the pharmaceutical composition comprises at least one compound of
• the pharmaceutical composition comprises at least Compound A and a BTK inhibitor.
• the pharmaceutical composition comprises at least Compound B and a BTK inhibitor.
• the pharmaceutical composition comprises at least Compound C and a BTK inhibitor.
• the pharmaceutical composition comprises at least Compound D and a BTK inhibitor. In embodiments, the pharmaceutical composition comprises at least one compound of Formula I and ibrutinib.
• the pharmaceutical composition comprises at least one compound of Formula II and ibrutinib
• the pharmaceutical composition comprises at least Compound A and ibrutinib.
• the pharmaceutical composition comprises at least Compound B and ibrutinib.
• the pharmaceutical composition comprises at least Compound C and ibrutinib.
• the pharmaceutical composition comprises at least Compound D and ibrutinib.
• the HDAC inhibitor (a compound of Formula I or II) is administered simultaneously with the Bruton's tyrosine kinase (BTK) inhibitor.
• BTK Bruton's tyrosine kinase
• Simultaneous administration typically means that both compounds enter the patient at precisely the same time.
• simultaneous administration also includes the possibility that the HDAC inhibitor and the Bruton's tyrosine kinase (BTK) inhibitor enter the patient at different times, but the difference in time is sufficiently miniscule that the first administered compound is not provided the time to take effect on the patient before entry of the second administered compound.
• Such delayed times typically correspond to less than 1 minute, and more typically, less than 30 seconds.
• simultaneous administration can be achieved by administering a solution containing the combination of compounds.
• simultaneous administration of separate solutions one of which contains the HDAC inhibitor and the other of which contains the Bruton's tyrosine kinase (BTK) inhibitor
• simultaneous administration can be achieved by administering a composition containing the combination of compounds.
• simultaneous administration can be achieved by administering two separate compositions, one comprising the HDAC inhibitor and the other comprising the Bruton's tyrosine kinase (BTK) inhibitor.
• the HDAC inhibitor and the Bruton's tyrosine kinase (BTK) inhibitor are not administered simultaneously.
• the HDAC inhibitor is administered before the Bruton's tyrosine kinase (BTK) inhibitor.
• the Bruton's tyrosine kinase (BTK) inhibitor is administered before the HDAC inhibitor.
• the time difference in non-simultaneous administrations can be greater than 1 minute, five minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, two hours, three hours, six hours, nine hours, 12 hours, etc.
• the first administered compound is provided time to take effect on the patient before the second administered compound is administered. Generally, the difference in time does not extend beyond the time for the first administered compound to complete its effect in the patient, or beyond the time the first administered compound is completely or substantially eliminated or deactivated in the patient.
• one or both of the HDAC inhibitor and the Bruton's tyrosine kinase (BTK) inhibitor are administered in a therapeutically effective amount or dosage.
• a “therapeutically effective amount” is an amount of HDAC6 selective inhibitor (a compound of Formula I or II) or a Bruton's tyrosine kinase (BTK) inhibitor that, when administered to a patient by itself, effectively treats the chronic lymphocytic leukemia.
• An amount that proves to be a "therapeutically effective amount" in a given instance, for a particular subject may not be effective for 100% of subjects similarly treated for the disease or condition under consideration, even though such dosage is deemed a "therapeutically effective amount" by skilled practitioners.
• the amount of the compound that corresponds to a therapeutically effective amount is strongly dependent on the type of cancer, stage of the cancer, the age of the patient being treated, and other facts.
• therapeutically effective amounts of these compounds are well-known in the art, such as provided in the supporting references cited above.
• one or both of the HDAC inhibitor and the Bruton's tyrosine kinase (BTK) inhibitor are administered in a sub-therapeutically effective amount or dosage.
• a sub-therapeutically effective amount is an amount of HDAC inhibitor (for example, a compound of Formula I or II) or a Bruton's tyrosine kinase (BTK) inhibitor that, when administered to a patient by itself, does not completely inhibit over time the biological activity of the intended target.
• the combination of the HDAC inhibitor and the Bruton's tyrosine kinase (BTK) inhibitor should be effective in treating chronic lymphocytic leukemia.
