WO2020049564A1 - Compositions pharmaceutiques d'esters d'acide fumarique et dérivés de vitamine d et leurs utilisations - Google Patents

Compositions pharmaceutiques d'esters d'acide fumarique et dérivés de vitamine d et leurs utilisations Download PDF

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Publication number
WO2020049564A1
WO2020049564A1 PCT/IL2019/050992 IL2019050992W WO2020049564A1 WO 2020049564 A1 WO2020049564 A1 WO 2020049564A1 IL 2019050992 W IL2019050992 W IL 2019050992W WO 2020049564 A1 WO2020049564 A1 WO 2020049564A1
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Prior art keywords
fae
vdd
pharmaceutical composition
pri
composition
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PCT/IL2019/050992
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English (en)
Inventor
Michael Danilenko
Matan NACHLIELY
Andrzej Kutner
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B. G. Negev Technologies And Applications Ltd., At Ben-Gurion University
Lukasiewicz Research Network – Pharmaceutical Research Institute
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Publication of WO2020049564A1 publication Critical patent/WO2020049564A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/225—Polycarboxylic acids
    • 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
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • 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
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/592—9,10-Secoergostane derivatives, e.g. ergocalciferol, i.e. vitamin D2
    • 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
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/593—9,10-Secocholestane derivatives, e.g. cholecalciferol, i.e. vitamin D3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the present invention is in the field of disease treatment.
  • AML Acute myeloid leukemia
  • APL subtype - acute promyelocytic leukemia
  • ATRA all -trans retinoic acid
  • vitamin D, la, 25-dihydroxy vitamin D 3 (l,25D3)
  • l,25D3 The active form of vitamin D, la, 25-dihydroxy vitamin D 3 (l,25D3), is a well- known inducer of monocyte/macrophage differentiation and growth arrest in various subtypes of AML cells in culture but these effects were observed at supra-physiological concentrations of l,25D3 that may cause life-threatening hypercalcemia in vivo.
  • numerous analogs of both l,25D3 and la, 25-dihydroxy vitamin D 2 (l,25D2) have been synthesized and some of them were found to be less calcemic than their parent compounds. Still, the analogs that reached clinical trials haven’t demonstrated convincing anticancer effects, which may be due, at least in part, to their dose-limiting calcemic toxicity.
  • the present invention is directed to compositions and kits comprising vitamin D derivative (VDD) and fumaric acid ester (FAE), such as for enhancing the differentiation and/or maturation of myeloid cells.
  • VDD vitamin D derivative
  • FAE fumaric acid ester
  • the present invention is further directed to a composition and kit comprising VDD and FAE for treating cancer, including but not limited to AML, in a subject in need thereof.
  • the present invention is further directed to a composition and kit comprising VDD and FAE for treating multiple sclerosis.
  • the present invention is further directed to a composition and kit comprising VDD and FAE for treating psoriasis.
  • the present invention is further directed to a method for enhancing the therapeutic efficacy of FAE in a subject in need thereof, the method comprising administering to the subject VDD, thereby enhancing the therapeutic efficacy of FAE in the subject.
  • the subject is a diseased subject such as having multiple sclerosis or psoriasis.
  • the present invention is further directed to a method for enhancing the therapeutic efficacy of VDD in a subject in need thereof, the method comprising administering to the subject FAE, thereby enhancing the therapeutic efficacy of VDD in the subject.
  • a pharmaceutical composition comprising therapeutically effective amounts of vitamin D derivative (VDD) and fumaric acid ester (FAE), for use in the treatment of a proliferative disease.
  • VDD vitamin D derivative
  • FAE fumaric acid ester
  • VDD and FAE for use in the treatment of a disease selected from the group consisting of cancer, multiple sclerosis, and psoriasis.
  • a method for increasing or enhancing differentiation of a myeloid cell comprising contacting the myeloid cell with a composition comprising an effective amount of VDD and FAE.
  • a method for increasing or enhancing the therapeutic efficacy of FAE in a subject in need thereof comprising administering to the subject a pharmaceutical composition comprising VDD.
  • a method for increasing or enhancing the therapeutic efficacy of VDD in a subject in need thereof comprising administering to the subject a pharmaceutical composition comprising FAE.
  • the proliferating disease is selected from the group consisting of: cancer, multiple sclerosis, and psoriasis.
  • the composition comprises VDD and FAE in a ratio ranging from 1:100 to 1:10,000 (w/w), and further comprises a pharmaceutically acceptable carrier.
  • the composition comprises 0.1-20 pg/ml VDD and 1-50 mg/ml FAE. In some embodiments, the composition comprises 1-5 pg/ml VDD and 1- 10 mg/ml FAE. In some embodiments, the VDD and FAE are for a concurrent or a subsequent use.
  • the VDD is formulated within a first pharmaceutical composition and the FAE is formulated within a second pharmaceutical composition.
  • the composition comprises 0.1-20 pg/ml VDD.
  • the composition comprises 1-50 mg/ml FAE.
  • the VDD is administered at a dosage of 5-40 pg/kg.
  • the FAE is administered at a dosage of 10-50 mg/kg.
  • the VDD is administered at a dosage of 10-20 pg/kg and the FAE is administered at a dosage of 25-35 pg/kg.
  • the VDD is selected from the group consisting of: la, 25- dihydroxyvitamin D 2 (l,25D2), la, 25-dihydroxy vitamin D 3 (l,25D3), and the 19 -nor- l,25D2 analogs PRI-5202, PRI-5100 and PRI-1907.
  • the FAE comprises dimethyl fumarate and/or a metabolite thereof.
  • the subject is afflicted with a proliferative disease.
  • the subject is afflicted with a disease selected from multiple sclerosis and psoriasis.
  • cancer is leukemia or lymphoma.
  • leukemia is acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • Figs. 1A-1L are scatter plots and vertical bar graphs demonstrating that structurally distinct Nrf2 activators enhance the differentiation-inducing effects of VDDs on acute myeloid leukemia (AML) cells. Cells were treated with the indicated agents or vehicle (0.2 % ethanol) for 96 h.
