WO2014138485A1 - Ex vivo production of platelets from hematopoietic stem cells and the product thereof - Google Patents

Ex vivo production of platelets from hematopoietic stem cells and the product thereof Download PDF

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WO2014138485A1
WO2014138485A1 PCT/US2014/021418 US2014021418W WO2014138485A1 WO 2014138485 A1 WO2014138485 A1 WO 2014138485A1 US 2014021418 W US2014021418 W US 2014021418W WO 2014138485 A1 WO2014138485 A1 WO 2014138485A1
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ethyl
purin
platelets
alkyl
reagent
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PCT/US2014/021418
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French (fr)
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Anthony E. Boitano
Michael P. Cooke
Jennifer SNEAD
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Irm Llc
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0644—Platelets; Megakaryocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/145—Thrombopoietin [TPO]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2306—Interleukin-6 (IL-6)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/73—Hydrolases (EC 3.)
    • C12N2501/734—Proteases (EC 3.4.)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • the present invention relates to a method and reagents for producing platelets from hematopoietic stem cell ex vivo.
  • the invention further relates to a therapeutic composition comprising platelets produced by the ex vivo method and its use in autologous or allogeneic transplantation for the treatment of patients with thrombocytopenia.
  • HLA-alloimmunized patients are ideally given matched platelets to avoid immediate platelet rejection; however, identifying and obtaining matched donor platelets is often not feasible and is a current unmet medical need.
  • the present invention relates to a process and reagents for manufacturing platelets from hematopoietic stem cells ex vivo.
  • the process is adaptable to manufacture platelets in a patient specific manner by using HLA-matched hematopoietic stem cells.
  • the exPLTs potentially can be a source of HLA-matched platelets for alloimmunized thrombocytopenic patients without a donor platelet match and, as such, will increase post-transfusion platelet recovery compared to standard of care (unmatched platelets).
  • the possibility of using exPLT to supplement and/or replace standard platelet products will help to alleviate a reliance on donors, avert shortages and improve product safety.
  • the invention provides a process of ex vivo manufacturing clinically relevant quantities of ex-vivo produced platelets (exPLTs) from hematopoietic stem cells.
  • the process comprising a step of:
  • a quantity of megakaryocytes with a small molecule reagent and thrombopoietin (TPO) for a duration of time to yield a population of ex vivo produced platelets (exPLT), wherein the small molecule reagent is a compound of Formula II or a pharmaceutically acceptable salt, or stereoisomer thereof; wherein the variables are as described in the Detailed Description of the Invention, paragraphs [00138], infra.
  • TPO thrombopoietin
  • the invention provides a composition of platelets produced from human hematopoietic stem cells by the ex vivo manufacturing method described in the first aspect of the invention, supra.
  • the invention provides a composition comprising ex vivo produced megakaryotecytes (exMKs) in a cell culture medium containing Reagent B.
  • exMKs ex vivo produced megakaryotecytes
  • the invention provides a composition comprising ex vivo produced platelets and Reagent B.
  • the invention relates to a composition of isolated ex vivo produced platelets characterized by the presence of Reagent B.
  • the invention relates to a method of using the ex vivo produced platelets for treating thrombocytopenia; thereby treating, abating, ameliorating or alleviating the diseases and conditions and/or its attendant symptoms that are the underlying causes of the thrombocytopenia.
  • Fig 1 Flow chart of the two-stage process of the invention useful for the manufacturing of platelets from hematopoietic stem cells ex vivo.
  • Fig. 2 A two-stage protocol is optimal for exPLTs production. The contribution of the presence or absence of: a first stage with 500 nM of Compound A1 (Reagent A), a second stage with 500 nM of Compound B157S (Reagent B) or 750 nM of Compound B1 (Reagent B) to platelet yield (number count per initiating HSC) were measured. "+” means the indicated stage was present and "-" means the indicated stage was absence.
  • Fig 3 The exPLTs are phenotypically the same as whole blood platelets.
  • Flow cytometry analyses of cell cultures ex vivo produced platelets at day 18 (top row) is compared to whole blood platelets (bottom row). Platelets were distinguished from nucleated cells by size (FSC) and granularity (SSC) and were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate.
  • FSC size
  • SSC granularity
  • Fig. 4 The exPLTs are similarly viable as whole blood platelets. Viability of the platelets was assessed by flow cytometry analyses of Calcein AM+ stained platelets from culture derived platelets at day 18 (top row) and whole blood platelets (bottom row). Platelets were distinguished from nucleated cells by size (FSC) and granularity (SSC) and were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate.
  • FSC size
  • SSC granularity
  • Fig. 5 The culture-derived platelets contain significantly higher percentage of young platelets. The percentage of young platelets were assessed by flow cytometry analyses of thiazole orange+ stained platelets of culture-derived platelets at day 18 (top row) and whole blood platelets (bottom row). Platelets were distinguished from nucleated cells by size (FSC) and granularity (SSC) and were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate.
  • FSC size
  • SSC granularity
  • exPLTs are functioned in vivo. Residual human platelets (number counts) from ex vivo culture derived, donor derived, and damaged whole blood (treated with carbonyl cyanide 3-chlorophenylhydrazone) in mouse circulation were measured 4 hrs (right) and 24 hrs (left) after injection. Residual human platelets were detected for culture- derived and donor derived platelets and not for damaged platelets.
  • exPLTs are functioning in vitro. The spreading response of exPLTs to thrombin (agonist) stimulation; no agonist (right) and with agonist (left).
  • Platelet yields folds over culturing with the 4- cytokine cocktail and Reagent B (250 nM of Compound 157S)) in the presence or absence of: a 4-cytokine cocktail (TPO, IL-6, Flt3-L and SCF, 50 ng/mL each), 100 ng/mL of TPO only, and/or Reagent B (250 nM of Compound B157S) were measured.
  • a 4-cytokine cocktail TPO, IL-6, Flt3-L and SCF, 50 ng/mL each
  • 100 ng/mL of TPO only 100 ng/mL of TPO only
  • Reagent B 250 nM of Compound B157S
  • TPO is effective within a wide concentration range. Platelet yields
  • Fig. 10 The effective concentration of Reagent B is different for the various compounds suitable for use as Reagent B, and should be separately determined. Platelet yields (folds over vehicle control) in response to the presence of 0.1 to 10,000 nM of Reagent B (Compound B157S, B88, or B1 ) were measured.
  • Fig. 1 1 Matrix metalloproteinase inhibitor is not required for producing functional platelets. Residual human platelets (number count) from ex vivo culture derived, donor derived, and damaged whole blood (treated with carbonyl cyanide 3- chlorophenylhydrazone) in mouse circulation were measured 4 hours after infusion.
  • Residual human platelets were detected for culture-derived and donor derived platelets and not for damaged platelets.
  • Fig. 12 The presence of MMP inhibitor in the second phase improved platelet yield.
  • the yield of platelets (number count per initiating cell) were measured in culture containing vehicle, 25 ⁇ of GM6001 or 10 ⁇ of TAPI-1 ; an approximately 30% fold increase was observed.
  • Fig. 13 The optimal duration for the first culture phase is 14 days. Effect of the duration of the first culture phase on exPLT yields on day 18 (number count per initiating HSC cell) (top left), exMK yield on day 14 (number count per initiating HSC cell) (top right) and % exMK on day 14 (bottom left) were measured.
  • Fig. 14 Recovering of the megakaryocytes by pelleting and resuspending is superior to isolation. Platelet yields (number count per exMK on day12) in the presence or absence of: Reagent B, pelleted and resuspension of megakaryocytes after the first phase, and isolation of megakaryocytes after the first phase were measured. "+” means the indicated reagent/protocol was present and "-" means the indicated reagent/protocol was absent.
  • Post-isolation platelets are phenotypically the same as pre- isolation platelets and whole blood platelets. Flow cytometry characterization of platelets from: pre-isolation (top row), post-isolation (middle row), and whole blood (bottom row) were conducted. Platelets were distinguished from nucleated cells by size (FSC) and granularity (SSC) and were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate.
  • FSC size
  • SSC granularity
  • Alkoxy refers the radical -O-alkyl, wherein the alkyi is as defined herein.
  • Y alkoxy as used herein describe alkoxy groups where X and Y indicate the number of carbon atoms in the alkyi chain. Representative examples of d- !
  • oalkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert- butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy and decyloxy.
  • the alkyi portion of the alkoxy may be optionally substituted, and the substituents include those described for the alkyi group below.
  • Alkyi refers to a fully saturated branched or unbranched hydrocarbon chain having up to 10 carbon atoms.
  • C x alkyi and C X - Y alkyi as used herein describe alkyi groups where X and Y indicate the number of carbon atoms in the alkyi chain.
  • C H0 alkyi refers to an alkyi radical as defined above containing one to ten carbon atoms.
  • C H0 alkyi includes, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2- dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like.
  • Alkyi represented along with another radical like arylalkyl, heteroarylalkyl, alkoxyalkyl, alkoxyalkyl, alkylamino, where the alkyi portion shall have the same meaning as described for alkyi and is bonded to the other radical.
  • (C 6 - 10 )aryl(C 1 - 3 )alkyl includes, benzyl, phenylethyl, 1 -phenylethyl, 3-phenylpropyl, 2-thienylmethyl, 2-pyridinylmethyl and the like.
  • an alkyi group may be unsubstituted or substituted by one or more substituents to the extent that such substitution makes sense chemically.
  • substituents include, but are not limited to halo, hydroxyl, alkoxy, cyano, amino, acyl, aryl, arylalkyl, and cycloalkyl, or an heteroforms of one of these groups, and each of which can be substituted by the substituents that are appropriate for the particular group.
  • Alkenyl refers to a straight or branched, hydrocarbon chain having up to 10 carbon atoms and at least one carbon-carbon double bond.
  • C x alkenyl and C x C x alkenyl and C x .
  • Y alkenyl as used herein describe alkenyl groups where X and Y indicate the number of carbon atoms in the alkenyl chain.
  • Examples of C 2 - 7 alkenyl include vinyl, allyl, isopropenyl, pentenyl, hexenyl, heptenyl, 1 -propenyl, 2-butenyl, 2-methyl-2-butenyl, and the like.
  • the alkenyl may be optionally substituted, and the substituents include those described for the alkyi group descried herein.
  • Alkynyl refers to a straight or branched, hydrocarbon chain having up to 10 carbon atoms and at least one carbon-carbon triple bond.
  • Y alkenyl as used herein describe alkynyl groups, where X and Y indicate the number of carbon atoms in the alkynyl chain.
  • C 2 - 7 alkenyl include, but are not limited to, ethynyl, propargyl, 3-methyl-1 -pentynyl, 2-heptynyl and the like.
  • An alkynyl may be optionally substituted, and the substituents include those described for the alkyl group described herein.
  • Alkylene refers to a divalent alkyl group defined herein.
  • C ⁇ oalkylene includes, but are not limited to, methylene, ethylene, n-propylene, /so-propylene, n-butylene, sec-butylene, /so-butylene, ferf-butylene, n-pentylene, isopentylene, neopentylene, n-hexylene, 3-methylhexylene, 2,2- dimethylpentylene, 2,3- dimethylpentylene, n-heptylene, n-octylene, n-nonylene and n-decylene.
  • An alkylene group may be optionally substituted, and the substituents include those described for the alkyl group described herein.
  • Amino refers to the radical -NH2.
  • the term includes NR'R" wherein each R' and R" is independently H, or is an alkyl, alkenyl, alkynyl, acyl, aryl, aryl, cycloalkyl, arylalkyl cycloalkylalkyl group or a heteroform of one of these groups, and each of the alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl or groups or heteroforms of one of these groups, each of which is optionally substituted with the substituents described herein as suitable for the corresponding group.
  • amino also includes forms wherein R' and R" are linked together to form a 3-8 membered ring which may be saturated, unsaturated or aromatic and which contains 1 -3 heteroatoms independently selected from N, O and S as ring members, and which is optionally substituted with the substituents described as suitable for alkyl groups or, if NR'R" is an aromatic group, it is optionally substituted with the substituents described as typical for heteroaryl groups.
  • the compounds of the invention containing amino moieties may include protected derivatives thereof.
  • Suitable protecting groups for amino moieties include acetyl, tert-butoxycarbonyl, benzyloxycarbonyl, and the like.
  • Alkylamino refers to the radical -NR a R b , where at least one of, or both, R a and R b are an alkyl group as described herein.
  • An d ⁇ alkylamino group includes -NHC 1 _ 4 alkyl and -N(C 1 _ 4 alkyl) 2 ; e.g., -NHCH 3 , -N(CH 3 ) 2 , -NH(CH 2 CH 3 ), - N(CH 2 CH 3 ) 2 , and the like.
  • Aryl refers to a 6-14 membered monocyclic or polycyclic aromatic ring assembly where all the ring atoms are carbon atoms.
  • the aryl is a 6 membered monocyclic, a 10-12 membered bicyclic or a 14-membered fused tricyclic aromatic ring system.
  • C x aryl and C X - Y aryl as used herein describe an aryl group where X and Y indicate the number of carbon atoms in the ring system.
  • C 6 . 14 aryls include, but are not limited to, phenyl, biphenyl, naphthyl, azulenyl, and anthracenyl.
  • An aryl may be unsubstituted or substituted by 1 -5 (such as one, or two, or three) substituents independently selected from the group consisting of hydroxy, thiol, cyano, nitro, C1 -4alkyl, C1 -4alkenyl, C1 -4alkynyl, C1 -4alkoxy, thioC1 -4alkyl, C1 - 4alkenyloxy, C1 -4alkynyloxy, halogen, C1 -4alkylcarbonyl, carboxy, C1 -4alkoxycarbonyl, amino, C1 -4alkylamino, di-C1 -4alkylamino, C1 -4alkylaminocarbonyl, di-C1 - 4alkylaminocarbonyl, C1 -4alkylcarbonylamino, C1 -4alkylcarbonylamino, C1 -4alkylcarbonyl(C1 -4alkyl)amino
  • aryloxyalkyl aryloxycarbonyl
  • aryloxy-carbonylalkyl aryloxy-carbonylalkyl
  • Aryloxy refers to the radical -O-aryl, wherein aryl is as defined herein.
  • Cycloalkyl means a radical comprising a non-aromatic, saturated or partially unsaturated, monocyclic, bicyclic, tricyclic, fused, bridged or spiro polycyclic hydrocarbon ring system of 3-20 carbon atoms.
  • C x cycloalkyl and C X - Y cycloalkyl are typically used where X and Y indicate the number of carbon atoms in the ring assembly.
  • C 3 - 6 cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,5-cyclohexadienyl.
  • Exemplary monocyclic hydrocarbon groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl and the like.
  • a cycloalkyl may be unsubstituted or substituted by one, or two, or three, or more substituents independently selected from the group consisting of hydroxyl, thiol, cyano, nitro, oxo, alkylimino, d- 4 alkyl, C 1 - 4 alkenyl, d- 4 alkynyl, d- 4 alkoxy, C 1 - 4 thioalkyl, d- 4 alkenyloxy, C 1 - 4 alkynyloxy, halogen, d- 4 alkylcarbonyl, carboxy, d- 4 alkoxycarbonyl, amino, d- 4 alkylamino, di-d- 4 alkylamino, d- 4 alkylaminocarbonyl, di-d- 4 alkylaminocarbonyl, d- 4 alkylcarbonylamino, d ⁇ alkylcarbony d ⁇ alky amino, sulfonyl
  • Cycloalkylene refers to a divalent radical comprising a cycloalkyl ring assembly as defined herein.
  • C 3.12 cycloalklyoxy refers to -O-cycloalkyl, wherein the cycloalkyl is defined herein.
  • Representative examples of C 3.12 cycloalklyoxy include, but are not limited to, monocyclic groups such as cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclopentenyloxy, cyclohexyloxy and cyclohexenyloxy and the like.
  • bicyclic hydrocarbon groups include bornyloxy, indyloxy, hexahydroindyloxy, tetrahydronaphthyloxy, decahydronaphthyloxy, bicyclo[2.1 .1 ]hexyloxy, bicyclo[2.2.1 ]heptyloxy,
  • Exemplary tricyclic hydrocarbon groups include, for example, adamantyloxy.
  • Cyano refers to the radical -CN.
  • Disease specifically includes any unhealthy condition of an animal or part thereof and includes an unhealthy condition that may be caused by, or incident to, medical or veterinary therapy applied to that animal, i.e., the "side effects" of such therapy.
  • EC 50 refers to the molar concentration of an inhibitor that produces 50% efficacy.
  • Halo or "halogen” as used herein refers to fluoro, chloro, bromo, and iodo.
  • Haloalkyl refers to an alkyl as defined herein, which is substituted by one or more halo atoms defined herein.
  • the haloalkyl can be mono-haloalkyl, dihaloalkyl or polyhaloalkyl including perhaloalkyl.
  • a monohaloalkyi can have one iodo, bromo, chloro or fluoro within the alkyl group.
  • Dihaloalky and polyhaloalkyl groups can have two or more of the same halo atoms or a combination of different halo groups within the alkyl.
  • Ci_ 4 haloalkyl include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl,
  • a d- 4 perhaloalkyl group refers to a C _ 4 alkyl group having all hydrogen atoms replaced with halo atoms.
  • Heteroaryl refers to a 5-14 membered ring assembly
  • C x heteroaryl and C X - Y heteroaryl as used herein describe heteroaryls where X and Y indicate the number of ring atoms in the heteroaryl ring. Typical C 5 .
  • yheteroaryl groups include thienyl, furanyl, imidazolyl, pyrazolyl, pyrrolyl, pyrrolinyl, thiazolyl, 1 ,3,4-thiadiazolyl, isothiazolyl, oxazolyl, oxadiazole isoxazolyl, triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrazinyl, pyrazinyl, pyrimidinyl, and the like.
  • Bicyclic or tricyclic C 8 - 14 heteroaryls include, but are not limited to, those derived from benzo[b]furan,
  • a heteroaryl may be unsubstituted or substituted with one or more substituents independently selected from hydroxyl, thiol, cyano, nitro, d ⁇ alkyl, d ⁇ alkenyl, C _ 4 alkynyl, d- 4 alkoxy, thioC - 4 alkyl, d ⁇ alkenyloxy, d- 4 alkynyloxy, halogen, d- 4 alkylcarbonyl, carboxy, d- 4 alkoxycarbonyl, amino, d- 4 alkylamino, di-d- 4 alkylamino, d- 4 alkylaminocarbonyl, di-d- 4 alkylaminocarbonyl, d- 4 alkylcarbonylamino, d- 4 alkylcarbonylamino, d- 4 alkylcarbonyl(d- 4 alkyl)amino, sulfonyl, sulfamoyl, alkylsul
  • heteroaryloxy shall have the same meaning as described in the above-mentioned definition of “heteroaryl”.
  • Heteroaryloxy refers to an -O-heteroaryl group, wherein the heteroaryl is as defined in this Application.
  • Heteroatom refers to an atom that is not a carbon atom.
  • heteroatoms include, but are not limited to nitrogen, oxygen, and sulfur.
  • Heterocycloalkyl refers to a 4-20 membered, non- aromatic, saturated or partially unsaturated, monocyclic or polycyclic ring system, comprising 1 -8 heteroatoms as ring atoms and that the remaining ring atoms are carbon atoms.
  • the heteroatoms are selected from N, O, and S, preferably O and N.
  • the nitrogen atoms of the heterocycloalkyl can be optionally quaternerized and the sulfur atoms of the heterocycloalkyl can be optionally oxidized.
  • the heterocycloalkyl can include fused or bridged rings as well as spirocyclic rings.
  • C x heterocycloalkyl and C X - Y heterocycloalkyl are typically used where X and Y indicate the number of ring atoms in the ring.
  • the .heterocycloalkyl is 4-8-membered monocyclic ring containing 1 to 3 heteroatoms, a 7 to 12- membered bicyclic ring system containing 1 -5 heteroatoms, or a 10-15-membered tricyclic ring system containing 1 to 7 heteroatoms.
  • C 4 - 6 heterocycloalkyl examples include azetidinyl, tetrahydrofuran (THF), dihydrofuran, 1 , 4-dioxane, morpholine, 1 ,4-dithiane, piperazine, piperidine, 1 ,3-dioxolane, imidazolidine, imidazoline, pyrazolidinyl, pyrroline, pyrrolidine, tetrahydropyran, dihydropyran, oxathiolane, dithiolane, 1 ,3-dioxane, 1 ,3- dithiane, oxathiane, thiomorpholine, and the like
  • a heterocycloalkyl may be unsubstituted or substituted with 1 -5 substituents (such as one, or two, or three) each independently selected from hydroxyl, thiol, cyano, nitro, oxo, alkylimino, C 1 - 4 alkyl, C 1 - 4 alkenyl, C 1 - 4 alkynyl, d- 4 alkoxy, d- 4 thioalkyl, d- 4 alkenyloxy, d- 4 alkynyloxy, halogen, d- 4 alkylcarbonyl, carboxy, d- 4 alkoxycarbonyl, amino, d- 4 alkylamino, di- d- 4 alkylamino, d- 4 alkylaminocarbonyl, di-d- 4 alkylaminocarbonyl, d- 4 alkylcarbonylamino, d- 4 alkylcarbonyl(d- 4 alkyl)amino
  • heterocycloalkyl forms part of other groups like “heterocycloalkyl- alkyl”, “heterocycloalkoxy”, “heterocycloalkyl-aryl", the heteroaryl portion shall have the same meaning as described in the above-mentioned definition of "heteroaryl”
  • Haldroxy refers to the radical -OH.
  • Hydroxyalkyl or "hydroxyl-substituted alkyl” as used herein, refers to an alkyl as defined herein, having one or more of the available hydrogen of the alkyl replaced by a hydroxyl group.
  • a hydroxyd_ 4 alkyl includes, but are not limited to, - CH 2 CH 2 OH, -CH(OH)CH 2 CH 2 OH, - CH(OH)CH 2 CH(OH)CH 3 .
  • Stepoisomers refers to compounds having identical molecular formulae but differ in the arrangement of their atoms in space are termed
  • stereoisomers Stereoisomers that are nonsuperimposable mirror images are termed “enantiomers” or sometimes “optical isomers.” Stereoisomers that are not mirror images of one another are termed “diastereomers”. Conventions for stereochemical nomenclature, methods for the determination of stereochemistry and the separation of stereoisomers are well known in the art (e.g., see “Advanced Organic Chemistry", 4th edition, March, Jerry, John Wiley & Sons, New York, 1992).
  • a carbon atom bonded to four different substituents is termed a "chiral center.”
  • a compound with one chiral center has two enantiomeric forms of opposite chirality.
  • a mixture of equal amounts of the two enantiomeric forms is termed a "racemic mixture.”
  • a compound that has more than one chiral center has 2n-1 enantiomeric pairs, where n is the number of chiral centers.
  • Compounds with more than one chiral center may exist as either an individual diastereomer or as a mixture of diastereomers, termed a "diastereomeric mixture.”
  • a stereoisomer may be characterized by the absolute configuration of that chiral center.
  • Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center.
  • Enantiomers are characterized by the absolute configuration of their chiral centers and described by the R- and S-sequencing rules of Cahn, Ingold and Prelog.
  • Ni refers to the radical -N0 2 .
  • “Pharmaceutically acceptable”, as used herein, means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use.
  • Radar refers to an atom or group of atoms having an open valence for bonding with another atom or groups of atoms.
  • “optionally substituted” or “substituted” group may include, without limitation, one or more substituents independently selected from the group or designated subsets thereof, aldehyde, alkyl, alkylene, alkylidene, amide, amino, aminoalkyl, aryl, bicycloalkyl, bicycloaryl, carbamoyl, carbocyclyl, carboxyl, carbonyl group, cycloalkyl, cycloalkylene, ester, halo, heterobicycloalkyl, heterocycloalkylene, heteroaryl, heterobicycloaryl, heterocycloalkyl, oxo, hydroxy, iminoketone, ketone, nitro, oxaalkyl, and oxoalkyl moieties, each of which may optionally also be substituted or unsubstituted.
  • “Therapeutically effective amount”, as used herein, means that amount which, when administered to an animal for treating a disease, is sufficient to effect such treatment for the disease.
  • any definition herein may be used in combination with any other definition to describe a composite structural group.
  • the trailing element of any such definition is that which attaches to the parent moiety.
  • the composite group alkoxyalkyl would represent an alkoxy group attached to the parent molecule through an alkyl group.
  • a d alkyl indicates that there is one carbon atom but does not indicate what are the substituents on the carbon atom.
  • a dalkyl comprises methyl (i.e., -CH 3 ) as well as -CR a RbR c where R a , Rb, and R c may each independently be hydrogen or any other substituent where the atom attached to the carbon is not a hydrogen atom.
  • -CF 3 , -CH 2 OH and -CH 2 CN are all dalkyls.
  • Hematopoietic stem cells refer to immature blood cells having the capacity to self-renew and to differentiate into more mature blood cells comprising granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes, platelets), and monocytes (e.g., monocytes, macrophages).
  • HSCs are interchangeably described as stem cells throughout the specification. It is known in the art that such cells may or may not include CD34 + cells.
  • CD34 + cells are immature cells that express the CD34 cell surface marker.
  • CD34+ cells are believed to include a subpopulation of cells with the stem cell properties defined above.
  • HSCs include pluripotent stem cells, multipotent stem cells (e.g., a lymphoid stem cell), and/or stem cells committed to specific hematopoietic lineages.
  • the stem cells committed to specific hematopoietic lineages may be of T cell lineage, B cell lineage, dendritic cell lineage, Langerhans cell lineage and/or lymphoid tissue-specific macrophage cell lineage.
  • HSCs also refer to long term HSC (LT-HSC) and short term HSC (ST-HSC).
  • ST-HSCs are more active and more proliferative than LT-HSCs.
  • LT-HSC have unlimited self-renewal (i.e., they survive throughout adulthood), whereas ST-HSC have limited self-renewal (i.e., they survive for only a limited period of time). Any of these HSCs can be used in any of the methods described herein.
  • ST-HSCs are useful because they are highly proliferative and thus, quickly increase the number of HSCs and their progeny.
  • Hematopoietic stem cells are optionally obtained from blood products.
  • a blood product includes a product obtained from the body or an organ of the body containing cells of hematopoietic origin.
  • Such sources include un-fractionated bone marrow, umbilical cord, peripheral blood, liver, thymus, lymph and spleen. All of the aforementioned crude or un-fractionated blood products can be enriched for cells having hematopoietic stem cell characteristics in ways known to those of skill in the art.
  • Treatment refers to a method of alleviating or abating a disease and/or its attendant symptoms.
  • “Expansion” in the context of cells refers to increase in the number of a characteristic cell type, or cell types, from an initial cell population of cells, which may or may not be identical.
  • the initial cells used for expansion may not be the same as the cells generated from expansion.
  • Cell population refers to eukaryotic mammalian, preferably human, cells isolated from biological sources, for example, blood product or tissues and derived from more than one cell.
  • a population of cells is generally inhomogeneous in the sense that the stem cell could be in various developmental stages.
  • stem cells when apply to stem cells, a population of HSC cells typically means the number of HSC containing in two cord blood units, more typically one cord blood unit.
  • Enriched when used in the context of cell population refers to a cell population selected based on the presence of one or more markers, for example, CD34+.
  • CD34+ cells refers to cells that express at their surface CD34 marker. CD34+ cells can be detected and counted using for example flow cytometry and fluorescently labeled anti-CD34 antibodies.
  • CD34+ cells “Enriched in CD34+ cells” means that a cell population has been selected based on the presence of CD34 marker. Accordingly, the percentage of CD34+ cells in the cell population after selection method is higher than the percentage of CD34+ cells in the initial cell population before selecting step based on CD34 markers.
  • CD34+ cells may represent at least 50%, 60%, 70%, 80% or at least 90% of the cells in a cell population enriched in CD34+ cells.
  • Cord blood unit refers to the blood collected from umbilical cord of a single birth.
  • Megakaryopoiesis as used herein means the differentiation of hematopoietic stem cells to megakaryocytes, which under current model of hematopoiesis, involves differentiation of the multipotent HSC to the common myeloid progenitor which then differentiates into the megakaryocyte/erythrocyte progenitors, and then into
  • Plate biogenesis as used herein means the formation of platelets.
  • the process which under current model of hematopoiesis, includes the maturing of megakaryocytes towards the formation of protoplatelet, and the release of platelets
  • the invention relates to a process for manufacturing platelets from hematopoietic stem cells ex vivo.
  • the process uses known and proprietary agents to manufacture platelets using a two-stage GMP compatible culture.
  • the process can produce platelets in a patient specific manner by using HLA-matched CD34+ cells derived from a CB unit.
  • the process may be adapted for large scale production of clinical relevant number of platelets to meet clinical needs.
  • the invention also relates to compositions of ex vivo manufactured platelets (exPLTs).
  • the exPLTs composition is as obtained by the process of the invention.
  • these exPLTs compositions are suitable for autologous or allogeneic transplantation.
  • the cell therapy product is characterized by the presence of a proprietary Reagent B (defined below) used in the production.
  • Reagent B is an antagonist of the aryl hydrocarbon receptor.
  • the cell therapy product is HLA-matched to a specific patient.
  • the invention further relates to use of the exPLT for treating patient suffering from thrombocytopenia, thereby treating the underlying diseases and conditions that caused the thrombocytopenia.
  • the exPLTs are HLA- matched for treating an alloimmunized thrombocytopenic patients.
  • FIG. 1 shows a schematic of the process of the invention.
  • the process of the invention is a two-stage process. The process first generates megakaryocytes by directed differentiation of hematopoietic stem cells and progenitor cell (HSPC) and second promotes platelet biogenesis from the megakaryocytes. Each of the stages requires a low molecular weight reagent and a combination of cytokines to achieve the desirable result.
  • the required Reagent A is a known platelet-derived growth factor receptor (PDGFR) antagonist.
  • the required Reagent B is an aryl hydrocarbon receptor (AHR) antagonist.
  • the process may optionally employ a third low molecular weight reagent, which is a matrix metalloproteinase receptor (MMP) antagonist.
  • MMP matrix metalloproteinase receptor
  • the invention is related to an ex vivo process for producing a population of ex vivo produced platelets (exPLTs) comprising a Step C, which comprises:
  • TP B is a compound of Formula Ila:
  • the process of the invention further comprises a process of producing the exMKs from hematopoietic stem cells (HSC), wherein Step A comprising:
  • Reagent A is a compound of Formula I:
  • Steps B1 and B2 which comprise:
  • Step B1 gathering the quantity of exMKs; and Step B2: re-suspending the quantity of exMKs.
  • ex vivo process for producing a population of ex vivo produced platelets (exPLTs) of the invention comprising the steps of:
  • HSC hematopoietic stem cells
  • Regent A is 3-(7-((2,6-dimethylpyridin-3-yl)amino)-1 -methyl-2-oxo-1 ,2- dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N-(3-(trifluoromethyl)phenyl)-4- vinylbenzamide;
  • Reagent B is of Formula lib:
  • the process of the invention further comprises Steps B1 and B2, which comprising:
  • Step B1 pelleting the population of exMKs.
  • Step B2 re-suspending the population of exMKs.
  • MK1 platelet-derived growth factor receptor
  • PDGFR platelet-derived growth factor receptor
  • PDGFR platelet-derived growth factor receptor
  • Example D we demonstrated that a PDGFR inhibition alone contributed a 2.5-fold yield increase, an AHR inhibition or alone contributed a 3-fold yield increase, and combining PDGFR inhibition in a first phase culture with AHR inhibition in a second phase culture provided 7.5-fold yield increase ( Figure 2).
  • Example E Our optimized process with MK1 at stage 1 and AHR antagonists along with a matrix metalloprotease (MMP) inhibitor and TPO as the sole cytokine at stage 2 is illustrated in Example E.
  • HSC were cultured for a total of 18 days, according to protocol.
  • exPLTs are functional. Using a mouse model, Example F demonstrated that ex-vivo culture-derived platelets and donor plateles persisted in mouse circulation with similar kinetics, whereas damaged platelets were more efficiently cleared from circulation with significantly decreased levels at 4 hr and 24 hr after injection (Figure 6).
  • Example G shows that exPLTs exhibited cell spreading on fibrinogen-coated surface in response to thrombin activation (Figure 7).
  • Step A1 or A The Starting Hematopoietic Stem Cell Population (Step A1 or A)
  • a starting cell population comprising hematopoietic stem cells will be selected by the person skilled in the art depending on the envisaged use.
  • Various sources of cells comprising HSC have been described in the art, including bone marrow, peripheral blood, neonatal umbilical cord blood, placenta or other sources such as liver, particularly fetal liver.
  • a population of the HSC may be obtained from commercial sources, e.g., Bioreclamation.
  • the HSC population may first be subjected to enrichment or purification steps, including negative and/or positive selection of cells based on specific cellular markers, in order to provide the starting HSC population.
  • Methods for isolating the starting cell population based on specific cellular markers may use fluorescent activated cell sorting (FACS) technology also called flow cytometry or solid or insoluble substrate to which is bound antibodies or ligands that interact with specific cell surface markers.
  • FACS fluorescent activated cell sorting
  • cells may be contacted with a solid substrate (e.g., column of beads, flasks, magnetic particles) containing the antibodies and any unbound cells are removed.
  • a solid substrate comprising magnetic or paramagnetic beads
  • cells bound to the beads can be readily isolated by a magnetic separator.
  • the starting cell population is enriched in a desirable cell marker phenotype (e.g., CD34+, CD133+, CD90+) or based on efflux of dyes such as rhodamine, Hoechst or aldehyde dehydrogenase activity.
  • a desirable cell marker phenotype e.g., CD34+, CD133+, CD90+
  • the starting cell population is enriched in CD34+ cells.
  • Methods for enriching blood cell population in CD34+ cells include kits commercialized by Miltenyi Biotec (CD34+ direct isolation kit, Miltenyi Biotec, Bergisch, Gladbach, Germany) or by Baxter (Isolex 3000).
  • the starting HSC population is derived from neonatal umbilical cord blood cells which have been enriched in CD34+ cells.
  • the starting HSC population is consisting essentially of CD34+ cells purified from one or two cord blood units.
  • the starting HSC population is derived from bone marrow blood cells.
  • the starting HSC population is derived from human mobilized peripheral blood cells which have been enriched in CD34+ cells.
  • said starting HSC population is derived from a single mammalian subject.
  • the starting HSC population is blood cells derived from human.
  • the starting HSC population may preferably contain at least 50% CD34+ cells, in some embodiments, more than 90% of CD34+ cells, and may comprise between 10 5 and 10 9 nucleated cells.
  • the starting HSC population may be used directly in the culture or frozen and stored for use at a later date.
  • the culturing of the HSC population may be carried out in a basal medium, which is supplemented with the mixtures of cytokines and growth factors described above.
  • a basal medium typically comprises amino acids, carbon sources, vitamins, serum proteins (e.g. albumin), inorganic salts, divalent cations, buffers and any other element suitable for use in culturing of HSC.
  • basal medium appropriate for a method of culturing HSC include, without limitation, StemSpan ® SFEM - Serum-Free Expansion Medium (StemCell Technologies, Vancouver, Canada), StemSpan ® H3000 - Defined Medium (StemCell Technologies, Vancouver, Canada), CellGro ® SCGM
  • the culture medium used is StemSpan SFEM
  • the medium is further supplemented with a recombinant human cytokines.
  • the recombination of cytokines is different for each of steps of the process, and will be further discussed in the next section.
  • the culture media is preferably prepared fresh the day of use.
  • Conditions for culturing the starting cell population for HSC expansion and megakaryocytepoesis will vary depending, inter alia, on the starting cell population, the desired final number and ploidy of the megakaryocytes.
  • TPO thrombopoietin
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • GCSF granulocyte colony-stimulating factor
  • IL-3 interleukin-3
  • IL-1 IL-6
  • IL- 1 1 IL- 1 1
  • Flt3-L which in synergy with other growth factors, stimulates the proliferation and differentiation of various blood cell progenitors.
  • Stem cell factor has been shown to increase the survival of HSCs in vitro and contributes to the self-renewal and maintenance of HSCs in vitro.
  • TPO that acts throughout megakaryopoiesis
  • these cytokines affect megakaryopoiesis during the early stages of megakaryocyte development.
  • Flt3-L or FLT-3 Ligand also referred as FL is a factor that binds to flt3- receptor, has been described by Hannum C, Nature 368 (6472): 643-8.
  • Stem cell factor or Human SCF also known as c-kit ligand, mast cell growth factor, or Steel factor has been described by Smith, MA et al. 2001 ACTA Haematologica 105, 3:143, 2001 .
  • Human IL-6 or interleukin-6, also known as B-cell stimulatory factor 2 has been described by Kishimoto, Ann. review of Imm. 23:1 2005.
  • TPO or thrombopoietin also known as megakarayocyte growth factor (MGDF) or c-Mpl ligand has been described (Kaushansky K (2006). N. Engl. J. Med. 354 (19) : 2034-45).
  • MGDF megakarayocyte growth factor
  • c-Mpl ligand has been described (Kaushansky K (2006). N. Engl. J. Med. 354 (19) : 2034-45).
  • TPO TPO
  • SCF SCF
  • Flt-3 Flt-3
  • IL-6 All of TPO, SCF, Flt-3 and IL-6 are commercially available.
  • the culturing comprises the use of cytokines and growth factors, generally known in the art for hematopoietic stem cell expansion and differentiation.
  • cytokines and growth factors include without limitation IL-1 , IL-3, IL-6, IL-1 1 , G-CSF, GM-CSF, SCF, Flt3-L, thrombopoietin (TPO), erythropoeitin, and analogs thereof.
  • analogs include any structural variants of the cytokines and growth factors having the biological activity as the naturally occurring forms, including without limitation, variants with enhanced or decreased biological activity when compared to the naturally occurring forms or cytokine receptor agonists such as an agonist antibody against the TPO receptor (for example, VB22B sc(Fv)2 as detailed in patent publication WO 2007/145227, and the like).
  • Cytokine and growth factor combinations are chosen to support HSC expansion and to induce HSC and progenitor cells differentiation, and preferably down the megakaryocyte/erythrocyte progenitor pathway.
  • Step A the contacting of the starting population of HSC with Reagent A occurs in a cell culturing medium.
  • the medium is
  • TPO, IL-6, Flt3-L, and SCF are each present in a concentration between 10 and 100 ng/mL.
  • TPO, IL6, Flt3-L, and SCF are each present in a concentration between 25 and 75 ng/mL.
  • TPO, IL-6, Flt3-L, and SCF are each present in a concentration of about 50 ng/mL.
  • Step C the contacting of the populations of megakaryocytes with Reagent B occurs in a cell culturing medium supplemented with cytokines.
  • cytokines we showed that supplement with TPO, IL-6, Flt3-L, and SCF would support support platelet biogenesis with an acceptable yield (Example B).
  • the cell culturing medium is supplement with TPO, IL-6, Flt3-L, and SCF, each of an effective amount to support platelet biogenesis.
  • TPO, IL-6, Flt3-L, and SCF are each at a concentration of about 50 g/mL.
  • TPO was dosed at 12 concentration points between 50pg/mL and 100 ng/mL and cultured to day 18 before platelets yield was assessed. TPO was effective over the entired test range, and could be effective at the concentration between 1 and 500 ng/mL. We determined the optimal working range is about 50 to 100 ng/mL (Figure 9).
  • the medium is supplement with TPO alone.
  • TPO was present in a concentration between 5 ng/mL and 200 ng/mL.
  • TPO is present at a concentration of about 100 ng/mL.
  • TPO is present at a concentration of about 50 ng/mL.
  • Reagent A is an agent capable of promoting megakaryocytepoies.
  • Reagent A is a low molecular weight compound of Formula I :
  • L is -NHC(O)- or -C(0)NH-;
  • RT is selected from hydrogen, d ⁇ alkyl, phenyl, and C 5 - 6 heteroaryl, wherein the d- 4 alkyl, phenyl or C 5 - 6 heteroaryl is unsubstituted or substituted by 1 to 2 substituents independently selected from halo, cyano, C 1 -4 alkyl, halo-substituted C 1 -4 alkyl, C ⁇ alkoxyCV 4 alkyl, d ⁇ alkoxy, C 3 - 6 cycloalkyl and C 4 - 6 heterocycloalkyl, wherein the C 3 - 6 cycloalkyl or C 4- eheterocycloalkyl is further unsubstituted or substituted by 1 to 2 substituents independently selected from halo, hydroxy, oxo, d ⁇ alkylcarbonyl, or d_ 4 alkyoxy;
  • R 2 is d- 4 alkyl or C 3 - 6 cycloalkyl
  • R 3 is d- 4 alkyl or d- 4 alkenyl
  • R 4 is hydrogen or C 5 - 6 heteroary, unsubstituted or substituted by d_ 4 alkyl; and R 5 is halo, d_ 4 alkyl, halo-substituted d- 4 alkyl, d_ 4 alkoxy-substituted Ci -4 alkyl, d- 4 alkoxy, and halo-substituted d_ 4 alkoxy.