• a sub-therapeutic amount of the Bruton's tyrosine kinase (BTK) inhibitor can be an effective amount if, when combined with a compound a compound of Formula I or II (HDAC6 selective inhibitor), the combination is effective in the treatment of chronic lymphocytic leukemia.
• the combination of compounds exhibits a synergistic effect (i.e., greater than additive effect) in the treatment of the chronic lymphocytic leukemia.
• synergistic effect refers to the action of two agents, such as, for example, a HDAC inhibitor and a Bruton's tyrosine kinase (BTK) inhibitor, producing an effect, for example, slowing the symptomatic progression of cancer or symptoms thereof, which is greater than the simple addition of the effects of each drug administered alone.
• a synergistic effect can be calculated, for example, using suitable methods such as the Sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)), the equation of Loewe additivity (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol.
• the combination of compounds exhibits a synergistic effect (i.e., greater than additive effect) in the treatment of chronic lymphocytic leukemia (CLL).
• CLL chronic lymphocytic leukemia
• the combination of compounds can inhibit cancer growth, achieve cancer stasis, or even achieve substantial or complete cancer regression.
• the amounts of a HDAC inhibitor and a Bruton's tyrosine kinase (BTK) inhibitor should result in the effective treatment of the chronic lymphocytic leukemia
• the amounts, when combined, are preferably not excessively toxic to the patient (i.e., the amounts are preferably within toxicity limits as established by medical guidelines).
• a limitation on the total administered dosage is provided.
• the amounts considered herein are per day; however, half-day and two- day or three-day cycles also are considered herein.
• a daily dosage such as any of the exemplary dosages described above, is administered once, twice, three times, or four times a day for three, four, five, six, seven, eight, nine, or ten days.
• a shorter treatment time e.g., up to five days
• a longer treatment time e.g., ten or more days, or weeks, or a month, or longer
• a once- or twice-daily dosage is administered every other day.
• each dosage contains both an HDAC inhibitor and a Bruton's tyrosine kinase (BTK) inhibitor to be delivered as a single dosage, while in other
• each dosage contains either a HDAC inhibitor and a Bruton's tyrosine kinase (BTK) inhibitor to be delivered as separate dosages.
• BTK Bruton's tyrosine kinase
• Compounds of Formula I or II, or their pharmaceutically acceptable salts or solvate forms, in pure form or in an appropriate pharmaceutical composition, can be administered via any of the accepted modes of administration or agents known in the art.
• the compounds can be administered, for example, orally, nasally, parenterally (intravenous, intramuscular, or subcutaneous), topically, transdermally, intravaginally, intravesically, intracistemally, or rectally.
• the dosage form can be, for example, a solid, semi-solid, lyophilized powder, or liquid dosage forms, such as for example, tablets, pills, soft elastic or hard gelatin capsules, powders, solutions, suspensions, suppositories, aerosols, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.
• a solid, semi-solid, lyophilized powder, or liquid dosage forms such as for example, tablets, pills, soft elastic or hard gelatin capsules, powders, solutions, suspensions, suppositories, aerosols, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.
• administration is oral, particularly one in which a convenient daily dosage regimen can be adjusted according to the degree of severity of the disease to be treated.
• the HDAC inhibitor and the BTK inhibitor of the pharmaceutical combination can be administered in a single unit dose or separate dosage forms.
• the phrase "pharmaceutical combination” includes a combination of two drugs in either a single dosage form or a separate dosage forms, i.e., the pharmaceutically acceptable carriers and excipients described throughout the application can be combined with an HDAC inhibitor and a BTK inhibitor in a single unit dose, as well as individually combined with a HDAC inhibitor and a BTK inhibitor when these compounds are administered separately.
• Auxiliary and adjuvant agents may include, for example, preserving, wetting, suspending, sweetening, flavoring, perfuming, emulsifying, and dispensing agents.
• antibacterial and antifungal agents such as, parabens, chlorobutanol, phenol, sorbic acid, and the like.
• Isotonic agents such as sugars, sodium chloride, and the like, may also be included.
• Prolonged absorption of an injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
• the auxiliary agents also can include wetting agents, emulsifying agents, pH buffering agents, and antioxidants, such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene, and the like.