  • (1A-1I) is scatter plots showing primary flow cytometry data exemplifying the enhancing effects of ieri-butylhydroquinone (/BHQ) and dimethyl fumarate (DMF) on l,25D3- and PRI-5202-induced differentiation of HL60 cells. The values displayed in the upper right quadrants of the dot plots indicate the percentages of CDl4 + CDl lb + -double positive cells.
  • (1J-1K) are vertical bar graphs showing the averaged percentages of CDl4 + CDl lb + HL60 (1J) and U937 (IK) cells determined following different treatments, as shown in Figs. 1A-1I.
  • (1L) is a vertical bar graph showing TPA- stimulated superoxide production measured by the ferricytochrome c reduction assay.
  • the data are the means ⁇ SD. *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001 and ****, p ⁇ 0.001 vs. vehicle. ⁇ , p ⁇ 0.05; ⁇ ⁇ , p ⁇ 0.01; ⁇ ⁇ ⁇ , p ⁇ 0.001 and ⁇ ⁇ ⁇ ⁇ , p ⁇ 0.0001 vs. VDD alone.
  • FIG. 2 is micrographs demonstrating that vitamin D derivatives and Nrf2 activators cooperatively upregulate protein levels of VDR and RXRa as well as Nrf2 and TrxRl.
  • HL60 cells were incubated with the indicated compounds for 48 h. Protein levels were determined by Western blotting.
  • b-Actin was used as a protein loading control. A representative image of western blot analyses of three similar experiments is shown. Absorbance values, normalized to those of b-actin, are indicated below each specific protein band.
  • Figs. 3A-3D are vertical bar graphs showing that VDDs and Nrf2 activators cooperate in inducing the expression of VDR and Nrf2 target genes.
  • HL60 cells were treated with test agents, alone or in combination, for 48 h.
  • mRNA transcript levels of the VDR target genes CYP24A1 (3A), CAMP (3B), the Nrf2 target gene TXNRD1 (3C), and the CDl lb myeloid marker gene ITGAM (3D) were measured by RT-qPCR.
  • the expression of specific genes was normalized by an average C t of three reference genes: GAPDH, TBP1, and GUSB. The data are the means ⁇ SD.
  • Figs. 4A-4D are graphs showing that PRI-5202 has the lowest toxicity in mice among the VDDs tested.
  • Female ICR mice (5/group) were injected i.p. with vehicle (PBS + 1% ethanol) or the indicated VDDs, 3 times per week, for about 7 weeks. The compounds were injected at 0.05, 0.1 or 0.25 pg/mouse. Control mice were left untreated.
  • (4A) Changes in the body weight over the period of 42 days. Serum calcium (4B) ALT (4C) and urea (4D) levels determined on Day 46.
  • mice designated x
  • mice treated with a high concentration of l,25D3 or PRI-1907 (0.25 pg/mouse) were euthanized within 1-2 weeks due to severe hypercalcemia symptoms.
  • Figs. 5A-5F demonstrate that PRI-5202 and the Nrf2 activator DMF cooperate in reducing leukemia tumor growth in mice.
  • SCID/Beige mice were inoculated i.p. with HL60 cells on Day 0. Starting on Day 7, mice received i.p. injections of vehicle (2.5 % DMSO and 1 % ethanol), PRI-5202 (0.25 pg/mouse), DMF (0.6 mg/mouse) or PRI-5202 + DMF, 3 times per week.
  • 5A is images of typical tumors excised from control (vehicle) and treated animals on Day 28.
  • (5B) is a vertical bar graph showing the averaged tumor wet weights.
  • (5C) is a vertical bar graph showing the changes in the body weight over the period of 28 days.
  • (5D) is a graph showing serum calcium.
  • Figs. 6A-6D are micrographs demonstrating that the antileukemic effect of the PRI-5202 + DMF combination is associated with reduced proliferation of AML cells in vivo.
  • Tumors were excised from mice treated with vehicle (6A) and PRI-5202 + DMF (6B), as described above (Fig. 5).
  • (6A and 6C) show Hematoxylin-eosin (H&E) staining. Apoptotic cells and/or bodies are indicated (Arrows).
  • (6B and 6D) show Ki-67 immunostaining (averaged Ki-76 labeling index is presented in Table 1). Scale bars indicate 50 pm.
  • Fig. 7 is a graph showing that Nrf2 activators differ in the ability to enhance VDD-induced differentiation of acute myeloid leukemia cells.
  • HL60 cells were exposed to the indicated compounds, alone or in combination, for 48 h followed by the analysis of the expression of surface myeloid markers (CD1 lb and CD 14) using flow cytometry. The data are the means ⁇ SD (n > 3). l,25D 3 , la, 25-dihydroxy vitamin D 3 .
  • Nrf2 activators CA, carnosic acid; tBHQ, le rl-b uty 1 h y droq u i no nc ; DMF. dimethyl fumarate; MMF, monomethyl fumarate; MEF, monoethyl fumarate; CSCS, chlorophyllin sodium copper salt; EGCG, epigallocatechin-3-gallate; RA, rosmarinic acid.
  • Figs. 8A-8C are graphs showing that the Nrf2 activators DMF and CA potentiate VDD-induced cell differentiation in a concentration-dependent manner.
  • the differentiation-enhancing effect of CA is nearly saturated at 25 pM and is then reduced at higher concentrations (8B). The latter reduction may be explained by a concentration-dependent cytotoxicity of CA (8C).
  • l,25D 3 la, 25-dihydroxy vitamin D 3 ; DMF, dimethyl fumarate; CA, carnosic acid.
  • Figs. 9A-9B are a western blot image and a vertical bar graph showing that transcriptionally active Nrf2 mediates the differentiation-enhancing effect of CA and MMF in AML cells.
  • U937 human AML cells stably transfected with either empty plasmid (Vector) or a plasmid expressing dominant- negative mutant of Nrf2 (DN-Nrf2) were exposed to the indicated compounds, alone or in combination, for 48 h followed by western blotting and the analysis of CDl lb and CD 14 expression.
  • Fig. 10 is a vertical bar graph showing that DMF and l,25D3 cooperate in inhibiting proliferation of AML cells.