  • L is -NHC(O)- or -C(0)NH-;
  • RT is hydrogen, d_ 4 alkyl or C 5 - 6 heteroaryl, wherein the alkyl is substituted by oxo- substitutedC 4 . 6 heterocycloalkyl, and the C 5 - 6 heteroaryl is substituted by two d_ 4 alkyl;
  • R 2 is d- 4 alkyl or C 3 . 6 cycloalkyl
  • R 3 is d- 4 alkyl or d_ 4 alkenyl
  • R 4 is hydrogen or C 5 - 6 heteroary, unsubstituted or substituted by d_ 4 alkyl.
  • R 4 is selected from hydrogen, 2.6-dimethylpyridin-3-yl, 1 ,3 dimethyl-1 /-/-pyrazol-5- yl, and 2-oxo-pyrrolidin-1 -yl-propyl.
  • R 2 is methyl or cyclopropyl.
  • R 3 is methyl or vinyl.
  • R 4 is hydrogen or 4-methyl-1 - -imidazol-1 -yl.
  • Reagent A is selected from the following compounds:
  • Reagent A is 3-(7-((2,6-dimethylpyridin-3- yl)amino)-1 -methyl-2-oxo-1 ,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N-(3- (trifluoromethyl)phenyl)-4-vinylbenzamide.
  • Step A Reagent A is present in present in the culturing medium at a concentration between 50 nM and 5 ⁇ . In one variation, in Step A, Reagent A is present in a concentration between 100 nM and 1000 nM.
  • Reagent A is present in a concentration about 250 nM, 500 nM or 750nM.
  • Step A Reagent A is present in a concentration about 250 nM. In anther variation, in Step A, Reagent A is present in a concentration about 500 nM.
  • Reagent A is capable of antagonizing the activity and/or the expression of the platelet-derived growth factor receptor (PDGFR) and/or a down-stream effector of the PDGFR pathway.
  • the PDGFR inhibitory activity (IC 50 ) of the compounds of Formula I are between 2 to 13 nM and the EC 50 concentration of the compounds for promoting megakaryopoiesis is between 10 to 50 nM (Table 1 ).
  • Boitano, et al. has demonstrated that Compound A1 (also referred to as MK1 ) out this class of naphthyridinones induces the selective differentiation of common myeloid progenitors (CMP) to megakaryocytes.
  • CMP common myeloid progenitors
  • PDGFR platelet-derived growth factor receptor
  • Reagent B is an agent capable of promoting platelet biogenesis.
  • Reagent B is a low molecular weight compound of Formula II :
  • Gi is selected from N and CR 3 ;
  • G 2 , G 3 and G 4 are independently selected from CH and N ; with the proviso that at least 1 of G3 and G4 is N ; with the proviso that Gi and G 2 are not both N;
  • L is selected from -NR 5A (CH 2 )2-3- (0-3 herein means 0, 1 , 2 or 3), - NR 5A CH(C(0)OCH 3 )CH 2 -, -N R 5A (CH 2 ) 2 NR 5B -, -NR 5A (CH 2 ) 2 S- -NR 5A CH 2 CH(CH 3 )CH 2 -, - NR 5A CH 2 CH(OH)- and -NR 5A CH(CH 3 )CH 2 -; wherein R 5A and R 5B are independently selected from hydrogen and d ⁇ alkyl;
  • RT is selected from thiophenyl, furanyl, benzoimidazolyl, isoquinolinyl,
  • imidazopyridinyl benzothiophenyl, pyrimidinyl, pyrazolyl, pyridinyl, imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, pyrrolyl and thiazolyl; wherein said thiophenyl, furanyl,
  • benzoimidazolyl isoquinolinyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyrazolyl, pyridinyl, imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, pyrrolyl and thiazolyl of can be optionally substituted by 1 to 3 radicals independently selected from cyano, hydroxy, d- 4 alkyl, C 1 -4 alkoxy, halo, halo-substituted-C 1 -4 alkyl, halo-substituted-C 1 -4 alkoxy, hydroxy, amino, -C(0) R 8A , -S(O) 0 - 2 R 8 a, -C(0)OR 8A and -C(0)N R 8A R 8B ; wherein R 8A and R 8B are independently selected from hydrogen and d- 4 alkyl; with the proviso that and R
  • R 6a , R 6 b and R 6c are independently selected from hydrogen and d ⁇ alkyl; the phenyl, pyrrolopyridinyl, indolyl, thiophenyl, pyridinyl, triazolyl, 2- oxoimidazolidinyl, pyrazolyl, 2-oxo-2,3-dihydro-benzoimidazolyl, indazolyl, imidazolyl, benzo[d]imidazolyl, or piperidinyl of R 2 is optionally substituted with 1 to 3 radicals independently selected from hydroxy, halo, methyl, methoxy, amino, - 0(CH 2 ) n NR 7a R 7b , -S(0) 2 NR 7a R 7b , -OS(0) 2 NR 7a R 7b and -NR 7a S(0) 2 R 7b ; wherein R 7a and R 7b are independently selected from hydrogen and C 1 -4 alkyl;
  • R 3 is selected from hydrogen, d ⁇ alkyl and biphenyl
  • R 4 is selected from Ci-i 0 alkyl, C 2 . 6 alkenyl,, cyclohexyl, cyclopropyl,
  • alkenyl proposition cyclohexyl, cyclopropyl, (oxopyrrolidinyl)ethyl, oxetanyl, benzhydryl, tetrahydropyranyl, tetrahydropyranyl, phenyl, tetrahydrofuranyl, or benzyl of R 4 can be optionally substituted with 1 to 3 radicals independently selected from hydroxy, d ⁇ alkyl and halo-substituted-d ⁇ alkyl; or the N-oxide derivatives, prodrug derivatives, protected derivatives, individual isomers and mixture of isomers thereof; or the salts (preferably the pharmaceutically acceptable salts) and solvates (e.g. hydrates) of such compounds.
  • Reagent B is a compound of Formula Ila:
  • L is selected from -NR 5a (CH 2 ) 2 _ 3 -, -NR 5a (CH 2 ) 2 N R 5B - -NR 5a (CH 2 ) 2 S- -
  • R 5A and R 5B are independently selected from hydrogen and d_ 4 alkyl;
  • RT is selected thiophenyl, furanyl, benzoimidazolyl, isoquinolinyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyrazolyl, pyridinyl, imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, pyrrolyl and thiazolyl; wherein
  • said thiophenyl, furanyl, benzoimidazolyl, isoquinolinyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyrazolyl, pyridinyl, imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, pyrrolyl and thiazolyl of can be optionally substituted by 1 to 3 radicals independently selected from halo, cyano, C 1-4 alkyl, halo-substituted-d- 4alkyl, C 1 - 4 alkoxy,-S(O) 0 -2R8a, and -C(0)OR 8a , wherein R 8a is selected from hydrogen and d ⁇ alkyl;
  • R 2 is selected from -S(0) 2 NR 6a R 6b , -NR 6a C(0)NR 6b R6c, phenyl, pyrrolopyridinyl, indolyl, thiophenyl, pyridinyl, I triazolyl, 2-oxoimidazolidinyl, pyrazolyl, and indazolyl; wherein
  • R 6a , R 6b and R 6c are independently selected from hydrogen and d ⁇ alkyl; and said phenyl, pyrrolopyridinyl, indolyl, thiophenyl, pyridinyl, triazolyl, oxoimidazolidinyl, pyrazolyl, and indazolyl of R 2 is optionally substituted with 1 to 3 radicals independently selected from hydroxy, halo, methyl, methoxy, amino, -
  • R 7a and R 7b are independently selected from hydrogen and d ⁇ alkyl
  • R 3 is selected from hydrogen, d ⁇ alkyl and biphenyl
  • R 4 is selected from d- 10 alkyl, d_ 4 alkenyl, oxetanyl, tetrahydrofuranyl, cyclohexyl, (oxopyrrolidinyl)ethyl, tetrahydropyranyl, phenyl, and benzyl, wherein said d.
  • R 4 10 alkyl, d- 4 alkenyl, oxetanyl, tetrahydrofuranyl, cyclohexyl, (oxopyrrolidinyl)ethyl, tetrahydropyranyl, phenyl, and benzyl of R 4 can be optionally substituted with 1 to 3 radicals independently selected from hydroxy, d_ 4 alkyl and halo-substituted-d- 4 alkyl.
  • Reagenat B is a compound of Formula lib:
  • L is selected from -NH(CH 2 ) 2 _ 3 - -NH(CH 2 ) 2 S- -NHCH(CH 3 )CH 2 - and - NHCH 2 CH(OH)-;
  • RT is selected from thiophenyl, benzoimidazolyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyridinyl, imidazolyl, and pyridazinyl; each of which is unsubstituted or substituted by 1 to 2 radicals independently selected from halo, cyano, d_ 4 alkyl, halo- substituted-d- 4 alkyl, d_ 4 alkoxy, and -S(0)o- 2 Rs a , wherein R 8a is d_ 4 alkyl;
  • R 2 is selected from phenyl, indolyl and pyrrolopyridinyl, each of which is
  • R 7a and R 7b are independently selected from hydrogen and d_ 4 alkyl;
  • R 4 is selected from d- 10 alkyl, d_ 4 alkenyl, oxetanyl, tetrahydrofuranyl, wherein the d- 10 alkyl or d_ 4 alkenyl is unsubstituted or substituted with hydroxyl.
  • L is selected from -NH(CH 2 ) 2 - -NH(CH 2 ) 2 S- and -NHCH 2 CH(OH)-. In another variation, L
  • any one of the above embodiments and variations is selected from 1 H-imidazol-1 -yl, thiophen-2-yl, thiophen-3-yl, pyridin-3-yl, pyridin-4-yl, pyrimidin-5-yl, pyridazin-4-yl, 1 H-benzo[d]imidazol-1 -yl, 3H-imidazo[4,5-b]pyridin-3-yl, and benzo[b]thiophen-3-yl, each of which is unsubstituted or substituted by 1 to 2 radicals independently selected from fluoro, chloro, cyano, methyl, trifluoromethyl, methoxy, and - S(O) 0 - 2 R 8a ; wherein R 8a is selected from methyl and ethyl.
  • R ⁇ is pyridin-3-yl or benzo[b]thiophen-3-yl. In still another variation, R ⁇ is pyridin-3-yl. In yet another variation, R ⁇ is benzo[b]thiophen-3-yl.
  • R 2 is selected from phenyl, 1 H-indol-3-yl and 1 H-pyrropyridin-3-yl, each of which is
  • R 7a and R 7b are independently selected from hydrogen and d- 4 alkyl.
  • R 2 is phenyl or 1 H-indol-3-yl, each unsubstituted or substituted with 1 to 2 radicals independently selected from halo, hydroxyl, amino, methyl, methoxy, diethylaminoethoxy, methylsulfonylamino, and aminosulfonyloxy.
  • R 2 is phenyl, unsubstituted or substituted with 1 to
  • R 2 radicals independently selected from fluoro, chloro, hydroxyl, amino, methyl, methoxy, methylsulfonylamino, and aminosulfonyloxy.
  • R 2 is phenyl substituted with hydroxyl.
  • R 2 is 1 H-indol-3-yl, unsubstituted or substituted with 1 to 2 radicals independently selected from fluoro, chloro, hydroxy, methyl, methoxy, and diethylaminoethoxy.
  • Compound B, R 4 is selected from isopropyl, methyl, ethyl, prop-1 -en-2-yl, isobutyl, sec- butyl, (S)-sec-butyl, (R)-sec-butyl, 1 -hydroxypropan-2-yl, (S)-1 -hydroxypropan-2-yl, (R)-1 - hydroxypropan-2-yl, nonan-2-yl, oxetan-3-yl, oxetan-2-yl, and tetrahydrofuran-3-yl.
  • R 4 is selected from isopropyl, methyl, ethyl, prop-1 - en-2-yl, isobutyl, sec-butyl, 1 -hydroxypropan-2-yl, nonan-2-yl.
  • R 4 is (S)-1 -hydroxypropan-2-yl or -hydroxypropan-2-yl.
  • R 4 is isopropyl.
  • Compound B is of Formula lie:
  • R 2 is selected from 1 H-indol-3-yl and phenyl, each unsubstituted or substituted with from halo, hydroxyl, amino, methyl, methoxy, diethylaminoethoxy, methylsulfonylamino, and aminosulfonyloxy.;
  • R 4 is selected from isopropyl, methyl, ethyl, prop-1 -en-2-yl, isobutyl, sec-butyl, (S)- sec-butyl, (R)-sec-butyl, 1 -hydroxypropan-2-yl, (S)-1 -hydroxypropan-2-yl, (R)-1 - hydroxypropan-2-yl, nonan-2-yl, oxetan-3-yl, and tetrahydrofuran-3-yl.
  • R 2 is selected from 1 H-indol-3-yl, unsubstituted or substituted with chloro, fluoro, hydroxyl, methyl, methoxy, and diethylaminoethoxy, methylsulfonylamino, and aminosulfonyloxy
  • R 4 is selected from isopropyl, sec-butyl, (S)-sec-butyl,
  • R 4 sec-butyl, nonan-2-yl, oxetan-3-yl, and tetrahydrofuran-3-yl.
  • R 4 is isopropyl.
  • R 4 is oxetan-3-yl.
  • R 4 is sec-butyl.
  • Reagenat B is a compound of formula
  • R 2 is selected from: 1 H-pyrrolo[2,3-b]pyridin-3-yl; 1 H-indol-3-yl optionally substituted with 1 to 2 radicals independently selected from halo, methyl and methoxy; and phenyl optionally substituted with 1 to 2 radicals independently selected from methyl, halo and hydroxy;
  • R 4 is selected from isopropyl, sec-butyl, 1 -hydroxypropan-2-yl, prop-1 -en-2-yl, benzhydryl, nonan-2-yl, oxetan-3-yl and tetrahydrofuran-3-yl; and
  • R a , R b and R c are independently selected from hydrogen, cyano, methyl, halo, -S0 2 CH 3 and trifluoromethyl.
  • Reagent B is selected from the group of compounds listed in Table II. [00154] In one embodiment, Reagent B is selected from the list of compounds below:
  • Regent B is 4-(2-(2-(benzo[b]thiophen-3-yl)-9- isopropyl-9H-purin-6-ylamino)ethyl)phenol.
  • Step C the contacting of the megakaryocytes occurs in a cell culture medium wherein Reagent B is present in a concentration effective for promoting platelet biogenesis.
  • Reagent B is present in a concentration effective for promoting platelet biogenesis.
  • Example J a dose response experiment was conducted. It was determined that the effective range for Compound B157S is between 1 to 5000 nM, and for Compound B1 , is between 50 to 5000 nM ( Figure 10).
  • Reagent B is present in the medium at a concentration between 1 nM and 5 ⁇ . In one variation, Reagent B is present in a concentration between 100 nM and 5000 nM. In another variation, Reagent B is present in a concentration between about 250 nM and about 750 nM.
  • Reagent B is present in a concentration of about 250 nM. In another embodiment of Step C, Reagent B is present in a
  • Reagent B is present in a concentration of about 750 nM.
  • Regent B is capable of antagonizing the activity and/or expression of the aryl hydrocarbon receptor (AHR) and/or a down-stream effector of the AHR pathway.
  • Reagent B is capable of antagonizing the activity and/or expression of the aryl hydrocarbon receptor.
  • Reagent is capable of antagonizing the activity and/or expression of a down-stream effector of the AHR pathway which is selected from Cyp1 B1 , Cyp1 A1 , Beta catenin, AHRR, STAT5 and STAT1 .
  • AHR aryl hydrocarbon (dioxin) receptor
  • phase I xenobiotic-metabolizing enzymes such as the cytochromes P450 CYP1 A1 , CYP1 A2, CYP1 B1 and CYP2S1 , and the phase II enzymes UDP-glucuronosyltransferase UGT1 A6, NAD(P)H-dependent quinone oxidoreductase-1 (NQ01 ), the aldehyde dehydrogenase ALDH3A1 , and several glutathione-S-transferase.
  • phase I xenobiotic-metabolizing enzymes such as the cytochromes P450 CYP1 A1 , CYP1 A2, CYP1 B1 and CYP2S1 , and the phase II enzymes UDP-glucuronosyltransferase UGT1 A6, NAD(P)H-dependent quinone oxidoreductase-1 (NQ01 ), the aldehyde dehydrogenase ALDH3A
  • Compound B1 To determine the effects of Compound B1 in AHR transcription the ability of Compound B1 to inhibit a dioxin-induced AHR dependent luciferase reporter gene assay was tested. Inclusion of Compound B1 (1 ⁇ ) completely abolished dioxin-induced AHR dependent transcription when used on cells expressing human AHR. Titration of Compound B1 revealed an EC 50 of 127 nM, demonstrating that Compound B1 is a potent AHR antagonist. Interestingly, Compound B1 only weakly inhibited dioxin induced transcription in murine cells and had no activity on rat cells, suggesting that Compound B1 preferentially inhibits human AHR.
  • Compound B1 had only weak agonist activity on murine or rat cells, and failed to induce AHR dependent transcription in human cells.
  • the ex vivo process of the invention may further include a matrix metallproteinase (MMP) inhibitor.
  • MMP matrix metallproteinase
  • the MMP inhibitor is not known to directly involve in platelet biogenesis. It has been reported that these inhibitors have both positiive and negative functional consequences in platelet survival.
  • Example K shows, when compared to donor-derived platelets, culture-derived "untreated" platelets is equally viable (99% vs 98% viable platelet counts), and includes a much higer percentage of young platelets (60% vs 1 %).
  • Example L shows, the exPLTs and donor derived platelets persisted in circulation with similar kinetics ( Figure 1 1 ).
  • the MMP inhibitors have a slight effect in platelet yield.
  • Two MMP inhibitors were tested in separate cultures (Example M), the platelet yields for cultures with MMP inhibitors were at least 20% higher for cultures with the MMP inhibitors over cultures with vehicle ( Figure 12).
  • the MMP inhibitor is included as an optional reagent in the optimized protocol.
  • the cell culturing medium optionally further comprising a matrix metalloproteinases (MMP) inhibitor.
  • MMP matrix metalloproteinases
  • the MMP inhibitor is selected from (N-(R)-[2-(Hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-L- naphthylalanyl-L-alanine, 2-aminoethyl amide (TAPI-1 ) and (N-[(2R)-2- (Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM6001 ).
  • Step C wherein the MMP inhibitor is administered to the culture medium two days before the completion of the contacting.
  • the MMP inhibitor in Step C, is present in a concentration between 1 and 75 ⁇ .
  • the MMP inhibitor is (N-[(2R)-2-(Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM6001 ), which is present in a concentration of about 25 ⁇ .
  • the MMP inhibitor is (N-(R)-[2-(Hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-L-naphthylalanyl-L- alanine, 2-aminoethyl amide (TAPI-1 ), which is present in a concentration of about 10 ⁇ .
  • Example N The optimal duration for culturing the starting cell population in the presence of the inhibitors was determined experimentally.
  • starting cell populations of HSC were exposed to Reagent A for 4, 7, 10, or 14 days, during 18 days culturing protocol, and platelet counts were determined. It was found the the optimal time for contacting with Reagent A is 14 days ( Figure 13).
  • the contacting with reagent A is for a duration between 2 days and 16 days. In one variation, the contacting is for a duration between 4 days and 14 days. In another variation, in Step A, the duration is for about 14 days.
  • Step C can be similarily determined.
  • the contacting in Step C, is for a duration between 2 days and 10 days.
  • the contacting of the exMKs with Reagent B and TPO is for a duration between 3 days and 5 days.
  • the contacting of the exMKs with Reagent B and TPO is for a duration of about 4 days.
  • Step A After the completion of the first stage (Step A), it is necessary to collect and transfer the megakaryocytes from the first stage culture to second phase culture.
  • Example O we compared the effect on platelet yield of transferring the megakaryocyte by "pelleting and resuspension" to "isolation".
  • pelleting and resuspension we mean collecting the cells in the Stage 1 culture without separating the megakaryocytes from othere cells; centrifugation would be the method of choice. Isolation meant separate the megakaryocyte from other cells and cell fragments.
  • the process further comprising Step B1 comprising: gathering the population of exMKs, and Step B2 comprising: re-suspending the population of exMKs.
  • Platelets were culture from isolated megakaryocytes (Example P).
  • the cell population obtained after the production process may be used without further purification or may be subject to further purification or selection steps.
  • the cell population may be washed to remove the reagents and/or any other components of the cell culture and resuspended in an appropriate cell suspension medium ready for use.
  • the platelets were purified by isolation over a BSA gradient (Example Q).
  • the post-isolation population contains 100% platelets (calculated as the number of platelets divided by the sum of the number of platelets and nucleated cells), with a yield averaging 34%.
  • the isolated cell population is resuspended in a pharmaceutically acceptable medium suitable for administration to a mammalian host, thereby providing a therapeutic composition.
  • Example E demonstrated the optimized protocol can be practiced entirely in flask and culture bags. Thus, further scale up practical for clinical is feasible.
  • exPLTs ex vivo manufactured platelets
  • the platelets are appropriate for intravenous administration as an infusion cell therapy.
  • the platelets are patient specific, and suitable for allogeneic infusion.
  • the cell population comprising exPLTs is obtained by the methods of described in the above section.
  • the cell population is purified and re-suspended in a pharmaceutically acceptable medium.
  • the exPLTs expressing CD41 a+ CD42b+ composed at least 20% of the sum of the number of platelets and viable nucleated cells.
  • the invention relates to a composition comprising exPLTs and Reagent B in a pharmaceutically acceptable medium wherein Reagent B is a compound as described in paragraph [00139], supra.
  • Reagent B is selected from the following compounds:
  • composition comprising exPLTs and
  • Reagent B of the invention Reagent B is present at a concentration between 1 nM and 1000 nM. In one variation, Reagent B is present at a concentration between 100 nM and 750 nM. In another variation, Reagent B is present at a concentration between 1 nM and 750 nM.
  • Another aspect of the invention relates to a cell composition comprising isolated exPLTs. In one embodiment, the cell composition comprising isolated exPLTs is characterized by the presence of Reagent B, wherein Reagent B is a compound of Formula lib as described in paragraph [00139].
  • Reagent B is selected from
  • the cell composition contains about to 10 12 exPLTs. In one variation, the composition contains aboufl O 9 to 5 x 10 11 exPLTs.
  • the exPLTs expressing CD41 a+ CD42b+ represent between 80 to 100% of the sum of the number of exPLTs and other viable nucleated cells in the population.
  • the exPLTs expressing CD41 a+ CD42b+ represent >95% of the sum of the number of exPLTs and viable nucleated cells in the population.
  • the cell composition comprising isolated exPLTs, the exPLTs expressing CD41 a+ CD42b+ is HLA-matched to a patient in need of a platelet infusion.
  • the starting composition comprising ex vivo produced megakaryocytes and Reagent B, wherein Reagent B is a compound according to paragraph [00129].
  • Reagent B is present in a concentration between 250 nM and 750 nM. In one varitation, Reagent B is present in a concentration about 250 nM. In another variation, Reagent B is present in a concentration about 750 nM. III. Use of the Therapeutic Compositions
  • the invention is related to the use ex vivo produced platelets for treating thrombocytopenia in a subject in need thereof, by administering a sufficient amount of ex vivo produced platelets. It is believe that by treating the
  • thrombocytopenia can also be treated, abated, ameliorated or eradicated.
  • a non-limiting list of diseases and conditions that can be treated follows, infra.
  • the exPLTs are HLA-matched to the subject.
  • the subject is alloimmunized.
  • the administration is an autologous
  • the administration is an allogeneic transplanation
  • the exPLTs are used to treat thrombocytopenia resulting from chemotherapy, radiation therapy, dengue fever, an autoimmune disorder, an hereditary syndrome, an inherited immunodeficient disease, or an hematopoietic disorder.
  • the autoimmune disorder is systemic lupus erythematosus
  • SLE systemic sclerosis
  • hereditary syndromes are selected from congenital amegakaryocytic thrombocytopenia, thrombocytopenia absent radius syndrome, Fanconi anemia, Bernard-Soulier syndrome, May-Hegglin anomaly, Grey platelet syndrome, Alport syndrome, and Wiskott-Aldrich syndrome.
  • the hematopoietic disorder is selected from Acute myeloid leukemia, Acute lymphoblastic leukemia, Amyloidosis Aplastic anemia, Chronic myeloid leukemia, Chronic lymphocytic leukemia, Germ cell tumors, Hemophagocytic lymphohistiocytosis, Hodgkin disease, inborn errors of metabolism, Myeloproliferative disorders, Myelodysplastic syndromes, Multiple myeloma, Neuroblastoma, Non-Hodgkin lymphoma, Paroxysmal nocturnal hemoglobinuria, Pure red cell aplasia, Severe combined immunodeficiency, Sickle cell anemia, and Thalassemia major.
  • the inborn errors of metabolism are selected from mucopolysaccharidosis, Gaucher disease, metachromatic leukodystrophies and
  • kits comprising one or more containers filled with one or more of the ingredients described herein.
  • kits optionally comprise solutions and buffers as needed or desired.
  • the invention provides a kit for for manufacture platelets according to the process of the invention, comprising Reagent B as defined in paragraph [00139] and instructions for use of Reagent B in a method for manufacture platelets and, optionally, one ore more cytokines or growth factors, or media for cell growth, in particular media for platelet biogenesis as described above.
  • the kit may further comprise Reagent A as defined in paragraph [00124], supra.
  • the kit may further comprise antibodies for monitoring production of the cells, such as anti-CD34, anti-CD41 a, anti-CD42b, anti-CD45RA and/or anti-CD61 .
  • such kit further include one or more cytokines or growth factors selected from the group consisting of IL6, Flt3-L, SCF and TPO.
  • the kit may further include a matrix metalloproteinase inhibitor.
  • the MMP inhibitor is selected from N-[(2R)-2-
  • Platelets or thrombocytes are small, irregularly shaped clear cell fragments (i.e. cells that do not have a nucleus), 2-3 ⁇ in diameter, which are derived from fragmentation of precursor megakaryocytes.
  • the average lifespan of a platelet is normally just 5 to 9 days. Platelets are a natural source of growth factors. They circulate in the blood of mammals and are involved in hemostasis, leading to the formation of blood clots.
  • a normal platelet count in a healthy individual is between 150,000 and
  • Thrombocytopenia is a condition of low number of platelets. Thrombocytopenia may be the result of diseases or treatments, for examply chemotherapy, radiation therapy, dengue fever, an autoimmune disorder, an hereditary syndrome, an inherited immunodeficient disease, or an hematopoietic disorder.
  • Transfusion is generally used to correct unusually low platelet counts
  • Platelets are either isolated from collected units of whole blood and pooled to make a therapeutic dose or collected by apheresis, sometimes concurrently with plasma or red blood cells,
  • Apheresis platelets are collected during a blood draw using a mechanical device that separates out the platelets and other components to be collected and the remaining blood is returned to the donor.
  • the advantage to this method is that a single donation provides at least one therapeutic dose, as opposed to the multiple donations for whole-blood platelets.
  • Platelets collected by either method have a very short shelf life, typically five days. This results in frequent problems with short supply, as testing the donations often requires up to a full day. Since there are no effective preservative solutions for platelets, they lose potency quickly and are best when fresh.
  • Hematology patients undergoing high dose chemotherapy account for up to 67% of platelet transfusions in the US. Because of prolonged thrombocytopenia, these patients normally require multiple platelet transfusions over a period of weeks or months.
  • HLA-alloimmunization is a common risk for these patients which renders them refractory to platelet transfusion.
  • TRAP clinical trial 8% of patients had baseline anti-HLA antibodies and another 17-45% developed them following chemotherapy.
  • HLA-alloimmunized patients are ideally given matched platelets to avoid immediate platelet rejection; however, identifying and obtaining matched donor platelets is often not feasible and is a current unmet medical need.
  • a method of manufacturing platelet ex vivo in a clinical relevant amount would have a great impact on the supply of the platelets for transfusion, and also address the unmet need for patient specific platelets.
  • Umbilical cord blood banks currently exist and cover the broad racial make-up of the general population.
  • An ex vivo method would enable the manufacture of platelets by using HLA-matched hematopoietic stem cell isolated from the cord blood.
  • the exPLTs potentially can be a source of HLA-matched platelets for alloimmunized thrombocytopenic patients without a donor platelet match and, as such, will increase post-transfusion platelet recovery compared to standard of care (unmatched platelets).
  • the possibility of using exPLT to supplement and/or replace standard platelet products will help to alleviate a reliance on donors, avert shortages and improve product safety.
  • the invention further relates to the cell population with exPLTs or its composition for use in allogeneic or autologous platelet infusion in a mammalian subject.
  • the manufactured platelets can be uses as is or could be isolated from the culture prior to use.
  • the isolated cell are pelleted by centrifugation for 10 minutes at 1000g and resuspended in infusion buffer e.g., a 5% HSA (Baxter) at a concentration of between 10 7 to 10 10 platelets /ml_.
  • infusion buffer e.g., a 5% HSA (Baxter) at a concentration of between 10 7 to 10 10 platelets /ml_.
  • the number of cells transfused will take into consideration factors such as sex, age, weight, the types of disease or disorder, stage of the disorder, the percentage of the desired cells in the cell population and the amount of cells needed to produce a therapeutic benefit.
  • the composition is administered by intravenous infusion and comprises at least 10 8 platelets/kg, from 10 9 to 10 10 platelets/kg or more if needed.
  • a transfusion dose is typical about 3 to 5 x 10 11 platelets.
  • subjects include, for example, domesticated animals, such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, and goats), laboratory animals (e.g., mice, rabbits, rats, and guinea pigs), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal.
  • livestock e.g., cattle, horses, pigs, sheep, and goats
  • laboratory animals e.g., mice, rabbits, rats, and guinea pigs
  • mammals non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal.
  • the subject is optionally a mammal such as a primate or a human.
  • compositions including the cell therapy product described herein that are generally safe, relatively nontoxic and neither biologically nor otherwise undesirable are generally safe, relatively nontoxic and neither biologically nor otherwise undesirable.
  • These compositions optionally include pharmaceutically acceptable carriers or stabilizers that are nontoxic to the cell or subject being exposed thereto at the dosages and concentrations employed.
  • the carrier or excipient is selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject or cell.
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt- forming counterions such as sodium ; and/or nonionic surfactants such as TWEENTM (Uniqema, United Kingdom), polyethylene glycol (PEG), and PLURONICSTM (BASF, Germany).
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • compositions are formulated in any conventional manner for use in the methods described herein. Administration is via any route known to be effective by one of ordinary skill.
  • the preferred method of administration is intravenous infusion.
  • a pharmaceutically acceptable carrier for infusion of a composition comprising cells into a patient typically comprise buffered saline with 5%HSA or unsupplemented basal medium or medium as known in the art.
  • the invention also relates to using other molecules in the process of invention to manufacturing platelets ex vivo.
  • AHR antagonists for example, small interference RNA (siRNA) and antisense oligonucleotide, can also be used in the ex vivo production of platelets.
  • siRNA small interference RNA
  • antisense oligonucleotide can also be used in the ex vivo production of platelets.
  • RNAi molecules suitable for use with the present invention can be affected as follows: First, the AHR mRNA sequence (or one or more of its downstream effectors) is scanned downstream of the AUG start codon for AA-dinucleotide sequences. Occurrence of each AA and the 19 3'-adjacent is recorded as a potential siRNA target site. Then, potential target sites are compared to an appropriate genomic database (e.g, human, mouse, rat, etc.) using any sequence alignment software. Putative target site that exhibit significant homology to other coding sequences are filtered out. Preferred sequences are then those including low G/C content, in particular sequences with G/C content lower than 55%.
  • an appropriate genomic database e.g, human, mouse, rat, etc.
  • target sites are then selected along the length of the target gene. Methods or algorithms to identify putative target site of siRNA are described for example in (Tilesi, et al., Curr. Opin. Mol. Ther. 1 1 :156, 2009).
  • siRNA molecules which are capable of down-regulating the expression of AHR are: AHR 1 1 1 S, 5' GCG GCA TAG AGA CCG ACT TAA TTT CAA GAG AAT TAA GTC GGT CTC TAT GCC GCT TTT TTG G 3' (SEQ ID NO: 1 ); AHR 1 1 1 AS, 5' CGC GCC AAA AAA GCG GCA TAG AGA CCG ACT TAA TTC TCT TGA AAT TAA GTC GGT CTC TAT GCC GC 3' (SEQ ID NO: 2); AHR 242S, 5' GGC TTC TTT GAT GTT GCA TTA ATT CAA GAG ATT AAT GCA ACA TCA AAG AAG CCT TTT TTG G 3' (SEQ ID NO: 3); AHR 242AS, 5' CGC GCC AAA AAA GGC TTC TTT GAT GTT GCA TTA ATC TCT TGA ATT AAT GCA ACA TCA AAG AAG CCT TTT TTG G 3
  • the invention provides an ex vivo process for producing a population of ex vivo produced platelets (exPLTs) comprising a Step C, which comprises:
  • exMKs ex vivo produced megakaryocytes
  • TPO thrombopoietin
  • L is selected from -NR 5a (CH 2 ) 2 _ 3 -, -NR 5 a(CH 2 ) 2 NR 5 b-, -NR 5a (CH 2 ) 2 S- - NR 5a CH 2 CH(OH)- and -NR 5a CH(CH 3 )CH 2 -; wherein R 5a and R 5b are independently selected from hydrogen and d- 4 alkyl;
  • RT is selected thiophenyl, furanyl, benzoimidazolyl, isoquinolinyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyrazolyl, pyridinyl, imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, pyrrolyl and thiazolyl; wherein said thiophenyl, furanyl, benzoimidazolyl, isoquinolinyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyrazolyl, pyridinyl, imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, pyrrolyl or thiazolyl of is optionally substituted by 1 to 3 radicals independently selected from halo, cyano, d- 4 alkyl, halo-sub
  • R 6a , R 6b and R 6c are independently selected from hydrogen and d ⁇ alkyl; and said phenyl, pyrrolopyridinyl, indolyl, thiophenyl, pyridinyl, triazolyl, oxoimidazolidinyl, pyrazolyl, or indazolyl of R 2 is optionally substituted with 1 to 3 radicals independently selected from hydroxy, halo, methyl, methoxy, amino, -
  • R 7a and R 7b are independently selected from hydrogen and d ⁇ alkyl
  • R 3 is selected from hydrogen, d ⁇ alkyl and biphenyl
  • R 4 is selected from Ci_i 0 alkyl, d_ 4 alkenyl, oxetanyl, tetrahydrofuranyl, cyclohexyl, (oxopyrrolidinyl)ethyl, tetrahydropyranyl, phenyl, and benzyl, wherein said d-ioalkyl, d- 4 alkenyl, oxetanyl, tetrahydrofuranyl, cyclohexyl, (oxopyrrolidinyl)ethyl, tetrahydropyranyl, phenyl, and benzyl of R 4 can be optionally substituted with 1 to 3 radicals independently selected from hydroxy, d_ 4 alkyl and halo-substituted-d- 4 alkyl.
  • Embodiment 2 The process according to Embodiment 1 , wherein the population of exPLTs represents at least a 2-fold increase of exPLTs per exMKs when compared to no contacting with Reagent B.
  • Embodiment s The process according to Embodiment 1 or
  • Embodiment 2 in Step C, wherein Regent B is capable of antagonizing the activity and/or expression of the aryl hydrocarbon receptor (AHR) and/or a down-stream effector of the AHR pathway.
  • AHR aryl hydrocarbon receptor
  • Embodiment 4 The process according to Embodiment 3, in Step C, wherein a down-stream effector of aryl hydrocarbon receptor pathway is selected from Cyp1 B1 , Cyp1 A1 , Beta catenin, AHRR, STAT5 and STAT1 .
  • Embodiment 5 The process according to any one of Embodiments 1 to 4, further comprising a Step A for producing the exMKs from hematopoietic stem cells (HSC), wherein Step A comprising:
  • Reagent A is of Formula I:
  • L is -NHC(O)- or -C(0)NH-; is selected from hydrogen, d- 4 alkyl, phenyl, and C 5 - 6 heteroaryl, wherein the d_ 4 alkyl, phenyl or C 5 . 6 heteroaryl is unsubstituted or substituted by 1 to 2 substituents independently selected from halo, cyano, d- 4 alkyl, halo-substituted d- 4 alkyl, d- 4 alkoxyd- 4 alkyl, d_ 4 alkoxy, C 3 - 6 cycloalkyl and C 4 . 6 heterocycloalkyl, wherein the C 3 - 6 cycloalkyl or C 4 . eheterocycloalkyl is further unsubstituted or substituted by 1 to 2 substituents independently selected from halo, hydroxy, oxo, d_ 4 alkylcarbonyl, or d_ 4 alkyoxy;
  • R 2 is d- 4 alkyl or C 3 - 6 cycloalkyl
  • R 3 is d- 4 alkyl or d_ 4 alkenyl
  • R 4 is hydrogen or C 5 - 6 heteroary, unsubstituted or substituted by d_ 4 alkyl; and R 5 is halo, d_ 4 alkyl, halo-substituted d- 4 alkyl, d- 4 alkoxy-substituted d- 4 alkyl, d_ 4 alkoxy, and halo-substituted d. 4 alkoxy.
  • Embodiment 6 The process according to Embodiment 5, in Step A, wherein Reagent A is capable of antagonizing the activity and/or the expression of the platelet-derived growth factor receptor (PDGFR).
  • PDGFR platelet-derived growth factor receptor
  • Embodiment 7 The process according to Embodiment 5 or
  • Embodiment 6 in Step A, wherein the population of exMKs represents about a 2.5-fold increase of exMKs per HSC when compared to no contacting with Compound A.
  • Embodiment 8 The process according to any one of Embodiments 1 to 7, further comprising:
  • Step B1 gathering the quantity of exMKs.
  • Step B2 re-suspending the quantity of exMKs.
  • Embodiment 9 The process according to any one of Embodiments 5 to 8, in Step A, wherein
  • L is -NHC(O)- or -C(0)NH-;
  • RT is hydrogen, d- 4 alkyl or C 5 - 6 heteroaryl, wherein the alkyl is substituted by substitutedC 4 . 6 heterocycloalkyl, and the C 5 - 6 heteroaryl is substituted by 2 d- 4 alkyl;
  • R 2 is d- 4 alkyl or C 3 . 6 cycloalkyl
  • R 3 is d- 4 alkyl or d_ 4 alkenyl
  • Embodiment 10 is hydrogen or C 5 - 6 heteroary, unsubstituted or substituted by d- 4 alky.
  • Step A is selected from hydrogen, 2.6-dimethylpyridin-3-yl, 1 ,3 dimethyl- 1 /-/-pyrazol-5-yl, and 2-oxo-pyrrolidin-1 -yl-propyl.
  • Embodiment 1 1 The process according to any one of Embodiments 5 to 10, in Step A, wherein R 2 is methyl or cyclopropyl.
  • Embodiment 12 The process according to any one of Embodiments 5 to 1 1 , in Step A, wherein R 3 is methyl or vinyl.
  • Embodiment 13 The process according to any one of Embodiments 5 to 12, in Step A, wherein R 4 is hydrogen or 4-methyl-1 /-/-imidazol-1 -yl.
  • Embodiment 14 The process according to Embodiment 5, in Step A, wherein Reagent A is selected from:
  • Embodiment 15 The process according to Embodiment 5, in Step A, wherein Reagent A is 3-(7-((2,6-dimethylpyridin-3-yl)amino)-1 -methyl-2-oxo-1 ,2- dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N-(3-(trifluoromethyl)phenyl)-4-vinylbenzamide.