• Solid dosage forms can be prepared with coatings and shells, such as enteric coatings and others well-known in the art. They can contain pacifying agents and can be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedded compositions that can be used are polymeric substances and waxes. The active compounds also can be in
• microencapsulated form if appropriate, with one or more of the above-mentioned excipients.
• Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. Such dosage forms are prepared, for example, by dissolving, dispersing, etc., the HDAC inhibitors or Bruton's tyrosine kinase (BTK) inhibitors described herein, or a pharmaceutically acceptable salt thereof, and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like; solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethyl formamide; oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame
• the pharmaceutically acceptable compositions will contain about 1% to about 99% by weight of the compounds described herein, or a pharmaceutically acceptable salt thereof, and 99% to 1% by weight of a pharmaceutically acceptable excipient.
• the composition will be between about 5% and about 75% by weight of a compound described herein, or a pharmaceutically acceptable salt thereof, with the rest being suitable pharmaceutical excipients.
• kits for treating chronic lymphocytic leukemia in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an HDAC inhibitor, such as an HDAC6 selective inhibitor, or a combination comprising an HDAC inhibitor, such as a HDAC6 selective inhibitor, and a Bruton's tyrosine kinase (BTK) inhibitor.
• an HDAC inhibitor such as an HDAC6 selective inhibitor
• a combination comprising an HDAC inhibitor such as a HDAC6 selective inhibitor
• BTK Bruton's tyrosine kinase
• the subject is typically a human. However, the subject can be any mammal for which treatment is desired. Thus, the methods described herein can be applied to both human and veterinary applications.
• treating or “treatment” indicate that the method has, at the least, mitigated abnormal cellular proliferation.
• the method can reduce the rate of cellular growth in a patient, or prevent the continued growth or spread of chronic
• lymphocytic leukemia or even reduce the overall reach of the chronic lymphocytic leukemia. Inhibition of abnormal cell growth can occur by a variety of mechanisms including, but not limited to, cell death, apoptosis, arrest of mitosis, inhibition of cell division, transcription, translation, transduction, etc.
• provided herein is a method for treating chronic lymphocytic leukemia in a subject in need thereof comprising administering to the subject a
• a method for treating chronic lymphocytic leukemia in a subject in need thereof comprising administering to the subject a
• lymphocytic leukemia in a subject in need thereof comprising administering to the subject a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof.
• provided herein is a method for treating chronic lymphocytic leukemia in a subject in need thereof comprising administering to the subject a
• provided herein is a method for treating chronic lymphocytic leukemia in a subject in need thereof comprising administering to the subject a
• provided herein is a method for treating chronic lymphocytic leukemia in a subject in need thereof comprising administering to the subject a
• provided herein is a method for treating chronic lymphocytic leukemia in a subject in need thereof comprising administering to the subject a
• provided herein is a method for treating chronic lymphocytic leukemia in a subject in need thereof comprising administering to the subject a
• provided herein is a method for treating chronic lymphocytic leukemia in a subject in need thereof comprising administering to the subject a
• provided herein is a method for treating chronic lymphocytic leukemia in a subject in need thereof comprising administering to the subject a
• a method for treating chronic lymphocytic leukemia in a subject in need thereof comprising administering to the subject a
• provided herein is a method for treating chronic lymphocytic leukemia in a subject in need thereof comprising administering to the subject a
• cell viability of chronic lymphocytic leukemia cells are decreased in a subject with chronic lymphocytic leukemia by administering an HDAC inhibitor, or a combination comprising an HDAC inhibitor and a BTK inhibitor, to the subject.
• the HDAC inhibitor is an HDAC6 selective inhibitor.
• the HDAC6 selective inhibitor can comprise one of the Compounds A-D.
• the BTK inhibitor is ibrutinib.
• apoptosis of chronic lymphocytic leukemia cells is synergistically increased by administering to a subject with chronic lymphocytic leukemia a combination comprising an HDAC inhibitor and a BTK inhibitor.
• the HDAC inhibitor is an HDAC6 selective inhibitor.
• the HDAC6 selective inhibitor can comprise one of the
• the BTK inhibitor is ibrutinib.