  • HL60 cells were exposed to the indicated compounds, alone or in combination, for 96 h followed by cell enumeration using the Trypan blue exclusion assay.
  • Figs. 11A-11B are graphs showing the antileukemic effect of the DMF + PRI- 5202 combination in a systemic model of murine AML.
  • C57BL/6 mice were inoculated intravenously (i.v.) with 10 5 oncogene (MLL-AF9)-transformed murine hematopoietic stem/progenitor cells. Seven days later, mice were randomly divided into 2 groups (18 mice in each) and treated intraperitoneally (i.p.) with vehicle (1% ethanol in PBS) or a combination of DMF (0.6 mg/mouse) plus PRI-5202 (0.25 pg/mouse), 3 times per week.
  • MLL-AF9 oncogene
  • the present invention is directed to a method for increasing or enhancing the differentiation and/or maturation of a myeloid cell comprising contacting the myeloid cell with a composition comprising vitamin D derivative (VDD) and fumaric acid ester (FAE).
  • VDD vitamin D derivative
  • FAE fumaric acid ester
  • the present invention is also directed to a method for increasing or enhancing the therapeutic efficacy of VDD administered to a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising FAE.
  • the present invention is also directed to a method for increasing or enhancing the therapeutic efficacy of FAE administered to a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising VDD.
  • the present invention is based, in part, on the finding that a combination of DMF and VDD exhibited a marked anti-leukemic activity, both in vitro and in vivo.
  • the invention is further based, in part, on the findings that the synergy of FAE and VDD did not enhance calcemic toxicity of VDD.
  • the present invention is directed to a combined therapy comprising a pharmaceutical composition comprising vitamin D, and methods of use thereof.
  • vitamin D comprises vitamin D 2 , or vitamin D 3 , or any combination thereof.
  • vitamin D 3 is cholecalciferol.
  • vitamin D 2 is ergocalciferol.
  • the terms "vitamin D”, “vitamin D 2 ", vitamin D 3 , “cholecalciferol” and “ergocalciferol” are interchangeable.
  • vitamin D according to the present invention comprises a vitamin D derivative (VDD).
  • vitamin D or VDD according to the present invention comprises synthetic or biosynthetic vitamin D or VDD.
  • VDD include, but are not limited to, la,25-dihydroxyvitamin D 2 (l,25D2), la, 25 -dihydroxy vitamin D 3 (l,25D3), the 19 -nor l,25D2 analogs PRI-5202, PRI-5100 (e.g., paricalcitol) and PRI-1907 (having the herein below structures), and other analogs of l,25D2 or l,25D3.
  • the 19 -nor l,25D2 analogs PRI-5202, PRI-5100 (e.g., paricalcitol) and PRI- 1907, have the following structures:
  • Methods of vitamin D biosynthesis are well known within the art, and can include, but are not limited to, production in a cell culture or enzymatic cell-free production.
  • vitamin D derivative or “VDD” encompasses any compound having a vitamin D activity that is generated from a similar compound by a chemical reaction, or any compound produced from another compound by substitution of one or more atoms.
  • the derivative comprises a structural analog.
  • the VDD of the invention is obtained by any chemical modification of vitamin D, as long as it maintains a vitamin D activity as described above.
  • a chemical modification comprises substitution.
  • the modification comprises the addition of a chemical group vitamin D or a VDD.
  • Non-limiting examples of chemical groups include but are not limited to acetyl, methyl, phosphate, amid, or others.
  • vitamin D activity is expressed by the ability of a compound to induce differentiation of acute myeloid leukemia cells.
  • vitamin D activity comprises increased expression of surface myeloid markers.
  • vitamin D activity comprises increased ability of cells to generate superoxide.
  • vitamin D activity comprises over expression of the vitamin D receptor (VDR).
  • the present invention is directed to a combined therapy comprising a pharmaceutical composition comprising FAE, and methods of use thereof.
  • FAE encompasses any fumaric acid (i.e., fumarate) comprising at least one ester bond and/or a metabolite thereof as well as FAE mixtures or a plurality of two or more FAEs as disclosed hereinbelow.
  • FAE is a monoester. In some embodiments, the FAE is a diester. In some embodiments, the FAE is a metabolite of FAE. In some embodiments, the FAE is a mixture of FAEs, comprising a plurality (e.g., two or more types) of FAE. In some embodiments, the FAE mixture comprises a FAE metabolite. In some embodiments, the FAE mixture comprises at least one fumarate comprising at least one ester bond, and a metabolite of fumaric acid ester. In some embodiments, the FAE mixture comprises one or more FAE metabolites. In some embodiments, the FAE mixture comprises one or more types of FAE and one or more metabolites thereof. In some embodiments, the FAE mixture comprises two or more metabolites of FAE.
  • FAE according to the present invention comprises synthetic or biosynthetic FAE.
  • Types of FAE and metabolites thereof are known in the art, non-limiting examples of which include, but are not limited to, dimethyl fumarate (DMF), monomethyl fumarate (MMF), monoethyl fumarate (MEF), and the like.
  • the term "metabolite” encompasses any intermediate or an end product of a metabolic process having a FAE activity, as disclosed herein.
  • FAE activity comprises activation of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2).
  • activation of Nrf2 comprises activation of Nrf2 transcription.
  • Non-limiting example for determining gene expression levels include, but is not limited to, quantitative PCR.
  • Non limiting example for determining protein expression levels include, but is not limited to immunoassays, such as enzyme linked immunosorbent assay (ELISA) and Western blotting.
  • ELISA enzyme linked immunosorbent assay
  • compositions and combinations of VDD and FAE Compositions and combinations of VDD and FAE
  • the invention is directed to a composition comprising as an active ingredient an effective amount of VDD and FAE, and an acceptable carrier and/or diluent. In some embodiments, the invention is directed to a composition comprising as an active ingredient an effective amount of VDD and FAE, and an acceptable carrier and/or diluent. In some embodiments, the invention is directed to a composition comprising as an active ingredient an effective amount of l9-nor-l,25O2 (paricalcitol) or its analog and FAE, and an acceptable carrier and/or diluent.
  • the invention is directed to a composition
  • a composition comprising as an active ingredient an effective amount of PRI-5202 and FAE, and an acceptable carrier and/or diluent.