  • Embodiment 16 The process according to any one of Embodiments 1 to 15, in Step C, wherein Reagent B is of Formula lib:
  • L is selected from -NH(CH 2 ) 2 _ 3 - -NH(CH 2 ) 2 S- and -NHCH 2 CH(OH)- RT is selected from thiophenyl, benzoimidazolyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyridinyl, imidazolyl, and pyridazinyl; each of which is unsubstituted or substituted by 1 to 2 radicals independently selected from halo, cyano, d ⁇ alkyl, halo- substituted-d ⁇ alkyl, d ⁇ alkoxy, and -S(O) 0 - 2 R8a, wherein R 8a is d ⁇ alkyl; R 2 is selected from phenyl, indolyl and pyrrolopyridinyl, each of which is
  • R 7a and R 7b are independently selected from hydrogen and d ⁇ alkyl
  • R 4 is selected from Ci-i 0 alkyl, d ⁇ alkenyl, oxetanyl, tetrahydrofuranyl, wherein the CV ⁇ alkyl or C 1-4 alkenyl is unsubstituted or substituted with hydroxyl.
  • Embodiment 17 The process according to any one of Embodiments 1 to 16, in Step C, wherein L is -NH(CH 2 ) 2 -.
  • Embodiment 18 The process according to any one of Embodiments 1 to 17, in Step C, wherein is selected from 1 H-imidazol-1 -yl, thiophen-2-yl, thiophen-3-yl, pyridin-3-yl, pyridin-4-yl, pyrimidin-5-yl, pyridazin-4-yl, 1 H-benzo[d]imidazol-1 -yl, 3H- imidazo[4,5-b]pyridin-3-yl, and benzo[b]thiophen-3-yl, each of which is unsubstituted or substituted by 1 to 2 radicals independently selected from fluoro, chloro, cyano, methyl, trifluoromethyl, methoxy, and -S(0)o- 2 Rs a ; wherein R 8a is selected from methyl and ethyl.
  • Embodiment 19 The process according to any one of Embodiments 1 to 17, in Step C, wherein is benzo[b]thiophen-3-yl or pyridin-3-yl, each unsubstituted or substituted by halo.
  • Embodiment 20 The process according to any one of Embodiments 1 to 18, in Step C, wherein R2 is selected from phenyl, 1 H-indol-3-yl and 1 H-pyrropyridin-3-yl, each of which is unsubstituted or substituted substituted with 1 to 2 radicals independently selected from halo, hydroxy, methyl, methoxy, dimethyaminoethoxy, and amino-sulfonyloxy, and -NR7aS(0)2R7b; wherein R7a and R7b are independently selected from hydrogen and C1 -4alkyl.
  • Embodiment 21 The process according to any one of Embodiments 1 to 19, in Step C, wherein R4 is selected from isopropyl, methyl, ethyl, prop-1 -en-2-yl, isobutyl, sec-butyl, (S)-sec-butyl, (R)-sec-butyl, 1 -hydroxypropan-2-yl, (S)-l -hydroxypropan- 2-yl, (R)-1 -hydroxypropan-2-yl, nonan-2-yl, oxetan-3-yl, oxetan-2-yl, and tetrahydrofuran-3- yi-
  • Embodiment 22 The process according to any one of Embodiments 1 to 19, in Step C, wherein R4 is selected from isopropyl, methyl, ethyl, prop-1 -en-2-yl, isobutyl, sec-butyl, 1 -hydroxypropan-2-yl, and nonan-2-yl.
  • Embodiment 23 The process according to any one of Embodiments 1 to 15, in Step C, wherein Reagent B is selected from the group consisting of compounds listed in Table II.
  • Embodiment 24 The process according to any one of Embodiments 1 to 15, in Step C, wherein reagent B is selected from the group consisting of: 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H ⁇ urin-6-ylamino)ethyl)phenol;
  • Embodiment 25 The process according to any one of Embodiments 5 to 24, in Step A, wherein the population of hematopoietic stem cells is enriched in CD34+ cells.
  • Embodiment 26 The process according to any one of Embodiments 5 to 25, in Step A, wherein the population of hematopoietic stem cells is derived from umbilical cord blood cells.
  • Embodiment 27 The process according to any one of Embodiments 5 to 25, in Step A, wherein the population of hematopoietic stem cells consisting essentially of CD34+ cells purified from one or two cord blood units.
  • Embodiment 28 The process according to any one of Embodiments 5 to 25, in Step A, wherein the population of hematopoietic stem cells is derived from bone marrow blood cells.
  • Embodiment 29 The process according to any one of Embodiments 5 to 25, in Step A, wherein the population of hematopoietic stem cells is derived from mobilized peripheral blood cells.
  • Embodiment 30 The process according to any one of Embodiments 5 to 29, in Step A, wherein the population of hematopoietic stem cells is derived from a single mammalian subject.
  • Embodiment 31 The process according to any one of Embodiments 5 to 30, in Step A, wherein the population of hematopoietic stem cells is derived from human.
  • Embodiment 32 The process according to any one of Embodiments 5 to 31 , in Step A, wherein the contacting of the population of hematopoietic stem cells with Reagent A and TPO occurs in a cell culturing medium further comprising interleukin-6 (IL- 6), Fms-liked tyrosine-protein kinase 3 ligand (Flt3-L), and stem cell factor (SCF), wherein Reagent A, TPO, IL-6, Flt3-L and SCF are each present in a concentration effective for promoting megakaryopoiesis.
  • IL-6 interleukin-6
  • Flt3-L Fms-liked tyrosine-protein kinase 3 ligand
  • SCF stem cell factor
  • Embodiment 33 The process according to Embodiment 32, in Step A, wherein TPO, IL6, Flt3-L, and SCF are each present in a concentration between 10 and 100 ng/mL.
  • Embodiment 34 The process according to Embodiment 32, in Step A, wherein TPO, IL6, Flt3-L, and SCF are each present in a concentration between 25 and 75 ng/mL.
  • Embodiment 35 The process according to Embodiment 32, in Step A, wherein TPO, IL6, Flt3-L, and SCF are each present in a concentration of about 50 ng/mL.
  • Embodiment 36 The process according to any one of Embodiments 5 to 35, in Step A, wherein Reagent A is present in a concentration between 50 nM and 5 ⁇ .
  • Embodiment 37 The process according to any one of Embodiments 5 to 35, in Step A, wherein Reagent A is present in a concentration between 100 nM and 1000 nM.
  • Embodiment 38 The process according to any one of Embodiments 5 to 35, in Step A, wherein Reagent A is present in a concentration about 250 nM, 500 nM or 750nM.
  • Embodiment 39 The process according to any one of Embodiments 5 to 35, in Step A, wherein Reagent A is present in a concentration of about 250 nM.
  • Embodiment 40 The process according to any one of Embodiments 5 to 39, in Step A, wherein the contacting is for a duration between 2 days and 16 days.
  • Embodiment 41 The process according to any one of Embodiments 5 to 39, in Step A, wherein the contacting is for a duration between 4 days and 14 days.
  • Embodiment 42 The process according to any one of Embodiments 5 to 39, in Step A, wherein the duration is about 14 days.
  • Embodiment 43 The process according to any one of Embodiments 1 to 43, in Step C, wherein the contacting of the exMKs with Compound B and TPO occurs in a cell culturing medium, wherein Reagent B and TPO are each present in a concentration effective for promoting platelet biogenesis.
  • Embodiment 44 The process according to any one of Embodiments 1 to 43, in Step C, wherein TPO is present in a concentration between 5 ng/mL and 500 ng/mL.
  • Embodiment 45 The process according to any one of Embodiments 1 to 43, in Step C, wherein TPO is present in a concentration of about 100 ng/mL.
  • Embodiment 46 The process according to any one of Embodiments 1 to 43, in Step C, wherein TPO is present in a concentration of about 50 ng/mL.
  • Embodiment 47 The process according to Embodiments 46, in Step C, wherein the medium further comprising IL-6, Flt3-L and SCF, each present in a
  • Embodiment 48 The process according to any one of Embodiments 1 to 47, in Step C, wherein Reagent B is present in a concentration between 1 nM and 5 ⁇ .
  • Embodiment 49 The process according to any one of Embodiments 1 to 47, in Step C, wherein Reagent B is present in a concentration between 100 nM and 5000 nM.
  • Embodiment 50 The process according to any one of Embodiments 1 to 47, in Step C, wherein Reagent B is present in a concentration between about 250 nM and about 750 nM.
  • Embodiment 51 The process according to any one of Embodiments 1 to 47, in Step C, wherein Reagent B is present in a concentration of about 250 nM.
  • Embodiment 52 The process according to any one of Embodiments 1 to 47, in Step C, wherein Reagent B is present in a concentration of about 500 nM.
  • Embodiment 53 The process according to any one of Embodiments 1 to 47, in Step C, wherein Reagent B is present in a concentration of about 750 nM.
  • Embodiment 54 The process according to any one of Embodiments 1 to 53, in Step C, wherein the cell culturing medium optionally further comprising a matrix metalloproteinases (MMP) inhibitor.
  • MMP matrix metalloproteinases
  • Embodiment 55 The process according to Embodiment 54, in Step C, wherein the MMP inhibitor is selected from (N-(R)-[2-(Hydroxyaminocarbonyl)methyl]-4- methylpentanoyl-L-naphthylalanyl-L-alanine, 2-aminoethyl amide (TAPI-1 ) and (N-[(2R)-2- (Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM6001 ).
  • the MMP inhibitor is selected from (N-(R)-[2-(Hydroxyaminocarbonyl)methyl]-4- methylpentanoyl-L-naphthylalanyl-L-alanine, 2-aminoethyl amide (TAPI-1 ) and (N-[(2R)-2- (Hydroxamidocarbonylmethyl)-4-methyl
  • Embodiment 56 The process according to Embodiment 54, in Step C, wherein the MMP inhibitor is administer to the culture medium two days before the completion of the contacting.
  • Embodiment 57 The process according to Embodiment 54 or
  • Embodiment 55 in Step C, wherein the MMP inhibitor is present in a concentration between 1 and 75 ⁇ .
  • Embodiment 58 The process according to Embodiment 54 or
  • Embodiment 55 in Step C, wherein the MMP inhibitor is (N-[(2R)-2- (Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM6001 ), which is present in a concentration of about 25 ⁇ .
  • the MMP inhibitor is (N-[(2R)-2- (Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM6001 ), which is present in a concentration of about 25 ⁇ .
  • Embodiment 59 The process according to Embodiment 54 or
  • Embodiment 55 in Step C, wherein the MMP inhibitor is (N-(R)-[2- (Hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-L-naphthylalanyl-L-alanine, 2- aminoethyl amide (TAPI-1 ), which is present in a concentration of about 10 ⁇ .
  • the MMP inhibitor is (N-(R)-[2- (Hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-L-naphthylalanyl-L-alanine, 2- aminoethyl amide (TAPI-1 ), which is present in a concentration of about 10 ⁇ .
  • Embodiment 60 The process according to any one of Embodiments 1 to 59, in Step C, wherein the contacting of the exMKs with Reagent B and TPO is for a duration between 2 days to 10 days.
  • Embodiment 61 The process according to any one of Embodiments 1 to 59, in Step C, wherein the contacting of the exMKs with Reagent B and TPO is for a duration of about 4 days.
  • Embodiment 62 An ex vivo process for producing a population of ex vivo produced platelets (exPLTs), wherein the process comprising:
  • HSC hematopoietic stem cells
  • exPLTs ex vivo produced platelets
  • Regent A is 3-(7-((2,6-dimethylpyridin-3-yl)amino)-1 -methyl-2-oxo-1 ,2- dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N-(3-(trifluoromethyl)phenyl)-4-vinylbenzamide;
  • Reagent B is of Formula lib:
  • L is selected from -NH(CH 2 ) 2 _ 3 - -NH(CH 2 ) 2 S- and -NHCH 2 CH(OH)-
  • R T is selected from thiophenyl, benzoimidazolyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyridinyl, imidazolyl, and pyridazinyl; each of which is unsubstituted or substituted by 1 to 2 radicals independently selected from halo, cyano, d ⁇ alkyl, halo- substituted-d ⁇ alkyl, d ⁇ alkoxy, and -S(O) 0 - 2 R8a, wherein R 8A is d ⁇ alkyl;
  • R 2 is selected from phenyl, indolyl and pyrrolopyridinyl, each of which is
  • R 7A and R 7B are independently selected from hydrogen and d_ 4 alkyl; and R 4 is selected from Ci-i 0 alkyl, d ⁇ alkenyl, oxetanyl, tetrahydrofuranyl, wherein the C ⁇ oalkyl or C 1-4 alkenyl is unsubstituted or substituted with hydroxyl.
  • Embodiment 63 The process according to Embodiment 62, wherein
  • Reagent B is selected from
  • Embodiment 64 The process according to Embodiment 62 or
  • Embodiment 63 further comprising the steps of:
  • Embodiment 65 The process according to any one of Embodiments 62 to 64, wherein the population of exPLTs represents at least about 2000 exPLT per HSC.
  • Embodiment 66 The process according to any one of Embodiments 62 to 65, wherein the population of exPLTs represents at least about 7.5-fold increase in yield of ex-platelets per HSC when compared to no contacting with Reagent A and Reagent B. .
  • Embodiment 67 The process according to any one of Embodiments 62 to 66, in Step A1 , wherein the population of hematopoietic stem cells is enriched in CD34+ cells and is derived from umbilical cord blood cells.
  • Embodiment 68 The process according to any one of Embodiments 62 to 67, in Step A, wherein the contacting of the population of HSC with Reagent A occurs in a cell culturing medium further comprising thrombopoietin (TPO), interleukin-6 (IL-6), Fms- liked tyrosine-protein kinase 3 ligand (Flt3-L), and stem cell factor (SCF), wherein TPO, IL- 6, Flt3-L and SCF are each present in a concentration of about 50 ng/mL.
  • TPO thrombopoietin
  • IL-6 interleukin-6
  • SCF stem cell factor
  • Embodiment 69 The process according to Embodiments 62 to 68, in
  • Step A wherein Reagent A is present in a concentration of about 250 nM.
  • Embodiment 70 The process according to any one of Embodiments 62 to 69, in Step A, wherein the contacting of HSC and Reagent A is for a duration of about 14 days.
  • Embodiment 71 The process according to any one of Embodiments 62 to 70, in Step C, wherein the contacting of the exMKs with Reagent B occurs in a cell culturing medium supplemented with TPO, wherein Reagent B and TPO are each present in a concentration effective for promoting platelet biogenesis.
  • Embodiment 72 The process according to Embodiment 71 , in Step C, wherein TPO is present in a concentration of about 100 ng/mL.
  • Embodiment 73 The process according to any one of Embodiments 62 to 72, in Step C, wherein Reagent B is present in a concentration between 250 to 750 nM.
  • Embodiment 74 The process according to any one of Embodiments 62 to 73, in Step C, wherein the contacting is for about 4 days.
  • Embodiment 75 The process according to any one of Embodiments 62 to 74, in Step C, wherein the cell culturing medium is optionally further supplemented with a matrix metalloproteinases (MMP) inhibitor on day 16.
  • MMP matrix metalloproteinases
  • Embodiment 76 The process according to Embodiment 75, in Step C, wherein the MMP inhibitor is N-[(2R)-2-(Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L- tryptophan methylamide (GM6001 ) supplemented to a concentration of about 25 ⁇ .
  • Embodiment 77 The process according to Embodiment 75, in Step C, wherein the MMP inhibitor is (N-(R)-[2-(Hydroxyaminocarbonyl)methyl]-4-methylpentanoyl- L-naphthylalanyl-L-alanine, 2-aminoethyl amide (TAPI-1 ) supplemented to a concentration of about 10 ⁇ .
  • the MMP inhibitor is (N-(R)-[2-(Hydroxyaminocarbonyl)methyl]-4-methylpentanoyl- L-naphthylalanyl-L-alanine, 2-aminoethyl amide (TAPI-1 ) supplemented to a concentration of about 10 ⁇ .
  • Embodiment 78 A cell population comprising exPLTs obtained by the method of any one of Embodiments 1 to 77.
  • Embodiment 79 The cell population according to Embodiment 78, wherein the composition is purified and re-suspended in a pharmaceutically acceptable medium.
  • Embodiment 80 The composition according to Embodiment 78, wherein exPLTs expressing CD41 a+ CD42b+ composed at least 20% of the sum of the number of platelets and viable nucleated cells.
  • Embodiment 81 A composition comprising exPLTs and Reagent B in a pharmaceutically acceptable medium wherein Reagent B is a compound according to Embodiment 16.
  • Embodiment 82 The composition according to Embodiment 81 , wherein Reagent B is selected from 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H ⁇ urin-6-ylamino)ethyl)phenol;
  • Embodiment 83 The composition according to Embodiment 82, wherein Reagent B is present at a concentration between 1 nM and 1000 nM.
  • Embodiment 84 The composition according to Embodiment 82, wherein Reagent B is present at a concentration between 100 nM and 750 nM.
  • Embodiment 85 The composition according to Embodiment 83, wherein Reagent B is present at a concentration between 1 nM and 750 nM.
  • Embodiment 86 A cell composition comprising isolated exPLTs, wherein the composition is characterized by the presence of Reagent B, wherein Reagent B is a compound according to Embodiment 16.
  • Embodiment 87 The composition according to Embodiment 88, wherein Reagent B is selected from
  • Embodiment 88 The composition according to Embodiment 86 or
  • Embodiment 87 wherein the composition contains about 10 9 to 10 12 exPLTs.
  • Embodiment 89 The composition according to Embodiment 86 or
  • Embodiment 87 wherein the composition contains about 5 x 10 11 exPLTs.
  • Embodiment 90 The composition according to Embodiment 86 or
  • Embodiment 87 wherein the exPLTs represent between 80 to 100% of the sum of the number of exPLTs and viable nucleated cells.
  • Embodiment 91 The composition according to Embodiment 86 or
  • Embodiment 87 wherein the exPLTs represent >95% of the sum of the number of exPLTs and viable nucleated cells.
  • Embodiment 92 The cell population according to any one of
  • Embodiments 88 to 91 wherein the exPLTs is HLA-matched to a patient in need of a platelet infusion.
  • Embodiment 93 A starting composition comprising ex vivo produced megakaryocytes and Reagent B, wherein Reagent B is a compound according to
  • Embodiment 94 The composition according to Embodiment 93, wherein Reagent B is present in a concentration between 250 nM and 750 nM.
  • Embodiment 95 A method of treating thrombocytopenia in a subject in need thereof, comprising administering a sufficient amount of exPLTs produced by the process of any one of claims 1 to 77.
  • Embodiment 96 The method of Embodiment 95, wherein the subject is alloimmunized and the exPLTs are HLA-matched to the subject.
  • Embodiment 97 The method of Embodiment 95 or Embodiment 96, wherein the thrombocytopenia is resulted from chemotherapy, radiation therapy, dengue fever, an autoimmune disorder, an hereditary syndrome, an-inherited immunodeficient disease, or an hematopoietic disorder.
  • Embodiment 98 The method of Embodiment 97, wherein the autoimmune disorders are selected from Systemic lupus erythematosus and systemic sclerosis.
  • Embodiment 99 The method of Embodiment 97, wherein the hereditary syndromes is selected from congenital amegakaryocytic thrombocytopenia,
  • thrombocytopenia absent radius syndrome Fanconi anemia, Bernard-Soulier syndrome, May-Hegglin anomaly, Grey platelet syndrome, Alport syndrome, and Wiskott-Aldrich syndrome.
  • Embodiment 100 The method of Embodiment 97, wherein said hematopoietic disorder is selected from Acute myeloid leukemia, Acute lymphoblastic leukemia, Amyloidosis Aplastic anemia, Chronic myeloid leukemia, Chronic lymphocytic leukemia, Germ cell tumors, Hemophagocytic lymphohistiocytosis, Hodgkin disease, inborn errors of metabolism.
  • Myeloproliferative disorders Myelodysplastic syndromes, Multiple myeloma, Neuroblastoma, Non-Hodgkin lymphoma, Paroxysmal nocturnal hemoglobinuria, Pure red cell aplasia, Severe combined immunodeficiency, Sickle cell anemia, and
  • Embodiment 101 The method of Embodiment 100, wherein the inborn errors of metabolism are selected from mucopolysaccharidosis, Gaucher disease, metachromatic leukodystrophies and adrenoleukodystrophies.
  • SFEM StemCell Technologies
  • Recombinant human cytokines were purchased from R & D Systems or Life Technologies.
  • Human CD34 + cells were purified from fresh human umbilical cord blood (Bioreclamation), as noted, using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols.
  • CD34 + cells were seeded at 3-5 x 10 4 viable nucleated cells/mL. Cells were cultured at 37 °C in 5% C0 2 and fresh medium (containing cytokines and reagent) was added as needed to keep the cell density between 3 x 10 4 and 1 x 10 6 cells/mL.
  • the cells are washed with staining media (Hanks balanced salt solution containing FBS (2%) and EDTA (2mM)) and stained (at room temperature for 30 minutes) with indicated primary conjugated antibodies.
  • the cells are washed in the previously described buffer and analyzed using a BD LSR II flow cytometer (Becton Dickinson, San Jose, CA) using 488-nm argon and 633-nm HeNe laser beams as the light source for excitation. Emission is measured using logarithmic amplification and analyzed using FlowJo software (TreeStar Inc. Ashland, OR).
  • Multicolor analysis for cell phenotyping was performed on a LSR II flow cytometer (Becton Dickinson). Cell sorting was perfomed on a FACSAria (Becton
  • Cells were stained with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC).
  • Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate.
  • Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
  • Custom curve-fitting software was used to produce a logistic fit of percent cell viability as a function of the logarithm of inhibitor concentration.
  • the IC 50 was interpolated as the concentration of compounded needed to reduce cell viability to 50% of a DMSO control.
  • CD34+ cells were resuspended in HSC expansion medium (5 x 104 cells/mL) before being aliquoted in 384 well plates (Greiner Bio-One). Compounds were added immediately after plating. Cells were cultured at 37 °C in 5% C0 2 . Multicolor analysis for cell phenotyping was performed on a LSR II flow cytometer (Becton Dickinson). Cells were stained in staining media (HBSS supplemented with FBS [2%] and EDTA [2 mM]) at 4 °C for 1 h with APC anti-human CD1 10 (BD
  • AHR inhibitory activity of a compound may be determined by its ability to block 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin)-mediated CYP1 B1 mRNA expression by qPCR in mPB-derived CD34 + cells (the dioxin-induced AHR dependent luciferase reporter gene assay).
  • Treatment with TCDD (3nM) caused a 4.5-fold increase in the level of CYP1 B1 mRNA compared with the vehicle control (0.01 % toluene).
  • the test compound is assayed in a dose response format (1 n M to 10 ⁇ ) to determine the effective concentration that produced the desired effect in 50% of the cells (EC 50 ).
  • An inhibitor of AHR activity is a compound that has an EC50 of less than 10 ⁇ , preferably less than 5 ⁇ as measured by this assay. Inclusion of Compound B1 (1 ⁇ ) completely abolished dioxin- induced AHR dependent transcription when used on cells expressing human AHR.
  • Compound B may be prepared by Schemes 1 -5 below.
  • reactive functional groups for example hydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, to avoid their unwanted participation in the reactions.
  • Conventional protecting groups can be used in accordance with standard practice, for example, see T.W. Greene and P. G. M. Wuts in
  • G G 2 , G 3 , G 4 , R 2 and R 4 are as defined for Formula I in the Summary of the Invention and L of Formula I is defined in the reaction scheme as -NH4_ which is equivalent to, for example, -NR 5a (CH 2 )o- 3 - where R 5a is hydrogen and -(CH 2 ) 0 - 3 - is
  • Formula 2 with a compound of Formula 3 in the presence of a suitable catalyst (e.g., Pd2(dba)3, or the like) in the presence of an appropriate ligand (e.g., 1 ,3-bis(2,4,6- trimethylphenyl) imidazolium chloride), a suitable base (e.g., Cs 2 C0 3 , or the like) and an appropriate solvent (e.g., 1 ,4-dioxane) at a temperature of about 80 to 100 °C for 2 to about 48 hours.
  • a suitable catalyst e.g., Pd2(dba)3, or the like
  • an appropriate ligand e.g., 1 ,3-bis(2,4,6- trimethylphenyl) imidazolium chloride
  • a suitable base e.g., Cs 2 C0 3 , or the like
  • an appropriate solvent e.g., 1 ,4-dioxane
  • Compounds of Formula 4 can be prepared by alkylation of a compound of Formula 6 with a suitable alkylating agent 7, in which is chlorine, bromine, iodine, or a sulfonate ester, in the presence of a suitable base (e.g. sodium hydride or potassium carbonate), in a suitable solvent (e.g. DMF), at a temperature of about 0 °C to about 80 °C.
  • a suitable base e.g. sodium hydride or potassium carbonate
  • a suitable solvent e.g. DMF
  • the reaction can be performed under Mitsunobu conditions using a suitable alcohol R 4 -OH in the presence of a suitable phosphine (e.g.
  • triphenylphosphine e.g. diethylazodicarboxylate
  • azodicarboxylate e.g. diethylazodicarboxylate
  • inert solvent such as THF or toluene
  • Compounds of Formula 8 can in turn be prepared by reacting a compound of Formula 9 with a compound of Formula 3 in the presence of a suitable catalyst (e.g., Pd(Ph 3 P) 4 , Pd 2 (dba) 3 , or the like), optionally in the presence of an appropriate ligand (e.g., 1 ,3-bis(2,4,6-trimethylphenyl) imidazolium chloride), a suitable base (e.g., Cs 2 C0 3 , or the like) and an appropriate solvent (e.g., 1 ,4-dioxane) at a temperature of about 80 to 100 °C for 2 to about 48 hours.
  • a suitable catalyst e.g., Pd(Ph 3 P) 4 , Pd 2 (dba) 3 , or the like
  • an appropriate ligand e.g., 1 ,3-bis(2,4,6-trimethylphenyl) imidazolium chloride
  • a suitable base e
  • Compounds of Formula 9 in turn can be prepared by reacting a compound of Formula 10 with a mixture of di-iodomethane, copper(l) iodide, and an alkyl nitrite (e.g. isoamylnitrite), optionally in the presence of an inert solvent, at a temperature of about 50 to 100 °C.
  • a compound of Formula 10 with a mixture of di-iodomethane, copper(l) iodide, and an alkyl nitrite (e.g. isoamylnitrite), optionally in the presence of an inert solvent, at a temperature of about 50 to 100 °C.
  • an alkyl nitrite e.g. isoamylnitrite
  • Compounds of Formula 10 can be prepared by alkylation of a compound of Formula 1 1 with a suitable alkylating agent 7, in which is chlorine, bromine, iodine, or a sulfonate ester, in the presence of a suitable base (e.g. sodium hydride or potassium carbonate), in a suitable solvent (e.g. DMF), at a temperature of about 0 °C to about 80 °C.
  • a suitable base e.g. sodium hydride or potassium carbonate
  • a suitable solvent e.g. DMF
  • the reaction can be performed under Mitsunobu conditions using a suitable alcohol R 4 -OH in the presence of a suitable phosphine (e.g. triphenylphosphine) and azodicarboxylate (e.g. diethylazodicarboxylate), in an inert solvent such as THF or toluene, at a temperature from about 0 °C to about room temperature.
  • Gi , G 2 , G 3 , G 4 , Ri , R 2 and R 4 are as defined for Formula I in the Summary of the Invention and L of Formula I is defined in the reaction scheme as -NH-L which is equivalent to, for example, -NR 5a (CH 2 )o- 3 - where R 5a is hydrogen and -(CH 2 )o-3- is .
  • Compounds of Formula II can be prepared by reacting a compound of Formula 2 with a compound of Formula 20 in the presence of an excess of cyclic amine or NH-bearing heterocycle (for example, substituted pyrazole, substituted imidazole, and the like), at a temperature of about 50 °C to about 250 °C, for about 1 to about 24 hours, optionally in the presence of a base such as sodium hydride or DBU.
  • an excess of cyclic amine or NH-bearing heterocycle for example, substituted pyrazole, substituted imidazole, and the like
  • R 3 and R 4 are as defined for Formula I in the Summary of the Invention.
  • Compounds of Formula 10 can be prepared according to procedures described in J. Med. Chem, 1972, 456, and J. Med. Chem., 1992, 4180.
  • An orthoester compound of Formula 21 is reacted with a compound of Formula 22, optionally in the presence of an acid such as acetic acid, at a temperature of about room temperature to about 150 oC, for about 1 to about 24 nr..
  • a compound of Formula 22 can in turn be prepared by reacting a compound of Formula 23 with a primary amine compound of Formula 24, optionally in the presence of an acid such as pTSA, or a base such as triethylamine or DBU, at a temperature of about 50 to about 200 oC.
  • R 20 and R 21 are independently selected from hydrogen and d ⁇ alkyl.
  • a compound of Formula IV, in which R 21 is hydrogen can be prepared from a compound of Formula III by treatment with a suitable reducing agent such as lithium aluminum hydride or di-isobutyl aluminum hydride, in a suitable solvent such as THF or toluene, at a temperature of about - 78°C to about 50°C. The reaction takes about 0.5 to about 16 hr to complete.
  • a compound of Formula IV, in which R 21 is lower alkyl can be prepared by treatment of a compound of Formula III with an alkyl lithium or Grignard reagent, in a suitable solvent such as ether or tetrahydrofuran, at a temperature of about -78°C to about 50°C. The reaction takes about 0.5 to about 16 hr to complete.
  • a compound of the invention can be prepared as a pharmaceutically acceptable acid addition salt by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid.
  • a pharmaceutically acceptable base addition salt of a compound of the invention can be prepared by reacting the free acid form of the compound with a pharmaceutically acceptable inorganic or organic base.
  • the salt forms of the compounds of the invention can be prepared using salts of the starting materials or intermediates.
  • the material has a melting point at about 233 °C with a melting enthalpy of 98g/J.
  • the material produced exhibited a loss on drying of 0.2%.
  • the water uptake was estimated by thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. A water uptake of 0.4% was observed.
  • the free acid or free base forms of the compounds of Formula I or II can be prepared from the corresponding base addition salt or acid addition salt form, respectively.
  • a compound of the invention in an acid addition salt form can be converted to the corresponding free base by treating with a suitable base (e.g., ammonium hydroxide solution, sodium hydroxide, and the like).
  • a compound of Formula I or Formula II in a base addition salt form can be converted to the corresponding free acid by treating with a suitable acid (e.g., hydrochloric acid, etc.).
  • a suitable acid e.g., hydrochloric acid, etc.
  • the nitrate salt of the compound of example 1 can be made using methods known to the skilled person.
  • Compounds of Formula I or II can be prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the
  • diastereomers and recovering the optically pure enantiomers. While resolution of enantiomers can be carried out using covalent diastereomeric derivatives of the compounds of the invention, dissociable complexes are preferred (e.g., crystalline diastereomeric salts). Diastereomers have distinct physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and can be readily separated by taking advantage of these dissimilarities. The diastereomers can be separated by chromatography, or preferably, by separation/resolution techniques based upon differences in solubility. The optically pure enantiomer is then recovered, along with the resolving agent, by any practical means that would not result in racemization.
  • dissociable complexes are preferred (e.g., crystalline diastereomeric salts).
  • Diastereomers have distinct physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and can be readily separated by taking advantage
  • CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols.
  • CD34+ cells were cultured at 37 °C for 18-20 days in 5% C0 2 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and Reagent B (250 nM of (S)-2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9- yl)propan-1 -ol (Example B157S)) and the following recombinant human cytokines:
  • TPO thrombopoietin
  • IL6 Flt3 ligand
  • stem cell factor each 50 ng/mL, R&D Systems.
  • CD34+ cells were seeded at 1 -25x10 4 viable nucleated cells per ml (count prior to cryopreservation), and cultures were maintained in culture plates and supplemented with culture media as necessary.
  • Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by analysis of a defined volume by flow cytometry. Final platelet counts were obtained at day 18 or day 20 (one sample).
  • Example B Reagent B Presence in 2 nd Stage Promotes Platelet Biogenesis
  • CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols.
  • CD34+ cells were cultured at 37 °C in 5% C0 2 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems).
  • CD34+ cells were seeded at 20-25x10 4 viable nucleated cells per ml_ (count prior to cryopreservation).
  • Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate.
  • Platelet concentration in the final sample was determined by analysis of a defined volume by flow cytometry and normalized to the number of CD34+ cells at day 1 in a defined volume by flow cytometry. Final platelet counts were obtained at day 18. Yields, baseline protocol with 250 nM Reagent B (Compound
  • CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols.
  • CD34+ cells were cultured at 37 °C in 5% C0 2 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows.
  • the first culture phase (days 0 to 14) contained the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems).
  • CD34+ cells were seeded at 3-5x10 4 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 3x10 4 and 1 x10 6 viable nucleated cells per ml, as determined by trypan blue exclusion.
  • Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
  • Reagent A at day 14 change of cytokine composition to TPO alone at day14 and addition of MMP inhibitor at day 16) increased the platelet yield from 281 platelets per seeded cell for the cytokines only baseline protocol or to 481 platelets per seeded cell (1 .7 fold effect) in large scale culture.
  • platelets were further assessed for viability by fluorescence with the dye Calcein, AM (Life Technologies). Cells were stained with 2.5 ⁇ Calcein AM for 15 minutes at 37 °C, along with cell surface antibodies, washed and analyzed by flow cytometry. The % Calcein AM+ cells was assessed in the platelet population as shown for Example E.
  • platelets were further assessed for young platelets (reticulocytes) by staining with the dye thiazole orange (Sigma). Cells were stained with 25 ⁇ g/ml thiazole orange for 15 minutes at 37 °C, along with cell surface antibodies, washed and analyzed by flow cytometry. Gating on whole blood platelets was based on the normal circulating value of 3-16%, mean 9% (Kienast J, Schmitz G. Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation of thrombocytopenic disorders. Blood 1990;75:1 16-121 ) as shown for Example E.
  • CD34+ cells were purchased as a CD34+ selected cord blood mixed donor vial (Allcells). CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows.
  • the first culture phase (days 0 to 14) contained the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems) with or without Reagent A (500 nM of 3-(7-((2,6- dimethylpyridin-3-yl)amino)-1 -methyl-2-oxo-1 ,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N- (3-(trifluoromethyl)phenyl)-4-vinylbenzamide (Compound A1 or MK1 ).
  • TPO thrombopoietin
  • IL6 Flt3 ligand Flt3 ligand
  • stem cell factor each 50 ng/mL, R&D Systems
  • Reagent A 500 nM of 3-(7-((2,6- dimethylpyridin-3-yl)a
  • CD34+ cells were seeded at 3x10 5 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 9x10 4 and 1 .4x10 6 viable nucleated cells per ml, as determined by trypan blue exclusion.
  • Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
  • Reagent B were each shown to contribute to increases in platelet yield, both individually and in combination. Addition of Compound A1 (Reagent A) only resulted in a 2.5 fold increase in platelet yield over no compound addition. Addition of Compound B157S (Reagent B) resulted in a further 3-fold increase, for a total increase of 7-fold.
  • CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols. CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows.
  • the first culture phase (days 0 to 14) contained Reagent A (250 nM of 3-(7-((2,6- dimethylpyridin-3-yl)amino)-1 -methyl-2-oxo-1 ,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N- (3-(trifluoromethyl)phenyl)-4-vinylbenzamide (Example A1 )) and the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems).
  • TPO thrombopoietin
  • IL6 Flt3 ligand IL6, Flt3 ligand
  • stem cell factor each 50 ng/mL, R&D Systems.
  • CD34+ cells were seeded at 3-5x10 4 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 3x10 4 and 1 x10 6 viable nucleated cells per ml, as determined by trypan blue exclusion.
  • MMP matrix metalloproteinase
  • Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate.
  • Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
  • Figure 3 shows the pseudocolor density plots displaying fluorescence intensity of particles analyzed by flow cytometry.
  • Gating scheme for platelets is defined by scatter properties and CD41 a and CD42b bound fluorescence intensity.
  • Ex vivo cultured platelets were defined by gates which encompass whole blood platelets shows that that ex vivo cultured platelets are phenotypically same as whole blood derived platelet.
  • platelets were further assessed for viability by fluorescence with the dye Calcein, AM (Life Technologies). Cells were stained with 2.5 ⁇ Calcein AM for 15 minutes at 37C, along with cell surface antibodies, washed and analyzed by flow cytometry.
  • Fig. 4 shows the pseudocolor density plots displaying fluorescence intensity of particles analyzed by flow cytometry. Platelets gated as in Fig 3 were further assessed for Calcein fluorescence, an indication of viability. Blood platelets were used to set the Calcein+ viability gate. shows flow cytometry chart for Calcein, AM stained cells.
  • platelets were further assessed for young platelets (reticulocytes) by staining with the dye thiazole orange (Sigma). Cells were stained with 25 ⁇ g/ml thiazole orange for 15 minutes at 37C, along with cell surface antibodies, washed and analyzed by flow cytometry. Gating on whole blood platelets was based on the normal circulating value of 3-16%, mean 9% (Kienast J, Schmitz G. Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation of thrombocytopenic disorders. S/ooc/ 1990 75:1 16-121 ).
  • Fig. 5 shows the pseudocolor density plots displaying fluorescence intensity of particles analyzed by flow cytometry.-Platelets gated as defined in Fig 3 were further assessed for thiazole orange binding, an indication of a newly formed platelet. Blood platelets were used to set the thiazole orange+ gate, as based on the normal circulating value of 3-16%, mean 9% (Kienast J, Schmitz G. Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation of thrombocytopenic disorders. Blood 1990;75:1 16-121 ) shows the thiazole orange stained cells.
  • Example F In vivo Functional Assay of Ex Vivo Manufactured Platelets
  • CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols. CD34+ cells were cultured at 37 °C in 5% C0 2 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows.
  • the first culture phase (days 0 to 14) contained Reagent A (250 nM of 3-(7-((2,6- dimethylpyridin-3-yl)amino)-1 -methyl-2-oxo-1 ,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N- (3-(trifluoromethyl)phenyl)-4-vinylbenzamide (Example A1 , also referred to as MK1 )) and the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems).
  • TPO thrombopoietin
  • IL6 Flt3 ligand IL6, Flt3 ligand
  • stem cell factor each 50 ng/mL, R&D Systems.
  • CD34+ cells were seeded at 3x10 4 viable nucleated cells per ml, and cultures were maintained in culture bags (American Fluoroseal Corp) and supplemented with culture media to maintain cell density between 3x10 4 and 1 x10 6 viable nucleated cells per ml, as determined by trypan blue exclusion.
  • MMP matrix metalloproteinase
  • Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
  • Platelets were isolated using a modification of a published protocol
  • ACD-anticoagulated whole blood platelets were isolated in a similar manner for comparison. Platelet rich plasma supernatant was collected after centrifugation at 200xg. Platelets were washed in citrate wash buffer before loading on the 2% to 12% BSA gradient and processed as above. An aliquot of the final isolated platelets in HBSS were treated with 100 ⁇ CCCP (Carbonyl cyanide 3-chlorophenylhydrazone, Sigma) for 1 hr at 37 °C to model platelet damage, then washed to remove CCCP. Untreated whole blood platelets were stored for 1 hr at 37 °C.
  • CCCP Carbonyl cyanide 3-chlorophenylhydrazone
  • Example G In Vitro Functional Assay - Platelet Spreading
  • Human CD34+ cells were purchased as a CD34+ selected cord blood mixed donor vial (Allcells).
  • CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media
  • the first culture phase (days 0 to 14) contained the following
  • CD34+ cells were seeded at 8x10 4 viable nucleated cells per ml, and cultures were maintained in culture bags (American Fluoroseal Corp) and supplemented with culture media to maintain cell density between 1 x10 5 and 6x10 5 viable nucleated cells per ml, as determined by trypan blue exclusion.
  • MMP matrix metalloproteinase
  • Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
  • Platelets were isolated using a modification of a published protocol
  • the platelet-containing upper phase was collected after spinning at 80xg for 15 min with no brake or acceleration and washed with DPBS containing 1 % BSA and 50 ng/ml PGE1 .
  • the washed platelets were pooled in serum free TPO containing media.
  • Platelets were added to fibrinogen coated chamber slides in the presence or absence of 1 U/ml thrombin and incubated for 30 min at 37C. After washing gently with warm PBS, adherent platelets were fixed by addition of 4% formaldehyde in PBS. Platelets were permeabilized with 0.1 % TritonX-100 and blocked with 1 %BSA in PBS before staining with an a-tubulin directed primary antibody (clone DM1 A, Sigma) and fluorescently conjugated goat anti-mouse F(ab')2 secondary antibody (Invitrogen). Slides were mounted with Prolong Gold (Invitrogen) and visualized by immunofluorescence microscopy.