• an inhibitory checkpoint molecule in a T- and/or B-cell compartment in a subject with chronic lymphocytic leukemia is altered by
• these checkpoint molecules are selected from the group consisting of CD274 (PDL-1), CD273 (PDL-2), CD80 (B7-1), CD86 (B7-2), CD 152 (CTLA4), CD275 (B7RP1), CD276 (B7-H3), B7-H4 (VTCN1), CD270 (HVEM), BLTA, GAL9, CD366 (TEVI3), A2aR, CD279 (PD-1), KIR, and CD223 (LAG3).
• these checkpoint molecules are CD274 (PDL-1) and CD273 (PDL-2).
• the checkpoint molecule is reduced on regulatory T lymphocytes (Tregs) in yet further embodiments.
• the HDAC inhibitor is an HDAC6 selective inhibitor.
• the HDAC6 selective inhibitor can comprise one of the Compounds A-D.
• the BTK inhibitor is ibrutinib.
• the expression of an antigen presenting complex is altered in a subject with chronic lymphocytic leukemia by administering to the subject a therapeutically effective amount of a HDAC inhibitor, or a combination comprising a HDAC inhibitor and a BTK inhibitor.
• the antigen presenting complex molecule is MHC I or MHC II, and expression is increased. In further embodiments, MHC II is increased.
• the HDAC inhibitor is an HDAC6 selective inhibitor.
• the HDAC6 selective inhibitor can comprise one of the Compounds A-D.
• the BTK inhibitor is ibrutinib.
• circulating regulatory T lymphocytes are reduced in a subject with chronic lymphocytic leukemia by administering to the subject a therapeutically effective amount of a HDAC inhibitor, or a combination comprising a HDAC inhibitor and a BTK inhibitor.
• the HDAC inhibitor is an HDAC6 selective inhibitor.
• the HDAC6 selective inhibitor can comprise one of the Compounds A-D.
• the BTK inhibitor is ibrutinib.
• IL-10 expression is reduced in chronic lymphocytic leukemia cells in a subject with chronic lymphocytic leukemia by administering to the subject a therapeutically effective amount of a HDAC inhibitor, or a combination comprising a HDAC inhibitor and a BTK inhibitor.
• the HDAC inhibitor is an HDAC6 selective inhibitor.
• the HDAC6 selective inhibitor can comprise one of the Compounds A-D.
• the BTK inhibitor is ibrutinib.
• cell proliferation is decreased in a subject with chronic lymphocytic leukemia by administering to the subject a therapeutically effective amount of a HDAC inhibitor, or a combination comprising a HDAC inhibitor and a BTK inhibitor.
• the HDAC inhibitor is an HDAC6 selective inhibitor.
• the HDAC6 selective inhibitor can comprise one of the Compounds A-D.
• the BTK inhibitor is ibrutinib.
• circulating tumor lymphocytes are reduced in a subject with chronic lymphocytic leukemia by administering to the subject a therapeutically effective amount of a HDAC inhibitor, or a combination comprising a HDAC inhibitor and a BTK inhibitor.
• the HDAC inhibitor is an HDAC6 selective inhibitor.
• the HDAC6 selective inhibitor can comprise one of the Compounds A-D.
• the BTK inhibitor is ibrutinib.
• a method for treating chronic lymphocytic leukemia in a subject wherein the subject was previously ineffectively treated for chronic lymphocytic leukemia with a HDAC6 selective inhibitor, the method comprising administering to the subject a therapeutically effective amount of a HDAC6 selective inhibitor and a therapeutically effective amount of a BTK inhibitor.
• the HDAC6 selective inhibitor can comprise one of the Compounds A-D.
• the BTK inhibitor can be ibrutinib.
• a method for treating chronic lymphocytic leukemia in a subject in need thereof, wherein the subject was previously ineffectively treated for chronic lymphocytic leukemia with a BTK inhibitor comprising administering to the subject a therapeutically effective amount of a HDAC6 selective inhibitor and a therapeutically effective amount of a BTK inhibitor.
• the HDAC6 selective inhibitor can comprise one of the Compounds A-D.
• the BTK inhibitor can be ibrutinib.