  • the acceptable carrier facilitates incorporation or coating of the active ingredient a substrate.
  • the term "pharmaceutically acceptable” can mean approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • the composition described herein comprises VDD and FAE in the ratio of 1: 100 to 1: 1,000 (w/w). In some embodiments, the composition described herein comprises VDD and FAE in the ratio of 1:200 to 1:5,000 (w/w). In some embodiments, the composition described herein comprises VDD and FAE in the ratio of 1:300 to 1:7,500 (w/w). In some embodiments, the composition described herein comprises VDD and FAE in the ratio of 1:50 to 1:2,000 (w/w). In some embodiments, the composition described herein comprises VDD and FAE in the ratio of 1 :700 to 1: 10,000 (w/w).
  • the composition described herein comprises VDD and FAE in the ratio of 1: 100 to 1: 10,000 (w/w). In some embodiments, the composition described herein comprises VDD and FAE in the ratio of 1: 1,000 to 1:9,000 (w/w). In some embodiments, the composition described herein comprises VDD and FAE in the ratio of 1:800 to 1:8,500 (w/w). In some embodiments, the composition described herein comprises VDD and FAE in the ratio of 1:2,000 to 1:9,500 (w/w). In some embodiments, the composition described herein comprises VDD and FAE in the ratio of 1:750 to 1:6,000 (w/w). In some embodiments, the composition described herein comprises VDD and FAE in the ratio of 1:100 to 1:20,000 (w/w). Each possibility represents a separate embodiment of the invention.
  • VDD is present at a concentration of at least 0.01 pg/ml, at least 0.1 pg/ml, at least 0.5 pg/ml, at least 1 pg/ml, at least 2 pg/ml, at least 3 pg/ml, at least 5 pg/ml, at least 7 pg/ml, at least 9 pg/ml, at least 10 pg/ml, at least 11 pg/ml, at least 12 pg/ml, at least 13 pg/ml, at least 14 pg/ml, at least 15 pg/ml, at least 16 pg/ml, at least 17 pg/ml, at least 18 pg/ml, at least 19 pg/ml, at least 20 pg/ml, or any range therebetween, within the composition.
  • VDD is present at a concentration of 0.01-0.1 pg/ml, 0.05-0.5 pg/ml, 0.4-1 pg/ml, 0.8-2 pg/ml, 1.5-3 pg/ml, 2-5 pg/ml, 4-7 pg/ml, 6-9 pg/ml, 7-10 pg/ml, 8-11 pg/ml, 9-12 pg/ml, 10-13 pg/ml, 11- 14 pg/ml, 12-15 pg/ml, 13-16 pg/ml, 14-17 pg/ml, or 16-20 pg/ml, within the composition.
  • Each possibility represents a separate embodiment of the invention.
  • FAE is present at a concentration of at least 1 mg/ml, at least 5 mg/ml, at least, at least 10 mg/ml, at least 15 mg/ml, at least 20 mg/ml, at least 25 mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 45 mg/ml, or at least 50 mg/ml within the composition.
  • FAE is present at a concentration of 0.1- 1 mg/ml, 0.05-1.5 mg/ml, 1-5 mg/ml, 4-10 mg/ml, 6-12 mg/ml, 11-15 mg/ml, 12-20 mg/ml, 15-25 mg/ml, 20-35 mg/ml, 30-45 mg/ml, or 40-60 mg/ml.
  • concentration 0.1- 1 mg/ml, 0.05-1.5 mg/ml, 1-5 mg/ml, 4-10 mg/ml, 6-12 mg/ml, 11-15 mg/ml, 12-20 mg/ml, 15-25 mg/ml, 20-35 mg/ml, 30-45 mg/ml, or 40-60 mg/ml.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the active ingredient is administered.
  • Such carriers can be sterile liquids, such as water-based and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents.
  • Water may be used as a carrier such as when the active ingredient is comprised by a pharmaceutical composition being administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.
  • composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates.
  • Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned.
  • the carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the compositions presented herein.
  • An embodiment of the invention relates to VDD and FAE, presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.
  • the unit dosage form is in the form of a tablet, capsule, lozenge, wafer, patch, ampoule, vial or pre-filled syringe.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the nature of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in-vitro or in-vivo animal model test bioassays or systems.
  • the composition of the present invention is administered in the form of a pharmaceutical composition comprising at least one of the active ingredients of this invention (e.g., VDD and FAE) together with a pharmaceutically acceptable carrier or diluent.
  • the composition of the invention can be administered either individually or together in any conventional oral, parenteral or transdermal dosage form.
  • the terms“administering”,“administration”, and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect.
  • the pharmaceutical composition described herein are administered via oral (i.e., enteral), rectal, vaginal, topical, nasal, ophthalmic, transdermal, subcutaneous, intramuscular, intraperitoneal or intravenous routes of administration.
  • oral i.e., enteral
  • vaginal topical
  • nasal ophthalmic
  • transdermal subcutaneous
  • intramuscular intraperitoneal
  • intravenous routes of administration e.g., transdermal, subcutaneous, intramuscular, intraperitoneal or intravenous routes of administration.
  • routes of administration of the pharmaceutical composition will depend on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injections, e.g., intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other mode of injection as known in the art.
  • intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer.
  • VDD and/or FAE may be combined with a pharmaceutically acceptable carrier so that an effective dosage is delivered, based on the desired activity.
  • the carrier can be in the form of, for example, and not by way of limitation, an ointment, cream, gel, paste, foam, aerosol, suppository, pad or gelled stick.
  • the pharmaceutical composition may be in the form of tablets or capsules, which can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate; or a glidant such as colloidal silicon dioxide.
  • a liquid carrier such as fatty oil.
  • dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents.
  • the tablets of the invention can further be film coated.
  • oral application of the pharmaceutical composition may be in the form of drinkable liquid.
  • oral application of the pharmaceutical composition may be in the form of an edible product.
  • solutions in sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water-soluble salts.
  • Such aqueous solutions may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes.
  • composition or pharmaceutical composition also include incorporation of the active ingredient into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc., or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.
  • polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc.