  • the culture-derived platelets are function in vitro. As shown in Figure 7, the culture-derived platelets exhibited cell spreading on fibrinogen-coated surface in response to thrombin activation.
  • CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols.
  • CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems).
  • CD34+ cells were seeded at 5x10 4 viable nucleated cells per ml (count prior to cryopreservation).
  • Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate.
  • CD34+ cells were purchased as a CD34+ selected cord blood mixed donor vial (Allcells). CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows.
  • the first culture phase (days 0 to 14) contained the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems).
  • CD34+ cells were seeded at 5x10 4 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 5x10 4 and 2.4x10 6 viable nucleated cells per ml, as determined by trypan blue exclusion. At day 14, cultures were 18% megakaryocytes. For the second culture phase (days 14 to 18), cells were pelleted and resuspended on day 14 at 3x10 5 viable nucleated cells per ml in serum-free media containing Reagent B (750 nM of Compound B157S). Cultures were plated in non-tissue culture treated plates. TPO was added in a 12 point dose response at 2-fold dilutions from 100 ng/ml to 50 pg/ml and cultured to day 18 before platelet analysis.
  • Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
  • CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols or purchased as a CD34+ selected mixed cord blood donor vial (Allcells).
  • CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems).
  • Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate.
  • CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols or purchased as a CD34+ selected cord blood obtained from mixed donors or a single donor (Allcells). CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows.
  • the first culture phase (days 0 to 14) contained Reagent A (500 nM of Compound A1 (also referred to as MK1 ) and the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems).
  • CD34+ cells were seeded at 5-50x10 4 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 9x10 4 and 1 .4x10 6 viable nucleated cells per ml, as determined by trypan blue exclusion.
  • Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
  • Platelet yields with this protocol were on average 3-fold increased over the baseline protocol with cytokines only in Example A.
  • Platelets were isolated using a modification of a published protocol (Robert, et al.
  • the platelet-containing upper phase was collected after spinning at 80xg for 15 min with no brake or acceleration and washed with DPBS containing 1 % BSA and 50 ng/ml PGE1 .
  • the washed platelets were pooled in HBSS and stored for 1 hr at 37C.
  • ACD-anticoagulated whole blood platelets were isolated in a similar manner for comparison. Platelet rich plasma supernatant was collected after centrifugation at 200xg. Platelets were washed in citrate wash buffer before loading on the 2% to 12% BSA gradient and processed as above.
  • Calcein, AM (Life Technologies). Cells were stained with 2 ⁇ Calcein AM for 15 minutes at 37C, along with cell surface antibodies, washed and analyzed by flow cytometry. The % Calcein AM+ cells was assessed in the platelet population as shown for Example E.
  • Platelets were assessed for young platelets (reticulocytes) by staining with the dye thiazole orange (Sigma). Cells were stained with 20 ⁇ g/ml thiazole orange for 15 minutes at 37C, along with cell surface antibodies, washed and analyzed by flow cytometry. % young platelets (thiazole orange+) at day 18
  • Human CD34+ cells were purchased as CD34+ selected cord blood obtained from a single donor (Allcells). CD34+ cells were cultured at 37°C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows.
  • the first culture phase (days 0 to 14) contained Reagent A, 500 nM of Compound A1 (also referred to as MK1 ) and the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems).
  • CD34+ cells were seeded at 2x10 5 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 1 x10 5 and 1 x10 6 viable nucleated cells per ml, as determined by trypan blue exclusion.
  • days 14 to 18 For the second culture phase (days 14 to 18), cells were pelleted and resuspended on day 14 at 9x10 5 viable nucleated cells per ml in serum- free media containing TPO (100 ng/ml) and Reagent B, 750 nM of Compound A1 . Cultures were maintained in culture bags (American Fluoroseal Corp).
  • Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate.
  • Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer. [00415] Platelets were isolated using a modification of a published protocol
  • Platelets were released by pipetting each aliquot up and down 12 times with a 50 ml serological pipette fitted with a P1000 pippete tip to the end. Each aliquot was pelleted at 1000xg for 15 min and resuspended in 2 ml citrate wash buffer (0.5% Citrate and 0.35% BSA in HBSS, adjusted to pH 6.5, with 50 ng/ml PGE1 added just before use), which was then layered on a 2% to 12% BSA step gradient. The platelet-containing upper phase was collected after spinning at 80xg for 15 min with no brake or acceleration and washed with DPBS containing 1 % BSA and 50 ng/ml PGE1 . The washed platelets were pooled in HBSS and stored for 1 hr at 37C.
  • ACD-anticoagulated whole blood platelets were isolated in a similar manner for comparison. Platelet rich plasma supernatant was collected after centrifugation at 200xg. Platelets were washed in citrate wash buffer before loading on the 2% to 12% BSA gradient and processed as above. An aliquot of the final isolated platelets in HBSS were treated with 100 ⁇ CCCP (Carbonyl cyanide 3-chlorophenylhydrazone, Sigma) for 1 hr at 37C to model platelet damage, then washed to remove CCCP. Untreated whole blood platelets were stored for 1 hr at 37C.
  • CCCP Carbonyl cyanide 3-chlorophenylhydrazone
  • CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols.
  • CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows.
  • the first culture phase (days 0 to 14) contained Reagent A, 500 nM of Compound A1 (also referred to as MK1 ) and the following recombinant human cytokines:
  • TPO thrombopoietin
  • IL6 Flt3 ligand
  • stem cell factor stem cell factor
  • CD34+ cells were seeded at 8.5x10 4 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 1 -10x10 5 viable nucleated cells per ml, as determined by trypan blue exclusion.
  • days 14 to 18 For the second culture phase (days 14 to 18), cells were pelleted and resuspended on day 14 at 5x10 5 viable nucleated cells per ml in serum-free media containing TPO (100 ng/ml) and Reagent B, 750 nM of Compound B1 .
  • MMP matrix metalloproteinases
  • Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
  • Example N Duration of the contacting with Reagent A
  • CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols.
  • CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows.
  • the first culture phase (days 0 to 14) contained the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems).
  • Reagent A Compound A1 , or MK1
  • CD34+ cells were seeded at 8.5x10 4 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media.
  • the cultures were pelleted and resuspended at 1 x10 5 viable nucleated cells per ml, as determined by trypan blue exclusion, with the first culture phase media above. An aliquot at each time point was set aside for further culture without MK1 .
  • MK1 was added at 500 nM to the remaining culture at each time point.
  • Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
  • Fig. 13 shows the optimal duration for the contacting with Reagent A is 14 day.
  • Example O Evaluate Method of Transferring Megakaryocyte between Stage 1 and Stage 2 Culture.
  • CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols.
  • CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows.
  • the first culture phase (days 0 to 12) contained the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems).
  • CD34+ cells were seeded at 8x10 4 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 5x10 4 and 6x10 5 viable nucleated cells per ml, as determined by trypan blue exclusion.
  • cultures were pelleted and either (for 2-stage cultures) resuspended at 4x10 5 viable nucleated cells per ml in serum-free media containing TPO (100 ng/ml) or (for isolation cultures) CD61 + cells were isolated using magnetic beads per the manufacturer's instructions (Milteni) and resuspended at 4x10 5 viable nucleated cells per ml in serum-free media containing TPO (100 ng/ml). Vehicle or Reagent B, 250 nM of Compound B157S, was added to each condition. Cells were cultured to day 17 in non- tissue culture treated plates before analysis for platelets.
  • Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
  • CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols or purchased as a CD34+ selected mixed cord blood donor vial (Allcells).
  • CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems).
  • CD34+ cells were seeded at 2.5-25x10 4 viable nucleated cells per ml (count prior to cryopreservation). Cultures were maintained in non-tissue culture treated culture plates and supplemented with culture media as necessary prior to megakaryocyte isolation (maintained between 3 x10 4 and 1 .4x10 6 when monitored).
  • Day 10 or day 1 1 cultures were pelleted and stained with antibodies as below.
  • CD45RA-CD41 + cells (one experiment) or CD45RA-CD41 +CD42+ (remaining experiments) were sorted by flow cytometry. Sorted cells were pelleted and resuspended in culture media as above, with addition of vehicle control and Reagent B: 750 nM Compound B1 , 750 nM Compound B88 control, or 250 nM of Compound B157S. Cells were then cultured as above to day 16. 2. CD61 + cells from day 12 or day 13 cultures were isolated using magnetic beads per the manufacturer's instructions (Milteni). Isolated cells were pelleted and resuspended in culture media as above, with addition of vehicle control, Reagent B: 750 nM of
  • CD61 + cells from day 12 or day 13 cultures were isolated using magnetic beads per the manufacturer's instructions (Milteni). Isolated cells were pelleted and resuspended in culture media as above except that 100 ng/ml TPO as the only cytokine, with addition of vehicle control, Reagent B: 750 nM of Compound B1 or 250 nM of Compound B157S. Cells were then cultured as above to day 17 or day 18.
  • Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Fold increases in platelet yield over control from the same donor in multiple experiments were averaged, prior to the analysis below. N indicates the number of unique donors.
  • Example Q Isolation of Platelets by Centrifugation over a BSA Gradient
  • Human CD34+ cells were purchased as a CD34+ selected cord blood mixed or single donor vials (Allcells).
  • CD34+ cells were cultured at 37 °C in 5% C0 2 in serum-free media
  • the first culture phase (days 0 to 14) contained the following
  • recombinant human cytokines thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems) with or without Reagent A, 250-500 nM of Compound A1 (also referred to as MK1 ).
  • CD34+ cells were seeded at 3-20x10 4 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 3x10 4 and 1 x10 6 viable nucleated cells per ml, as determined by trypan blue exclusion.
  • Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience).
  • Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
  • Platelets were isolated using a modification of a published protocol
  • the platelet-containing upper phase was collected after spinning at 80xg for 15 min with no brake or acceleration and washed with DPBS containing 1 % BSA and 50 ng/ml PGE1 .
  • the washed platelets were pooled in serum free TPO containing media or HBSS.
  • the percent platelets were calculated as the number platelets divided by the sum of the number of platelets and nucleated cells. Platelet isolation at day 18
  • FIG. 15 A representative pseudocolor density plots displaying fluorescence intensity of particles analyzed by flow cytometry of Sample 1 was shown in Fig. 15. Culture- derived platelets before and after isolation were analyzed in comparison with isolated blood platelets. Gating is as shown. Viable nucleated cells were gated based on scatter properties. Platelets were gated based on scatter properties and CD41 a and CD42b bound fluorescence intensity. These gates were used to calculate the %platelets and platelet recovery.
  • the data shows the isolation method effectively purified platelets from nucleated cells in the culture.
  • Example A1 3-(7-((2,6-dimethylpyridin-3-yl)amino)-1 -methyl-2-oxo-1 ,2- dihydropyrimido[4,5-d rimidin-3(4H)-yl)-N-(3-(trifluoromethyl)phenyl)-4-vinylbenzamide
  • a solution of methyl 3-nitro-4-vinylbenzoate (40 mg, 0.21 mmol) in THF is treated with 2N aqueous sodium hydroxide solution (0.5 ml, 1 .0 mmol) and the mixture is stirred at rt for 2 hr.
  • the mixture is concentrated under reduced pressure and washed with chloroform.
  • the remaining aqueous layer is acidified with 1 N hydrochloric acid and the mixture is extracted 3x with chloroform.
  • the combined acidic chloroform extracts are dried over sodium sulfate and concentrated to provide 3-nitro-4-vinylbenzoic acid, which is used without further purification.
  • the residue is purified by silica gel column chromatography (5:1 hexanes/ethyl acetate eluant) to provide 3-nitro-N-(3- (trifluoromethyl)phenyl)-4-vinylbenzamide.
  • the residue is purified by silica gel chromatography (4:1 hexanes/ethyl acetate eluant) to provide 3-amino- N-(3-(trifluoromethyl)phenyl)-4-vinylbenzamide.
  • Example A2 N-(4-Methyl-3-(1 -methyl-2-oxo-7-((3-(2-oxopyrrolidin-1 -yl)propyl)amino)-1 ,2- dihydropyrimido[4,5-d rimidin-3(4H)-yl)phenyl)-3-(trifluoromethyl)benzamide
  • Example A3 N-(3-(2-Amino-8-methyl-7-oxo-7,8-dihydropteridin-6-yl)-4-methylphenyl)-3-(4- methyl-1 H-imidazol-1 -yl)-5-(trifluoromethyl)benzamide
  • reaction mixture After stirring for 16 hours at about 65°C, the reaction mixture is cooled to room temperature and stirred with a 10% aqueous solution of potassium fluoride (800 mL) and diethyl ether (600 mL) for 1 hour before filtering through a pad of Celite.
  • the pad of Celite is rinsed with a further portion of diethyl ether (200 mL).
  • the aqueous layer is separated and extracted with CHCI 3 .
  • Ozone is bubbled through a solution of (2-chloro-5-vinyl-pyrimidin-4-yl)- methylamine (2.50 g, 14.7 mmol) in CHCI 3 /MeOH (15 mL/15 mL) for 30 minutes at -78°C and then a stream of argon is passed through the solution for 3 minutes at the same temperature.
  • the reaction mixture is allowed to warm up to room temperature and treated with dimethyl sulfide (3.24 mL, 44.1 mmol).
  • Example B1 4-(2-(2-(benzo[b]thio hen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol
  • the flask was evacuated and backfilled with N 2 and anhydrous 1 ,4-dioxane (2 ml_) was added. The flask was sealed and the reaction mixture was stirred at 80 °C for 24 hours. The reaction mixture was concentrated and purified directly by column chromatography on silica gel, eluting with hexane/EtOAc (20:1 to 1 :4) to afford the title compound as a yellowish solid.
  • Example B1 can be recrystallised using a
  • Example B15 4-(2-(Pyridin-3-yl)- -isopropyl-9H-purin-6-ylamino)ethyl)phenol
  • 6-Chloro-2-iodo-9-isopropyl-9/-/-purine (c): 6-chloro-9- isopropyl-9H-purin-2-amine (2.68 g, 12.7 mmol) was dissolved in THF (64 mL) at rt. Iodine (1 .61 g, 6.25 mmol), CH 2 I 2 (10.6 mL) and Cul (1 .27 g, 6.66 mmol) were added. The mixture was stirred for 5min at room temperature. Isopentyl nitrite (5.33 mL) was added. The reaction mixture was refluxed for 45 min, and was then cooled to room temperature.
  • Example B123 4-(2-(9-lsopropyl-2-(2-methyl-1 H-imidazol-1 -yl)-9H-purin-6- ylamino)ethyl)phenol
  • Example B134 4-(2-(6-(5-Fluoropyridin-3-yl)-1 -isopropyl-1 H-pyrazolo[3,4-d]pyrimidin-4- ylamino)ethyl)phenol
  • Example B141 4-(2-(2-(5-Fluoropyridin-3-yl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4- ylamino)ethyl)phenol
  • Example B153b using 2,6-dichloro-9H-purine and (S)-1 -(benzyloxy)propan-2-ol as reactants
  • Example B153c using tryptamine as reactant
  • Example B153d using 5- fluoropyridin-3-ylboronic acid as reactant

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Abstract

The present invention relates to a method of producing platelets (characterized by CD41+ and CD42+) from hematopoietic stem cells ex vivo. The invention further relates to a composition comprising the ex vivo produced platelets and a reagent comprising an aryl hydrocarbon antagonist and its use in autologous or allogeneic transfusion for the treatment of thrombocytopenia, thereby treating disease and conditions that caused the thrombocytopenia.

Description

EX VIVO PRODUCTION OF PLATELETS FROM HEMATOPOIETIC STEM CELLS AND
THE PRODUCT THEREOF
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] The present invention relates to a method and reagents for producing platelets from hematopoietic stem cell ex vivo. The invention further relates to a therapeutic composition comprising platelets produced by the ex vivo method and its use in autologous or allogeneic transplantation for the treatment of patients with thrombocytopenia.
Background
[0002] Over 2 million units of platelets platelet are transfused per year in the US
(2008 blood utilization survey report). Platelets have a limited 5 day shelf-life, and platelet shortages are an ongoing problem for the medical community. Hematology patients undergoing high dose chemotherapy account for up to 67% of platelet transfusions in the US. Because of prolonged thrombocytopenia, these patients normally require multiple platelet transfusions over a period of weeks or months. Current medical practice does not require human leukocyte antigen (HLA)-matched platelets, and alloimmunization is a common risk for these patients which renders them refractory to platelet transfusion. In the TRAP clinical trial 8% of patients had baseline anti-HLA antibodies and another 17-45% developed them following chemotherapy. HLA-alloimmunized patients are ideally given matched platelets to avoid immediate platelet rejection; however, identifying and obtaining matched donor platelets is often not feasible and is a current unmet medical need.
[0003] Ex vivo platelet generation could address this need. The present invention relates to a process and reagents for manufacturing platelets from hematopoietic stem cells ex vivo. The process is adaptable to manufacture platelets in a patient specific manner by using HLA-matched hematopoietic stem cells. The exPLTs potentially can be a source of HLA-matched platelets for alloimmunized thrombocytopenic patients without a donor platelet match and, as such, will increase post-transfusion platelet recovery compared to standard of care (unmatched platelets). In addition, the possibility of using exPLT to supplement and/or replace standard platelet products will help to alleviate a reliance on donors, avert shortages and improve product safety.
SUMMARY OF THE INVENTION
[0004] In one aspect, the invention provides a process of ex vivo manufacturing clinically relevant quantities of ex-vivo produced platelets (exPLTs) from hematopoietic stem cells. The process comprising a step of:
contacting a quantity of megakaryocytes with a small molecule reagent and thrombopoietin (TPO) for a duration of time to yield a population of ex vivo produced platelets (exPLT), wherein the small molecule reagent is a compound of Formula II
Figure imgf000004_0001
or a pharmaceutically acceptable salt, or stereoisomer thereof; wherein the variables are as described in the Detailed Description of the Invention, paragraphs [00138], infra.
[0005] In a second aspect, the invention provides a composition of platelets produced from human hematopoietic stem cells by the ex vivo manufacturing method described in the first aspect of the invention, supra.
[0006] In a third aspect, the invention provides a composition comprising ex vivo produced megakaryotecytes (exMKs) in a cell culture medium containing Reagent B.
[0007] In a fourth aspect, the invention provides a composition comprising ex vivo produced platelets and Reagent B.
[0008] In a fifth aspect, the invention relates to a composition of isolated ex vivo produced platelets characterized by the presence of Reagent B.
[0009] In a sixth aspect, the invention relates to a method of using the ex vivo produced platelets for treating thrombocytopenia; thereby treating, abating, ameliorating or alleviating the diseases and conditions and/or its attendant symptoms that are the underlying causes of the thrombocytopenia.
BRIEF DESCRIPTION OF DRAWINGS
[0010] Fig 1 . Flow chart of the two-stage process of the invention useful for the manufacturing of platelets from hematopoietic stem cells ex vivo.
[0011] Fig. 2. A two-stage protocol is optimal for exPLTs production. The contribution of the presence or absence of: a first stage with 500 nM of Compound A1 (Reagent A), a second stage with 500 nM of Compound B157S (Reagent B) or 750 nM of Compound B1 (Reagent B) to platelet yield (number count per initiating HSC) were measured. "+" means the indicated stage was present and "-" means the indicated stage was absence.
[0012] Fig 3. The exPLTs are phenotypically the same as whole blood platelets.
Flow cytometry analyses of cell cultures: ex vivo produced platelets at day 18 (top row) is compared to whole blood platelets (bottom row). Platelets were distinguished from nucleated cells by size (FSC) and granularity (SSC) and were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate.
[0013] Fig. 4. The exPLTs are similarly viable as whole blood platelets. Viability of the platelets was assessed by flow cytometry analyses of Calcein AM+ stained platelets from culture derived platelets at day 18 (top row) and whole blood platelets (bottom row). Platelets were distinguished from nucleated cells by size (FSC) and granularity (SSC) and were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate.
[0014] Fig. 5. The culture-derived platelets contain significantly higher percentage of young platelets. The percentage of young platelets were assessed by flow cytometry analyses of thiazole orange+ stained platelets of culture-derived platelets at day 18 (top row) and whole blood platelets (bottom row). Platelets were distinguished from nucleated cells by size (FSC) and granularity (SSC) and were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate.
[0015] Fig. 6. exPLTs are functioned in vivo. Residual human platelets (number counts) from ex vivo culture derived, donor derived, and damaged whole blood (treated with carbonyl cyanide 3-chlorophenylhydrazone) in mouse circulation were measured 4 hrs (right) and 24 hrs (left) after injection. Residual human platelets were detected for culture- derived and donor derived platelets and not for damaged platelets.
[0016] Fig. 7. exPLTs are functioning in vitro. The spreading response of exPLTs to thrombin (agonist) stimulation; no agonist (right) and with agonist (left).
[0017] Fig. 8. TPO in combination with Reagent B (250 nM of Compound
B157S) produced the highest platelet yield. Platelet yields (folds over culturing with the 4- cytokine cocktail and Reagent B (250 nM of Compound 157S)) in the presence or absence of: a 4-cytokine cocktail (TPO, IL-6, Flt3-L and SCF, 50 ng/mL each), 100 ng/mL of TPO only, and/or Reagent B (250 nM of Compound B157S) were measured. "+" means the indicated reagent was present and "-" means the indicated reagent was absent.
[0018] Fig. 9. TPO is effective within a wide concentration range. Platelet yields
(% of maximum) in response to TPO (0.1 to 10,000 ng/mL) were measured. [0019] Fig. 10. The effective concentration of Reagent B is different for the various compounds suitable for use as Reagent B, and should be separately determined. Platelet yields (folds over vehicle control) in response to the presence of 0.1 to 10,000 nM of Reagent B (Compound B157S, B88, or B1 ) were measured.
[0020] Fig. 1 1 . Matrix metalloproteinase inhibitor is not required for producing functional platelets. Residual human platelets (number count) from ex vivo culture derived, donor derived, and damaged whole blood (treated with carbonyl cyanide 3- chlorophenylhydrazone) in mouse circulation were measured 4 hours after infusion.
Residual human platelets were detected for culture-derived and donor derived platelets and not for damaged platelets.
[0021] Fig. 12. The presence of MMP inhibitor in the second phase improved platelet yield. The yield of platelets (number count per initiating cell) were measured in culture containing vehicle, 25 μΜ of GM6001 or 10 μΜ of TAPI-1 ; an approximately 30% fold increase was observed.
[0022] Fig. 13. The optimal duration for the first culture phase is 14 days. Effect of the duration of the first culture phase on exPLT yields on day 18 (number count per initiating HSC cell) (top left), exMK yield on day 14 (number count per initiating HSC cell) (top right) and % exMK on day 14 (bottom left) were measured.
[0023] Fig. 14. Recovering of the megakaryocytes by pelleting and resuspending is superior to isolation. Platelet yields (number count per exMK on day12) in the presence or absence of: Reagent B, pelleted and resuspension of megakaryocytes after the first phase, and isolation of megakaryocytes after the first phase were measured. "+" means the indicated reagent/protocol was present and "-" means the indicated reagent/protocol was absent.
[0024] Fig 15. Post-isolation platelets are phenotypically the same as pre- isolation platelets and whole blood platelets. Flow cytometry characterization of platelets from: pre-isolation (top row), post-isolation (middle row), and whole blood (bottom row) were conducted. Platelets were distinguished from nucleated cells by size (FSC) and granularity (SSC) and were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0025] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. [0026] "Alkoxy" as used herein refers the radical -O-alkyl, wherein the alkyi is as defined herein. Cxalkoxy and Cx.Yalkoxy as used herein describe alkoxy groups where X and Y indicate the number of carbon atoms in the alkyi chain. Representative examples of d-!oalkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert- butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy and decyloxy. The alkyi portion of the alkoxy may be optionally substituted, and the substituents include those described for the alkyi group below.
[0027] "Alkyi" as used herein refers to a fully saturated branched or unbranched hydrocarbon chain having up to 10 carbon atoms. Cx alkyi and CX-Y alkyi as used herein describe alkyi groups where X and Y indicate the number of carbon atoms in the alkyi chain. For example, CH0 alkyi refers to an alkyi radical as defined above containing one to ten carbon atoms. CH0 alkyi includes, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2- dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like. Alkyi represented along with another radical like arylalkyl, heteroarylalkyl, alkoxyalkyl, alkoxyalkyl, alkylamino, where the alkyi portion shall have the same meaning as described for alkyi and is bonded to the other radical. For example, (C6-10)aryl(C1-3)alkyl includes, benzyl, phenylethyl, 1 -phenylethyl, 3-phenylpropyl, 2-thienylmethyl, 2-pyridinylmethyl and the like.
[0028] Unless stated otherwise specifically in the specification, an alkyi group may be unsubstituted or substituted by one or more substituents to the extent that such substitution makes sense chemically. Typical substituents include, but are not limited to halo, hydroxyl, alkoxy, cyano, amino, acyl, aryl, arylalkyl, and cycloalkyl, or an heteroforms of one of these groups, and each of which can be substituted by the substituents that are appropriate for the particular group.
[0029] "Alkenyl" as used herein refers to a straight or branched, hydrocarbon chain having up to 10 carbon atoms and at least one carbon-carbon double bond.
Cxalkenyl and Cx.Yalkenyl as used herein describe alkenyl groups where X and Y indicate the number of carbon atoms in the alkenyl chain. Examples of C2-7alkenyl include vinyl, allyl, isopropenyl, pentenyl, hexenyl, heptenyl, 1 -propenyl, 2-butenyl, 2-methyl-2-butenyl, and the like. The alkenyl may be optionally substituted, and the substituents include those described for the alkyi group descried herein.
[0030] "Alkynyl" as used herein refers to a straight or branched, hydrocarbon chain having up to 10 carbon atoms and at least one carbon-carbon triple bond. Cxalkenyl and Cx.Yalkenyl as used herein describe alkynyl groups, where X and Y indicate the number of carbon atoms in the alkynyl chain. For example, C2-7alkenyl include, but are not limited to, ethynyl, propargyl, 3-methyl-1 -pentynyl, 2-heptynyl and the like. An alkynyl may be optionally substituted, and the substituents include those described for the alkyl group described herein.
[0031] "Alkylene" as used herein refers to a divalent alkyl group defined herein.
Examples of C^oalkylene includes, but are not limited to, methylene, ethylene, n-propylene, /so-propylene, n-butylene, sec-butylene, /so-butylene, ferf-butylene, n-pentylene, isopentylene, neopentylene, n-hexylene, 3-methylhexylene, 2,2- dimethylpentylene, 2,3- dimethylpentylene, n-heptylene, n-octylene, n-nonylene and n-decylene. An alkylene group may be optionally substituted, and the substituents include those described for the alkyl group described herein.
[0032] "Amino" as used herein refers to the radical -NH2. When an amino is described as "substituted" or "optionally substituted", the term includes NR'R" wherein each R' and R" is independently H, or is an alkyl, alkenyl, alkynyl, acyl, aryl, aryl, cycloalkyl, arylalkyl cycloalkylalkyl group or a heteroform of one of these groups, and each of the alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl or groups or heteroforms of one of these groups, each of which is optionally substituted with the substituents described herein as suitable for the corresponding group.
[0033] The term "amino" also includes forms wherein R' and R" are linked together to form a 3-8 membered ring which may be saturated, unsaturated or aromatic and which contains 1 -3 heteroatoms independently selected from N, O and S as ring members, and which is optionally substituted with the substituents described as suitable for alkyl groups or, if NR'R" is an aromatic group, it is optionally substituted with the substituents described as typical for heteroaryl groups.
[0034] Unless indicated otherwise, the compounds of the invention containing amino moieties may include protected derivatives thereof. Suitable protecting groups for amino moieties include acetyl, tert-butoxycarbonyl, benzyloxycarbonyl, and the like.
[0035] "Alkylamino" as used herein refers to the radical -NRaRb, where at least one of, or both, Ra and Rb are an alkyl group as described herein. An d^alkylamino group includes -NHC1_4alkyl and -N(C1_4alkyl)2; e.g., -NHCH3, -N(CH3)2, -NH(CH2CH3), - N(CH2CH3)2, and the like.
[0036] "Aryl" as used herein refers to a 6-14 membered monocyclic or polycyclic aromatic ring assembly where all the ring atoms are carbon atoms. Typically, the aryl is a 6 membered monocyclic, a 10-12 membered bicyclic or a 14-membered fused tricyclic aromatic ring system. Cxaryl and CX-Yaryl as used herein describe an aryl group where X and Y indicate the number of carbon atoms in the ring system. C6.14aryls include, but are not limited to, phenyl, biphenyl, naphthyl, azulenyl, and anthracenyl. [0037] An aryl may be unsubstituted or substituted by 1 -5 (such as one, or two, or three) substituents independently selected from the group consisting of hydroxy, thiol, cyano, nitro, C1 -4alkyl, C1 -4alkenyl, C1 -4alkynyl, C1 -4alkoxy, thioC1 -4alkyl, C1 - 4alkenyloxy, C1 -4alkynyloxy, halogen, C1 -4alkylcarbonyl, carboxy, C1 -4alkoxycarbonyl, amino, C1 -4alkylamino, di-C1 -4alkylamino, C1 -4alkylaminocarbonyl, di-C1 - 4alkylaminocarbonyl, C1 -4alkylcarbonylamino, C1 -4alkylcarbonyl(C1 -4alkyl)amino, sulfonyl, sulfamoyl, alkylsulfamoyl, C1 -4alkylaminosulfonyl, aryl, heteroaryl, cycloalkyl and heterocycloalkyl, wherein each of the afore-mentioned substitutents may be further substituted by one or more substituents independently selected from halogen, alkyl, hydroxyl or C1 -4alkoxy groups.
[0038] When an "aryl" is represented along with another radical like "arylalkyl",
"aryloxyalkyl", "aryloxycarbonyl", "aryloxy-carbonylalkyl", the aryl portion shall have the same meaning as described in the above-mentioned definition of "aryl".
[0039] "Aryloxy" as used herein, refers to the radical -O-aryl, wherein aryl is as defined herein.
[0040] "Carbonyl", as used herein, refers to a divalent radical -C(=0)-. It is noted that the term "carbonyl" when referring to a monovalent substituent can alternatively refer to a substituted carbonyl or acyl group, -C(=0)Ra, where Ra is hydrogen or a non- hydrogen substituent on the carbonyl carbon, forming different carbonyl-containing groups including acids, acid halides, aldehydes, amides, esters, and ketones.
[0041] "Cycloalkyl", as used herein, means a radical comprising a non-aromatic, saturated or partially unsaturated, monocyclic, bicyclic, tricyclic, fused, bridged or spiro polycyclic hydrocarbon ring system of 3-20 carbon atoms. Cxcycloalkyl and CX-Ycycloalkyl are typically used where X and Y indicate the number of carbon atoms in the ring assembly. For example, C3-6cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,5-cyclohexadienyl.
Exemplary monocyclic hydrocarbon groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl and the like.
[0042] A cycloalkyl may be unsubstituted or substituted by one, or two, or three, or more substituents independently selected from the group consisting of hydroxyl, thiol, cyano, nitro, oxo, alkylimino, d-4alkyl, C1-4alkenyl, d-4alkynyl, d-4alkoxy, C1-4thioalkyl, d- 4alkenyloxy, C1-4alkynyloxy, halogen, d-4alkylcarbonyl, carboxy, d-4alkoxycarbonyl, amino, d-4alkylamino, di-d-4alkylamino, d-4alkylaminocarbonyl, di-d- 4alkylaminocarbonyl, d-4alkylcarbonylamino, d^alkylcarbony d^alky amino, sulfonyl, sulfamoyl, alkylsulfamoyl, d-4alkylaminosulfonyl where each of the afore-mentioned hydrocarbon groups (e.g., alkyl, alkenyl, alkynyl, alkoxy residues) may be further substituted by one or more residues independently selected at each occurrence from halogen, hydroxyl or d-4alkoxy groups.
[0043] "Cycloalkylene", as used herein, refers to a divalent radical comprising a cycloalkyl ring assembly as defined herein.
[0044] "Cycloalkoxy", as used herein, refers to -O-cycloalkyl, wherein the cycloalkyl is defined herein. Representative examples of C3.12cycloalklyoxy include, but are not limited to, monocyclic groups such as cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclopentenyloxy, cyclohexyloxy and cyclohexenyloxy and the like. Exemplary bicyclic hydrocarbon groups include bornyloxy, indyloxy, hexahydroindyloxy, tetrahydronaphthyloxy, decahydronaphthyloxy, bicyclo[2.1 .1 ]hexyloxy, bicyclo[2.2.1 ]heptyloxy,
bicyclo[2.2.1 ]heptenyloxy, 6,6-dimethylbicyclo[3.1 .1]heptyloxy, 2,6,6- trimethylbicyclo[3.1 .1 ]heptyloxy, bicyclo[2.2.2]octyloxy and the like. Exemplary tricyclic hydrocarbon groups include, for example, adamantyloxy.
[0045] "Cyano", as used herein, refers to the radical -CN.
[0046] "Disease" specifically includes any unhealthy condition of an animal or part thereof and includes an unhealthy condition that may be caused by, or incident to, medical or veterinary therapy applied to that animal, i.e., the "side effects" of such therapy.
[0047] "EC50", refers to the molar concentration of an inhibitor that produces 50% efficacy.
[0048] "Halo" or "halogen" as used herein refers to fluoro, chloro, bromo, and iodo.
[0049] "Haloalkyl", or halo-substituted-alkyl" as used herein, refers to an alkyl as defined herein, which is substituted by one or more halo atoms defined herein. The haloalkyl can be mono-haloalkyl, dihaloalkyl or polyhaloalkyl including perhaloalkyl. A monohaloalkyi can have one iodo, bromo, chloro or fluoro within the alkyl group. Dihaloalky and polyhaloalkyl groups can have two or more of the same halo atoms or a combination of different halo groups within the alkyl. Cxhaloalkyl and CX-Yhaloalkyl are typically used where X and Y indicate the number of carbon atoms in the alkyl chain. Non-limiting examples of Ci_4haloalkyl include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl,
dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl. A d- 4perhaloalkyl group refers to a C _4alkyl group having all hydrogen atoms replaced with halo atoms.
[0050] "Heteroaryl", as used herein, refers to a 5-14 membered ring assembly
(e.g., a 5-7 membered monocycle, an 8-10 membered bicycle, or a 13-14 membered tricyclic ring system) having 1 to 8 heteroatoms selected from N, O and S as ring atoms and the remaining ring atoms are carbon atoms. The nitrogen atoms of such heteroaryl rings can be optionally quaternerized and the sulfur atoms of such heteroaryl rings can be optionally oxidized. Cxheteroaryl and CX-Yheteroaryl as used herein describe heteroaryls where X and Y indicate the number of ring atoms in the heteroaryl ring. Typical C5.
yheteroaryl groups include thienyl, furanyl, imidazolyl, pyrazolyl, pyrrolyl, pyrrolinyl, thiazolyl, 1 ,3,4-thiadiazolyl, isothiazolyl, oxazolyl, oxadiazole isoxazolyl, triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrazinyl, pyrazinyl, pyrimidinyl, and the like. Bicyclic or tricyclic C8- 14heteroaryls include, but are not limited to, those derived from benzo[b]furan,
benzo[b]thiophene, benzimidazole, imidazo[4,5-c]pyridine, quinazoline, thieno[2,3- c]pyridine, thieno[3,2-b]pyridine, thieno[2,3-b]pyridine, quinazolinyle, pteridinyl, indolizine, imidazo[1 ,2a]pyridine, quinoline, quinolinyl, isoquinoline, phthalazine, quinoxaline, naphthyridine, naphthyridinyl, quinolizine, indolyl, indole, isoindole, indazole, indoline, benzoxazole, benzopyrazole, benzothiazole, imidazo[1 ,5-a]pyridine, pyrazolo[1 ,5-a]pyridine, imidazo[1 ,2-a]pyrimidine, imidazo[1 ,2-c]pyrimidine, imidazo[1 ,5-a]pyrimidine, imidazo[1 ,5- c]pyrimidine, pyrrolo[2,3-b]pyridine, pyrrolo[2,3-c]pyridine, pyrrolo[3,2-c]pyridine, pyrrolo[3,2- b]pyridine, pyrrolo[2,3-d]pyrimidine, pyrrolo[3,2-d]pyrimidine, pyrrolo[2,3-b]pyrazine, pyrazolo[1 ,5-a]pyridine, pyrrolo[1 ,2-b]pyridazine, pyrrolo[1 ,2-c]pyrimidine, pyrrolo[1 ,2- a]pyrimidine, pyrrolo[1 ,2-a]pyrazine, triazo[1 ,5-a]pyridine, pteridine, purine, purinyl, carbazole, acridine, phenazine, phenothiazene, phenoxazine, 1 ,2-dihydropyrrolo[3,2,1 -?/]indole, indolizine, pyrido[1 ,2-a]indole and 2(1 H)-pyridinone.
[0051] A heteroaryl may be unsubstituted or substituted with one or more substituents independently selected from hydroxyl, thiol, cyano, nitro, d^alkyl, d^alkenyl, C _4alkynyl, d-4alkoxy, thioC -4alkyl, d^alkenyloxy, d-4alkynyloxy, halogen, d- 4alkylcarbonyl, carboxy, d-4alkoxycarbonyl, amino, d-4alkylamino, di-d-4alkylamino, d- 4alkylaminocarbonyl, di-d-4alkylaminocarbonyl, d-4alkylcarbonylamino, d- 4alkylcarbonyl(d-4alkyl)amino, sulfonyl, sulfamoyl, alkylsulfamoyl, d-4alkylaminosulfonyl where each of the afore-mentioned hydrocarbon groups (e.g., alkyl, alkenyl, alkynyl, alkoxy residues) may be further substituted by one or more residues independently selected at each occurrence from halogen, hydroxyl or d-4alkoxy groups.
[0052] When a heteroaryl is represented along with another radical like
"heteroaryloxy", "heteroaryloxyalkyl", "heteroaryloxycarbonyl", the heteroaryl portion shall have the same meaning as described in the above-mentioned definition of "heteroaryl".
[0053] "Heteroaryloxy", as used herein, refers to an -O-heteroaryl group, wherein the heteroaryl is as defined in this Application. [0054] "Heteroatom", as used herein, refers to an atom that is not a carbon atom.
Particular examples of heteroatoms include, but are not limited to nitrogen, oxygen, and sulfur.
[0055] "Heterocycloalkyl", as used herein, refers to a 4-20 membered, non- aromatic, saturated or partially unsaturated, monocyclic or polycyclic ring system, comprising 1 -8 heteroatoms as ring atoms and that the remaining ring atoms are carbon atoms. The heteroatoms are selected from N, O, and S, preferably O and N. The nitrogen atoms of the heterocycloalkyl can be optionally quaternerized and the sulfur atoms of the heterocycloalkyl can be optionally oxidized. The heterocycloalkyl can include fused or bridged rings as well as spirocyclic rings. Cxheterocycloalkyl and CX-Yheterocycloalkyl are typically used where X and Y indicate the number of ring atoms in the ring. Typically, the .heterocycloalkyl is 4-8-membered monocyclic ring containing 1 to 3 heteroatoms, a 7 to 12- membered bicyclic ring system containing 1 -5 heteroatoms, or a 10-15-membered tricyclic ring system containing 1 to 7 heteroatoms. Examples of C4-6heterocycloalkyl include azetidinyl, tetrahydrofuran (THF), dihydrofuran, 1 , 4-dioxane, morpholine, 1 ,4-dithiane, piperazine, piperidine, 1 ,3-dioxolane, imidazolidine, imidazoline, pyrazolidinyl, pyrroline, pyrrolidine, tetrahydropyran, dihydropyran, oxathiolane, dithiolane, 1 ,3-dioxane, 1 ,3- dithiane, oxathiane, thiomorpholine, and the like
[0056] A heterocycloalkyl may be unsubstituted or substituted with 1 -5 substituents (such as one, or two, or three) each independently selected from hydroxyl, thiol, cyano, nitro, oxo, alkylimino, C1-4alkyl, C1-4alkenyl, C1-4alkynyl, d-4alkoxy, d- 4thioalkyl, d-4alkenyloxy, d-4alkynyloxy, halogen, d-4alkylcarbonyl, carboxy, d- 4alkoxycarbonyl, amino, d-4alkylamino, di- d-4alkylamino, d-4alkylaminocarbonyl, di-d- 4alkylaminocarbonyl, d-4alkylcarbonylamino, d-4alkylcarbonyl(d-4alkyl)amino, sulfonyl, sulfamoyl, alkylsulfamoyl, d-4alkylaminosulfonyl where each of the afore-mentioned hydrocarbon groups (e.g., alkyl, alkenyl, alkynyl, alkoxy residues) may be further substituted by one or more residues independently selected at each occurrence from halogen, hydroxyl or d-4alkoxy groups.