• a method of treating CLL in a subject in need thereof comprising administering to the patient a therapeutically effective amount of Formula I or Formula II, or a pharmaceutically acceptable salt thereof, and ibrutinib, or a
• Formula I or Formula II and ibrutinib are administered at dosages and over time intervals that produce a synergistic effect.
• provided herein is a method of treating CLL in a subject in need thereof comprising administering to the patient a therapeutically effective amount of
• Compound A or a pharmaceutically acceptable salt thereof, and ibrutinib, or a
• Compound A and ibrutinib are administered at dosages and over time intervals that produce a synergistic effect.
• provided herein is a method of treating CLL in a subject in need thereof comprising administering to the patient a therapeutically effective amount of
• Compound B or a pharmaceutically acceptable salt thereof, and ibrutinib, or a
• Compound B and ibrutinib are administered at dosages and over time intervals that produce a synergistic effect.
• provided herein is a method of treating CLL in a subject in need thereof comprising administering to the patient a therapeutically effective amount of
• Compound C and ibrutinib are administered at dosages and over time intervals that produce a synergistic effect.
• provided herein is a method of treating CLL in a subject in need thereof comprising administering to the patient a therapeutically effective amount of
• Compound D and ibrutinib are administered at dosages and over time intervals that produce a synergistic effect.
• a HDAC6 selective inhibitor and a BTK inhibitor for use in therapy.
• provided herein is a compound of Formula I and a BTK inhibitor for use in therapy. In an embodiment, provided herein is a compound of Formula II and a BTK inhibitor for use in therapy.
• a Compound A and a BTK inhibitor for use in therapy.
• a Compound B and a BTK inhibitor for use in therapy.
• a Compound C and a BTK inhibitor for use in therapy.
• a Compound D and a BTK inhibitor for use in therapy.
• a compound of Formula I and a BTK inhibitor for use in treating CLL in a subject in need thereof.
• a compound of Formula II and a BTK inhibitor for use in treating CLL in a subject in need thereof.
• a Compound A and a BTK inhibitor for use in treating CLL in a subject in need thereof.
• a Compound B and a BTK inhibitor for use in treating CLL in a subject in need thereof.
• a Compound C and a BTK inhibitor for use in treating CLL in a subject in need thereof.
• a Compound D and a BTK inhibitor for use in treating CLL in a subject in need thereof.
• an HDAC6 selective inhibitor for use in treating CLL in a subject in need thereof, wherein the HDAC6 selective inhibitor is for use in combination with a BTK inhibitor.
• a compound of Formula II for use in treating CLL in a subject in need thereof, wherein the compound of Formula I is for use in
• Compound A for use in treating CLL in a subject in need thereof, wherein Compound A is for use in combination with a BTK inhibitor.
• Compound B for use in treating CLL in a subject in need thereof, wherein Compound B is for use in combination with a BTK inhibitor.
• Compound C for use in treating CLL in a subject in need thereof, wherein Compound C is for use in combination with a BTK inhibitor.
• Compound D for use in treating CLL in a subject in need thereof, wherein Compound D is for use in combination with a BTK inhibitor.
• an HDAC6 selective inhibitor and a BTK inhibitor for use in combination therapy for treating CLL in a subject in need thereof, wherein the HDAC6 selective inhibitor and the BTK inhibitor are for concurrent, sequential or separate administration.
• a compound of Formula I and a BTK inhibitor for use in combination therapy for treating CLL in a subject in need thereof, wherein the compound of formula I and the BTK inhibitor are for concurrent, sequential or separate administration.
• a compound of Formula II and a BTK inhibitor for use in combination therapy for treating CLL in a subject in need thereof, wherein the compound of formula II and the BTK inhibitor are for concurrent, sequential or separate administration.
• Compound A and a BTK inhibitor for use in combination therapy for treating CLL in a subject in need thereof, wherein Compound A and the BTK inhibitor are for concurrent, sequential or separate administration.
• Compound B and a BTK inhibitor for use in combination therapy for treating CLL in a subject in need thereof, wherein Compound B and the BTK inhibitor are for concurrent, sequential or separate administration.
• Compound C and a BTK inhibitor for use in combination therapy for treating CLL in a subject in need thereof, wherein Compound C and the BTK inhibitor are for concurrent, sequential or separate administration.