  • liposomes such as polylactic acid, polyglycolic acid, hydrogels, etc.
  • microemulsions such as polylactic acid, polyglycolic acid, hydrogels, etc.
  • Such composition or pharmaceutical composition will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.
  • the present invention provides combined preparations.
  • “a combined preparation” defines especially a“kit of parts” in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners i.e., simultaneously, concurrently, separately or sequentially.
  • the parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts.
  • the ratio of the total amounts of the combination partners in some embodiments, can be administered in the combined preparation.
  • the combined preparation can be varied, e.g., in order to cope with the needs of a patient subpopulation to be treated or the needs of the single patient which different needs can be due to a particular disease, severity of a disease, age, sex, or body weight as can be readily made by a person skilled in the art.
  • a pharmaceutical composition comprising an effective amount of VDD and an effective amount of FAE, and a pharmaceutically effective carrier.
  • a combination of VDD and FAE for use in the treatment of a disease or a disorder.
  • a combination of a pharmaceutical composition comprising an effective amount of VDD and a pharmaceutical composition comprising an effective amount of FAE for use in the treatment of a disease or a disorder.
  • the combination of VDD and FAE is for use in the treatment of a disease selected from: cancer, multiple sclerosis, and psoriasis.
  • a pharmaceutical composition comprising an effective amount of VDD for use in enhancing the therapeutic efficacy of FAE.
  • a pharmaceutical composition comprising an effective amount of VDD for use in treatment of a disease in a subject amendable for treatment by FAE (e.g., multiple sclerosis or psoriasis).
  • a pharmaceutical composition comprising an effective amount of FAE for use in enhancing the therapeutic efficacy of VDD.
  • a pharmaceutical composition comprising an effective amount of FAE for use in treatment of a disease in a subject amendable for treatment by VDD.
  • VDD and FAE are administered concurrently. In some embodiments, VDD and FAE are administered sequentially. In some embodiments, VDD and FAE are administered subsequently.
  • VDD and FAE according to the present invention can be provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself.
  • measures e.g., dosing and selection of the complementary agent
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is affected or diminution of the disease state is achieved.
  • the composition of the preset invention is administered in a therapeutically safe and effective amount.
  • safe and effective amount refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects, including but not limited to toxicity, such as calcemic toxicity, irritation, or allergic response, commensurate with a reasonable benefit/risk ratio when used in the presently described manner.
  • a therapeutically effective amount of VDD and FAE is the amount of the mentioned herein VDD and FAE necessary for the in vivo measurable expected biological effect.
  • the actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g.
  • preparation of effective amount or dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.
  • toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro , in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosages vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 13* Ed., McGraw-Hill/Education, New York, NY (2017)].
  • compositions containing the presently described VDD and FAE as the active ingredient can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington: The Science and Practice of Pharmacy, 22 nd Ed., Pharmaceutical Press, Philadelphia, PA (2012).
  • compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier are prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • compositions of the present invention are presented in a pack or dispenser device, such as an FDA approved kit, which contains, one or more unit dosages forms containing the active ingredient.
  • the pack for example, comprises metal or plastic foil, such as a blister pack.
  • the pack or dispenser device is accompanied by instructions for administration.
  • the pack or dispenser is accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • Such notice is labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • the present invention is directed to a method for increasing or enhancing the differentiation of a myeloid cell, comprising contacting the myeloid cell with a composition comprising VDD and FAE.
  • a differentiated myeloid cell contacted with a composition as disclosed herein expresses CDl lb, CD14, or both.
  • a differentiated myeloid cell contacted with a composition as disclosed herein is classified as a CDl lb + CDl4 + cell.
  • a differentiated myeloid cell is less proliferative than an immature myeloid cell or is non-proliferative.
  • a differentiating myeloid cell is more susceptible to anti proliferative agents.
  • a differentiated myeloid cell is more susceptible to apoptotic, necrotic, cell death-inducing, chemotherapeutic or other toxic agents. In some embodiments, a differentiated myeloid cell has reduced survival. In some embodiments, a differentiated myeloid cell has reduced toxic resistance capabilities, such as for chemo therapeutics.
  • Non-limiting examples of a myeloid cell include, but are not limited to, multipotent hematopoietic stem cell, hemocytoblast, common myeloid progenitor cell, megakaryocyte, erythrocyte, mast cell, myeloblast, myeloid leukemia blast, basophil, neutrophil, eosinophil, monocyte and a macrophage.
  • Non-limiting examples of less differentiated myeloid cells include, but are not limited to multipotent hematopoietic stem cell, hemocytoblast, myeloblast, myeloid leukemia blast, and common myeloid progenitor cell.
  • Non-limiting examples of differentiated myeloid cells include but are not limited to megakaryocyte, erythrocyte, mast cell, basophil, neutrophil, eosinophil, monocyte, and a macrophage.
  • the present invention is directed to a method for increasing or enhancing the therapeutic efficacy of FAE administered to a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising VDD.
  • the present invention is directed to a method for increasing or enhancing the therapeutic efficacy of a VDD administered to a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising FAE.
  • increasing or enhancing is by at least 5%, by at least 15%, by at least 25%, by at least 40%, by at least 50%, by at least 65%, by at least 75%, by at least 85%, by at least 90%, by at least 95%, or by at least 99% more compared to control, or any value and range therebetween.
  • increasing or enhancing is by 1-5%, 7-15%, 10- 25%, 20-40%, 35-50%, 45-65%, 55-75%, 70-85%, 80-90%, 87-95%, or 92-100% more compared to control.
  • Each possibility represents a separate embodiment of the invention.
  • a control comprises a subject administered with VDD or with FAE, and not with both or a sample derived therefrom.
  • a control comprises a subject not administered with VDD or with FAE, or a sample derived therefrom.
  • a control comprises a naive myeloid cell.
  • a naive myeloid cell encompasses any myeloid cell which was not contacted with VDD, or FAE, or any combination thereof.
  • a sample refers to a physical specimen obtained from a mammal.
  • a sample is obtained from a human.
  • a sample is obtained well within the capabilities of those skilled in the art.