[0057] When a heterocycloalkyl forms part of other groups like "heterocycloalkyl- alkyl", "heterocycloalkoxy", "heterocycloalkyl-aryl", the heteroaryl portion shall have the same meaning as described in the above-mentioned definition of "heteroaryl"
[0058] "Hydroxy", as used herein, refers to the radical -OH.
[0059] "Hydroxyalkyl" or "hydroxyl-substituted alkyl" as used herein, refers to an alkyl as defined herein, having one or more of the available hydrogen of the alkyl replaced by a hydroxyl group. For example, a hydroxyd_4alkyl includes, but are not limited to, - CH2CH2OH, -CH(OH)CH2CH2OH, - CH(OH)CH2CH(OH)CH3. [0060] "Stereoisomers", as used herein, refers to compounds having identical molecular formulae but differ in the arrangement of their atoms in space are termed
"stereoisomers." Stereoisomers that are nonsuperimposable mirror images are termed "enantiomers" or sometimes "optical isomers." Stereoisomers that are not mirror images of one another are termed "diastereomers". Conventions for stereochemical nomenclature, methods for the determination of stereochemistry and the separation of stereoisomers are well known in the art (e.g., see "Advanced Organic Chemistry", 4th edition, March, Jerry, John Wiley & Sons, New York, 1992).
[0061] A carbon atom bonded to four different substituents (where no two are the same) is termed a "chiral center." A compound with one chiral center has two enantiomeric forms of opposite chirality. A mixture of equal amounts of the two enantiomeric forms is termed a "racemic mixture." A compound that has more than one chiral center has 2n-1 enantiomeric pairs, where n is the number of chiral centers. Compounds with more than one chiral center may exist as either an individual diastereomer or as a mixture of diastereomers, termed a "diastereomeric mixture." When one chiral center is present a stereoisomer may be characterized by the absolute configuration of that chiral center.
Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. Enantiomers are characterized by the absolute configuration of their chiral centers and described by the R- and S-sequencing rules of Cahn, Ingold and Prelog.
[0062] "Nitro", as used herein, refers to the radical -N02.
[0063] "Oxo", as used herein, refers to the divalent radical =0
[0064] "Pharmaceutically acceptable", as used herein, means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use.
[0065] "Radical" as used herein refers to an atom or group of atoms having an open valence for bonding with another atom or groups of atoms.
[0066] "Unsubstituted or substituted" or "optionally substituted" as used herein indicate the substituent bound on the available valance of a named group or radical.
"Unsubstituted" as used herein indicates that the named group or radical will have no further non-hydrogen substituents. "Substituted" or "optionally substituted" as used herein indicates that at least one of the available hydrogen atoms of named group or radical has been (or may be) replaced by a non-hydrogen substituent. The substituents of an
"optionally substituted" or "substituted" group may include, without limitation, one or more substituents independently selected from the group or designated subsets thereof, aldehyde, alkyl, alkylene, alkylidene, amide, amino, aminoalkyl, aryl, bicycloalkyl, bicycloaryl, carbamoyl, carbocyclyl, carboxyl, carbonyl group, cycloalkyl, cycloalkylene, ester, halo, heterobicycloalkyl, heterocycloalkylene, heteroaryl, heterobicycloaryl, heterocycloalkyl, oxo, hydroxy, iminoketone, ketone, nitro, oxaalkyl, and oxoalkyl moieties, each of which may optionally also be substituted or unsubstituted.
[0067] "Sulfonyl", as used herein, means the radical -S(0)2- It is noted that the term "sulfonyl" when referring to a monovalent substituent can alternatively refer to a substituted sulfonyl group, -S(=0)2R, where R is hydrogen or a non-hydrogen substituent on the sulfur atom forming different sulfonyl groups including sulfonic acids, sulfonamides, sulfonate esters, and sulfones.
[0068] "Therapeutically effective amount", as used herein, means that amount which, when administered to an animal for treating a disease, is sufficient to effect such treatment for the disease.
V
[0069] "X " and "x" " are symbols denoting the point of attachment of X, to other part of the molecule.
[0070] Any definition herein may be used in combination with any other definition to describe a composite structural group. By convention, the trailing element of any such definition is that which attaches to the parent moiety. For example, the composite group alkoxyalkyl would represent an alkoxy group attached to the parent molecule through an alkyl group.
[0071] It is noted in regard to all of the definitions provided herein that the definitions should be interpreted as being open ended in the sense that further substituents beyond those specified may be included. Hence, a d alkyl indicates that there is one carbon atom but does not indicate what are the substituents on the carbon atom. Hence, a dalkyl comprises methyl (i.e., -CH3) as well as -CRaRbRc where Ra, Rb, and Rc may each independently be hydrogen or any other substituent where the atom attached to the carbon is not a hydrogen atom. Hence, -CF3, -CH2OH and -CH2CN, for example, are all dalkyls.
[0072] "Hematopoietic stem cells" (HSCs) as used herein refer to immature blood cells having the capacity to self-renew and to differentiate into more mature blood cells comprising granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes, platelets), and monocytes (e.g., monocytes, macrophages). HSCs are interchangeably described as stem cells throughout the specification. It is known in the art that such cells may or may not include CD34+ cells. CD34+ cells are immature cells that express the CD34 cell surface marker. CD34+ cells are believed to include a subpopulation of cells with the stem cell properties defined above. It is well known in the art that HSCs include pluripotent stem cells, multipotent stem cells (e.g., a lymphoid stem cell), and/or stem cells committed to specific hematopoietic lineages. The stem cells committed to specific hematopoietic lineages may be of T cell lineage, B cell lineage, dendritic cell lineage, Langerhans cell lineage and/or lymphoid tissue-specific macrophage cell lineage. In addition, HSCs also refer to long term HSC (LT-HSC) and short term HSC (ST-HSC). ST-HSCs are more active and more proliferative than LT-HSCs. However, LT-HSC have unlimited self-renewal (i.e., they survive throughout adulthood), whereas ST-HSC have limited self-renewal (i.e., they survive for only a limited period of time). Any of these HSCs can be used in any of the methods described herein. Optionally, ST-HSCs are useful because they are highly proliferative and thus, quickly increase the number of HSCs and their progeny. Hematopoietic stem cells are optionally obtained from blood products. A blood product includes a product obtained from the body or an organ of the body containing cells of hematopoietic origin. Such sources include un-fractionated bone marrow, umbilical cord, peripheral blood, liver, thymus, lymph and spleen. All of the aforementioned crude or un-fractionated blood products can be enriched for cells having hematopoietic stem cell characteristics in ways known to those of skill in the art.
[0073] "Treat", "treating" and "treatment" refer to a method of alleviating or abating a disease and/or its attendant symptoms.
[0074] "About" refer to +/- 5 percent of the number indicate.
[0075] "Expansion" in the context of cells refers to increase in the number of a characteristic cell type, or cell types, from an initial cell population of cells, which may or may not be identical. The initial cells used for expansion may not be the same as the cells generated from expansion.
[0076] "Cell population" refers to eukaryotic mammalian, preferably human, cells isolated from biological sources, for example, blood product or tissues and derived from more than one cell. A population of cells is generally inhomogeneous in the sense that the stem cell could be in various developmental stages. As used herein, when apply to stem cells, a population of HSC cells typically means the number of HSC containing in two cord blood units, more typically one cord blood unit.
[0077] "Enriched" when used in the context of cell population refers to a cell population selected based on the presence of one or more markers, for example, CD34+.
[0078] The term "CD34+ cells" refers to cells that express at their surface CD34 marker. CD34+ cells can be detected and counted using for example flow cytometry and fluorescently labeled anti-CD34 antibodies.
[0079] "Enriched in CD34+ cells" means that a cell population has been selected based on the presence of CD34 marker. Accordingly, the percentage of CD34+ cells in the cell population after selection method is higher than the percentage of CD34+ cells in the initial cell population before selecting step based on CD34 markers. For example, CD34+ cells may represent at least 50%, 60%, 70%, 80% or at least 90% of the cells in a cell population enriched in CD34+ cells.
[0080] "Cord blood unit" refers to the blood collected from umbilical cord of a single birth.
[0081] "Megakaryopoiesis" as used herein means the differentiation of hematopoietic stem cells to megakaryocytes, which under current model of hematopoiesis, involves differentiation of the multipotent HSC to the common myeloid progenitor which then differentiates into the megakaryocyte/erythrocyte progenitors, and then into
megakaryocytes.
[0082] "Platelet biogenesis" as used herein means the formation of platelets.
The process, which under current model of hematopoiesis, includes the maturing of megakaryocytes towards the formation of protoplatelet, and the release of platelets
Description of the Preferred Embodiments
[0083] The invention relates to a process for manufacturing platelets from hematopoietic stem cells ex vivo. The process uses known and proprietary agents to manufacture platelets using a two-stage GMP compatible culture. The process can produce platelets in a patient specific manner by using HLA-matched CD34+ cells derived from a CB unit. The process may be adapted for large scale production of clinical relevant number of platelets to meet clinical needs.
[0084] The invention also relates to compositions of ex vivo manufactured platelets (exPLTs). In one embodiment, the exPLTs composition is as obtained by the process of the invention. In another embodiment, these exPLTs compositions are suitable for autologous or allogeneic transplantation. In another embodiment, the cell therapy product is characterized by the presence of a proprietary Reagent B (defined below) used in the production. Reagent B is an antagonist of the aryl hydrocarbon receptor. In still another embodiment, the cell therapy product is HLA-matched to a specific patient.
[0085] The invention further relates to use of the exPLT for treating patient suffering from thrombocytopenia, thereby treating the underlying diseases and conditions that caused the thrombocytopenia. In one particular embodiment, the exPLTs are HLA- matched for treating an alloimmunized thrombocytopenic patients.
Methods for Ex Vivo Manufacturing of Platelets
[0086] Figure 1 shows a schematic of the process of the invention. [0087] The process of the invention is a two-stage process. The process first generates megakaryocytes by directed differentiation of hematopoietic stem cells and progenitor cell (HSPC) and second promotes platelet biogenesis from the megakaryocytes. Each of the stages requires a low molecular weight reagent and a combination of cytokines to achieve the desirable result. In the first stage, the required Reagent A is a known platelet-derived growth factor receptor (PDGFR) antagonist. In the second stage, the required Reagent B is an aryl hydrocarbon receptor (AHR) antagonist. The process may optionally employ a third low molecular weight reagent, which is a matrix metalloproteinase receptor (MMP) antagonist.
[0088] In one embodiment, the invention is related to an ex vivo process for producing a population of ex vivo produced platelets (exPLTs) comprising a Step C, which comprises:
contacting a population of ex vivo produced megakaryocytes (exMKs) with Reagent B and thrombopoietin (TP B is a compound of Formula Ila:
Figure imgf000017_0001
or a pharmaceutically acceptable salt, or stereoisomer thereof; wherein the variables of Formula Ila are recited in paragraph [00138], infra.
[0089] In one variation of the above embodiment, the process of the invention further comprises a process of producing the exMKs from hematopoietic stem cells (HSC), wherein Step A comprising:
contacting a population of HSC with Reagent A and TPO, wherein Reagent A is a compound of Formula I:
Figure imgf000017_0002
or a pharmaceutically acceptable salt, or stereoisomer thereof; wherein the variables of Formula I are recited in paragraph [00124], infra.
[0090] In another variation of the above embodiment and variation, the process further comprising Steps B1 and B2, which comprise:
Step B1 : gathering the quantity of exMKs; and Step B2: re-suspending the quantity of exMKs.
[0091] In a particular embodiment, the ex vivo process for producing a population of ex vivo produced platelets (exPLTs) of the invention comprising the steps of:
A1 ) providing a population of hematopoietic stem cells (HSC);
A) contacting HSC with Reagent A to yield a population of ex vivo produced megakaryocytes (exMKs), and
C) contacting the population of exMKs with ex vivo produced platelets
(exPLTs);
wherein
Regent A is 3-(7-((2,6-dimethylpyridin-3-yl)amino)-1 -methyl-2-oxo-1 ,2- dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N-(3-(trifluoromethyl)phenyl)-4- vinylbenzamide; and
Reagent B is of Formula lib:
Figure imgf000018_0001
or a pharmaceutically acceptable salt, or stereoisomer thereof; wherein the variables of Formula lib are recited in paragraph [00139] infra.
[0092] In one variation of the particular embodiment, the process of the invention further comprises Steps B1 and B2, which comprising:
Step B1 : pelleting the population of exMKs; and
Step B2: re-suspending the population of exMKs.
[0093] The two-stage protocol was the result of continued optimization.
[0094] HSPCs differentiate into MK, which then undergo maturation culminating in formation of proplatelet projections that pinch off into anucleate platelets. This process has been replicated ex vivo with some success; however, protocols with highest platelet yields were unsuitable for clinical use due to scale-up constraints or the presence of serum and feeder cells. Platelet generation under preferable serum-free and feeder-free defined culture conditions requires cytokines to promote progenitor proliferation and/or lineage specification. While thrombopoietin (TPO) alone supports MK differentiation, culture with TPO, stem cell factor, Flt-3 ligand and interleukin-6 balances purity with proliferation to maximize MK number in a suitable culture volume and generate a reported 142 platelets per CB-HSPC. In our laboratory this approach yielded a comparable 281 +/- 37 exPLT per HSPC (N=5). [0095] The exPLT manufacturing process was further optimized into two stages:
1 ) formation of MK from HSPCs and 2) conversion of MK into platelets. We first identified a platelet-derived growth factor receptor (PDGFR) inhibitor, 3-(7-((2,6-dimethylpyridin-3- yl)amino)-1 -methyl-2-oxo-1 ,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N-(3- (trifluoromethyl)phenyl)-4-vinylbenzamide (MK1 ), in a screen for enhanced ex vivo differentiation of HSPCs into MK (Boitano, 2012 PNAS). In CB-HSPC culture, addition of MK1 increased MK yield 4 +/- 2 fold (N=4).
[0096] We next focused on identifying factors to enhance platelet biogenesis from MK, since each MK produces hundreds to thousands less platelets in ex vivo defined culture than in vivo (Boitano, 2010 Science).
[0097] Building on our experience with AHR inhibitors, originally identified to enhance HSC expansion, (Boitano, 2010 Science), we demonstrated that antagonizing the AHR receptor and/or the downstream pathway produces viable platelets, in either a single stage (see, Experiment A) or two stage process (Examples B and C), of about 400 platelets per seated HSC, or about 2 fold increases over background. Our unpublished results show that addition of aryl hydrocarbon receptor (AHR) antagonists to isolated MK increased exPLT yield 17 +/- 6 fold (N=7).
[0098] We recognized that a protocol which includes both PDGFR inhibition and
AHR inhibition would improve platelet yields. In Example D, we demonstrated that a PDGFR inhibition alone contributed a 2.5-fold yield increase, an AHR inhibition or alone contributed a 3-fold yield increase, and combining PDGFR inhibition in a first phase culture with AHR inhibition in a second phase culture provided 7.5-fold yield increase (Figure 2).
[0099] Our optimized process with MK1 at stage 1 and AHR antagonists along with a matrix metalloprotease (MMP) inhibitor and TPO as the sole cytokine at stage 2 is illustrated in Example E. HSC were cultured for a total of 18 days, according to protocol. The platelets yield was about 2321 +/- 556 platelets per seeded HSC (N=4), which represents an 8.4 fold increase when compared to culturing with cytokine alone (281 +/- 37 exPLT per HSPC (N=5)).
[00100] Assay data suggests ex vivo produced platelets (exPLT) are
phenotypically and functionally comparable to platelets derived from fresh whole blood (donor platelets). Flow cytometric characterization shows similar size, granularity, and expression of the platelets markers CD41 a and CD42a (Figure 3) in exPLTs and donor platelets. The ex vivo derived cells contain slightly fewer viable cells by Calcein AM staining (89.1 % vs 99.8%) than donor derived platelets (Figure 4), but contains significantly higher percentage of thiozole orange staining (a measure of RNA content), a characteristic of newly formed platelets (55.8% vs 10%) (Figure 5). This is significant, because newly formed platelets should have a longer in vivo lifespan than donor platelets.
[00101] exPLTs are functional. Using a mouse model, Example F demonstrated that ex-vivo culture-derived platelets and donor plateles persisted in mouse circulation with similar kinetics, whereas damaged platelets were more efficiently cleared from circulation with significantly decreased levels at 4 hr and 24 hr after injection (Figure 6). Example G shows that exPLTs exhibited cell spreading on fibrinogen-coated surface in response to thrombin activation (Figure 7).
[00102] Together, these experimental results demonstrate proof-of-concept for the process of the invention for the generation of exPLTs. The following sections describe the each of the components of the process.
The Starting Hematopoietic Stem Cell Population (Step A1 or A)
[00103] A starting cell population comprising hematopoietic stem cells (HSC) will be selected by the person skilled in the art depending on the envisaged use. Various sources of cells comprising HSC have been described in the art, including bone marrow, peripheral blood, neonatal umbilical cord blood, placenta or other sources such as liver, particularly fetal liver. A population of the HSC may be obtained from commercial sources, e.g., Bioreclamation.
[00104] The HSC population may first be subjected to enrichment or purification steps, including negative and/or positive selection of cells based on specific cellular markers, in order to provide the starting HSC population. Methods for isolating the starting cell population based on specific cellular markers may use fluorescent activated cell sorting (FACS) technology also called flow cytometry or solid or insoluble substrate to which is bound antibodies or ligands that interact with specific cell surface markers. For example, cells may be contacted with a solid substrate (e.g., column of beads, flasks, magnetic particles) containing the antibodies and any unbound cells are removed. When a solid substrate comprising magnetic or paramagnetic beads is used, cells bound to the beads can be readily isolated by a magnetic separator.
[00105] In one embodiment, the starting cell population is enriched in a desirable cell marker phenotype (e.g., CD34+, CD133+, CD90+) or based on efflux of dyes such as rhodamine, Hoechst or aldehyde dehydrogenase activity. In one specific embodiment, the starting cell population is enriched in CD34+ cells. Methods for enriching blood cell population in CD34+ cells include kits commercialized by Miltenyi Biotec (CD34+ direct isolation kit, Miltenyi Biotec, Bergisch, Gladbach, Germany) or by Baxter (Isolex 3000). [00106] In one embodiment, the starting HSC population is derived from neonatal umbilical cord blood cells which have been enriched in CD34+ cells. In one related embodiment, the starting HSC population is consisting essentially of CD34+ cells purified from one or two cord blood units.
[00107] In another embodiment, the starting HSC population is derived from bone marrow blood cells.
[00108] In yet another embodiment, the starting HSC population is derived from human mobilized peripheral blood cells which have been enriched in CD34+ cells. In one related embodiment, said starting HSC population is derived from a single mammalian subject. In another related embodiment, the starting HSC population is blood cells derived from human.
[00109] The starting HSC population may preferably contain at least 50% CD34+ cells, in some embodiments, more than 90% of CD34+ cells, and may comprise between 105 and 109 nucleated cells.
[00110] The starting HSC population may be used directly in the culture or frozen and stored for use at a later date.
Culture Medium
[00111 ] The culturing of the HSC population may be carried out in a basal medium, which is supplemented with the mixtures of cytokines and growth factors described above. A basal medium typically comprises amino acids, carbon sources, vitamins, serum proteins (e.g. albumin), inorganic salts, divalent cations, buffers and any other element suitable for use in culturing of HSC. Examples of such basal medium appropriate for a method of culturing HSC include, without limitation, StemSpan® SFEM - Serum-Free Expansion Medium (StemCell Technologies, Vancouver, Canada), StemSpan® H3000 - Defined Medium (StemCell Technologies, Vancouver, Canada), CellGro® SCGM
(CellGenix, Freiburg Germany), StemPro®-34 SFM (Invitrogen).
[00112] In one embodiment, the culture medium used is StemSpan SFEM
(StemCell Technologies, Cat. #09650) which is supplemented with 1 x antibiotics. In one variation, the medium is further supplemented with a recombinant human cytokines. The recombination of cytokines is different for each of steps of the process, and will be further discussed in the next section. The culture media is preferably prepared fresh the day of use. Cytokines
[00113] Conditions for culturing the starting cell population for HSC expansion and megakaryocytepoesis will vary depending, inter alia, on the starting cell population, the desired final number and ploidy of the megakaryocytes.
[00114] Extensive research using cocktails of hematopoietic growth factors have been employed for ex vivo megakaryocyte growth and expansion. The primary signal for megakaryocyte production is thrombopoietin (TPO). TPO is sufficient, but not absolutely necessary for inducing differentiation of progenitor cells towards a final megakaryocyte phenotype. Bunting, et al. 1997, Blood 90(9) : 3423-3429. Other molecular signals for megakaryocyte differentiation include granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (GCSF), interleukin-3 (IL-3), IL-1 , IL-6, IL- 1 1 , have been shown to be important for normal megakaryopoiesis. Szalai, et al. 2006 Cell Mol Life Sci 63, 2460-76. In addition, Fms-liked tyrosine-protein kinase 3 ligand (Flt3-L). and stem cell factor (SCF) have been shown to play an important role in hematopoiesis. Flt3-L, which in synergy with other growth factors, stimulates the proliferation and differentiation of various blood cell progenitors. Stem cell factor has been shown to increase the survival of HSCs in vitro and contributes to the self-renewal and maintenance of HSCs in vitro. In contrast to TPO, that acts throughout megakaryopoiesis, these cytokines affect megakaryopoiesis during the early stages of megakaryocyte development.
[00115] Flt3-L or FLT-3 Ligand, also referred as FL is a factor that binds to flt3- receptor, has been described by Hannum C, Nature 368 (6472): 643-8. Stem cell factor or Human SCF, also known as c-kit ligand, mast cell growth factor, or Steel factor has been described by Smith, MA et al. 2001 ACTA Haematologica 105, 3:143, 2001 . Human IL-6 or interleukin-6, also known as B-cell stimulatory factor 2 has been described by Kishimoto, Ann. review of Imm. 23:1 2005. TPO or thrombopoietin, also known as megakarayocyte growth factor (MGDF) or c-Mpl ligand has been described (Kaushansky K (2006). N. Engl. J. Med. 354 (19) : 2034-45).
[00116] All of TPO, SCF, Flt-3 and IL-6 are commercially available.
[00117] In the current process, in Step A, the culturing comprises the use of cytokines and growth factors, generally known in the art for hematopoietic stem cell expansion and differentiation. Such cytokines and growth factors include without limitation IL-1 , IL-3, IL-6, IL-1 1 , G-CSF, GM-CSF, SCF, Flt3-L, thrombopoietin (TPO), erythropoeitin, and analogs thereof. As used herein, "analogs" include any structural variants of the cytokines and growth factors having the biological activity as the naturally occurring forms, including without limitation, variants with enhanced or decreased biological activity when compared to the naturally occurring forms or cytokine receptor agonists such as an agonist antibody against the TPO receptor (for example, VB22B sc(Fv)2 as detailed in patent publication WO 2007/145227, and the like). Cytokine and growth factor combinations are chosen to support HSC expansion and to induce HSC and progenitor cells differentiation, and preferably down the megakaryocyte/erythrocyte progenitor pathway.
[00118] In Step A, the contacting of the starting population of HSC with Reagent A occurs in a cell culturing medium. In one embodiment of Step A, the medium is
supplemented with TPO, IL-6, Flt3-L and SCF, each of which is present in a concentration effective for promoting megakaryopoiesis.
[00119] In one variation of the above embodiment of Step A, TPO, IL-6, Flt3-L, and SCF are each present in a concentration between 10 and 100 ng/mL. In another variation, TPO, IL6, Flt3-L, and SCF are each present in a concentration between 25 and 75 ng/mL. In another variation, TPO, IL-6, Flt3-L, and SCF are each present in a concentration of about 50 ng/mL.
[00120] Similarly, in Step C, the contacting of the populations of megakaryocytes with Reagent B occurs in a cell culturing medium supplemented with cytokines. We showed that supplement with TPO, IL-6, Flt3-L, and SCF would support support platelet biogenesis with an acceptable yield (Example B). In one embodiment of Step C, the cell culturing medium is supplement with TPO, IL-6, Flt3-L, and SCF, each of an effective amount to support platelet biogenesis. In one variation of the above embodiment, TPO, IL-6, Flt3-L, and SCF are each at a concentration of about 50 g/mL.
[00121] However, we also observed that TPO alone supports plateless
biogenesis. We compared the effectiveness of TPO alone to the four cytokine combined in supporting platelet biogenesis (Example H), we found that supplement with TPO alone increases platelet production by about 1 .5 fold over the combination of TPO, IL-6, Flt3-L, and SCF.
[00122] The operationally range for TPO was established experimentally (Example
I). TPO was dosed at 12 concentration points between 50pg/mL and 100 ng/mL and cultured to day 18 before platelets yield was assessed. TPO was effective over the entired test range, and could be effective at the concentration between 1 and 500 ng/mL. We determined the optimal working range is about 50 to 100 ng/mL (Figure 9).
[00123] Accordingly, in one embodiment of Step C, the medium is supplement with TPO alone. In one variation, TPO was present in a concentration between 5 ng/mL and 200 ng/mL. In another variation, TPO is present at a concentration of about 100 ng/mL. In still another variation, TPO is present at a concentration of about 50 ng/mL. Reagent A
[00124] Reagent A is an agent capable of promoting megakaryocytepoies. In one embodiment of the process of the invention, Reagent A is a low molecular weight compound of Formula I :
Figure imgf000024_0001
or a pharmaceutically acceptable salt, or stereoisomer thereof; wherein
L is -NHC(O)- or -C(0)NH-;
RT is selected from hydrogen, d^alkyl, phenyl, and C5-6heteroaryl, wherein the d- 4alkyl, phenyl or C5-6heteroaryl is unsubstituted or substituted by 1 to 2 substituents independently selected from halo, cyano, C1 -4alkyl, halo-substituted C1 -4alkyl, C^alkoxyCV 4alkyl, d^alkoxy, C3-6cycloalkyl and C4-6heterocycloalkyl, wherein the C3-6cycloalkyl or C4- eheterocycloalkyl is further unsubstituted or substituted by 1 to 2 substituents independently selected from halo, hydroxy, oxo, d^alkylcarbonyl, or d_4alkyoxy;
R2 is d-4alkyl or C3-6cycloalkyl;
R3 is d-4alkyl or d-4alkenyl;
R4 is hydrogen or C5-6heteroary, unsubstituted or substituted by d_4alkyl; and R5 is halo, d_4alkyl, halo-substituted d-4alkyl, d_4alkoxy-substituted Ci-4alkyl, d- 4alkoxy, and halo-substituted d_4alkoxy.
[00125] In anoth is of Formula la:
Figure imgf000024_0002
whererin
L is -NHC(O)- or -C(0)NH-;
RT is hydrogen, d_4alkyl or C5-6heteroaryl, wherein the alkyl is substituted by oxo- substitutedC4.6heterocycloalkyl, and the C5-6heteroaryl is substituted by two d_4alkyl;
R2 is d-4alkyl or C3.6cycloalkyl;
R3 is d-4alkyl or d_4alkenyl; and
R4 is hydrogen or C5-6heteroary, unsubstituted or substituted by d_4alkyl. [00126] In one variation of the above two embodiments of the process of the invention, is selected from hydrogen, 2.6-dimethylpyridin-3-yl, 1 ,3 dimethyl-1 /-/-pyrazol-5- yl, and 2-oxo-pyrrolidin-1 -yl-propyl.
[00127] In another embodiment of the above embodiments and variation of the process, R2 is methyl or cyclopropyl.
[00128] In another embodiment of the above embodiments and variations of the process, R3 is methyl or vinyl.
[00129] In still another embodiment of the above embodiments and variations of the process, R4 is hydrogen or 4-methyl-1 - -imidazol-1 -yl.
[00130] In one embodiment, Reagent A is selected from the following compounds:
3-(7-((2,6-dimethylpyridin-3-yl)amino)-1 -methyl-2-oxo-1 ,2-dihydropyrimido[4,5- d]pyrimidin-3(4H)-yl)-N-(3-(trifluoromethyl)phenyl)-4-vinylbenzamide;
N-(4-methyl-3-(1 -methyl-2-oxo-7-((3-(2-oxopyrrolidin-1 -yl)propyl)amino)-1 ,2- dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)phenyl)-3-(trifluoromethyl)benzamide;
N-(3-(2-amino-8-methyl-7-oxo-7,8-dihydropteridin-6-yl)-4-methylphenyl)-3-(4-methyl- 1 H-imidazol-1 -yl)-5-(trifluoromethyl)benzamide;
N-(3-(7-((1 ,3-dimethyl-1 H-pyrazol-5-yl)amino)-1 -methyl-2-oxo-1 ,2- dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-4-methylphenyl)-3-(trifluoromethyl)benzamide; and
N-(3-(7-amino-1 -methyl-2-oxo-1 ,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-4- methylphenyl)-3-(trifluoromethyl)benzamide.
[00131] In another embodiment, Reagent A is 3-(7-((2,6-dimethylpyridin-3- yl)amino)-1 -methyl-2-oxo-1 ,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N-(3- (trifluoromethyl)phenyl)-4-vinylbenzamide.
[00132] In one embodiment, in Step A, Reagent A is present in present in the culturing medium at a concentration between 50 nM and 5 μΜ. In one variation, in Step A, Reagent A is present in a concentration between 100 nM and 1000 nM.
[00133] In anther variation, in Step A, Reagent A is present in a concentration about 250 nM, 500 nM or 750nM.
[00134] In anther variation, in Step A, Reagent A is present in a concentration about 250 nM. In anther variation, in Step A, Reagent A is present in a concentration about 500 nM.
[00135] Reagent A is capable of antagonizing the activity and/or the expression of the platelet-derived growth factor receptor (PDGFR) and/or a down-stream effector of the PDGFR pathway. The PDGFR inhibitory activity (IC50) of the compounds of Formula I are between 2 to 13 nM and the EC50 concentration of the compounds for promoting megakaryopoiesis is between 10 to 50 nM (Table 1 ). Boitano, et al. has demonstrated that Compound A1 (also referred to as MK1 ) out this class of naphthyridinones induces the selective differentiation of common myeloid progenitors (CMP) to megakaryocytes. Kinase profiling and subsequent functional assays revealed that these compounds act through inhibition of platelet-derived growth factor receptor (PDGFR) signaling in CMPs. Boitano, et al. 2012 PNAS 109(35) : 14019-14023.
[00136] A listing of the compounds of Formula I and their PDGFR inhibitory activity
(IC5o in nM) and megarkaryopoieses potential (EC50 in nM) are summaried in Table I infra,
Reagent B
[00137] Reagent B is an agent capable of promoting platelet biogenesis. In one embodiment of the process of the invention, Reagent B is a low molecular weight compound of Formula II :
Figure imgf000026_0001
or a pharmaceutically acceptable salt, or stereoisomer thereof; wherein :
Gi is selected from N and CR3;
G2, G3 and G4 are independently selected from CH and N ; with the proviso that at least 1 of G3 and G4 is N ; with the proviso that Gi and G2 are not both N;
L is selected from -NR5A(CH2)2-3- (0-3 herein means 0, 1 , 2 or 3), - NR5ACH(C(0)OCH3)CH2-, -N R5A(CH2)2NR5B-, -NR5A(CH2)2S- -NR5ACH2CH(CH3)CH2-, - NR5ACH2CH(OH)- and -NR5ACH(CH3)CH2-; wherein R5A and R5B are independently selected from hydrogen and d^alkyl;
RT is selected from thiophenyl, furanyl, benzoimidazolyl, isoquinolinyl,
imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyrazolyl, pyridinyl, imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, pyrrolyl and thiazolyl; wherein said thiophenyl, furanyl,
benzoimidazolyl, isoquinolinyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyrazolyl, pyridinyl, imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, pyrrolyl and thiazolyl of can be optionally substituted by 1 to 3 radicals independently selected from cyano, hydroxy, d- 4alkyl, C1 -4alkoxy, halo, halo-substituted-C1 -4alkyl, halo-substituted-C1 -4alkoxy, hydroxy, amino, -C(0) R8A, -S(O)0-2R8a, -C(0)OR8A and -C(0)N R8AR8B; wherein R8A and R8B are independently selected from hydrogen and d-4alkyl; with the proviso that and R3 are not both hydrogen; R2 is selected from -S(0)2NR6aR6b, -NR6aC(0)R6b, -NR6aC(0)NR6bR6c, phenyl, pyrrolopyridinyl, indolyl, thiophenyl, pyridinyl, triazolyl, 2-oxoimidazolidinyl, pyrazolyl, 2-oxo- 2,3-dihydro-benzoimidazolyl, indazolyl, imidazolyl, benzo[d]imidazolyl, and piperidinyl, wherein
R6a, R6b and R6c are independently selected from hydrogen and d^alkyl; the phenyl, pyrrolopyridinyl, indolyl, thiophenyl, pyridinyl, triazolyl, 2- oxoimidazolidinyl, pyrazolyl, 2-oxo-2,3-dihydro-benzoimidazolyl, indazolyl, imidazolyl, benzo[d]imidazolyl, or piperidinyl of R2 is optionally substituted with 1 to 3 radicals independently selected from hydroxy, halo, methyl, methoxy, amino, - 0(CH2)nNR7aR7b, -S(0)2NR7aR7b, -OS(0)2NR7aR7b and -NR7aS(0)2R7b; wherein R7a and R7b are independently selected from hydrogen and C1 -4alkyl;
R3 is selected from hydrogen, d^alkyl and biphenyl; and
R4 is selected from Ci-i0alkyl, C2.6alkenyl,, cyclohexyl, cyclopropyl,
(oxopyrrolidinyl)ethyl, oxetanyl, benzhydryl, tetrahydropyranyl, tetrahydropyranyl, phenyl, tetrahydrofuranyl, and benzyl; wherein said C1 -10alkyl, C2.6alkenyl„ cyclohexyl, cyclopropyl, (oxopyrrolidinyl)ethyl, oxetanyl, benzhydryl, tetrahydropyranyl, tetrahydropyranyl, phenyl, tetrahydrofuranyl, or benzyl of R4 can be optionally substituted with 1 to 3 radicals independently selected from hydroxy, d^alkyl and halo-substituted-d^alkyl; or the N-oxide derivatives, prodrug derivatives, protected derivatives, individual isomers and mixture of isomers thereof; or the salts (preferably the pharmaceutically acceptable salts) and solvates (e.g. hydrates) of such compounds.
[00138] In one embodiment of the process of the invention, Reagent B is a compound of Formula Ila:
Figure imgf000027_0001
or a pharmaceutically acceptable salt, or stereoisomer thereof; wherein
L is selected from -NR5a(CH2)2_3-, -NR5a(CH2)2N R5B- -NR5a(CH2)2S- -
NR5ACH2CH(OH)- and -NR5ACH(CH3)CH2-; wherein R5A and R5B are independently selected from hydrogen and d_4alkyl;
RT is selected thiophenyl, furanyl, benzoimidazolyl, isoquinolinyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyrazolyl, pyridinyl, imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, pyrrolyl and thiazolyl; wherein
said thiophenyl, furanyl, benzoimidazolyl, isoquinolinyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyrazolyl, pyridinyl, imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, pyrrolyl and thiazolyl of can be optionally substituted by 1 to 3 radicals independently selected from halo, cyano, C1-4alkyl, halo-substituted-d- 4alkyl, C1-4alkoxy,-S(O)0-2R8a, and -C(0)OR8a, wherein R8a is selected from hydrogen and d^alkyl;
R2 is selected from -S(0)2NR6aR6b, -NR6aC(0)NR6bR6c, phenyl, pyrrolopyridinyl, indolyl, thiophenyl, pyridinyl, I triazolyl, 2-oxoimidazolidinyl, pyrazolyl, and indazolyl; wherein
R6a, R6b and R6c are independently selected from hydrogen and d^alkyl; and said phenyl, pyrrolopyridinyl, indolyl, thiophenyl, pyridinyl, triazolyl, oxoimidazolidinyl, pyrazolyl, and indazolyl of R2 is optionally substituted with 1 to 3 radicals independently selected from hydroxy, halo, methyl, methoxy, amino, -
0(CH2)nNR7aR7b, -OS(0)2NR7aR7b and -NR7aS(0)2R7b; wherein R7a and R7b are independently selected from hydrogen and d^alkyl;
R3 is selected from hydrogen, d^alkyl and biphenyl; and
R4 is selected from d-10alkyl, d_4alkenyl, oxetanyl, tetrahydrofuranyl, cyclohexyl, (oxopyrrolidinyl)ethyl, tetrahydropyranyl, phenyl, and benzyl, wherein said d.10alkyl, d- 4alkenyl, oxetanyl, tetrahydrofuranyl, cyclohexyl, (oxopyrrolidinyl)ethyl, tetrahydropyranyl, phenyl, and benzyl of R4 can be optionally substituted with 1 to 3 radicals independently selected from hydroxy, d_4alkyl and halo-substituted-d-4alkyl.
[00139] In still another embodiment of the process of the invention, Reagenat B is a compound of Formula lib:
Figure imgf000028_0001
L is selected from -NH(CH2)2_3- -NH(CH2)2S- -NHCH(CH3)CH2- and - NHCH2CH(OH)-;
RT is selected from thiophenyl, benzoimidazolyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyridinyl, imidazolyl, and pyridazinyl; each of which is unsubstituted or substituted by 1 to 2 radicals independently selected from halo, cyano, d_4alkyl, halo- substituted-d-4alkyl, d_4alkoxy, and -S(0)o-2Rsa, wherein R8a is d_4alkyl;
R2 is selected from phenyl, indolyl and pyrrolopyridinyl, each of which is
unsubstituted or substituted with 1 to 2 radicals independently selected from halo, hydroxy, methyl, methoxy, -0(CH2)nNR7aR7b, -OS(0)2NR7aR7b, and -NR7aS(0)2R7b; wherein R7a and R7b are independently selected from hydrogen and d_4alkyl; and
R4 is selected from d-10alkyl, d_4alkenyl, oxetanyl, tetrahydrofuranyl, wherein the d-10alkyl or d_4alkenyl is unsubstituted or substituted with hydroxyl. [00140] In one variation of the compounds of Formula II, lla or lib, L is selected from -NH(CH2)2- -NH(CH2)2S- and -NHCH2CH(OH)-. In another variation, L
is -NH(CH2)2.
[00141 ] In one variation of any one of the above embodiments and variations, is selected from 1 H-imidazol-1 -yl, thiophen-2-yl, thiophen-3-yl, pyridin-3-yl, pyridin-4-yl, pyrimidin-5-yl, pyridazin-4-yl, 1 H-benzo[d]imidazol-1 -yl, 3H-imidazo[4,5-b]pyridin-3-yl, and benzo[b]thiophen-3-yl, each of which is unsubstituted or substituted by 1 to 2 radicals independently selected from fluoro, chloro, cyano, methyl, trifluoromethyl, methoxy, and - S(O)0-2R8a; wherein R8a is selected from methyl and ethyl.
[00142] In another variation, R^ is pyridin-3-yl or benzo[b]thiophen-3-yl. In still another variation, R^ is pyridin-3-yl. In yet another variation, R^ is benzo[b]thiophen-3-yl.
[00143] In one variation of any one of the above embodiments and variations, R2 is selected from phenyl, 1 H-indol-3-yl and 1 H-pyrropyridin-3-yl, each of which is
unsubstituted or substituted substituted with 1 to 2 radicals independently selected from halo, hydroxy, methyl, methoxy, dimethyaminoethoxy, and amino-sulfonyloxy, and - NR7aS(0)2R7b; wherein R7a and R7b are independently selected from hydrogen and d-4alkyl.
[00144] In another variation, R2 is phenyl or 1 H-indol-3-yl, each unsubstituted or substituted with 1 to 2 radicals independently selected from halo, hydroxyl, amino, methyl, methoxy, diethylaminoethoxy, methylsulfonylamino, and aminosulfonyloxy.