• Compound D and a BTK inhibitor for use in combination therapy for treating CLL in a subject in need thereof, wherein Compound D and the BTK inhibitor are for concurrent, sequential or separate administration.
• the ratio of HDAC6 selective inhibitor to BTK inhibitor is in the range of 700: 1 - 1 AO.
• the ratio of HDAC6 selective inhibitor to BTK inhibitor is in the range of 2: 1 to 1 :2, for example, 2: 1, 1 : 1, or 1 :2; 170: 1 to 150: 1, for example, 170: 1, 160: 1 or 150: 1 ; 3 : 1 to 1 : 1, for example, 3 : 1, 2: 1 or 1 : 1 ; or 30: 1 to 10: 1, for example, 30: 1, 20: 1 or 10: 1.
• the ratio of Formula I to BTK inhibitor is in the range of 700: 1 - 1 AO.
• the ratio of Formula I to BTK inhibitor is in the range of 2: 1 to 1 :2, for example, 2:1, 1:1, or 1:2; 170:1 to 150:1, for example, 170:1, 160:1 or 150:1; 3:1 to 1:1, for example, 3:1, 2:1 or 1:1; or 30:1 to 10:1, for example, 30:1, 20:1 or 10:1.
• the ratio of Formula II to BTK inhibitor is in the range of 700: 1 - 1 AO. In another embodiment of the pharmaceutical combination, the ratio of Formula II to BTK inhibitor is in the range of 2: 1 to 1 :2, for example, 2:1, 1:1, or 1:2; 170:1 to 150:1, for example, 170:1, 160:1 or 150:1; 3:1 to 1:1, for example, 3:1, 2:1 or 1:1; or 30:1 to 10:1, for example, 30:1, 20:1 or 10:1.
• the ratio of Compound A to BTK inhibitor is in the range of 700: 1 - 1 AO. In another embodiment of the pharmaceutical combination, the ratio of Compound A to BTK inhibitor is in the range of 2: 1 to 1 :2, for example, 2:1, 1:1, or 1:2; 170:1 to 150:1, for example, 170:1, 160:1 or 150:1; 3:1 to 1:1, for example, 3:1, 2:1 or 1:1; or 30:1 to 10:1, for example, 30:1, 20:1 or 10:1.
• the ratio of Compound B to BTK inhibitor is in the range of 700: 1 - 1 AO. In another embodiment of the pharmaceutical combination, the ratio of Compound B to BTK inhibitor is in the range of 2: 1 to 1 :2, for example, 2:1, 1:1, or 1:2; 170:1 to 150:1, for example, 170:1, 160:1 or 150:1; 3:1 to 1:1, for example, 3:1, 2:1 or 1:1; or 30:1 to 10:1, for example, 30:1, 20:1 or 10:1.
• the ratio of Compound C to BTK inhibitor is in the range of 700: 1 - 1 AO. In another embodiment of the pharmaceutical combination, the ratio of Compound C to BTK inhibitor is in the range of 2: 1 to 1 :2, for example, 2:1, 1:1, or 1:2; 170:1 to 150:1, for example, 170:1, 160:1 or 150:1; 3:1 to 1:1, for example, 3:1, 2:1 or 1:1; or 30:1 to 10:1, for example, 30:1, 20:1 or 10:1.
• the ratio of Compound D to BTK inhibitor is in the range of 700: 1 - 1 AO. In another embodiment of the pharmaceutical combination, the ratio of Compound D to BTK inhibitor is in the range of 2: 1 to 1 :2, for example, 2:1, 1:1, or 1:2; 170:1 to 150:1, for example, 170:1, 160:1 or 150:1; 3:1 to 1:1, for example, 3:1, 2:1 or 1:1; or 30:1 to 10:1, for example, 30:1, 20:1 or 10:1.
• the ratio of Compound A to ibrutinib is in the range of 700: 1 - 1 AO. In another embodiment of the pharmaceutical combination, the ratio of Compound A to ibrutinib is in the range of 2: 1 to 1 :2, for example, 2:1, 1:1, or 1:2; 170:1 to 150:1, for example, 170:1, 160:1 or 150:1; 3:1 to 1:1, for example, 3:1, 2:1 or 1:1; or 30:1 to 10:1, for example, 30:1, 20:1 or 10:1.