  • a sample comprises biological fluids selected from: serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, urine, cerebrospinal fluid, saliva, sputum, tears, perspiration, mucus, and tissue culture media, including tissue extracts such as homogenized tissue, and cellular extracts.
  • the sample comprises a tissue, a tissue fragment, a portion thereof, any combination thereof, or a biopsy.
  • a subject in need thereof is afflicted with multiple sclerosis (MS).
  • multiple sclerosis refers to a disease characterized by disseminated patches of demyelination in the brain and spinal cord.
  • Methods for diagnosing MS would be apparent to one of ordinary skill in the art, non-limiting examples of which, include, but are not limited to clinical criteria, brain or spinal MRI, or both, and cerebrospinal fluid (CSF) immunoglobulin G (IgG) levels.
  • CSF cerebrospinal fluid
  • IgG immunoglobulin G
  • a subject in need thereof is afflicted by psoriasis.
  • psoriasis refers to an inflammatory disease that manifests most commonly as well-circumscribed, erythematous papules and plaques covered with silvery scales. Diagnosing psoriasis would be apparent to one of ordinary skill in the art and may be based on clinical appearance and distribution of lesions.
  • a subject in need thereof is afflicted with a condition associated with increased cell proliferation.
  • the subject is suspected to develop cancer. In some embodiments, the subject is diagnosed with cancer. In some embodiments, the subject is at risk of developing cancer.
  • cancer is a solid cancer (i.e., comprises a solid tumor).
  • cancer is a liquid cancer.
  • liquid cancer comprises hematopoietic malignancy.
  • hematopoietic malignancy comprises hematopoietic cells undergone or undergoing cancerous transformation.
  • Non limiting examples of phenotypes observed in cancerous transformation include, but are not limited to, increased proliferation rate, dysregulated cell cycle, increased amount of DNA compared to naive cells, or any combination thereof.
  • liquid cancer comprises any disease selected from Leukemia and Lymphoma.
  • Leukemia is acute myeloid leukemia (AML).
  • the term "subject" refers to an animal, more particularly to non human mammals and human organism.
  • Non-human animal subjects may also include prenatal forms of animals, such as, e.g., embryos or fetuses.
  • Non-limiting examples of non-human animals include, but are not limited to, horse, cow, camel, goat, sheep, dog, cat, non-human primate, mouse, rat, rabbit, hamster, guinea pig, pig.
  • the subject is a human. Human subjects may also include fetuses.
  • the method of the present invention is further directed to treating a subject afflicted by a disease as disclosed hereinabove.
  • treatment or “treating” of a disease, disorder, or condition encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured.
  • a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life.
  • the method of the present invention is further directed to preventing a disease as disclosed hereinabove in a subject.
  • preventing or “prevention” of a disease, disorder, or condition encompasses the delay, prevention, suppression, or inhibition of the onset of a disease, disorder, or condition.
  • prevention relates to a process of prophylaxis in which a subject is exposed to the presently described composition comprising VDD and FAE prior the induction or onset of the disease/disorder process. This could be done where an individual has a genetic pedigree indicating a predisposition toward occurrence of the disease/disorder to be prevented.
  • suppression is used to describe a condition wherein the disease/disorder process has already begun but obvious symptoms of the condition have yet to be realized.
  • the cells of an individual may have the disease or disorder, but no outside signs of the disease/disorder have yet been clinically recognized.
  • prophylaxis can be applied to encompass both prevention and suppression.
  • treatment refers to the clinical application of active agents to combat an already existing condition whose clinical presentation has already been realized in a patient.
  • the term "condition” includes anatomic and physiological deviations from the normal that constitute an impairment of the normal state of the living animal or one of its parts, that interrupts or modifies the performance of the bodily functions.
  • the administered composition comprises VDD in a dose range comprising of 0.001 to 50 pg/kg human body.
  • the administered composition comprises a VDD dose of 0.001-0.01 pg/kg human body, 0.01-0.1 pg/kg human body, 0.1-1 pg/kg human body, 1-5 pg/kg human body, 2-8 pg/kg human body, 7-12 pg/kg human body, 10-15 pg/kg human body, 14-25 pg/kg human body, 18-30 pg/kg human body, 25-35 pg/kg human body, 30-38 pg/kg human body, 36-45 pg/kg human body, or 44-60 pg/kg human body.
  • a VDD dose of 0.001-0.01 pg/kg human body, 0.01-0.1 pg/kg human body, 0.1-1 pg/kg human body, 1-5 pg/kg human body, 2-8 pg/kg human body, 7-12 pg/kg human body, 10-15 pg/kg human body, 14-25 pg/kg human body, 18-30 pg/kg human body, 25
  • the administered composition comprises FAE in a dose range comprising of 5 to 100 mg/kg human body.
  • the administered composition comprises a FAE dose of 1-10 pg/kg human body, 5-20 pg/kg human body, 15-30 pg/kg human body, 25-40 pg/kg human body, 35-60 pg/kg human body, 45-55 pg/kg human body, 50-75 pg/kg human body, 70-95 pg/kg human body, 75-85 pg/kg human body, or 80-100 pg/kg human body.
  • Each possibility represents a separate embodiment of the invention.
  • concentration ranges, percentage range, dose/dosage or ratio range recited herein are to be understood to include concentrations, percentages or ratios of any integer within that range and fractions thereof, such as one tenth and one hundredth of an integer, unless otherwise indicated.
  • each of the verbs, “comprise”,“include” and“have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.
  • l,25D2 and its analogs PRI-1907, PRI-5100 and PRI-5202 were synthesized at the Chemistry Department of the Pharmaceutical Research Institute (Warsaw, Poland).
  • l,25D 3 (>99%) was purchased from Selleck Chemicals (Houston, TX).
  • Carnosic acid (98%) was obtained from Nanjing Chemlin Chemical Industry Co. (Nanjing, China).
  • Dimethyl fumarate, monomethyl fumarate, DMSO, ieri-butylhydroquinone, chlorophyllin sodium copper salt, cytochrome c and 12-O-tctradccanoylphorbol- 13- acetate (TPA) were obtained from Sigma Chemical Co. (St. Louis, MO).