[00145] In still another variation, R2 is phenyl, unsubstituted or substituted with 1 to
2 radicals independently selected from fluoro, chloro, hydroxyl, amino, methyl, methoxy, methylsulfonylamino, and aminosulfonyloxy. In yet still another variation, R2 is phenyl substituted with hydroxyl.
[00146] In yet another further variation, R2 is 1 H-indol-3-yl, unsubstituted or substituted with 1 to 2 radicals independently selected from fluoro, chloro, hydroxy, methyl, methoxy, and diethylaminoethoxy.
[00147] In one variation of any one of the above embodiments and variations of
Compound B, R4 is selected from isopropyl, methyl, ethyl, prop-1 -en-2-yl, isobutyl, sec- butyl, (S)-sec-butyl, (R)-sec-butyl, 1 -hydroxypropan-2-yl, (S)-1 -hydroxypropan-2-yl, (R)-1 - hydroxypropan-2-yl, nonan-2-yl, oxetan-3-yl, oxetan-2-yl, and tetrahydrofuran-3-yl.
[00148] In another variation, R4 is selected from isopropyl, methyl, ethyl, prop-1 - en-2-yl, isobutyl, sec-butyl, 1 -hydroxypropan-2-yl, nonan-2-yl. In still another variation, R4 is (S)-1 -hydroxypropan-2-yl or -hydroxypropan-2-yl. In yet another variation, R4 is isopropyl.
[00149] In a preferred embodiment, Compound B is of Formula lie:
Figure imgf000030_0001
in which:
R2 is selected from 1 H-indol-3-yl and phenyl, each unsubstituted or substituted with from halo, hydroxyl, amino, methyl, methoxy, diethylaminoethoxy, methylsulfonylamino, and aminosulfonyloxy.; and
R4 is selected from isopropyl, methyl, ethyl, prop-1 -en-2-yl, isobutyl, sec-butyl, (S)- sec-butyl, (R)-sec-butyl, 1 -hydroxypropan-2-yl, (S)-1 -hydroxypropan-2-yl, (R)-1 - hydroxypropan-2-yl, nonan-2-yl, oxetan-3-yl, and tetrahydrofuran-3-yl..
[00150] In one variation, R2 is selected from 1 H-indol-3-yl, unsubstituted or substituted with chloro, fluoro, hydroxyl, methyl, methoxy, and diethylaminoethoxy, methylsulfonylamino, and aminosulfonyloxy
[00151] In another variation, R4 is selected from isopropyl, sec-butyl, (S)-sec-butyl,
(R)-sec-butyl, nonan-2-yl, oxetan-3-yl, and tetrahydrofuran-3-yl. In another variation, R4 is isopropyl. In another variation, R4 is oxetan-3-yl. In still another variation, R4 is sec-butyl.
[00152] In another preferred embodiment, Reagenat B is a compound of formula
1 d:
Figure imgf000030_0002
in which: R2 is selected from: 1 H-pyrrolo[2,3-b]pyridin-3-yl; 1 H-indol-3-yl optionally substituted with 1 to 2 radicals independently selected from halo, methyl and methoxy; and phenyl optionally substituted with 1 to 2 radicals independently selected from methyl, halo and hydroxy; R4 is selected from isopropyl, sec-butyl, 1 -hydroxypropan-2-yl, prop-1 -en-2-yl, benzhydryl, nonan-2-yl, oxetan-3-yl and tetrahydrofuran-3-yl; and Ra, Rb and Rc are independently selected from hydrogen, cyano, methyl, halo, -S02CH3 and trifluoromethyl.
[00153] In one embodiment, Reagent B is selected from the group of compounds listed in Table II. [00154] In one embodiment, Reagent B is selected from the list of compounds below:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)phenol; N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- amine;
N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6- amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin-6-amine;
2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1 - ol;
N-(2-(1 H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; N-(2-(1 H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine; and
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(2-methyl-1 H-indol-3-yl)ethyl)-9H-purin- 6-amine.
[00155] In another embodiment, Regent B is 4-(2-(2-(benzo[b]thiophen-3-yl)-9- isopropyl-9H-purin-6-ylamino)ethyl)phenol.
[00156] In one embodiment of the process of the invention, in Step C, the contacting of the megakaryocytes occurs in a cell culture medium wherein Reagent B is present in a concentration effective for promoting platelet biogenesis. To determine the optimal concentration for Reagent B, a dose response experiment (Example J) was conducted. It was determined that the effective range for Compound B157S is between 1 to 5000 nM, and for Compound B1 , is between 50 to 5000 nM (Figure 10).
[00157] Accordingly, in one embodiment of Step C, Reagent B is present in the medium at a concentration between 1 nM and 5 μΜ. In one variation, Reagent B is present in a concentration between 100 nM and 5000 nM. In another variation, Reagent B is present in a concentration between about 250 nM and about 750 nM.
[00158] In another embodiment of Step C, Reagent B is present in a concentration of about 250 nM. In another embodiment of Step C, Reagent B is present in a
concentration of about 500 nM. In another embodiment of Step C, Reagent B is present in a concentration of about 750 nM.
[00159] Without being bound by theory, it is believed that compounds of formula
I la promotes platelet biogenesis by down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a down-stream effector of aryl hydrocarbon receptor pathway. Accordinly, in one embodiment of the process of the invention, Regent B is capable of antagonizing the activity and/or expression of the aryl hydrocarbon receptor (AHR) and/or a down-stream effector of the AHR pathway. In another embodiment, Reagent B is capable of antagonizing the activity and/or expression of the aryl hydrocarbon receptor. In still another embodiment, Reagent is capable of antagonizing the activity and/or expression of a down-stream effector of the AHR pathway which is selected from Cyp1 B1 , Cyp1 A1 , Beta catenin, AHRR, STAT5 and STAT1 .
[00160] The aryl hydrocarbon (dioxin) receptor (AHR) is a cytosolic ligand- activated transcription factor known to mediate a large number of toxic and carcinogenic effects in animals and possible in human (Safe S 2001 Toxicol Lett 120: 1 -7). As a consequence of AHR activation by its ligands, many detoxification genes are
transcriptionally induced, including those coding for phase I xenobiotic-metabolizing enzymes, such as the cytochromes P450 CYP1 A1 , CYP1 A2, CYP1 B1 and CYP2S1 , and the phase II enzymes UDP-glucuronosyltransferase UGT1 A6, NAD(P)H-dependent quinone oxidoreductase-1 (NQ01 ), the aldehyde dehydrogenase ALDH3A1 , and several glutathione-S-transferase.
[00161] Compounds of Formula II have been previously characterized as antagonist of the aryl hydrocarbon receptor. Boitano, et al. 2010 (WO2010/059401 A2). A genome-wide transcriptional profiling of mPB-derived CD34+ cells treated for 24 hours with Compound B1 (4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol) and Compound B88 (2-(1 -benzo[b]thiophen-3-yl)-N-(3-(3,5-dimethyl- 1 H-pyrazol-4-yl)propyl)-9-isopropyl-9H-purin-6-amine), a less active analog (~20-fold). Of the >50,000 probe sets analyzed, only 5 genes were up-regulated greater than 3-fold upon treatment with Compound B1 and most were also induced to some degree by Compound 88. In addition, 5 genes were down-regulated by >70% upon treatment with 1 μΜ of Compound A1 . All were down-regulated in a dose dependent fashion and none were significantly affected by the less active analog. The two genes that were the most highly repressed by treatment with Compound B1 (cytochrome P450 1 B1 [CYP1 B1 ] and the aryl hydrocarbon receptor repressor [AHRR]) are transcriptionally regulated by the aryl hydrocarbon receptor (AHR). Therefore, compounds of the invention could be acting as an antagonist of AHR signaling.
[00162] Further, the ability of Compound A1 to block 2,3,7, 8-tetrachlorodibenzo-p- dioxin (TCDD, dioxin)-mediated CYP1 B1 mRNA expression by qPCR in mPB-derived CD34+ cells was determined. Treatment with TCDD (3nM) caused a 4.5-fold increase in the level of CYP1 B1 mRNA compared with the vehicle control (0.01 % toluene). This increase was inhibited by Compound B1 in a dose-dependent manner indicating that compounds of the invention can antagonize AHR signaling. To determine the effects of Compound B1 in AHR transcription the ability of Compound B1 to inhibit a dioxin-induced AHR dependent luciferase reporter gene assay was tested. Inclusion of Compound B1 (1 μΜ) completely abolished dioxin-induced AHR dependent transcription when used on cells expressing human AHR. Titration of Compound B1 revealed an EC50 of 127 nM, demonstrating that Compound B1 is a potent AHR antagonist. Interestingly, Compound B1 only weakly inhibited dioxin induced transcription in murine cells and had no activity on rat cells, suggesting that Compound B1 preferentially inhibits human AHR. This correlates with a lack of activity of Compound B1 on murine HSC, and can explain the species selectivity of Compound B1 . Finally, Compound B1 had only weak agonist activity on murine or rat cells, and failed to induce AHR dependent transcription in human cells.
[00163] Studies to establish the mechanistic underpinning of this result are ongoing.
Matrix Metalloproteinase Inhibitor
[00164] The ex vivo process of the invention may further include a matrix metallproteinase (MMP) inhibitor. The MMP inhibitor is not known to directly involve in platelet biogenesis. It has been reported that these inhibitors have both positiive and negative functional consequences in platelet survival.
[00165] The effect of culturing without the inhibitors was investigated (Examples K and L). It was found that the absence of MMP inhibitor has little effect on the phyical and function properties of the exPLTs. Example K shows, when compared to donor-derived platelets, culture-derived "untreated" platelets is equally viable (99% vs 98% viable platelet counts), and includes a much higer percentage of young platelets (60% vs 1 %). Example L shows, the exPLTs and donor derived platelets persisted in circulation with similar kinetics (Figure 1 1 ).
[00166] The MMP inhibitors have a slight effect in platelet yield. Two MMP inhibitors were tested in separate cultures (Example M), the platelet yields for cultures with MMP inhibitors were at least 20% higher for cultures with the MMP inhibitors over cultures with vehicle (Figure 12). The data support including MMP inhibitors in the optimized protocol. However, before better understood other the functional effects MMP inhibitors might have platelets, the MMP inhibitor is included as an optional reagent in the optimized protocol.
[00167] Accordingly, in one embodiment of the process, in Step C, the cell culturing medium optionally further comprising a matrix metalloproteinases (MMP) inhibitor. [00168] In one embodiment of the process, in Step C, the MMP inhibitor is selected from (N-(R)-[2-(Hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-L- naphthylalanyl-L-alanine, 2-aminoethyl amide (TAPI-1 ) and (N-[(2R)-2- (Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM6001 ).
[00169] In one embodiment of the process, in Step C, wherein the MMP inhibitor is administered to the culture medium two days before the completion of the contacting.
[00170] In one embodiment of the process, in Step C, the MMP inhibitor is present in a concentration between 1 and 75 μΜ.
[00171] In one variation of the above embodiment and variation, the MMP inhibitor is (N-[(2R)-2-(Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM6001 ), which is present in a concentration of about 25 μΜ.
[00172] In another variation of the above embodiment and variation, the MMP inhibitor is (N-(R)-[2-(Hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-L-naphthylalanyl-L- alanine, 2-aminoethyl amide (TAPI-1 ), which is present in a concentration of about 10 μΜ.
Duration
[00173] The optimal duration for culturing the starting cell population in the presence of the inhibitors was determined experimentally. In Example N, starting cell populations of HSC were exposed to Reagent A for 4, 7, 10, or 14 days, during 18 days culturing protocol, and platelet counts were determined. It was found the the optimal time for contacting with Reagent A is 14 days (Figure 13).
[00174] Accordingly, in one embodiment of the process, in Step A, the contacting with reagent A is for a duration between 2 days and 16 days. In one variation, the contacting is for a duration between 4 days and 14 days. In another variation, in Step A, the duration is for about 14 days.
[00175] The duration of the contacting of the exMKs with Reagent B and TPO in
Step C can be similarily determined. In one embodiment of the process of the invention, in Step C, the contacting is for a duration between 2 days and 10 days In another embodiment, in Step C, the contacting of the exMKs with Reagent B and TPO is for a duration between 3 days and 5 days. In another embodiment, in Step C, the contacting of the exMKs with Reagent B and TPO is for a duration of about 4 days.
[00176] After the completion of the first stage (Step A), it is necessary to collect and transfer the megakaryocytes from the first stage culture to second phase culture. In Example O, we compared the effect on platelet yield of transferring the megakaryocyte by "pelleting and resuspension" to "isolation". By "pelleting and resuspension", we mean collecting the cells in the Stage 1 culture without separating the megakaryocytes from othere cells; centrifugation would be the method of choice. Isolation meant separate the megakaryocyte from other cells and cell fragments. We isolated the megakaryocytes using magnetic beads (Milteni). Other methods may be used; those methods are generally known by those skilled in the art. We found pelleting and resuspension produced an about 2-fold higher platelet yield per megakaryocyte on day 12 (Figure 14). This transferring method has shown to be consistently reproducible. We incorporated the pelleting and resuspension into the optimized protocol for the transferring the megakaryocytes. Accordingly, in one embodiment of the process of the invention, the process further comprising Step B1 comprising: gathering the population of exMKs, and Step B2 comprising: re-suspending the population of exMKs.
[00177] Platelets were culture from isolated megakaryocytes (Example P).
However, the platelets yields were highly variable. Without being bound by theory, we hypothesize that damage to the megarkaryocyte during isolation and donor variability are the contributing factors.
Isolation of the platelets.
[00178] The cell population obtained after the production process may be used without further purification or may be subject to further purification or selection steps. The cell population may be washed to remove the reagents and/or any other components of the cell culture and resuspended in an appropriate cell suspension medium ready for use.
[00179] In one embodiment of the process, the platelets were purified by isolation over a BSA gradient (Example Q). In three samples, the post-isolation population contains 100% platelets (calculated as the number of platelets divided by the sum of the number of platelets and nucleated cells), with a yield averaging 34%.
[00180] Flow cytometric characterization of the pre-isolation, post-isolation and whole blood shows similar size, granularity, and expression of the platelets markers CD41 a and CD42a for all three cell population. Figure 15 shows the flow cytometric
characterization of Sample 1 .
[00181] In one specific embodiment, the isolated cell population is resuspended in a pharmaceutically acceptable medium suitable for administration to a mammalian host, thereby providing a therapeutic composition.
Large scale production
[00182] The process of the invention can be adopted for large scale production.
The process was validated in large scale culture in tissue culture flasks and culture bags and is amenable to culture entirely in culture bags, thus appropriate for cultures exceeding 1 L final volume. For example, Example E demonstrated the optimized protocol can be practiced entirely in flask and culture bags. Thus, further scale up practical for clinical is feasible.
II. Products of the Invention
[00183] In another aspect of the invention relates to a cell population comprising ex vivo manufactured platelets (exPLTs). The platelets are appropriate for intravenous administration as an infusion cell therapy. In particular embodiments, the platelets are patient specific, and suitable for allogeneic infusion.
[00184] In one embodiment of the product of the invention, the cell population comprising exPLTs is obtained by the methods of described in the above section. In one variation of the above embodiment, the cell population is purified and re-suspended in a pharmaceutically acceptable medium. In another variation, the exPLTs expressing CD41 a+ CD42b+ composed at least 20% of the sum of the number of platelets and viable nucleated cells.
[00185] In another aspect, the invention relates to a composition comprising exPLTs and Reagent B in a pharmaceutically acceptable medium wherein Reagent B is a compound as described in paragraph [00139], supra.
[00186] In one variation of the compositon of the above embodiment, Reagent B is selected from the following compounds:
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)phenol;
N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin-6-amine;
2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1 -ol;
N-(2-(1 H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine; and
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(2-methyl-1 H-indol-3-yl)ethyl)-9H-purin-6- amine.
[00187] In another embodiment of the composition comprising exPLTs and
Reagent B of the invention, Reagent B is present at a concentration between 1 nM and 1000 nM. In one variation, Reagent B is present at a concentration between 100 nM and 750 nM. In another variation, Reagent B is present at a concentration between 1 nM and 750 nM. [00188] Another aspect of the invention relates to a cell composition comprising isolated exPLTs. In one embodiment, the cell composition comprising isolated exPLTs is characterized by the presence of Reagent B, wherein Reagent B is a compound of Formula lib as described in paragraph [00139].
[00189] In one variation of the above embodiments, Reagent B is selected from
4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)phenol;
N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-amine; N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-amine; N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine; N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin-6-amine; 2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1 -ol; N-(2-(1 H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; N-(2-(1 H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine; and 2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(2-methyl-1 H-indol-3-yl)ethyl)-9H-purin-6- amine.
[00190] In another embodiment of the above embodiment and variations of cell composition comprising isolated exPLTs, the cell composition contains about to 1012 exPLTs. In one variation, the composition contains aboufl O9 to 5 x 1011 exPLTs.
[00191] In yet another embodiment of the above embodiment and variations of cell composition comprising isolated exPLTs, the exPLTs expressing CD41 a+ CD42b+ represent between 80 to 100% of the sum of the number of exPLTs and other viable nucleated cells in the population. In another variation, the exPLTs expressing CD41 a+ CD42b+ represent >95% of the sum of the number of exPLTs and viable nucleated cells in the population.
[00192] In a particular embodiment of the above embodiments and variations, the cell composition comprising isolated exPLTs, the exPLTs expressing CD41 a+ CD42b+ is HLA-matched to a patient in need of a platelet infusion.
[00193] In still another aspect of the invention relates to starting compositions. In one embodiment, the starting composition comprising ex vivo produced megakaryocytes and Reagent B, wherein Reagent B is a compound according to paragraph [00129].
[00194] In one embodiment of the starting composition, Reagent B is present in a concentration between 250 nM and 750 nM. In one varitation, Reagent B is present in a concentration about 250 nM. In another variation, Reagent B is present in a concentration about 750 nM. III. Use of the Therapeutic Compositions
[00195] In another aspect, the invention is related to the use ex vivo produced platelets for treating thrombocytopenia in a subject in need thereof, by administering a sufficient amount of ex vivo produced platelets. It is believe that by treating the
thrombocytopenia, the underlying diseases and conditions which caused the
thrombocytopenia can also be treated, abated, ameliorated or eradicated. A non-limiting list of diseases and conditions that can be treated follows, infra.
[00196] In one embodiment, the exPLTs are HLA-matched to the subject. In another embodiment, the subject is alloimmunized.
[00197] In another embodiment, the administration is an autologous
transplantation. In another embodiment, the administration is an allogeneic transplanation
[00198] In one embodiment the exPLTs are used to treat thrombocytopenia resulting from chemotherapy, radiation therapy, dengue fever, an autoimmune disorder, an hereditary syndrome, an inherited immunodeficient disease, or an hematopoietic disorder.
[00199] In one variation the autoimmune disorder is systemic lupus erythematosus
(SLE) or systemic sclerosis.
[00200] In one variation the hereditary syndromes are selected from congenital amegakaryocytic thrombocytopenia, thrombocytopenia absent radius syndrome, Fanconi anemia, Bernard-Soulier syndrome, May-Hegglin anomaly, Grey platelet syndrome, Alport syndrome, and Wiskott-Aldrich syndrome.
[00201] In one variation, the hematopoietic disorder is selected from Acute myeloid leukemia, Acute lymphoblastic leukemia, Amyloidosis Aplastic anemia, Chronic myeloid leukemia, Chronic lymphocytic leukemia, Germ cell tumors, Hemophagocytic lymphohistiocytosis, Hodgkin disease, inborn errors of metabolism, Myeloproliferative disorders, Myelodysplastic syndromes, Multiple myeloma, Neuroblastoma, Non-Hodgkin lymphoma, Paroxysmal nocturnal hemoglobinuria, Pure red cell aplasia, Severe combined immunodeficiency, Sickle cell anemia, and Thalassemia major.
[00202] In a further variation, the inborn errors of metabolism are selected from mucopolysaccharidosis, Gaucher disease, metachromatic leukodystrophies and
adrenoleukodystrophies.
KIT
[00203] Also provided herein is a pack or a kit comprising one or more containers filled with one or more of the ingredients described herein. Such kits optionally comprise solutions and buffers as needed or desired. In particular, the invention provides a kit for for manufacture platelets according to the process of the invention, comprising Reagent B as defined in paragraph [00139] and instructions for use of Reagent B in a method for manufacture platelets and, optionally, one ore more cytokines or growth factors, or media for cell growth, in particular media for platelet biogenesis as described above. The kit may further comprise Reagent A as defined in paragraph [00124], supra. The kit may further comprise antibodies for monitoring production of the cells, such as anti-CD34, anti-CD41 a, anti-CD42b, anti-CD45RA and/or anti-CD61 . In one specific embodiment, such kit further include one or more cytokines or growth factors selected from the group consisting of IL6, Flt3-L, SCF and TPO. The kit may further include a matrix metalloproteinase inhibitor. In one embodiment, the MMP inhibitor is selected from N-[(2R)-2-
(Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan Methylamide; (GM 6001 , EMD Chemicals) or N-(R)-[2-(Hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-L- naphthylalanyl-L-alanine, 2-aminoethyl Amide (TAPI-1 , EMD Chemicals). Optionally associated with such pack(s) or kit(s) are instructions for use.
Utility
[00204] Platelets, or thrombocytes are small, irregularly shaped clear cell fragments (i.e. cells that do not have a nucleus), 2-3 μιη in diameter, which are derived from fragmentation of precursor megakaryocytes. The average lifespan of a platelet is normally just 5 to 9 days. Platelets are a natural source of growth factors. They circulate in the blood of mammals and are involved in hemostasis, leading to the formation of blood clots.
[00205] A normal platelet count in a healthy individual is between 150,000 and
450,000 per μΙ_ (microlitre) of blood ((150-450) χ 109/L). Ninety-five percent of healthy people will have platelet counts in this range. If the number of platelets is too low, excessive bleeding can occur. Thrombocytopenia is a condition of low number of platelets. Thrombocytopenia may be the result of diseases or treatments, for examply chemotherapy, radiation therapy, dengue fever, an autoimmune disorder, an hereditary syndrome, an inherited immunodeficient disease, or an hematopoietic disorder.
[00206] Transfusion is generally used to correct unusually low platelet counts
(typically below (1 0-1 5) x 1 09/L). Over 1 0 million platelet units are transfused per year in the US. Each transfused dose is about 5x 1011 platelets.
[00207] Platelets are either isolated from collected units of whole blood and pooled to make a therapeutic dose or collected by apheresis, sometimes concurrently with plasma or red blood cells,
[00208] Pooled whole-blood platelets, sometimes called "random" platelets,-as the name implies, could be pooled from multiple donations to make a single desired therapeutic dose. This means that a recipient is potentially exposed to many different donors and has increased risk of transfusion-transmitted disease and other complications.
[00209] Apheresis platelets are collected during a blood draw using a mechanical device that separates out the platelets and other components to be collected and the remaining blood is returned to the donor. The advantage to this method is that a single donation provides at least one therapeutic dose, as opposed to the multiple donations for whole-blood platelets. However, there is a shortage of donors.
[00210] Platelets collected by either method have a very short shelf life, typically five days. This results in frequent problems with short supply, as testing the donations often requires up to a full day. Since there are no effective preservative solutions for platelets, they lose potency quickly and are best when fresh.
[00211 ] Hematology patients undergoing high dose chemotherapy account for up to 67% of platelet transfusions in the US. Because of prolonged thrombocytopenia, these patients normally require multiple platelet transfusions over a period of weeks or months.
[00212] Current medical practice does not require human leukocyte antigen
(HLA)-matched platelets, and alloimmunization is a common risk for these patients which renders them refractory to platelet transfusion. In the TRAP clinical trial 8% of patients had baseline anti-HLA antibodies and another 17-45% developed them following chemotherapy. HLA-alloimmunized patients are ideally given matched platelets to avoid immediate platelet rejection; however, identifying and obtaining matched donor platelets is often not feasible and is a current unmet medical need.
[00213] A method of manufacturing platelet ex vivo in a clinical relevant amount would have a great impact on the supply of the platelets for transfusion, and also address the unmet need for patient specific platelets. Umbilical cord blood banks currently exist and cover the broad racial make-up of the general population. An ex vivo method would enable the manufacture of platelets by using HLA-matched hematopoietic stem cell isolated from the cord blood. The exPLTs potentially can be a source of HLA-matched platelets for alloimmunized thrombocytopenic patients without a donor platelet match and, as such, will increase post-transfusion platelet recovery compared to standard of care (unmatched platelets). In addition, the possibility of using exPLT to supplement and/or replace standard platelet products will help to alleviate a reliance on donors, avert shortages and improve product safety.
[00214] The availability of ex-vivo produced platelets, whether from adult, umbilical cord blood, fetal, or embryonic sources, would have a huge impact on the treatment of thrombocytopenia, and the underlying causes of the condition. Dosage, Adminstration, and Formulation
[00215] The invention further relates to the cell population with exPLTs or its composition for use in allogeneic or autologous platelet infusion in a mammalian subject. The manufactured platelets can be uses as is or could be isolated from the culture prior to use.
[00216] To prepare cells for infusion, the isolated cell are pelleted by centrifugation for 10 minutes at 1000g and resuspended in infusion buffer e.g., a 5% HSA (Baxter) at a concentration of between 107 to 1010 platelets /ml_.
[00217] The number of cells transfused will take into consideration factors such as sex, age, weight, the types of disease or disorder, stage of the disorder, the percentage of the desired cells in the cell population and the amount of cells needed to produce a therapeutic benefit. In one particular embodiment, the composition is administered by intravenous infusion and comprises at least 108 platelets/kg, from 109 to 1010 platelets/kg or more if needed. A transfusion dose is typical about 3 to 5 x 1011 platelets.
[00218] Thus, provided are methods of providing ex vivo produced platelets to a subject in need thereof, comprising administering to the subject the exPLTs described herein or made by the methods described herein.
[00219] As used throughout, by a subject is meant an individual. Thus, subjects include, for example, domesticated animals, such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, and goats), laboratory animals (e.g., mice, rabbits, rats, and guinea pigs), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. The subject is optionally a mammal such as a primate or a human.
[00220] The term pharmaceutically acceptable form refers to compositions including the cell therapy product described herein that are generally safe, relatively nontoxic and neither biologically nor otherwise undesirable. These compositions optionally include pharmaceutically acceptable carriers or stabilizers that are nontoxic to the cell or subject being exposed thereto at the dosages and concentrations employed. The carrier or excipient is selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject or cell. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt- forming counterions such as sodium ; and/or nonionic surfactants such as TWEEN™ (Uniqema, United Kingdom), polyethylene glycol (PEG), and PLURONICS™ (BASF, Germany).
[00221 ] The compositions are formulated in any conventional manner for use in the methods described herein. Administration is via any route known to be effective by one of ordinary skill.
[00222] The preferred method of administration is intravenous infusion.
[00223] A pharmaceutically acceptable carrier for infusion of a composition comprising cells into a patient typically comprise buffered saline with 5%HSA or unsupplemented basal medium or medium as known in the art.
Other AHR antagonists suitable for promoting platelet biogenesis
[00224] The invention also relates to using other molecules in the process of invention to manufacturing platelets ex vivo.
[00225] In addition to low molecular weight an organic molecules, other AHR antagonists, for example, small interference RNA (siRNA) and antisense oligonucleotide, can also be used in the ex vivo production of platelets.
[00226] Design of antisense oligonucleotides which can be used to efficiently inhibit the AHR protein expression must be effected in a way that such oligonucleotides specifically binds the designated mRNA within cells in a way which inhibits translation thereof. Sequence suitable for use in design and synthesis of antisense oligonucleotides which specifically bind to AHR mRNA, genomic DNA and/or its promoter or other control sequences are available in published sequence of AHR, in particular human AHR. In addition, algorithms for identifying sequences with the highest predicted binding affinity for their target mRNA based on thermodynamic cycle that accounts for the energetics of structural alterations in both the target mRNA and the oligonucleotides are also available.
[00227] Synthesis of RNAi molecules suitable for use with the present invention can be affected as follows: First, the AHR mRNA sequence (or one or more of its downstream effectors) is scanned downstream of the AUG start codon for AA-dinucleotide sequences. Occurrence of each AA and the 19 3'-adjacent is recorded as a potential siRNA target site. Then, potential target sites are compared to an appropriate genomic database (e.g, human, mouse, rat, etc.) using any sequence alignment software. Putative target site that exhibit significant homology to other coding sequences are filtered out. Preferred sequences are then those including low G/C content, in particular sequences with G/C content lower than 55%. Several target sites are then selected along the length of the target gene. Methods or algorithms to identify putative target site of siRNA are described for example in (Tilesi, et al., Curr. Opin. Mol. Ther. 1 1 :156, 2009). Examples of siRNA molecules which are capable of down-regulating the expression of AHR are: AHR 1 1 1 S, 5' GCG GCA TAG AGA CCG ACT TAA TTT CAA GAG AAT TAA GTC GGT CTC TAT GCC GCT TTT TTG G 3' (SEQ ID NO: 1 ); AHR 1 1 1 AS, 5' CGC GCC AAA AAA GCG GCA TAG AGA CCG ACT TAA TTC TCT TGA AAT TAA GTC GGT CTC TAT GCC GC 3' (SEQ ID NO: 2); AHR 242S, 5' GGC TTC TTT GAT GTT GCA TTA ATT CAA GAG ATT AAT GCA ACA TCA AAG AAG CCT TTT TTG G 3' (SEQ ID NO: 3); AHR 242AS, 5' CGC GCC AAA AAA GGC TTC TTT GAT GTT GCA TTA ATC TCT TGA ATT AAT GCA ACA TCA AAG AAG CC 3' (SEQ ID NO: 4).
ENUMERATED EMBODIMENTS
[00228] Various enumerated embodiments of the invention are described herein.
It will be recognized that features specified in each embodiment may be combined with other specified features to provide further embodiments of the present invention.
[00229] In a first embodiment, the invention provides an ex vivo process for producing a population of ex vivo produced platelets (exPLTs) comprising a Step C, which comprises:
contacting a population of ex vivo produced megakaryocytes (exMKs) with Reagent B and thrombopoietin (TPO), where a compound of Formula Ila:
Figure imgf000043_0001
or a pharmaceutically acceptable salt, or stereoisomer thereof; wherein
L is selected from -NR5a(CH2)2_3-, -NR5a(CH2)2NR5b-, -NR5a(CH2)2S- - NR5aCH2CH(OH)- and -NR5aCH(CH3)CH2-; wherein R5a and R5b are independently selected from hydrogen and d-4alkyl;
RT is selected thiophenyl, furanyl, benzoimidazolyl, isoquinolinyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyrazolyl, pyridinyl, imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, pyrrolyl and thiazolyl; wherein said thiophenyl, furanyl, benzoimidazolyl, isoquinolinyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyrazolyl, pyridinyl, imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, pyrrolyl or thiazolyl of is optionally substituted by 1 to 3 radicals independently selected from halo, cyano, d-4alkyl, halo-substituted-d-4alkyl, d_ 4alkoxy,-S(0)o-2R8a, and -C(Q)OR8a, wherein R8a is selected from hydrogen and d-4alkyl; R2 is selected from -S(0)2NR6aR6b, -NR6aC(0)NR6bR6c, phenyl, pyrrolopyridinyl, indolyl, thiophenyl, pyridinyl, triazolyl, 2-oxoimidazolidinyl, pyrazolyl, and indazolyl; wherein
R6a, R6b and R6c are independently selected from hydrogen and d^alkyl; and said phenyl, pyrrolopyridinyl, indolyl, thiophenyl, pyridinyl, triazolyl, oxoimidazolidinyl, pyrazolyl, or indazolyl of R2 is optionally substituted with 1 to 3 radicals independently selected from hydroxy, halo, methyl, methoxy, amino, -
0(CH2)nNR7aR7b, -OS(0)2NR7aR7b and -NR7aS(0)2R7b; wherein R7a and R7b are independently selected from hydrogen and d^alkyl;
R3 is selected from hydrogen, d^alkyl and biphenyl; and
R4 is selected from Ci_i0alkyl, d_4alkenyl, oxetanyl, tetrahydrofuranyl, cyclohexyl, (oxopyrrolidinyl)ethyl, tetrahydropyranyl, phenyl, and benzyl, wherein said d-ioalkyl, d- 4alkenyl, oxetanyl, tetrahydrofuranyl, cyclohexyl, (oxopyrrolidinyl)ethyl, tetrahydropyranyl, phenyl, and benzyl of R4 can be optionally substituted with 1 to 3 radicals independently selected from hydroxy, d_4alkyl and halo-substituted-d-4alkyl.
[00230] Embodiment 2. The process according to Embodiment 1 , wherein the population of exPLTs represents at least a 2-fold increase of exPLTs per exMKs when compared to no contacting with Reagent B.
[00231] Embodiment s. The process according to Embodiment 1 or
Embodiment 2, in Step C, wherein Regent B is capable of antagonizing the activity and/or expression of the aryl hydrocarbon receptor (AHR) and/or a down-stream effector of the AHR pathway.
[00232] Embodiment 4. The process according to Embodiment 3, in Step C, wherein a down-stream effector of aryl hydrocarbon receptor pathway is selected from Cyp1 B1 , Cyp1 A1 , Beta catenin, AHRR, STAT5 and STAT1 .
[00233] Embodiment 5. The process according to any one of Embodiments 1 to 4, further comprising a Step A for producing the exMKs from hematopoietic stem cells (HSC), wherein Step A comprising:
contacting a population of HSC with Reagent A and TPO, wherein Reagent A is of Formula I:
Figure imgf000044_0001
or a pharmaceutically acceptable salt, or stereoisomer thereof; wherein
L is -NHC(O)- or -C(0)NH-; is selected from hydrogen, d-4alkyl, phenyl, and C5-6heteroaryl, wherein the d_ 4alkyl, phenyl or C5.6heteroaryl is unsubstituted or substituted by 1 to 2 substituents independently selected from halo, cyano, d-4alkyl, halo-substituted d-4alkyl, d-4alkoxyd- 4alkyl, d_4alkoxy, C3-6cycloalkyl and C4.6heterocycloalkyl, wherein the C3-6cycloalkyl or C4. eheterocycloalkyl is further unsubstituted or substituted by 1 to 2 substituents independently selected from halo, hydroxy, oxo, d_4alkylcarbonyl, or d_4alkyoxy;
R2 is d-4alkyl or C3-6cycloalkyl;
R3 is d-4alkyl or d_4alkenyl;
R4 is hydrogen or C5-6heteroary, unsubstituted or substituted by d_4alkyl; and R5 is halo, d_4alkyl, halo-substituted d-4alkyl, d-4alkoxy-substituted d-4alkyl, d_ 4alkoxy, and halo-substituted d.4alkoxy.
[00234] Embodiment 6. The process according to Embodiment 5, in Step A, wherein Reagent A is capable of antagonizing the activity and/or the expression of the platelet-derived growth factor receptor (PDGFR).
[00235] Embodiment 7. The process according to Embodiment 5 or
Embodiment 6, in Step A, wherein the population of exMKs represents about a 2.5-fold increase of exMKs per HSC when compared to no contacting with Compound A.
[00236] Embodiment 8. The process according to any one of Embodiments 1 to 7, further comprising:
Step B1 : gathering the quantity of exMKs; and
Step B2: re-suspending the quantity of exMKs.
[00237] Embodiment 9. The process according to any one of Embodiments 5 to 8, in Step A, wherein
Figure imgf000045_0001
la
L is -NHC(O)- or -C(0)NH-;
RT is hydrogen, d-4alkyl or C5-6heteroaryl, wherein the alkyl is substituted by substitutedC4.6heterocycloalkyl, and the C5-6heteroaryl is substituted by 2 d-4alkyl;
R2 is d-4alkyl or C3.6cycloalkyl;
R3 is d-4alkyl or d_4alkenyl; and
R4 is hydrogen or C5-6heteroary, unsubstituted or substituted by d-4alky. [00238] Embodiment 10. The process according to any one of Embodiments 5 to 9, in Step A, wherein is selected from hydrogen, 2.6-dimethylpyridin-3-yl, 1 ,3 dimethyl- 1 /-/-pyrazol-5-yl, and 2-oxo-pyrrolidin-1 -yl-propyl.
[00239] Embodiment 1 1 . The process according to any one of Embodiments 5 to 10, in Step A, wherein R2 is methyl or cyclopropyl.
[00240] Embodiment 12. The process according to any one of Embodiments 5 to 1 1 , in Step A, wherein R3 is methyl or vinyl.
[00241] Embodiment 13. The process according to any one of Embodiments 5 to 12, in Step A, wherein R4 is hydrogen or 4-methyl-1 /-/-imidazol-1 -yl.
[00242] Embodiment 14. The process according to Embodiment 5, in Step A, wherein Reagent A is selected from:
3-(7-((2,6-dimethylpyridin-3-yl)amino)-1 -methyl-2-oxo-1 ,2-dihydropyrimido[4,5- d]pyrimidin-3(4H)-yl)-N-(3-(trifluoromethyl)phenyl)-4-vinylbenzamide;
N-(4-methyl-3-(1 -methyl-2-oxo-7-((3-(2-oxopyrrolidin-1 -yl)propyl)amino)-1 ,2- dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)phenyl)-3-(trifluoromethyl)benzamide;
N-(3-(2-amino-8-methyl-7-oxo-7,8-dihydropteridin-6-yl)-4-methylphenyl)-3-(4-methyl- 1 H-imidazol-1 -yl)-5-(trifluoromethyl)benzamide;
N-(3-(1 -Cyclopropyl-7-((1 ,3-dimethyl-1 H-pyrazol-5-yl)amino)-2-oxo-1 ,2- dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-4-methylphenyl)-3-(trifluoromethyl)benzamide; and
N-(3-(7-amino-1 -methyl-2-oxo-1 ,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-4- methylphenyl)-3-(trifluoromethyl)benzamide.
[00243] Embodiment 15. The process according to Embodiment 5, in Step A, wherein Reagent A is 3-(7-((2,6-dimethylpyridin-3-yl)amino)-1 -methyl-2-oxo-1 ,2- dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N-(3-(trifluoromethyl)phenyl)-4-vinylbenzamide.
[00244] Embodiment 16. The process according to any one of Embodiments 1 to 15, in Step C, wherein Reagent B is of Formula lib:
Figure imgf000046_0001
L is selected from -NH(CH2)2_3- -NH(CH2)2S- and -NHCH2CH(OH)- RT is selected from thiophenyl, benzoimidazolyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyridinyl, imidazolyl, and pyridazinyl; each of which is unsubstituted or substituted by 1 to 2 radicals independently selected from halo, cyano, d^alkyl, halo- substituted-d^alkyl, d^alkoxy, and -S(O)0-2R8a, wherein R8a is d^alkyl; R2 is selected from phenyl, indolyl and pyrrolopyridinyl, each of which is
unsubstituted or substituted with 1 to 2 radicals independently selected from halo, hydroxy, methyl, methoxy, -0(CH2)nNR7aR7b, -OS(0)2NR7aR7b, and -NR7aS(0)2R7b; wherein R7a and R7b are independently selected from hydrogen and d^alkyl; and
R4 is selected from Ci-i0alkyl, d^alkenyl, oxetanyl, tetrahydrofuranyl, wherein the CV^alkyl or C1-4alkenyl is unsubstituted or substituted with hydroxyl.
[00245] Embodiment 17. The process according to any one of Embodiments 1 to 16, in Step C, wherein L is -NH(CH2)2-.
[00246] Embodiment 18. The process according to any one of Embodiments 1 to 17, in Step C, wherein is selected from 1 H-imidazol-1 -yl, thiophen-2-yl, thiophen-3-yl, pyridin-3-yl, pyridin-4-yl, pyrimidin-5-yl, pyridazin-4-yl, 1 H-benzo[d]imidazol-1 -yl, 3H- imidazo[4,5-b]pyridin-3-yl, and benzo[b]thiophen-3-yl, each of which is unsubstituted or substituted by 1 to 2 radicals independently selected from fluoro, chloro, cyano, methyl, trifluoromethyl, methoxy, and -S(0)o-2Rsa; wherein R8a is selected from methyl and ethyl.
[00247] Embodiment 19. The process according to any one of Embodiments 1 to 17, in Step C, wherein is benzo[b]thiophen-3-yl or pyridin-3-yl, each unsubstituted or substituted by halo.