• the ratio of Compound B to ibrutinib is in the range of 700: 1 - 1 AO. In another embodiment of the pharmaceutical combination, the ratio of Compound B to ibrutinib is in the range of 2: 1 to 1 :2, for example, 2:1, 1:1, or 1:2; 170:1 to 150:1, for example, 170:1, 160:1 or 150:1; 3:1 to 1:1, for example, 3:1, 2:1 or 1:1; or 30:1 to 10:1, for example, 30:1, 20:1 or 10:1.
• the ratio of Compound C to ibrutinib is in the range of 700: 1 - 1 AO. In another embodiment of the pharmaceutical combination, the ratio of Compound C to ibrutinib is in the range of 2: 1 to 1 :2, for example, 2:1, 1:1, or 1:2; 170:1 to 150:1, for example, 170:1, 160:1 or 150:1; 3:1 to 1:1, for example, 3:1, 2:1 or 1:1; or 30:1 to 10:1, for example, 30:1, 20:1 or 10:1.
• the ratio of Compound D to ibrutinib is in the range of 700: 1 - 1 AO. In another embodiment of the pharmaceutical combination, the ratio of Compound D to ibrutinib is in the range of 2: 1 to 1 :2, for example, 2:1, 1:1, or 1:2; 170:1 to 150:1, for example, 170:1, 160:1 or 150:1; 3:1 to 1:1, for example, 3:1, 2:1 or 1:1; or 30:1 to 10:1, for example, 30:1, 20:1 or 10:1.
• kits include package(s) comprising compounds or compositions disclosed herein. In some embodiments, kits comprise a HDAC inhibitor or a pharmaceutically acceptable salt thereof, or a HDAC inhibitor or a
• the package can be a box or wrapping.
• Packaging materials for use in packaging pharmaceutical products are well-known to those of skill in the art. Examples of pharmaceutical packaging materials include, but are not limited to, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.
• the kit can also contain items that are not contained within the package, but are attached to the outside of the package, for example, pipettes.
• Kits can further contain instructions for administering the disclosed compounds or compositions to a patient. Kits also can comprise instructions for approved uses of compounds herein by regulatory agencies, such as the United States Food and Drug
• Kits can also contain labeling or product inserts for the compounds.
• the package(s) and/or any product insert(s) may themselves be approved by regulatory agencies.
• the kits can include compounds in the solid phase or in a liquid phase (such as buffers provided) in a package.
• the kits can also include buffers for preparing solutions for conducting the methods, and pipettes for transferring liquids from one container to another.
• BF 3 -ether (1300 ml, 2.0 equiv.) was added dropwise over a period of 60 min., while the inner temperature was maintained below 15 °C.
• the reaction mixture was stirred at 15-20 °C for 1-2 hr. and stopped when a low level of benzonitrile remained.
• IN HC1 (2500 ml) was added dropwise while maintaining the inner temperature below 30 °C.
• NaOH (20%, 3000 ml) was added dropwise to bring the pH to about 9.0, while still maintaining a temperature below 30 °C.
• reaction mixture was extracted with MTBE (3 L x 2) and EtOAc (3 L x 2), and the combined organic layers were dried with anhydrous Na 2 S0 4 and concentrated under reduced pressure (below 45 °C) to yield a red oil.
• MTBE (2500 ml) was added to the oil to give a clear solution, and upon bubbling with dry HC1 gas, a solid precipitated. This solid was filtered and dried in vacuum yielding 143 g of compound 2.
• the reaction mixture was adjusted to a pH of about 8.5-9 through addition of HC1 (6N), resulting in precipitation.
• the mixture was concentrated under reduced pressure. Water (3000 ml) was added to the residue with intense stirring and the precipitate was collected by filtration. The product was dried in an oven at 45 °C overnight (340 g, 79% yield).
• Example 5 Other materials and methods
• PBMCs peripheral blood mononuclear cells
• Compound A is an injectable (i.p.) form of HDAC6 inhibitor currently in use for clinical trials in multiple myeloma and lymphoma.
• Compound D is a selective HDAC6 inhibitor which can be made available in animal feed ("chow") which provides constant but low level exposure.