  • VDR The antibodies against VDR (C-20), Nrf2 (H-300), RXRa (D-20), and TrxRl (H-270) were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX).
  • Stock solutions of VDDs (0.25- 2.40 mM), and Nrf2 activators (10-50 mM) were prepared in absolute ethanol. The precise concentration of l,25D3 in ethanol solutions was verified spectrophotometrically at 264 nm (e 19,000).
  • HL60 myeloblastic leukemia cells ATCC-CCL- 240.
  • U937 promonocytic leukemia cells ATCC-CRL-1593.2 were purchased from American Type Culture Collection (Rockville, MD). Cells were grown in RPMI 1640 medium supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (0.1 mg/ml), and 10 mM HEPES (pH 7.4) in a humidified atmosphere of 95% air and 5% C0 2 at 37 °C.
  • Cells were seeded at 1 x 10 5 cells/ml and incubated with test agents or vehicle ( ⁇ 0.2% ethanol) for 48 h. Preparation of whole cell lysates and western blotting analysis were performed as follows. Briefly, cells were lysed in buffer containing 1% (v/v) Triton X-100 at 4 °C, subjected to SDS-PAGE and electroblotted into nitrocellulose membranes. The membranes were exposed to primary antibodies overnight at 4 °C. Blots were washed and incubated with horse-radish peroxidase-conjugated antibody. Membranes were then stripped and re-probed for calregulin, as the internal loading control. The protein bands were visualized using Western LightningTM Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Inc., Boston, MA). The absorbance of each band was determined using the Image Quant LAS 4000 system (GE Healthcare, Little Chalfont, UK).
  • RNA extraction RNA extraction, cDNA synthesis, and RT-qPCR
  • RNA was purified from cell cultures according to manufacturer’s instructions using an RNA extraction kit (GENEzolTM TriRNA Pure Kit+DNase I; Geneaid, New Taipei City, Taiwan).
  • a micro-volume spectrophotometer (NanoDrop; Wilmington, DE, USA) was used for RNA quantification.
  • First-strand cDNA was generated by reverse transcriptase kit (qScript cDNA synthesis kit; QUANTA Biosciences; Gaithersburg, MD, USA) using random oligo (dT) after a sample concentration was normalized.
  • Quantitative cDNA amplification was performed by real time PCR (StepOne Real-Time PCR System; Thermo Fisher Scientific; Wilmington, DE, USA) using SYBR green (Fast SYBR Green Master Mix, Thermo Fisher Scientific, Wilmington, DE, USA).
  • Relative mRNA expression levels were determined using the 2 (DDa) f orrnuia where ACt is Ct(tar g et gene) - mean of Ct( re ference genes).
  • the three reference genes used in this study were GAPDH, GUSB, and TBP1. Each experiment was performed using two to three biological replicates, each assayed in triplicate. Primers for qPCR were sensitized by Hylabs Ltd. (Rehovot, Israel).
  • the primer sequences used in this study were as follows:
  • Group 1 control: PBS; Group 2 (vehicle): 1% (v/v) ethanol in PBS; Groups 3-5: 0.05, 0.1, and 0.25 pg/mouse l,25D 3 in PBS; Groups 6-8: 0.05, 0.1, and 0.25 pg/mouse PRI-1907 in PBS; and Groups 9-11: 0.05, 0.1, and 0.25 pg/mouse PRI-5202 in PBS.
  • Mice were injected intraperitoneally (i.p.) with 100 pl of the above solutions, three times per week. Animal appearance, behavior, and body weight were then assessed three times per week. Blood was collected from the tail vein at two time points, on Day 23 and Day 46.
  • Serum calcium, alanine transaminase (ALT) and urea levels were measured in the Biochemistry Laboratory of Soroka University Medical Center (Beer Sheva, Israel). At the end of the experiment (Day 46) all mice were humanely sacrificed.
  • mice For the peritoneal AML tumor model, 24 female 6-week-old SCID/Beige mice (Envigo) were inoculated i.p. with 3 x 10 6 HL60 cells in 100 pl PBS. Seven days later, mice were randomly assigned to four groups (6 mice/group) and were started on the following treatments (100 pl, i.p.): Group 1 (vehicle): 2.5% (v/v) DMSO and 1% (v/v) ethanol in PBS; Group 2: 0.25 pg/mouse PRI-5202 in vehicle; Group 3: 0.6 mg/mouse DMF in vehicle; and Group 4: 0.25 pg/mouse PRI-5202 and 0.6 mg/mouse DMF in vehicle.
  • mice were monitored for another 22 days. Animal appearance and behavior were assessed daily, and the body weight was measured every three days. At the end of the experiment, blood was collected from the tail and blood chemistry tests were performed as described above. All mice were then humanely sacrificed and abdominal tumors were excised, weighed and fixed in 10% (v/v) neutral buffered formalin. Hematoxylin-eosin staining and Ki-67 immunostaining of tumor sections
  • Nrf2 activators synergistically potentiate the differentiation-inducing effects of low concentrations of VDD
  • CA camosic acid
  • HL60 and U937 cells were treated with VDDs (2.5 nM l,25D3, 5 nM PRI-5100 or 0.25 nM PRI-5202), Nrf2 activators at non-cytotoxic concentrations (10 mM CA, 50 mM DMF, 50 pM MMF, 10 pM /BHQ, and 50 pM CSCS), alone or in combination, for 96 h.
  • VDDs 2.5 nM l,25D3, 5 nM PRI-5100 or 0.25 nM PRI-5202
  • Nrf2 activators at non-cytotoxic concentrations (10 mM CA, 50 mM DMF, 50 pM MMF, 10 pM /BHQ, and 50 pM CSCS), alone or in combination, for 96 h.
  • the extent of myeloid differentiation was then assessed by bivariate analysis of the surface expression of the specific monocytic marker CD 14 and the general myeloid marker CDl lb.
  • cytochrome c reduction assay to determine whether, similar to CA, tBHQ and MMF can augment the ability of HL60 cells induced to differentiate by VDDs to produce superoxide anion (a measure of the respiratory burst activity of mature myeloid cells).
  • TP A- stimulated superoxide generation was low in untreated cells and in those incubated with Nrf2 activators alone.