[00248] Embodiment 20. The process according to any one of Embodiments 1 to 18, in Step C, wherein R2 is selected from phenyl, 1 H-indol-3-yl and 1 H-pyrropyridin-3-yl, each of which is unsubstituted or substituted substituted with 1 to 2 radicals independently selected from halo, hydroxy, methyl, methoxy, dimethyaminoethoxy, and amino-sulfonyloxy, and -NR7aS(0)2R7b; wherein R7a and R7b are independently selected from hydrogen and C1 -4alkyl.
[00249] Embodiment 21 . The process according to any one of Embodiments 1 to 19, in Step C, wherein R4 is selected from isopropyl, methyl, ethyl, prop-1 -en-2-yl, isobutyl, sec-butyl, (S)-sec-butyl, (R)-sec-butyl, 1 -hydroxypropan-2-yl, (S)-l -hydroxypropan- 2-yl, (R)-1 -hydroxypropan-2-yl, nonan-2-yl, oxetan-3-yl, oxetan-2-yl, and tetrahydrofuran-3- yi-
[00250] Embodiment 22. The process according to any one of Embodiments 1 to 19, in Step C, wherein R4 is selected from isopropyl, methyl, ethyl, prop-1 -en-2-yl, isobutyl, sec-butyl, 1 -hydroxypropan-2-yl, and nonan-2-yl.
[00251] Embodiment 23. The process according to any one of Embodiments 1 to 15, in Step C, wherein Reagent B is selected from the group consisting of compounds listed in Table II.
[00252] Embodiment 24. The process according to any one of Embodiments 1 to 15, in Step C, wherein reagent B is selected from the group consisting of: 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H^urin-6-ylamino)ethyl)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H^urin-6-ylamino)ethyl)phenol;
N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H^urin-6-amin
N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H^urin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-purin-6-am
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin-6-amine;
(S)-2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-
1 -ol;
N-(2-(1 H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; N-(2-(1 H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine and 2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(2-methyl-1 H-indol-3-yl)ethyl)-9H-purin-6- amine.
[00253] Embodiment 25. The process according to any one of Embodiments 5 to 24, in Step A, wherein the population of hematopoietic stem cells is enriched in CD34+ cells.
[00254] Embodiment 26. The process according to any one of Embodiments 5 to 25, in Step A, wherein the population of hematopoietic stem cells is derived from umbilical cord blood cells.
[00255] Embodiment 27. The process according to any one of Embodiments 5 to 25, in Step A, wherein the population of hematopoietic stem cells consisting essentially of CD34+ cells purified from one or two cord blood units.
[00256] Embodiment 28. The process according to any one of Embodiments 5 to 25, in Step A, wherein the population of hematopoietic stem cells is derived from bone marrow blood cells.
[00257] Embodiment 29. The process according to any one of Embodiments 5 to 25, in Step A, wherein the population of hematopoietic stem cells is derived from mobilized peripheral blood cells.
[00258] Embodiment 30. The process according to any one of Embodiments 5 to 29, in Step A, wherein the population of hematopoietic stem cells is derived from a single mammalian subject.
[00259] Embodiment 31 . The process according to any one of Embodiments 5 to 30, in Step A, wherein the population of hematopoietic stem cells is derived from human.
[00260] Embodiment 32. The process according to any one of Embodiments 5 to 31 , in Step A, wherein the contacting of the population of hematopoietic stem cells with Reagent A and TPO occurs in a cell culturing medium further comprising interleukin-6 (IL- 6), Fms-liked tyrosine-protein kinase 3 ligand (Flt3-L), and stem cell factor (SCF), wherein Reagent A, TPO, IL-6, Flt3-L and SCF are each present in a concentration effective for promoting megakaryopoiesis.
[00261] Embodiment 33. The process according to Embodiment 32, in Step A, wherein TPO, IL6, Flt3-L, and SCF are each present in a concentration between 10 and 100 ng/mL.
[00262] Embodiment 34. The process according to Embodiment 32, in Step A, wherein TPO, IL6, Flt3-L, and SCF are each present in a concentration between 25 and 75 ng/mL.
[00263] Embodiment 35. The process according to Embodiment 32, in Step A, wherein TPO, IL6, Flt3-L, and SCF are each present in a concentration of about 50 ng/mL.
[00264] Embodiment 36. The process according to any one of Embodiments 5 to 35, in Step A, wherein Reagent A is present in a concentration between 50 nM and 5 μΜ.
[00265] Embodiment 37. The process according to any one of Embodiments 5 to 35, in Step A, wherein Reagent A is present in a concentration between 100 nM and 1000 nM.
[00266] Embodiment 38. The process according to any one of Embodiments 5 to 35, in Step A, wherein Reagent A is present in a concentration about 250 nM, 500 nM or 750nM.
[00267] Embodiment 39. The process according to any one of Embodiments 5 to 35, in Step A, wherein Reagent A is present in a concentration of about 250 nM.
[00268] Embodiment 40. The process according to any one of Embodiments 5 to 39, in Step A, wherein the contacting is for a duration between 2 days and 16 days.
[00269] Embodiment 41 . The process according to any one of Embodiments 5 to 39, in Step A, wherein the contacting is for a duration between 4 days and 14 days.
[00270] Embodiment 42. The process according to any one of Embodiments 5 to 39, in Step A, wherein the duration is about 14 days.
[00271] Embodiment 43. The process according to any one of Embodiments 1 to 43, in Step C, wherein the contacting of the exMKs with Compound B and TPO occurs in a cell culturing medium, wherein Reagent B and TPO are each present in a concentration effective for promoting platelet biogenesis.
[00272] Embodiment 44. The process according to any one of Embodiments 1 to 43, in Step C, wherein TPO is present in a concentration between 5 ng/mL and 500 ng/mL.
[00273] Embodiment 45. The process according to any one of Embodiments 1 to 43, in Step C, wherein TPO is present in a concentration of about 100 ng/mL. [00274] Embodiment 46. The process according to any one of Embodiments 1 to 43, in Step C, wherein TPO is present in a concentration of about 50 ng/mL.
[00275] Embodiment 47. The process according to Embodiments 46, in Step C, wherein the medium further comprising IL-6, Flt3-L and SCF, each present in a
concentration of about 50 ng/mL.
[00276] Embodiment 48. The process according to any one of Embodiments 1 to 47, in Step C, wherein Reagent B is present in a concentration between 1 nM and 5 μΜ.
[00277] Embodiment 49. The process according to any one of Embodiments 1 to 47, in Step C, wherein Reagent B is present in a concentration between 100 nM and 5000 nM.
[00278] Embodiment 50. The process according to any one of Embodiments 1 to 47, in Step C, wherein Reagent B is present in a concentration between about 250 nM and about 750 nM.
[00279] Embodiment 51 . The process according to any one of Embodiments 1 to 47, in Step C, wherein Reagent B is present in a concentration of about 250 nM.
[00280] Embodiment 52. The process according to any one of Embodiments 1 to 47, in Step C, wherein Reagent B is present in a concentration of about 500 nM.
[00281] Embodiment 53. The process according to any one of Embodiments 1 to 47, in Step C, wherein Reagent B is present in a concentration of about 750 nM.
[00282] Embodiment 54. The process according to any one of Embodiments 1 to 53, in Step C, wherein the cell culturing medium optionally further comprising a matrix metalloproteinases (MMP) inhibitor.
[00283] Embodiment 55. The process according to Embodiment 54, in Step C, wherein the MMP inhibitor is selected from (N-(R)-[2-(Hydroxyaminocarbonyl)methyl]-4- methylpentanoyl-L-naphthylalanyl-L-alanine, 2-aminoethyl amide (TAPI-1 ) and (N-[(2R)-2- (Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM6001 ).
[00284] Embodiment 56. The process according to Embodiment 54, in Step C, wherein the MMP inhibitor is administer to the culture medium two days before the completion of the contacting.
[00285] Embodiment 57. The process according to Embodiment 54 or
Embodiment 55, in Step C, wherein the MMP inhibitor is present in a concentration between 1 and 75 μΜ.
[00286] Embodiment 58. The process according to Embodiment 54 or
Embodiment 55, in Step C, wherein the MMP inhibitor is (N-[(2R)-2- (Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM6001 ), which is present in a concentration of about 25 μΜ. [00287] Embodiment 59. The process according to Embodiment 54 or
Embodiment 55, in Step C, wherein the MMP inhibitor is (N-(R)-[2- (Hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-L-naphthylalanyl-L-alanine, 2- aminoethyl amide (TAPI-1 ), which is present in a concentration of about 10 μΜ.
[00288] Embodiment 60. The process according to any one of Embodiments 1 to 59, in Step C, wherein the contacting of the exMKs with Reagent B and TPO is for a duration between 2 days to 10 days.
[00289] Embodiment 61 . The process according to any one of Embodiments 1 to 59, in Step C, wherein the contacting of the exMKs with Reagent B and TPO is for a duration of about 4 days.
[00290] Embodiment 62. An ex vivo process for producing a population of ex vivo produced platelets (exPLTs), wherein the process comprising:
A1 ) providing a population of hematopoietic stem cells (HSC);
A) contacting HSC with Reagent A to yield a population of ex vivo produced megakaryocytes (exMKs), and
C) contacting the population of exMKs with ex vivo produced platelets (exPLTs); wherein
Regent A is 3-(7-((2,6-dimethylpyridin-3-yl)amino)-1 -methyl-2-oxo-1 ,2- dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N-(3-(trifluoromethyl)phenyl)-4-vinylbenzamide; and
Reagent B is of Formula lib:
Figure imgf000051_0001
or a pharmaceutically acceptable salt, or stereoisomer thereof; wherein
L is selected from -NH(CH2)2_3- -NH(CH2)2S- and -NHCH2CH(OH)- RT is selected from thiophenyl, benzoimidazolyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyridinyl, imidazolyl, and pyridazinyl; each of which is unsubstituted or substituted by 1 to 2 radicals independently selected from halo, cyano, d^alkyl, halo- substituted-d^alkyl, d^alkoxy, and -S(O)0-2R8a, wherein R8A is d^alkyl;
R2 is selected from phenyl, indolyl and pyrrolopyridinyl, each of which is
unsubstituted or substituted with 1 to 2 radicals independently selected from halo, hydroxy, methyl, methoxy, -0(CH2)NNR7AR7B, -OS(0)2NR7AR7B, and -NR7AS(0)2R7B; wherein R7A and R7B are independently selected from hydrogen and d_4alkyl; and R4 is selected from Ci-i0alkyl, d^alkenyl, oxetanyl, tetrahydrofuranyl, wherein the C^oalkyl or C1-4alkenyl is unsubstituted or substituted with hydroxyl.
[00291] Embodiment 63. The process according to Embodiment 62, wherein
Reagent B is selected from
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)phenol;
N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin-6-amine;
2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1 -ol;
N-(2-(1 H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine; and
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(2-methyl-1 H-indol-3-yl)ethyl)-9H-purin-6- amine.
[00292] Embodiment 64. The process according to Embodiment 62 or
Embodiment 63, further comprising the steps of:
B-1 ) pelleting the population of exMKs; and
B-2) re-suspending the population of exMKs in a medium suitable for Step C.
[00293] Embodiment 65. The process according to any one of Embodiments 62 to 64, wherein the population of exPLTs represents at least about 2000 exPLT per HSC.
[00294] Embodiment 66. The process according to any one of Embodiments 62 to 65, wherein the population of exPLTs represents at least about 7.5-fold increase in yield of ex-platelets per HSC when compared to no contacting with Reagent A and Reagent B. .
[00295] Embodiment 67. The process according to any one of Embodiments 62 to 66, in Step A1 , wherein the population of hematopoietic stem cells is enriched in CD34+ cells and is derived from umbilical cord blood cells.
[00296] Embodiment 68. The process according to any one of Embodiments 62 to 67, in Step A, wherein the contacting of the population of HSC with Reagent A occurs in a cell culturing medium further comprising thrombopoietin (TPO), interleukin-6 (IL-6), Fms- liked tyrosine-protein kinase 3 ligand (Flt3-L), and stem cell factor (SCF), wherein TPO, IL- 6, Flt3-L and SCF are each present in a concentration of about 50 ng/mL.
[00297] Embodiment 69. The process according to Embodiments 62 to 68, in
Step A, wherein Reagent A is present in a concentration of about 250 nM. [00298] Embodiment 70. The process according to any one of Embodiments 62 to 69, in Step A, wherein the contacting of HSC and Reagent A is for a duration of about 14 days.
[00299] Embodiment 71 . The process according to any one of Embodiments 62 to 70, in Step C, wherein the contacting of the exMKs with Reagent B occurs in a cell culturing medium supplemented with TPO, wherein Reagent B and TPO are each present in a concentration effective for promoting platelet biogenesis.
[00300] Embodiment 72. The process according to Embodiment 71 , in Step C, wherein TPO is present in a concentration of about 100 ng/mL.
[00301] Embodiment 73. The process according to any one of Embodiments 62 to 72, in Step C, wherein Reagent B is present in a concentration between 250 to 750 nM.
[00302] Embodiment 74. The process according to any one of Embodiments 62 to 73, in Step C, wherein the contacting is for about 4 days.
[00303] Embodiment 75. The process according to any one of Embodiments 62 to 74, in Step C, wherein the cell culturing medium is optionally further supplemented with a matrix metalloproteinases (MMP) inhibitor on day 16.
[00304] Embodiment 76. The process according to Embodiment 75, in Step C, wherein the MMP inhibitor is N-[(2R)-2-(Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L- tryptophan methylamide (GM6001 ) supplemented to a concentration of about 25 μΜ.
[00305] Embodiment 77. The process according to Embodiment 75, in Step C, wherein the MMP inhibitor is (N-(R)-[2-(Hydroxyaminocarbonyl)methyl]-4-methylpentanoyl- L-naphthylalanyl-L-alanine, 2-aminoethyl amide (TAPI-1 ) supplemented to a concentration of about 10 μΜ.
[00306] Embodiment 78. A cell population comprising exPLTs obtained by the method of any one of Embodiments 1 to 77.
[00307] Embodiment 79. The cell population according to Embodiment 78, wherein the composition is purified and re-suspended in a pharmaceutically acceptable medium.
[00308] Embodiment 80. The composition according to Embodiment 78, wherein exPLTs expressing CD41 a+ CD42b+ composed at least 20% of the sum of the number of platelets and viable nucleated cells.
[00309] Embodiment 81 . A composition comprising exPLTs and Reagent B in a pharmaceutically acceptable medium wherein Reagent B is a compound according to Embodiment 16.
[00310] Embodiment 82. The composition according to Embodiment 81 , wherein Reagent B is selected from 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H^urin-6-ylamino)ethyl)phenol;
4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H^urin-6-ylamino)ethyl)phenol;
N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H^urin-6-amin
N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H^urin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-purin-6-ami
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin-6-amine;
2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H^urin-9-y
N-(2-(1 H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H^urin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H^urin-6-amine; and
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(2-methyl-1 H-indol-3-yl)ethyl)-9H-purin-6- amine.
[00311] Embodiment 83. The composition according to Embodiment 82, wherein Reagent B is present at a concentration between 1 nM and 1000 nM.
[00312] Embodiment 84. The composition according to Embodiment 82, wherein Reagent B is present at a concentration between 100 nM and 750 nM.
[00313] Embodiment 85. The composition according to Embodiment 83, wherein Reagent B is present at a concentration between 1 nM and 750 nM.
[00314] Embodiment 86. A cell composition comprising isolated exPLTs, wherein the composition is characterized by the presence of Reagent B, wherein Reagent B is a compound according to Embodiment 16.
[00315] Embodiment 87. The composition according to Embodiment 88, wherein Reagent B is selected from
4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)phenol;
N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin-6-amine;
2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1 -ol;
N-(2-(1 H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine; and
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(2-methyl-1 H-indol-3-yl)ethyl)-9H-purin-6- amine.
[00316] Embodiment 88. The composition according to Embodiment 86 or
Embodiment 87, wherein the composition contains about 109 to 1012 exPLTs.
[00317] Embodiment 89. The composition according to Embodiment 86 or
Embodiment 87, wherein the composition contains about 5 x 1011 exPLTs. [00318] Embodiment 90. The composition according to Embodiment 86 or
Embodiment 87, wherein the exPLTs represent between 80 to 100% of the sum of the number of exPLTs and viable nucleated cells.
[00319] Embodiment 91 . The composition according to Embodiment 86 or
Embodiment 87, wherein the exPLTs represent >95% of the sum of the number of exPLTs and viable nucleated cells.
[00320] Embodiment 92. The cell population according to any one of
Embodiments 88 to 91 , wherein the exPLTs is HLA-matched to a patient in need of a platelet infusion.
[00321] Embodiment 93. A starting composition comprising ex vivo produced megakaryocytes and Reagent B, wherein Reagent B is a compound according to
Embodiment 16.
[00322] Embodiment 94. The composition according to Embodiment 93, wherein Reagent B is present in a concentration between 250 nM and 750 nM.
[00323] Embodiment 95. A method of treating thrombocytopenia in a subject in need thereof, comprising administering a sufficient amount of exPLTs produced by the process of any one of claims 1 to 77.
[00324] Embodiment 96. The method of Embodiment 95, wherein the subject is alloimmunized and the exPLTs are HLA-matched to the subject.
[00325] Embodiment 97. The method of Embodiment 95 or Embodiment 96, wherein the thrombocytopenia is resulted from chemotherapy, radiation therapy, dengue fever, an autoimmune disorder, an hereditary syndrome, an-inherited immunodeficient disease, or an hematopoietic disorder.
[00326] Embodiment 98. The method of Embodiment 97, wherein the autoimmune disorders are selected from Systemic lupus erythematosus and systemic sclerosis.
[00327] Embodiment 99. The method of Embodiment 97, wherein the hereditary syndromes is selected from congenital amegakaryocytic thrombocytopenia,
thrombocytopenia absent radius syndrome, Fanconi anemia, Bernard-Soulier syndrome, May-Hegglin anomaly, Grey platelet syndrome, Alport syndrome, and Wiskott-Aldrich syndrome.
[00328] Embodiment 100. The method of Embodiment 97, wherein said hematopoietic disorder is selected from Acute myeloid leukemia, Acute lymphoblastic leukemia, Amyloidosis Aplastic anemia, Chronic myeloid leukemia, Chronic lymphocytic leukemia, Germ cell tumors, Hemophagocytic lymphohistiocytosis, Hodgkin disease, inborn errors of metabolism. Myeloproliferative disorders, Myelodysplastic syndromes, Multiple myeloma, Neuroblastoma, Non-Hodgkin lymphoma, Paroxysmal nocturnal hemoglobinuria, Pure red cell aplasia, Severe combined immunodeficiency, Sickle cell anemia, and
Thalassemia major.
[00329] Embodiment 101 . The method of Embodiment 100, wherein the inborn errors of metabolism are selected from mucopolysaccharidosis, Gaucher disease, metachromatic leukodystrophies and adrenoleukodystrophies.
ASSAYS AND GENERAL PROCEDURES
CD34+ cell culture
[00330] All experiments were performed in HSC expansion media (StemSpan
SFEM [StemCell Technologies] supplemented with 1 x antibiotics and the appropriate combination of recombinant human cytokines. Recombinant human cytokines were purchased from R & D Systems or Life Technologies. Human CD34+ cells were purified from fresh human umbilical cord blood (Bioreclamation), as noted, using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols. CD34+ cells were seeded at 3-5 x 104 viable nucleated cells/mL. Cells were cultured at 37 °C in 5% C02 and fresh medium (containing cytokines and reagent) was added as needed to keep the cell density between 3 x 104 and 1 x 106 cells/mL.
Surface antigen analysis:
The cells are washed with staining media (Hanks balanced salt solution containing FBS (2%) and EDTA (2mM)) and stained (at room temperature for 30 minutes) with indicated primary conjugated antibodies. The cells are washed in the previously described buffer and analyzed using a BD LSR II flow cytometer (Becton Dickinson, San Jose, CA) using 488-nm argon and 633-nm HeNe laser beams as the light source for excitation. Emission is measured using logarithmic amplification and analyzed using FlowJo software (TreeStar Inc. Ashland, OR).
Cell phenotypinq by flow cytometry
[00331] Multicolor analysis for cell phenotyping was performed on a LSR II flow cytometer (Becton Dickinson). Cell sorting was perfomed on a FACSAria (Becton
Dickinson). Cells were stained with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
PDGFR Inhibition Assay
[00332] Exponentially growing Ba/F3 or Ba/F3 Tel-TK cells were diluted to
100,000 cells/ml and distributed into 384-well plates (Greiner, Nurtingen, Germany, custom white clear bottom) at 50 ml/well by using a mFill liquid dispenser (Bio-Tek, Burlington, NY). Kinase inhibitors were serially diluted with DMSO and arrayed in a polypropylene 384-well plate. Fifty nanoliters of compound was transferred into cell plates using the pin-transfer device and the plates incubated at 37 °C (5% C02) for 48 h. 25 ml Bright-Glo (Promega) was added and luminescence was quantified using an Analyst-GT (Molecular Devices). Custom curve-fitting software was used to produce a logistic fit of percent cell viability as a function of the logarithm of inhibitor concentration. The IC50 was interpolated as the concentration of compounded needed to reduce cell viability to 50% of a DMSO control.
[00333] The inhibitory activity against PDGFR of selected compounds of Formula I is listed in Table 1 .
Assay for the Capability of Compounds of Formula I in Promoting Meqakarvopoiesis:
[00334] All experiments were performed in HSC expansion media (StemSpan
SFEM [StemCell Technologies] supplemented with 1 x antibiotics and the following recombinant human cytokines: thrombopoietin, IL6, Flt3 ligand, and stem cell factor [100 ng/mL, R & D Systems]) unless otherwise indicated. CD34+ cells were resuspended in HSC expansion medium (5 x 104 cells/mL) before being aliquoted in 384 well plates (Greiner Bio-One). Compounds were added immediately after plating. Cells were cultured at 37 °C in 5% C02. Multicolor analysis for cell phenotyping was performed on a LSR II flow cytometer (Becton Dickinson). Cells were stained in staining media (HBSS supplemented with FBS [2%] and EDTA [2 mM]) at 4 °C for 1 h with APC anti-human CD1 10 (BD
Bioscience), PerCP anti-CD34 (BD Bioscience), FITC anti-CD45RA (e-Bioscience), PECy7 anti-CD41 (BD Bioscience), and PE anti-GlyA (BD Bioscience), then washed with staining media and analyzed. The percentages of indicated cell subsets were determined from aliquots of the cell culture at day 8. Flow cytometry results of these subsets are given as percentage of the total population. The total cell number in each treatment group was determined by trypan blue exclusion. Absolute numbers of each population in the culture were calculated from the total number of cells multiplied by the percentage of each population. [00335] The capability of selected compounds of Formula I in promoting megakaryopoiesis is listed in Table 1 .
AHR inhibition assay
[00336] AHR inhibitory activity of a compound may be determined by its ability to block 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin)-mediated CYP1 B1 mRNA expression by qPCR in mPB-derived CD34+ cells (the dioxin-induced AHR dependent luciferase reporter gene assay). Treatment with TCDD (3nM) caused a 4.5-fold increase in the level of CYP1 B1 mRNA compared with the vehicle control (0.01 % toluene). The test compound is assayed in a dose response format (1 n M to 10 μΜ) to determine the effective concentration that produced the desired effect in 50% of the cells (EC50). An inhibitor of AHR activity is a compound that has an EC50 of less than 10 μΜ, preferably less than 5μΜ as measured by this assay. Inclusion of Compound B1 (1 μΜ) completely abolished dioxin- induced AHR dependent transcription when used on cells expressing human AHR.
[00337] Titration of Compound B1 revealed an EC50 of 127nM, Compound B157 showed an EC50 of 8 nM, Compound B131 showed an EC50 of 5 nM, and Compound 184 showed an EC50 of 92 nM; demonstrating the compounds of Formula II are potent AHR antagonists.
General Procedure for Making Compound of Formula II (Compound B)
[00338] Compound B may be prepared by Schemes 1 -5 below. In the reactions described, it can be necessary to protect reactive functional groups, for example hydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, to avoid their unwanted participation in the reactions. Conventional protecting groups can be used in accordance with standard practice, for example, see T.W. Greene and P. G. M. Wuts in
"Protective Groups in Organic Chemistry", John Wiley and Sons, 1991 .
[0001] It will be appreciated by one skilled in the art that, following introduction by the methods detailed below, any of the groups R^ R2, R3, R4, and may optionally be further elaborated by known transformations to arrive at the desired final compounds of
Formula II.
[00339] Compounds of Formula II can be prepared according the following
Reaction Scheme 1 : Reaction Scheme 1
Figure imgf000059_0001
in which G G2, G3, G4, R2 and R4 are as defined for Formula I in the Summary of the Invention and L of Formula I is defined in the reaction scheme as -NH4_ which is equivalent to, for example, -NR5a(CH2)o-3- where R5a is hydrogen and -(CH2)0-3- is
[00340] Compounds of Formula II can be prepared by reacting a compound of
Formula 2 with a compound of Formula 3 in the presence of a suitable catalyst (e.g., Pd2(dba)3, or the like) in the presence of an appropriate ligand (e.g., 1 ,3-bis(2,4,6- trimethylphenyl) imidazolium chloride), a suitable base (e.g., Cs2C03, or the like) and an appropriate solvent (e.g., 1 ,4-dioxane) at a temperature of about 80 to 100 °C for 2 to about 48 hours. Compounds of Formula 2 in turn can be prepared by reacting a compound of Formula 4 with a slight excess of an amine compound of Formula 5 in an appropriate solvent (e.g. isopropanol) at a temperature of about room temperature to about 80 oC. Compounds of Formula 4 can be prepared by alkylation of a compound of Formula 6 with a suitable alkylating agent 7, in which is chlorine, bromine, iodine, or a sulfonate ester, in the presence of a suitable base (e.g. sodium hydride or potassium carbonate), in a suitable solvent (e.g. DMF), at a temperature of about 0 °C to about 80 °C. Alternatively, the reaction can be performed under Mitsunobu conditions using a suitable alcohol R4-OH in the presence of a suitable phosphine (e.g. triphenylphosphine) and azodicarboxylate (e.g. diethylazodicarboxylate), in an inert solvent such as THF or toluene, at a temperature from about 0 °C to about room temperature.
[00341 ] Compounds of Formula lla, in which Gi is CR3 and in which all other G groups are N, can also be prepared by proceeding as in the following Reaction Scheme 2: Reaction Scheme 2
Figure imgf000060_0001
(9) (8) (1 a) in which R2, R3 and R4 are as defined for Formula I in the Summary of the Invention and L of Formula I is defined in the reaction scheme as -NH-L which is equivalent to, for example, -NR5a(CH2)o-3- where R5a is hydrogen and -(CH2)o-3- is .
[00342] Compounds of Formula I can be prepared by reacting a compound of
Formula 8 with an amine compound of Formula 5 in an appropriate solvent (e.g.
isopropanol) at a temperature of about room temperature to about 100 oC. Compounds of Formula 8 can in turn be prepared by reacting a compound of Formula 9 with a compound of Formula 3 in the presence of a suitable catalyst (e.g., Pd(Ph3P)4 , Pd2(dba)3, or the like), optionally in the presence of an appropriate ligand (e.g., 1 ,3-bis(2,4,6-trimethylphenyl) imidazolium chloride), a suitable base (e.g., Cs2C03, or the like) and an appropriate solvent (e.g., 1 ,4-dioxane) at a temperature of about 80 to 100 °C for 2 to about 48 hours.
Compounds of Formula 9 in turn can be prepared by reacting a compound of Formula 10 with a mixture of di-iodomethane, copper(l) iodide, and an alkyl nitrite (e.g. isoamylnitrite), optionally in the presence of an inert solvent, at a temperature of about 50 to 100 °C.
Compounds of Formula 10 can be prepared by alkylation of a compound of Formula 1 1 with a suitable alkylating agent 7, in which is chlorine, bromine, iodine, or a sulfonate ester, in the presence of a suitable base (e.g. sodium hydride or potassium carbonate), in a suitable solvent (e.g. DMF), at a temperature of about 0 °C to about 80 °C. Alternatively, the reaction can be performed under Mitsunobu conditions using a suitable alcohol R4-OH in the presence of a suitable phosphine (e.g. triphenylphosphine) and azodicarboxylate (e.g. diethylazodicarboxylate), in an inert solvent such as THF or toluene, at a temperature from about 0 °C to about room temperature.
[00343] Compounds of Formula II, which are a subset of compounds of Formula I in which is N-linked heterocyclyl or N-linked heteroaryl, can be prepared as detailed in the following Reaction Scheme 3: Reaction Scheme 3
Figure imgf000061_0001
Gi , G2, G3, G4, Ri , R2 and R4 are as defined for Formula I in the Summary of the Invention and L of Formula I is defined in the reaction scheme as -NH-L which is equivalent to, for example, -NR5a(CH2)o-3- where R5a is hydrogen and -(CH2)o-3- is . Compounds of Formula II can be prepared by reacting a compound of Formula 2 with a compound of Formula 20 in the presence of an excess of cyclic amine or NH-bearing heterocycle (for example, substituted pyrazole, substituted imidazole, and the like), at a temperature of about 50 °C to about 250 °C, for about 1 to about 24 hours, optionally in the presence of a base such as sodium hydride or DBU.
[00344] Compounds of Formula 10 in which Gi is CR3, and in which all other G groups are N, can also be prepared by proceeding as in the following Reaction Scheme 4:
Reaction Scheme 4
Figure imgf000061_0002
in which R3 and R4 are as defined for Formula I in the Summary of the Invention.
[00345] Compounds of Formula 10 can be prepared according to procedures described in J. Med. Chem, 1972, 456, and J. Med. Chem., 1992, 4180. An orthoester compound of Formula 21 is reacted with a compound of Formula 22, optionally in the presence of an acid such as acetic acid, at a temperature of about room temperature to about 150 oC, for about 1 to about 24 nr.. A compound of Formula 22 can in turn be prepared by reacting a compound of Formula 23 with a primary amine compound of Formula 24, optionally in the presence of an acid such as pTSA, or a base such as triethylamine or DBU, at a temperature of about 50 to about 200 oC.
[00346] Compounds of Formula IV can be prepared as detailed in the following
Reaction Scheme 5: Reaction Scheme 5
Figure imgf000062_0001
[00347] in which Gi , G2, G3, G4, Ri and R2 are as defined for Formula I in the
Summary of the Invention and L of Formula II is defined in the reaction scheme as -NH-L which is equivalent to, for example, -NR5a(CH2)o-3- where R5a is hydrogen and -(CH2)M- is
R20 and R21 are independently selected from hydrogen and d^alkyl. A compound of Formula IV, in which R21 is hydrogen, can be prepared from a compound of Formula III by treatment with a suitable reducing agent such as lithium aluminum hydride or di-isobutyl aluminum hydride, in a suitable solvent such as THF or toluene, at a temperature of about - 78°C to about 50°C. The reaction takes about 0.5 to about 16 hr to complete. A compound of Formula IV, in which R21 is lower alkyl, can be prepared by treatment of a compound of Formula III with an alkyl lithium or Grignard reagent, in a suitable solvent such as ether or tetrahydrofuran, at a temperature of about -78°C to about 50°C. The reaction takes about 0.5 to about 16 hr to complete.
[00348] Detailed examples of the synthesis of a compound of Formula I can be found in the Examples, infra.
Additional Processes for Making Compounds of the Invention
[00349] A compound of the invention can be prepared as a pharmaceutically acceptable acid addition salt by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid. Alternatively, a pharmaceutically acceptable base addition salt of a compound of the invention can be prepared by reacting the free acid form of the compound with a pharmaceutically acceptable inorganic or organic base. Alternatively, the salt forms of the compounds of the invention can be prepared using salts of the starting materials or intermediates.
[00350] For example, salt forms of 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H- purin-6-ylamino)ethyl)phenol (Example 1 of Compound B, infra) were synthesized as follows:
[00351] Mesylate salt: 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol free base (0.60g; 1 .40 mmoles) are dissolved in 12 ml acetone at 50°C. Methanesulfonic acid (0.137 g; 1 .40 mmoles) is added drop wise. The crystallization takes place rapidly. The white suspension is allowed to cool over about 30 minutes with cooling to room temperature. The slurry is stirred for 18 hours at room temperature and filtered. The solid is washed with acetone (6 ml) in three portions and dried first for about 3 hours at 50 °C / ca. 10 mbar and then for about 16 hours at 80 °C /ca.10 mbar. The material has a melting point at about 233 °C with a melting enthalpy of 98g/J. The material produced exhibited a loss on drying of 0.2%.The water uptake was estimated by thermogravimetry after exposure to relative humidity (80%rh) during 24 hours. A water uptake of 0.4% was observed.
[00352] The free acid or free base forms of the compounds of Formula I or II can be prepared from the corresponding base addition salt or acid addition salt form, respectively. For example a compound of the invention in an acid addition salt form can be converted to the corresponding free base by treating with a suitable base (e.g., ammonium hydroxide solution, sodium hydroxide, and the like). A compound of Formula I or Formula II in a base addition salt form can be converted to the corresponding free acid by treating with a suitable acid (e.g., hydrochloric acid, etc.). The nitrate salt of the compound of example 1 can be made using methods known to the skilled person.
[00353] Compounds of Formula I or II can be prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the
diastereomers and recovering the optically pure enantiomers. While resolution of enantiomers can be carried out using covalent diastereomeric derivatives of the compounds of the invention, dissociable complexes are preferred (e.g., crystalline diastereomeric salts). Diastereomers have distinct physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and can be readily separated by taking advantage of these dissimilarities. The diastereomers can be separated by chromatography, or preferably, by separation/resolution techniques based upon differences in solubility. The optically pure enantiomer is then recovered, along with the resolving agent, by any practical means that would not result in racemization. A more detailed description of the techniques applicable to the resolution of stereoisomers of compounds from their racemic mixture can be found in Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and Resolutions", John Wiley And Sons, Inc., 1981 . Compounds of the invention can also be prepared as their individual stereoisomers by using chiral chromatography techniques, in particular, by use of HPLC or SFC chromatography using a chiral stationary phase. [00354] Insofar as the production of the starting materials is not particularly described, the compounds are known or can be prepared analogously to methods known in the art or as disclosed in the Examples hereinafter.
[00355] One of skill in the art will appreciate that the above transformations are only representative of methods for preparation of the compounds of the present invention, and that other well known methods can similarly be used.
EXAMPLES
Example A: Reagent B Promotes Platelet Biogenesis in a Single Stage Culture
[00356] Human CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols. CD34+ cells were cultured at 37 °C for 18-20 days in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and Reagent B (250 nM of (S)-2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9- yl)propan-1 -ol (Example B157S)) and the following recombinant human cytokines:
thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems). CD34+ cells were seeded at 1 -25x104 viable nucleated cells per ml (count prior to cryopreservation), and cultures were maintained in culture plates and supplemented with culture media as necessary.
[00357] Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by analysis of a defined volume by flow cytometry. Final platelet counts were obtained at day 18 or day 20 (one sample).
Platelet yield per seeded CD34+ cell,
with and without 250 nM of Reagent B (Example B157S)
N Mean +/- SEM Median Range
5 622 +/- 188 412 257-1240 Reagent B+
5 281 +/- 37 278 195-412 Cytokine only Platelet yield -fold over cytokines only control, baseline
protocol with 250 nM of Reagent B (Example B157S)
N Mean +/- SEM Median Range
5 2.1 +/- 0.4 1 .7 1 .1 -3.2
[00358] Thus, the data showed that on average, Reagent B increased platelet yield when added for the entire culture period.
Example B: Reagent B Presence in 2nd Stage Promotes Platelet Biogenesis
[00359] Human CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols. CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems). CD34+ cells were seeded at 20-25x104 viable nucleated cells per ml_ (count prior to cryopreservation). Cultures were maintained in non-tissue culture treated culture plates and supplemented with culture media as necessary for 10 days. After 10 days, cells were pelleted and resuspended in culture media as above, with or without addition of Reagent B (250 nM of (S)-2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5- fluoropyridin-3-yl)-9H-purin-9-yl)propan-1 -ol (Compound B157S)). Cells were then cultured as above to day 18.
[00360] Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by analysis of a defined volume by flow cytometry and normalized to the number of CD34+ cells at day 1 in a defined volume by flow cytometry. Final platelet counts were obtained at day 18. Yields, baseline protocol with 250 nM Reagent B (Compound
B157S) at day 10
% Platelet yield Platelet yield, fold
Sample Megakaryocytes per seeded over cytokines only at day 10 CD34+ cell control
1 24 366 1 .9
2 20 310 1 .4
[00361] The data demonstrated that Reagent B also increased platelet yield when present only from culture day 10 to 18, in contrast to Example A that Reagent B was present the entire culture period.
Example C: Optimized Protocol Without Reagent A in Stage 1
[00362] Human CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols. CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows. The first culture phase (days 0 to 14) contained the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems). CD34+ cells were seeded at 3-5x104 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 3x104 and 1 x106 viable nucleated cells per ml, as determined by trypan blue exclusion. For the second culture phase (days 14 to 18), cells were pelleted and resuspended on day 14 at 1 -5x105 viable nucleated cells per ml in serum-free media containing TPO (100 ng/ml) and Reagent B (500 nM of (S)-2-(6-(2-(1 H-indol-3- yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1 -ol (Compound 157S)). A matrix metalloproteinase (MMP) inhibitor, 25 μΜ of N-[(2R)-2-
(Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM 6001 ; EMD Chemicals), was added at day 16. Cultures were maintained in culture bags
(American Fluoroseal Corp).
[00363] Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
Megakaryocyte characterization at day 14,
optimized protocol without Reagent A
N Mean +/- SEM Median Range % Megakaryocytes (MK) 4 16 +/- 2 15 12-23
MK per seeded cell at day 0 4 206 +/- 102 1 16 82-51 1
Platelet yield per seeded cell at day 0, optimized protocol without Reagent A
N Mean +/- SEM Median Range
4 481 +/- 220 339 145-1 100
[00364] The data demonstrates that on average, these changes (addition of
Reagent A at day 14, change of cytokine composition to TPO alone at day14 and addition of MMP inhibitor at day 16) increased the platelet yield from 281 platelets per seeded cell for the cytokines only baseline protocol or to 481 platelets per seeded cell (1 .7 fold effect) in large scale culture.
[00365] For three of the samples, platelets were further assessed for viability by fluorescence with the dye Calcein, AM (Life Technologies). Cells were stained with 2.5 μΜ Calcein AM for 15 minutes at 37 °C, along with cell surface antibodies, washed and analyzed by flow cytometry. The % Calcein AM+ cells was assessed in the platelet population as shown for Example E.
% platelet viability (Calcein AM+) at day 18, optimized protocol without Reagent A
N Mean +/- SEM Median Range
3 89 +/- 3 91 83-93 [00366] The data thus demonstrates that the vast majority of platelets generated with this protocol were viable.
[00367] For three of these samples, platelets were further assessed for young platelets (reticulocytes) by staining with the dye thiazole orange (Sigma). Cells were stained with 25 μg/ml thiazole orange for 15 minutes at 37 °C, along with cell surface antibodies, washed and analyzed by flow cytometry. Gating on whole blood platelets was based on the normal circulating value of 3-16%, mean 9% (Kienast J, Schmitz G. Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation of thrombocytopenic disorders. Blood 1990;75:1 16-121 ) as shown for Example E.
% young platelets (thiazole orange+) at day 18,
optimized protocol without Reagent A
N Mean +/- SEM Median Range
3 64 +/- 7 63 53-77
[00368] It should be noted that a greater percentage of the platelets generated with this protocol were young platelets, compared with blood platelets (which on average will be at their half-life). This indicates the potential for a longer lifespan of culture-derived platelets compared with donor blood platelets.
Example D: Contribution of Reagent A vs. Reagent B to Platelet Yield
[00369] Human CD34+ cells were purchased as a CD34+ selected cord blood mixed donor vial (Allcells). CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows. The first culture phase (days 0 to 14) contained the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems) with or without Reagent A (500 nM of 3-(7-((2,6- dimethylpyridin-3-yl)amino)-1 -methyl-2-oxo-1 ,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N- (3-(trifluoromethyl)phenyl)-4-vinylbenzamide (Compound A1 or MK1 ). CD34+ cells were seeded at 3x105 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 9x104 and 1 .4x106 viable nucleated cells per ml, as determined by trypan blue exclusion. For the second culture phase (days 14 to 18), cells were pelleted and resuspended on day 14 at 5x105 viable nucleated cells per ml in serum-free media containing TPO (100 ng/ml) with or without Reagent B (500 nM (S)-2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)- 9H-purin-9-yl)propan-1 -ol (Compound B157S) or 750 nM of 4-(2-(2-(benzo[b]thiophen-3-yl)- 9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (Compound B1 ). Cultures were maintained in non-tissue culture treated plates.