• Example 6 HDAC6 is over expressed in human primary B-CLL and modulation of its expression alters cell viability in CLL cell lines
• FIDAC6 expression Using RT-PCR analysis of twenty B-CLL with Rai stages ranging from 0-4, fifteen of twenty patients show elevated FIDAC6 expression (Fig. 1A). Viability of CLL cell line Mecl in this experiment was determined using MTS CellTiter 96® (Promega) analysis, and the results are shown in Fig. IB. Two polyclonal FIDAC6KD cells were developed by stably transfecting them with HDAC6 shRNA (Sigma-Plasmid M_006044), and subsequently analyzed for viability and compared to non-targeted control counterpart (Fig. IB-Left part). Mecl cells were transfected with increasing concentrations of FIDAC6 over-expressing (H60E) plasmid following viability analysis. Next, Mecl cells were treated with two FIDAC6 inhibitors at various doses and assessed for viability by MTS (Fig. IB-Right part).
• OSU-CLL cells were treated with Compound A (Fig. 2A) or Compound D (Fig. 2B) for 48 hours and cells death was measured using cellTox assay SyTox® Green (Promega). Mecl cells treated with Compound A for 24 hours and stimulated with LPS show a dose- dependent decrease in IL-10 production (important B cell survival factor) (Fig. 2C).
• Example 8 Treatment ofCLL cell lines with Compound A and Ibrutinib, synergistically decreases viability and alters BCR signaling pathway
• the activity of the combination of drugs was determined using the CellTiter-Blue (Promega) cell viability assay after a 72 hour treatment in Mecl (Fig. 3A) and OSU-CLL (Fig. 3B) cells.
• the results were analyzed for synergism using the combination index (CI) method developed by Chou & Talalay (Chou TC. Cancer research 2010; 70(2): 440-6).
• Mecl cells were treated with Compound A at varying doses (0.5 and 1 ⁇ ) alone or in combination with ibrutinib (0.2 and 1 ⁇ ) for 24 hrs. and blotted for PARP and pERK (Fig. 3C).
• Example 9 Biological or pharmacological manipulation ofHDA C6 reduces disease burden and renders survival advantage
• mice from eu-TCLl and euTCLl/HDAC6KO groups were selected and aged up to 300 days (corresponding to the day the last euTCLl expired) (Fig. 4A).
• euTCLl mice succumbed to disease starting day 200 while ALL eu-TCLl/HDAC6KO mice survived (panel insert).
• Spleens from these mice were measured post-mortem and compared (p value ⁇ 0.0005***) (Fig. 4A).
• mice treated with Compound D showed significantly lower disease burden (9 months old/25 weeks post treatment start date) than mice treated without Compound D (Fig. 4C).
• mice receiving Compound D treatment have a more advantageous overall survival (Top of Fig. 4D) and significantly lower disease burden (Bottom of Fig. 4D).
• mice treated with Compound D have a more advantageous overall survival than mice not treated with Compound D (Fig. 4E). As shown, at around day 350, all mice not treated with Compound D (NT) have succumbed to the disease.
• Example 10 Inhibition of HDAC6 in vivo and in vitro alters the phenotypic profile ofB and T cell compartment in CLL
• B-cells malignant population
• CD223 LAG3
• CD279 PD-1
• CD274 PDL-1
• CD273 PDL-2
• Example 11 The Combination of BTK inhibitors and Compound D causes significantly lower disease burden in a mouse model .
• mice receiving a combination of Compound D and Ibrutinib demonstrated significantly lower disease burden (Fig. 6A).
• Fig. 6A mice receiving a combination of Compound D and Ibrutinib demonstrated significantly lower disease burden.
• a further decrease in tumor burden was found when compared to treatment with either compound alone. This observed effect on tumor burden occurred in conjuction with decreases in co-inhibitory molecules and circulating T-reg frequency.
• the combination was well tolerated and no significant toxicity was observed.
• mice receiving a combination of Compound D and Ibrutinib demonstrated significant advantageous overall survival when compared with mice receiving either Compound D or Ibrutinib alone (Fig. 6B). As shown, at the latest time point, all mice have succumbed to the disease except for the combination group.

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