  • Treatment with VDDs alone at the concentrations used for flow cytometric determination of myeloid differentiation markers resulted in small ( ⁇ 2- fold) but significant increases in the respiratory burst activity, which was further dramatically enhanced in the presence of either MMF or tBHQ.
  • Nrf2 is a positive regulator of myeloid differentiation of AML cells.
  • Vitamin D derivatives and Nrf2 activators mutually upregulate VDR and Nrf2 signaling pathways
  • the inventors further examined whether, similar to CA, the Nrf2 activators tBHQ, and DMF can cooperate with VDDs in regulating the expression of VDR/RXRa, Nrf2 and their target genes in HL60 cells.
  • VDR protein levels were moderately increased in HL60 cells treated with 2.5 nM l,25D3 or 0.25 nM PRI-5202 while CA, /BHQ or DMF alone had a slight or no effect. Nonetheless, all three Nrf2 activators were capable of further enhancing VDR upregulation induced by both VDDs, particularly by PRI-5202 (Fig. 2).
  • RXRa protein expression was not affected by single compounds, its levels were moderately upregulated by the tested combinations (Fig. 2).
  • Nrf2 protein levels As expected, treatment with CA, tBHQ or DMF alone resulted in elevation Nrf2 protein levels, with CA having a stronger effect, whereas the VDDs alone were practically ineffective.
  • VDDs particularly, PRI-5202 to the Nrf2 activators further enhanced Nrf2 upregulation (Fig. 2).
  • Fig. 2 A similar, though more pronounced cooperation between Nrf2 activators and VDDs was observed when protein levels of the Nrf2 target gene product, TrxRl, were measured (Fig. 2).
  • CD1 lb integrated subunit a-M; ITGAM
  • Fig. 3D The expression of CD1 lb (integrin subunit a-M; ITGAM) gene was also cooperatively upregulated in these experiments (Fig. 3D), further supporting the association between the level of VDR and Nrf2 functional activation and the extent of myeloid differentiation of AML cells.
  • PRI-5202 has the lowest toxicity in mice compared to its direct precursors
  • PRI-5202 acted as a highly potent differentiation-inducing VDD shown to have the ability to cooperate with Nrf2 activators, including the clinically approved drug DMF.
  • the inventors thus, selected PRI-5202 and DMF for in vivo testing of their potential combinatory antileukemic activity in an animal model.
  • the inventors first determined the maximum tolerated dose (MTD) of PRI-5202 in healthy ICR mice.
  • PRI-1907 the direct precursor of PRI-5202 and l,25D3 were used for comparison.
  • the VDDs were injected intra- peritoneally (i.p.) at 0.05, 0.1, and 0.25 pg/mouse, 3 times per week.
  • mice injected with a relatively high dose of PRI-1907 or l,25D3 (0.25 pg/mouse) became moribund within the first one or two weeks of the experiment, likely due to severe hypercalcemia, and were euthanized.
  • the animals treated with a moderate dose of PRI-1907 (0.1 pg/mouse) demonstrated a marked time-dependent decrease in the body weight (Fig. 4A), severe hypercalcemia (Fig. 4B) and other signs of toxicity. This group, thus, was euthanized after 25 days.
  • the high calcemic toxicity of PRI-1907 in mice has been reported and was found to be even greater than that of l,25D3 (Fig. 4B).
  • mice demonstrated somewhat slower body weight gain rates compared to the vehicle-treated group, which was more evident in mice injected with 0.1 pg/mouse l,25D3 (Fig. 4A).
  • the persistent hypercalcemia was observed only in mice injected with 0.1 pg/mouse l,25D3 and PRI-1907, whereas in all the groups of PRI-5202-treated mice serum calcium levels remained within the normal range (9.0-10.5 mg/dL) throughout the entire period of the experiment (Fig. 4B).
  • Serum levels of ALT (Fig. 4C) and urea (Fig. 4D) in VDD-treated mice measured at the end of the experiment did not significantly differ from either untreated or vehicle -treated animals.
  • PRI-5202 and DMF cooperatively suppress AML tumor growth in vivo
  • PRI-5202 (0.25 pg/mouse) and DMF (0.6 mg/mouse), alone or in combination, induced no measurable toxicity to treated mice (Figs. 5C-5F).
  • DMF did not enhance its potential calcemic activity in vivo (Fig. 5C).
  • VDD and FAE induces cell differentiation and inhibits cell growth
  • Nrf2 activators tested did not induce cell differentiation but potentiated (to a varying extent) the differentiation-inducing effect of a low, near physiologic concentration of l,25D 3 (Fig. 7). According to their potentiating activities, the activators were divided into 3 groups with the following order of potencies: Gr. I > Gr. II >> Gr. 3. Notably, while being an established Nrf2 activator, rosmarinic acid had only a negligible differentiation-enhancing effect.
  • Nrf2 Transcriptionally inactive dominant-negative (DN)-Nrf2, which lacks the transactivation domain, is known to compete with and thereby inhibit Nrf2 transcriptional activity. Therefore, the data provided herein, is a direct evidence that the differentiation-enhancing effects of CA and MMF are mediated by transcriptionally active Nrf2 (Fig. 9B). Further, these data suggest that Nrf2 may function as a positive regulator of VDD signaling in AML cells.
  • MLL-AF9-transformed stem/progenitor cells were derived from the spleen of mice bearing murine AML (Minzel et al. (2016)). The data demonstrated that treatment with the DMF + PRI-5202 combination significantly prolonged the life span of leukemia-bearing mice (Fig. 11A). No apparent decrease in the animal body weight was observed during the treatment, indicating that it was well tolerated (Fig. 11B).

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Abstract

La présente invention concerne un procédé d'augmentation ou d'amélioration de l'efficacité thérapeutique d'un dérivé de vitamine D (VDD) ou d'ester d'acide fumarique (FAE) administré à un sujet en ayant besoin. L'invention concerne également une composition pharmaceutique comprenant du VDD et du FAE.
PCT/IL2019/050992 2018-09-06 2019-09-05 Compositions pharmaceutiques d'esters d'acide fumarique et dérivés de vitamine d et leurs utilisations WO2020049564A1 (fr)

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