[00370] Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
[00371] The result is represented by a bar-graph in Figure 2. Reagent A and
Reagent B were each shown to contribute to increases in platelet yield, both individually and in combination. Addition of Compound A1 (Reagent A) only resulted in a 2.5 fold increase in platelet yield over no compound addition. Addition of Compound B157S (Reagent B) resulted in a further 3-fold increase, for a total increase of 7-fold.
Example E Optimized Protocol for the Manufacture of Platelets Ex Vivo
[00372] Human CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols. CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows. The first culture phase (days 0 to 14) contained Reagent A (250 nM of 3-(7-((2,6- dimethylpyridin-3-yl)amino)-1 -methyl-2-oxo-1 ,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N- (3-(trifluoromethyl)phenyl)-4-vinylbenzamide (Example A1 )) and the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems). CD34+ cells were seeded at 3-5x104 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 3x104 and 1 x106 viable nucleated cells per ml, as determined by trypan blue exclusion. For the second culture phase (days 14 to 18), cells were pelleted and resuspended on day 14 at 1 -5x105 viable nucleated cells per ml in serum- free media containing TPO (100 ng/ml) and Reagent B (500 nM of (S)-2-(6-(2-(1 H-indol-3- yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1 -ol (Compound B157S)). A matrix metalloproteinase (MMP) inhibitor, 25 μΜ of N-[(2R)-2- (Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM 6001 ); EMD Chemicals) was added at day 16. Cultures were maintained in culture bags
(American Fluoroseal Corp).
[00373] Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate.
[00374] Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
Megakaryocyte characterization at day 14
N Mean +/- SEM Median Range
% Megakaryocytes (MK) 4 23 +/- 5 24 1 1 -32
MK per seeded cell at day 0 4 635 +/- 278 407 267-1458 Fold increase in MK over - MK1
4 3.5 +/- 0.8 2.9 2.4-6.0 control
Platelet yield per seeded cell at day 0, optimized protocol
N Mean +/- SEM Median Range
4 2321 +/- 556 2000 1379-3906
[00375] Comparing to Example C, addition of Reagent A increased the average megakaryocyte number at day 14 by 3.5 fold, and also increased the platelet yield by 4.8 fold (from 481 to 2321 average yield per seeded cell). The platelet yield with this optimized protocol represents an 8.3 fold increase over the baseline cytokines only protocol in Example A.
[00376] Figure 3 shows the pseudocolor density plots displaying fluorescence intensity of particles analyzed by flow cytometry. Gating scheme for platelets is defined by scatter properties and CD41 a and CD42b bound fluorescence intensity. Ex vivo cultured platelets were defined by gates which encompass whole blood platelets shows that that ex vivo cultured platelets are phenotypically same as whole blood derived platelet.
[00377] For three of these samples, platelets were further assessed for viability by fluorescence with the dye Calcein, AM (Life Technologies). Cells were stained with 2.5 μΜ Calcein AM for 15 minutes at 37C, along with cell surface antibodies, washed and analyzed by flow cytometry.
% platelet viability (Calcein AM+) at day 18,
optimized protocol
N Mean +/- SEM Median Range
3 86 +/- 3 89 79-89
[00378] The data shows that the vast majority of platelets generated with this protocol were viable
[00379] Fig. 4 shows the pseudocolor density plots displaying fluorescence intensity of particles analyzed by flow cytometry. Platelets gated as in Fig 3 were further assessed for Calcein fluorescence, an indication of viability. Blood platelets were used to set the Calcein+ viability gate. shows flow cytometry chart for Calcein, AM stained cells.
[00380] For three of these samples, platelets were further assessed for young platelets (reticulocytes) by staining with the dye thiazole orange (Sigma). Cells were stained with 25 μg/ml thiazole orange for 15 minutes at 37C, along with cell surface antibodies, washed and analyzed by flow cytometry. Gating on whole blood platelets was based on the normal circulating value of 3-16%, mean 9% (Kienast J, Schmitz G. Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation of thrombocytopenic disorders. S/ooc/ 1990 75:1 16-121 ).
% young platelets (thiazole orange+) at day 18
N Mean +/- SEM Median Range
3 62 +/- 5 58 56-73
The data shows a greater percentage of the platelets generated with this protocol were young platelets, compared with blood platelets (which on average will be at their half-life). This indicates the potential for a longer lifespan of culture-derived platelets compared with donor blood platelets. [00382] Fig. 5 shows the pseudocolor density plots displaying fluorescence intensity of particles analyzed by flow cytometry.-Platelets gated as defined in Fig 3 were further assessed for thiazole orange binding, an indication of a newly formed platelet. Blood platelets were used to set the thiazole orange+ gate, as based on the normal circulating value of 3-16%, mean 9% (Kienast J, Schmitz G. Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation of thrombocytopenic disorders. Blood 1990;75:1 16-121 ) shows the thiazole orange stained cells.
Example F: In vivo Functional Assay of Ex Vivo Manufactured Platelets
[00383] Human CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols. CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows. The first culture phase (days 0 to 14) contained Reagent A (250 nM of 3-(7-((2,6- dimethylpyridin-3-yl)amino)-1 -methyl-2-oxo-1 ,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N- (3-(trifluoromethyl)phenyl)-4-vinylbenzamide (Example A1 , also referred to as MK1 )) and the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems). CD34+ cells were seeded at 3x104 viable nucleated cells per ml, and cultures were maintained in culture bags (American Fluoroseal Corp) and supplemented with culture media to maintain cell density between 3x104 and 1 x106 viable nucleated cells per ml, as determined by trypan blue exclusion. For the second culture phase (days 14 to 18), cells were pelleted and resuspended on day 14 at 4x105 viable nucleated cells per ml in serum-free media containing TPO (100 ng/ml) and Reagent B (500 nM of (S)-2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H- purin-9-yl)propan-1 ( Compound B157S)). A matrix metalloproteinase (MMP) inhibitor (25 μΜ of N-[(2R)-2-(Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM 6001 , EMD Chemicals)) was added at day 16. Cultures were maintained in culture bags (American Fluoroseal Corp).
[00384] Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
[00385] Platelets were isolated using a modification of a published protocol
(Robert, et al. Megakaryocyte and platelet production from human cord blood stem cells. Methods Mol Biol. 2012 788:219-47). The culture was aliquotted into 50 ml tubes. Platelets were released by pipetting each aliquot up and down 25 ml 10 times with a 25 ml serological pipette fitted with a P1000 pippete tip to the end. Each aliquot was pelleted at 1000xg for 15 min and resuspended in 1 ml citrate wash buffer (0.5% Citrate and 0.35% BSA in HBSS, adjusted to pH 6.5, with 50 ng/ml PGE1 added just before use). Sets of two aliquots were combined for 2 ml total, which was then layered on a 2% to 12% BSA step gradient. The platelet-containing upper phase was collected after spinning at 80xg for 15 min with no brake or acceleration and washed with DPBS containing 1 % BSA and 50 ng/ml PGE1 . The washed platelets were pooled in HBSS and stored for 1 hr at 37C.
[00386] ACD-anticoagulated whole blood platelets were isolated in a similar manner for comparison. Platelet rich plasma supernatant was collected after centrifugation at 200xg. Platelets were washed in citrate wash buffer before loading on the 2% to 12% BSA gradient and processed as above. An aliquot of the final isolated platelets in HBSS were treated with 100 μΜ CCCP (Carbonyl cyanide 3-chlorophenylhydrazone, Sigma) for 1 hr at 37 °C to model platelet damage, then washed to remove CCCP. Untreated whole blood platelets were stored for 1 hr at 37 °C.
[00387] Equal volumes of whole blood, isolated whole blood derived and culture derived platelets were analyzed by flow cytometry to determine relative platelet
concentrations, which were then used to equalize the platelets injected for each group. Absolute platelet concentration was measured for whole blood using a Cell Dyne 3700 hematology analyzer. 2x106 platelets from each group (ex vivo culture, whole blood or damaged whole blood) were injected per mouse i.v. in the retro-orbital plexus of NOD/Shi- scid/IL2PiY null (NSG) mice depleted of macrophages by injection of liposomal clondronate (Clophosome, FormuMax) 24 hours prior to platelet injection. Peripheral blood of injected mice was collected retro-orbitally from the non-injected eye after 30 min, 2 hr, 4 hr, and 24 hr. 25 μΙ blood was stained for the human platelet markers CD41 a and CD42b and the mouse platelet marker CD41 (BD Biosciences), resuspended in 150 μΙ, and 100 μΙ was analyzed by flow cytometry.
[00388] The results indicated that ex vivo culture-derived platelets and donor blood platelets persisted in circulation with similar kinetics, whereas damaged platelets were more efficiently cleared from circulation with significantly decreased levels at both 4 hr and 24 hr after injection. Comparisons were performed in Graphpad Prism using the unpaired t- test (** P < 0.01 , * P < 0.05, ns not significant). This result is graphically demonstrated in Fig. 6.
Example G: In Vitro Functional Assay - Platelet Spreading
[00389] Human CD34+ cells were purchased as a CD34+ selected cord blood mixed donor vial (Allcells).
[00390] CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media
(StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows. The first culture phase (days 0 to 14) contained the following
recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems). CD34+ cells were seeded at 8x104 viable nucleated cells per ml, and cultures were maintained in culture bags (American Fluoroseal Corp) and supplemented with culture media to maintain cell density between 1 x105 and 6x105 viable nucleated cells per ml, as determined by trypan blue exclusion. For the second culture phase (days 14 to 18), cells were pelleted and resuspended on day 14 at 3x105 viable nucleated cells per ml in serum-free media containing TPO (50 ng/ml) and Reagent A (500 nM of (S)-2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1 - ol (Compound B157S)). A matrix metalloproteinase (MMP) inhibitor (25 μΜ of N-[(2R)-2- (Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM 6001 , EMD Chemicals)) was added at day 16. Cultures were maintained in culture bags
(American Fluoroseal Corp). Platelets were assessed at day 18, and 235 platelets were produced per seeded CD34+ cell.
[00391] Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
[00392] Platelets were isolated using a modification of a published protocol
(Robert, et al. Megakaryocyte and platelet production from human cord blood stem cells. 2012 Methods Mol Biol. 788:219-47). The culture was aliquotted into 50 ml tubes. Platelets were released by pipetting each aliquot up and down 25 ml 10 times with a 25 ml serological pipette fitted with a P1000 pipette tip to the end. Each aliquot was pelleted at l OOOxg for 15 min and resuspended in 2 ml citrate wash buffer (0.5% Citrate and 0.35% BSA in HBSS, adjusted to pH 6.5, with 50 ng/ml PGE1 added just before use), which was then layered on a 2% to 12% BSA step gradient. The platelet-containing upper phase was collected after spinning at 80xg for 15 min with no brake or acceleration and washed with DPBS containing 1 % BSA and 50 ng/ml PGE1 . The washed platelets were pooled in serum free TPO containing media.
[00393] Platelets were added to fibrinogen coated chamber slides in the presence or absence of 1 U/ml thrombin and incubated for 30 min at 37C. After washing gently with warm PBS, adherent platelets were fixed by addition of 4% formaldehyde in PBS. Platelets were permeabilized with 0.1 % TritonX-100 and blocked with 1 %BSA in PBS before staining with an a-tubulin directed primary antibody (clone DM1 A, Sigma) and fluorescently conjugated goat anti-mouse F(ab')2 secondary antibody (Invitrogen). Slides were mounted with Prolong Gold (Invitrogen) and visualized by immunofluorescence microscopy.
[00394] The culture-derived platelets are function in vitro. As shown in Figure 7, the culture-derived platelets exhibited cell spreading on fibrinogen-coated surface in response to thrombin activation.
Example H: Comparing the Effectiveness of Cytokines In Phase 2 Culture: TPO Alone or Four Cytokines Combination
[00395] Human CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols. CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems). CD34+ cells were seeded at 5x104 viable nucleated cells per ml (count prior to cryopreservation). Cultures were maintained in non-tissue culture treated culture plates and supplemented with culture media as necessary prior to megakaryocyte isolation (maintained between 3 x104 and 1 .4x106 when monitored). CD61 + cells from day 12 cultures were isolated using magnetic beads per the manufacturer's instructions (Milteni). Isolated cells were pelleted and resuspended at 4x105 viable nucleated cells per ml in culture media as above (4 cytokines) or serum free media containing 100 ng/ml TPO, with addition of vehicle control or Reagent B (250 nM of Compound B157S). Cells were then cultured as above to day 18.
[00396] Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate.
[00397] As shown in Fig. 8, TPO alone with Reagent B (250 nM of Compound
B157S) in Stage 2 produced best platelet yield.
Example I: TPO Dose Response for Stage 2 Culture
[00398] Human CD34+ cells were purchased as a CD34+ selected cord blood mixed donor vial (Allcells). CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows. The first culture phase (days 0 to 14) contained the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems). CD34+ cells were seeded at 5x104 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 5x104 and 2.4x106 viable nucleated cells per ml, as determined by trypan blue exclusion. At day 14, cultures were 18% megakaryocytes. For the second culture phase (days 14 to 18), cells were pelleted and resuspended on day 14 at 3x105 viable nucleated cells per ml in serum-free media containing Reagent B (750 nM of Compound B157S). Cultures were plated in non-tissue culture treated plates. TPO was added in a 12 point dose response at 2-fold dilutions from 100 ng/ml to 50 pg/ml and cultured to day 18 before platelet analysis.
[00399] Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
[00400] As shown in Fig. 9, the effective range for TPO in Stage 2 culture was between 0.001 ng/mL and 200 ng/mL. Example J: Dose Response Curve for Reagent B.
[00401] Human CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols or purchased as a CD34+ selected mixed cord blood donor vial (Allcells). CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems). Cultures were maintained in non-tissue culture treated culture plates and supplemented with culture media as necessary prior to megakaryocyte isolation. Day 10 cultures were pelleted and stained with antibodies as below. CD45RA-CD41 +CD42+ cells were sorted by flow cytometry. Sorted cells were pelleted and resuspended at 2.8x105 cells/ml in culture media as above. Various concentration of vehicle control, Compound B1 , Compound B88 control and Compound B157S were added. Cells were then cultured as above to day 16.
[00402] Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate.
[00403] The response curves for the three compounds are shown in Fig. 10. It is demonstrated that among compounds of Formula II that are suitable for use as Reagent B, the effective concentration is significantly different; hence the effective concentration should be separately determined.
Example K: Optimized Protocol Without MMP Inhibitor
[00404] Human CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols or purchased as a CD34+ selected cord blood obtained from mixed donors or a single donor (Allcells). CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows. The first culture phase (days 0 to 14) contained Reagent A (500 nM of Compound A1 (also referred to as MK1 ) and the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems). CD34+ cells were seeded at 5-50x104 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 9x104 and 1 .4x106 viable nucleated cells per ml, as determined by trypan blue exclusion. For the second culture phase (days 14 to 18), cells were pelleted and resuspended on day 14 at 5-9x105 viable nucleated cells per ml in serum-free media containing TPO (100 ng/ml) and Reagent B (750 nM of Compound B1 ). Cultures were maintained in culture bags (American Fluoroseal Corp) or in non-tissue culture treated plates.
[00405] Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
Megakaryocyte characterization at day 14
N Mean +/- SEM Median Range
% Megakaryocytes (MK) 3 40 +/- 8 40 26-55
MK per seeded cell at day 0 3 505 +/- 177 428 267-1458 Fold increase in MK over - MK1
1 3.3
control
Platelet yield per seeded cell at day 0
N Mean +/- SEM Median Range
4 840 +/- 335 588 429-1503
[00406] Platelet yields with this protocol were on average 3-fold increased over the baseline protocol with cytokines only in Example A.
[00407] For two of these samples, platelets were isolated and further assessed.
Platelets were isolated using a modification of a published protocol (Robert, et al.
Megakaryocyte and platelet production from human cord blood stem cells. 2012 Methods Mol Biol. 788:219-47). The culture was aliquotted into tubes and platelets were released by pipetting each aliquot up and down with a serological pipette fitted with a P1000 pippete tip to the end. Each aliquot was pelleted at 10OOxg for 10 min and resuspended in 2 ml citrate wash buffer (0.5% Citrate and 0.35% BSA in HBSS, adjusted to pH 6.5, with 50 ng/ml PGE1 added just before use), which was then layered on a 2% to 12% BSA step gradient. The platelet-containing upper phase was collected after spinning at 80xg for 15 min with no brake or acceleration and washed with DPBS containing 1 % BSA and 50 ng/ml PGE1 . The washed platelets were pooled in HBSS and stored for 1 hr at 37C.
[00408] ACD-anticoagulated whole blood platelets were isolated in a similar manner for comparison. Platelet rich plasma supernatant was collected after centrifugation at 200xg. Platelets were washed in citrate wash buffer before loading on the 2% to 12% BSA gradient and processed as above.
[00409] Platelets were further assessed for viability by fluorescence with the dye
Calcein, AM (Life Technologies). Cells were stained with 2 μΜ Calcein AM for 15 minutes at 37C, along with cell surface antibodies, washed and analyzed by flow cytometry. The % Calcein AM+ cells was assessed in the platelet population as shown for Example E.
% platelet viability (Calcein AM+) at day 18
% of culture % of blood control
sample
platelets platelets
1 99 97
2 99 99
[00410] The data shows vast majority of platelets generated with this protocol were viable.
[00411] Platelets were assessed for young platelets (reticulocytes) by staining with the dye thiazole orange (Sigma). Cells were stained with 20 μg/ml thiazole orange for 15 minutes at 37C, along with cell surface antibodies, washed and analyzed by flow cytometry. % young platelets (thiazole orange+) at day 18
% of culture % of blood control
sample
platelets platelets
1 76 1
2 44 1
[00412] The data shows a greater percentage of these platelets generated with this protocol were young platelets, compared with blood platelets (which on average will be at their half-life). This indicates the potential for a longer lifespan of culture-derived platelets compared with donor blood platelets.
Example L
Functional Assay of Platelets from Optimized Protocol Without MMP Inhibitor
[00413] Human CD34+ cells were purchased as CD34+ selected cord blood obtained from a single donor (Allcells). CD34+ cells were cultured at 37°C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows. The first culture phase (days 0 to 14) contained Reagent A, 500 nM of Compound A1 (also referred to as MK1 ) and the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems). CD34+ cells were seeded at 2x105 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 1 x105 and 1 x106 viable nucleated cells per ml, as determined by trypan blue exclusion. For the second culture phase (days 14 to 18), cells were pelleted and resuspended on day 14 at 9x105 viable nucleated cells per ml in serum- free media containing TPO (100 ng/ml) and Reagent B, 750 nM of Compound A1 . Cultures were maintained in culture bags (American Fluoroseal Corp).
[00414] Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer. [00415] Platelets were isolated using a modification of a published protocol
(Robert, et al. Megakaryocyte and platelet production from human cord blood stem cells. 2012 Methods Mol Biol. 788:219-47). The culture was aliquotted into 200 ml tubes.
Platelets were released by pipetting each aliquot up and down 12 times with a 50 ml serological pipette fitted with a P1000 pippete tip to the end. Each aliquot was pelleted at 1000xg for 15 min and resuspended in 2 ml citrate wash buffer (0.5% Citrate and 0.35% BSA in HBSS, adjusted to pH 6.5, with 50 ng/ml PGE1 added just before use), which was then layered on a 2% to 12% BSA step gradient. The platelet-containing upper phase was collected after spinning at 80xg for 15 min with no brake or acceleration and washed with DPBS containing 1 % BSA and 50 ng/ml PGE1 . The washed platelets were pooled in HBSS and stored for 1 hr at 37C.
[00416] ACD-anticoagulated whole blood platelets were isolated in a similar manner for comparison. Platelet rich plasma supernatant was collected after centrifugation at 200xg. Platelets were washed in citrate wash buffer before loading on the 2% to 12% BSA gradient and processed as above. An aliquot of the final isolated platelets in HBSS were treated with 100 μΜ CCCP (Carbonyl cyanide 3-chlorophenylhydrazone, Sigma) for 1 hr at 37C to model platelet damage, then washed to remove CCCP. Untreated whole blood platelets were stored for 1 hr at 37C.
[00417] Equal volumes of whole blood, isolated whole blood derived and culture derived platelets were analyzed by flow cytometry to determine relative platelet
concentrations, which were then used to equalize the platelets injected for each group. Absolute platelet concentration was measured for whole blood using a Cell Dyne 3700 hematology analyzer. 2.6x107 platelets from each group (ex vivo culture, whole blood or damaged whole blood) were injected per mouse i.v. in the retro-orbital plexus of NOD/Shi- scid/IL2PiY null (NSG) mice. Peripheral blood of injected mice was collected retro-orbitally from the non-injected eye after 30 min, 2 hr, 4 hr, and 24 hr. 25 μΙ blood was stained for the human platelet markers CD41 a and CD42b and the mouse platelet marker CD41 (BD Biosciences), resuspended in 150 μΙ, and 100 μΙ was analyzed by flow cytometry.
[00418] The results indicated that ex vivo culture-derived platelets and donor blood platelets persisted in circulation with similar kinetics, whereas damaged platelets were more efficiently cleared from circulation with significantly decreased levels at 4 hr after injection. Comparisons were performed in Graphpad Prism using the unpaired t-test (** P < 0.01 , * P < 0.05, ns not significant). The results are graphically illustrated in Fig. 1 1 . Example M: Compare Platelet Yield With or Without MMP Inhibitors
[00419] Human CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols. CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows. The first culture phase (days 0 to 14) contained Reagent A, 500 nM of Compound A1 (also referred to as MK1 ) and the following recombinant human cytokines:
thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems). CD34+ cells were seeded at 8.5x104 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 1 -10x105 viable nucleated cells per ml, as determined by trypan blue exclusion. For the second culture phase (days 14 to 18), cells were pelleted and resuspended on day 14 at 5x105 viable nucleated cells per ml in serum-free media containing TPO (100 ng/ml) and Reagent B, 750 nM of Compound B1 . Cultures were maintained in non-tissue culture treated plates. A matrix metalloproteinases (MMP) inhibitor or vehicle control was added at day 16 as follows: 25 μΜ of N-[(2R)-2- (Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan Methylamide (GM 6001 , EMD Chemicals), or 10 μΜ of N-(R)-[2-(Hydroxyaminocarbonyl)methyl]-4-methylpentanoyl- L-naphthylalanyl-L-alanine, 2-aminoethyl Amide (TAPI-1 , EMD Chemicals).
[00420] Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
[00421] It is shown that addition of MMP to Stage 2 culture increase platelets yield by 33%. The result is illustrated in a bar-graph in Fig. 12
Example N: Duration of the contacting with Reagent A
[00422] Human CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols. CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows. The first culture phase (days 0 to 14) contained the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems). At day 0, 500 nM of Reagent A (Compound A1 , or MK1 ) was added. CD34+ cells were seeded at 8.5x104 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media. On days 4, 7, and 10, the cultures were pelleted and resuspended at 1 x105 viable nucleated cells per ml, as determined by trypan blue exclusion, with the first culture phase media above. An aliquot at each time point was set aside for further culture without MK1 . MK1 was added at 500 nM to the remaining culture at each time point. For the second culture phase (days 14 to 18), cells were pelleted and resuspended on day 14 at 5x105 viable nucleated cells per ml in serum-free media containing TPO (100 ng/ml) and Reagent B (750 nM of Compound B1 ). Cultures were maintained in non-tissue culture treated plates.
[00423] Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
[00424] Fig. 13 shows the optimal duration for the contacting with Reagent A is 14 day.
Example O: Evaluate Method of Transferring Megakaryocyte between Stage 1 and Stage 2 Culture.
[00425] Human CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols. CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows. The first culture phase (days 0 to 12) contained the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems). CD34+ cells were seeded at 8x104 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 5x104 and 6x105 viable nucleated cells per ml, as determined by trypan blue exclusion. At day 12, cultures were pelleted and either (for 2-stage cultures) resuspended at 4x105 viable nucleated cells per ml in serum-free media containing TPO (100 ng/ml) or (for isolation cultures) CD61 + cells were isolated using magnetic beads per the manufacturer's instructions (Milteni) and resuspended at 4x105 viable nucleated cells per ml in serum-free media containing TPO (100 ng/ml). Vehicle or Reagent B, 250 nM of Compound B157S, was added to each condition. Cells were cultured to day 17 in non- tissue culture treated plates before analysis for platelets.
[00426] Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
[00427] It is shown that the "pelleting and resuspension" method of transferring megakaryocytes produced the highest platelet yield. The result is illustrated in Figure 14.
Example P: Isolation of Megakaryocyte from Stage 1 Culture
[00428] Human CD34+ cells were purified from fresh cord blood (Bioreclamation) using direct CD34 progenitor cell isolation kits (Miltenyi Biotec) following manufacturers protocols or purchased as a CD34+ selected mixed cord blood donor vial (Allcells). CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media (StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and the following recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems). CD34+ cells were seeded at 2.5-25x104 viable nucleated cells per ml (count prior to cryopreservation). Cultures were maintained in non-tissue culture treated culture plates and supplemented with culture media as necessary prior to megakaryocyte isolation (maintained between 3 x104 and 1 .4x106 when monitored).
[00429] Megakaryocytes were isolated by one of the following methods:
1 . Day 10 or day 1 1 cultures were pelleted and stained with antibodies as below.
CD45RA-CD41 + cells (one experiment) or CD45RA-CD41 +CD42+ (remaining experiments) were sorted by flow cytometry. Sorted cells were pelleted and resuspended in culture media as above, with addition of vehicle control and Reagent B: 750 nM Compound B1 , 750 nM Compound B88 control, or 250 nM of Compound B157S. Cells were then cultured as above to day 16. 2. CD61 + cells from day 12 or day 13 cultures were isolated using magnetic beads per the manufacturer's instructions (Milteni). Isolated cells were pelleted and resuspended in culture media as above, with addition of vehicle control, Reagent B: 750 nM of
Compoudn B1 or 250 nM of Compound B157S. Cells were then cultured as above to day 17 or day 18.
3. CD61 + cells from day 12 or day 13 cultures were isolated using magnetic beads per the manufacturer's instructions (Milteni). Isolated cells were pelleted and resuspended in culture media as above except that 100 ng/ml TPO as the only cytokine, with addition of vehicle control, Reagent B: 750 nM of Compound B1 or 250 nM of Compound B157S. Cells were then cultured as above to day 17 or day 18.
[00430] Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Fold increases in platelet yield over control from the same donor in multiple experiments were averaged, prior to the analysis below. N indicates the number of unique donors.
Platelet yield with Reagent B - fold over control
Protocol N Mean +/- SEM Median Range
1 6 10 +/- 3 12 3 - 21
2 3 57 +/- 21 73 15 - 84
3 2 185 +/- 173 185 12 - 358
Example Q: Isolation of Platelets by Centrifugation over a BSA Gradient
[00431] Human CD34+ cells were purchased as a CD34+ selected cord blood mixed or single donor vials (Allcells).
[00432] CD34+ cells were cultured at 37 °C in 5% C02 in serum-free media
(StemSpan SFEM [StemCell Technologies] supplemented with 1 x antibiotics) and additional factors as follows. The first culture phase (days 0 to 14) contained the following
recombinant human cytokines: thrombopoietin (TPO), IL6, Flt3 ligand, and stem cell factor (each 50 ng/mL, R&D Systems) with or without Reagent A, 250-500 nM of Compound A1 (also referred to as MK1 ). CD34+ cells were seeded at 3-20x104 viable nucleated cells per ml, and cultures were maintained in tissue culture flasks and supplemented with culture media to maintain cell density between 3x104 and 1 x106 viable nucleated cells per ml, as determined by trypan blue exclusion. For the second culture phase (days 14 to 18), cells were pelleted and resuspended on day 14 at 1 -9x105 viable nucleated cells per ml in serum- free media containing TPO (50-100 ng/ml) and Reagent B: 500 nM of Compound B157S or 750 nM of Compound B1. A matrix metalloproteinase (MMP) inhibitor, 25 μΜ of N-[(2R)-2- (Hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan Methylamide (GM 6001 , EMD Chemicals) was added in two of the three experiments at day 16. Cultures were maintained in culture bags (American Fluoroseal Corp).
[00433] Cell phenotyping was performed by flow cytometry by staining with antibodies directed against the cell surface markers CD34 (clone 4H1 1 , eBioscience), CD41 a (clone HIP8, eBioscience), CD42b (clone HIP1 , eBioscience) and CD45RA (clone HI100, eBioscience). Nucleated cells were distinguished from platelets by size (FSC) and granularity (SSC). Megakaryocytes were defined as CD41 a+ CD45RA- cells in the nucleated cell FSC/SSC gate. Platelets were defined as CD41 a+ CD42b+ cells in the platelet FSC/SSC gate. Platelet concentration in the final sample was determined by multiplying the platelets per nucleated cell by flow cytometry by the viable nucleated cell concentration by trypan blue exclusion using a hemacytometer.
[00434] Platelets were isolated using a modification of a published protocol
(Robert, et al. Megakaryocyte and platelet production from human cord blood stem cells. Methods Mol Biol. 2012 788:219-47). The culture was aliquotted and platelets were released by pipetting each aliquot up and down with a serological pipette fitted with a P1000 pipette tip to the end. Each aliquot was pelleted at 10OOxg for 15 min and resuspended in 2 ml citrate wash buffer (0.5% Citrate and 0.35% BSA in HBSS, adjusted to pH 6.5, with 50 ng/ml PGE1 added just before use), which was then layered on a 2% to 12% BSA step gradient. The platelet-containing upper phase was collected after spinning at 80xg for 15 min with no brake or acceleration and washed with DPBS containing 1 % BSA and 50 ng/ml PGE1 . The washed platelets were pooled in serum free TPO containing media or HBSS.
[00435] For the purpose of the following analysis, the percent platelets were calculated as the number platelets divided by the sum of the number of platelets and nucleated cells. Platelet isolation at day 18
% platelet
% platelets % platelets
Sample recovery on
pre-isolation post-isolation
isolation
1 25 100 29
2 27 100 57
3 40 100 16
[00436] A representative pseudocolor density plots displaying fluorescence intensity of particles analyzed by flow cytometry of Sample 1 was shown in Fig. 15. Culture- derived platelets before and after isolation were analyzed in comparison with isolated blood platelets. Gating is as shown. Viable nucleated cells were gated based on scatter properties. Platelets were gated based on scatter properties and CD41 a and CD42b bound fluorescence intensity. These gates were used to calculate the %platelets and platelet recovery.
[00437] The data shows the isolation method effectively purified platelets from nucleated cells in the culture.
Examples of Reagent A (Compound of Formula I)
[00438] The present invention is further exemplified, but not limited, by the following examples that illustrate the compounds of Formula I. These examples are also summarized in Table I.
Example A1 : 3-(7-((2,6-dimethylpyridin-3-yl)amino)-1 -methyl-2-oxo-1 ,2- dihydropyrimido[4,5-d rimidin-3(4H)-yl)-N-(3-(trifluoromethyl)phenyl)-4-vinylbenzamide
Figure imgf000087_0001
[00439] To a stirred solution of methyltriphenylphosphonium bromide (402 mg,
1 .15 mmol, 1 .5 eq) in THF at 0 °C under nitrogen is added sodium hexamethyldisilazide
(1 .15 ml of 1 M solution in THF, 1 .5 eq) and the resulting mixture is stirred at the same temperature for 20 min. To this mixture is added a solution of 4-carboxymethyl-2- nitrobenzaldehyde (157 mg, 0.75 mmol) in THF and the resulting mixture is stirred at 0 °C for 1 hr. Saturated aqueous ammonium chloride solution is added, then the mixture is diluted with ethyl acetate and the layers separated. The organic layer is washed with brine, dried over sodium sulfate, and concentrated. The residue is purified by silica gel column chromatography (7:1 hexanes/ethyl acetate eluant) to provide methyl 4-vinyl-3- nitrobenzoate. TLC (5:1 hexanes/EtOAc): Rf -0.3
[00440] A solution of methyl 3-nitro-4-vinylbenzoate (40 mg, 0.21 mmol) in THF is treated with 2N aqueous sodium hydroxide solution (0.5 ml, 1 .0 mmol) and the mixture is stirred at rt for 2 hr. The mixture is concentrated under reduced pressure and washed with chloroform. The remaining aqueous layer is acidified with 1 N hydrochloric acid and the mixture is extracted 3x with chloroform. The combined acidic chloroform extracts are dried over sodium sulfate and concentrated to provide 3-nitro-4-vinylbenzoic acid, which is used without further purification.
[00441] To a solution of 3-nitro-4-vinylbenzoic acid (18 mg, 0.093 mmol), 3- trifluoromethylaniline (18 mg, 1 .2 eq)), DIEA (50 ul, 0.29 mmol), and DMF (1 ml) is added HATU (43 mg,1 .2 eq), and the mixture is stirred for 1 h at room temperature. The reaction mixture is diluted with EtOAc and washed with 5% aqueous Na2S203 solution, saturated aqueous NaHC03 solution, and brine. The organic layer is dried over MgS04 and concentrated under reduced pressure. The residue is purified by silica gel column chromatography (5:1 hexanes/ethyl acetate eluant) to provide 3-nitro-N-(3- (trifluoromethyl)phenyl)-4-vinylbenzamide.
[00442] A mixture of 3-nitro-N-(3-(trifluoromethyl)phenyl)-4-vinylbenzamide (1 eq), iron powder (4.2 mg, 5 eq), and ethanol/concentrated hydrochloric acid (5:1 ) is heated with stirring at 80 °C for 20 min. Upon completion of the reaction, the cooled mixture is poured into a mixture of ethyl acetate and 4N aqueous sodium hydroxide solution. The organic layer is washed with brine, dried over sodium sulfate, and concentrated. The residue is purified by silica gel chromatography (4:1 hexanes/ethyl acetate eluant) to provide 3-amino- N-(3-(trifluoromethyl)phenyl)-4-vinylbenzamide.
[00443] A solution of 2-chloro-4-methylamino-pyrimidine-5-carbaldehyde (80 mg,
0.47 mmol) and 3-amino-N-(3-(trifluoromethyl)phenyl)-4-vinylbenzamide (130 mg, 0.42 mmol) in MeOH (2 ml_) is stirred for 2 hours at rt, and then it is treated with sodium cyanoborohydride (80 mg, 1 .27 mmol), acetic acid (50 uL), and DMF (0.5 ml) sequentially. After stirring for 2 hours at 50 °C, the reaction mixture is diluted with CHCI3 and washed with saturated NaHC03. The organic layer is dried over MgS04 and concentrated under reduced pressure. The residue is purified by silica gel column chromatography (3:1 hexanes/ethyl acetate eluant) to give 3-(((2-chloro-4-(methylamino)pyrimidin-5-yl)methyl)amino)-N-(3- (trifluoromethyl)phenyl)-4-vinylbenzamide. [00444] To a stirred solution of 3-(((2-chloro-4-(methylamino)pyrimidin-5- yl)methyl)amino)-N-(3-(trifluoromethyl)phenyl)-4-vinylbenzamide (1 14 mg, 0.25 mmol) and diisopropylethylamine (95 μΙ_, 2.2 eq) in THF is added triphosgene (27 mg, 1 .1 eq) and the mixture is stirred at rt for 30 min. To the mixture is added sodium hexamethyldisilazide (1 M solution in THF, 0.5 ml, 2.0 eq), and the mixture is stirred for 2 hr at rt. The reaction mixture is then diluted with EtOAc and washed with saturated NaHC03. The organic layer is dried over MgS04 and concentrated. The residue is purified by crystallization from methanol to provide 3-(7-chloro-1 -methyl-2-oxo-1 ,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N-(3- (trifluoromethyl)phenyl)-4-vinylbenzamide.
[00445] A mixture of 3-(7-chloro-1 -methyl-2-oxo-1 ,2-dihydropyrimido[4,5- d]pyrimidin-3(4H)-yl)-N-(3-(trifluoromethyl)phenyl)-4-vinylbenzamide (20 mg, 0.041 mmol), 3-amino-2,6-dimethylpyridine (10 mg, 0.13 mmol), palladium acetate (2 mg, 0.009 mmol), xantphos (7 mg, 0.012 mmol), cesium carbonate (30 mg, 0.098 mmol), and dioxane (2 ml) is heated at 130 °C in a sealed vessel with stirring for 1 h. The cooled reaction mixture is subjected to preparative LCMS purification to provide the title compound..
[00446] MS m/z 574.4(M + 1 )
Example A2: N-(4-Methyl-3-(1 -methyl-2-oxo-7-((3-(2-oxopyrrolidin-1 -yl)propyl)amino)-1 ,2- dihydropyrimido[4,5-d rimidin-3(4H)-yl)phenyl)-3-(trifluoromethyl)benzamide
Figure imgf000089_0001
[00447] /V-[3-(7-Chloro-2-oxo-1 ,4-dihydro-2H-pyrimido[4,5-d]pyrimidin-3-yl)-4- methylphenyl]-3-trifluoromethylbenzamide (prepared in Example 1 ) is treated with 1 -(3- aminopropyl)-2-pyrolidinone (5 eq) with stirring at 60 °C for 5 hours. The cooled mixture was purified by prep LCMS to provide the title compound.
[0002] MS m/z 582.2 (M + 1 )
Example A3: N-(3-(2-Amino-8-methyl-7-oxo-7,8-dihydropteridin-6-yl)-4-methylphenyl)-3-(4- methyl-1 H-imidazol-1 -yl)-5-(trifluoromethyl)benzamide
Figure imgf000090_0001
[00448] 10% palladium on carbon (165 mg) is added to a mixture of 5-nitro-N4- methylpyrimidine-2,4-diamine (425 mg, 2.44 mmol), ethanol (50 ml), and THF (30 ml). The mixture is hydrogenated under a hydrogen atmosphere (1 atm) for 16 h. The mixture is filtered through a silica gel plug, eluting with 2-propanol. An excess of 6N hydrochloric acid is added to the filtrate. Concentration provides N4-methylpyrimidine-2,4,5-triamine.
[00449] To a solution of 3-bromo-4-methylaniline (1 .27 g, 6.82 mmol) in DCM (20 mL) at OoC is added acetic anhydride (0.676 mL, 7.16 mmol) dropwise. After 30 minutes, the mixture is partitioned between DCM and saturated sodium carbonate solution. The layers are separated and the aqueous layer is extracted with DCM. The combined organic extracts are washed with brine, dried over Na2S04, filtered and concentrated to afford N- acetyl-3-bromo-4-methylaniline (1 .55 g, 99%). The residue is used in the next step without further purification.
[00450] To a solution of N-acetyl-3-bromo-4-methylaniline (1 .43 g, 6.27 mmol) in
THF (50 mL) at -78oC is added 2M BuLi in pentane (7.83 mL, 15.66 mmol) dropwise. After a further 1 h, diethyl oxalate (4.26 mL, 31 .3 mmol) is added. After stirring for 4 hours at - 78°C, the reaction mixture is quenched with saturated NH4CI solution. The mixture is partitioned between ethyl acetate and water. The layer is separated and the aqueous layer is extracted with ethyl acetate. The combined organic extracts are washed with brine, dried over Na2S04, filtered and concentrated. The residue is purified by column chromatography (silica gel, eluting with ethyl acetate : hexanes from 1/1 to 2/1 ) to afford ethyl 2-(5- acetylamino-2-methylphenyl)-2-oxoacetate (0.82 g, 52%).
[00451] A mixture of ethyl 2-(5-acetylamino-2-methylphenyl)-2-oxoacetate (303 mg, 1 .2 mmol), N4-methylpyrimidine-2,4,5-triamine (300 mg, HCI salt, 1 .2 mmol) and ethanol (12 mL) is heated at 100oC in a sealed tube for 16 h. 5N HCI in isopropanol (5 mL) is added and the mixture is heated at 95 oC for an additional 6 h before the solvent is removed under vacuum. The mixture is treated with saturated sodium carbonate solution, resulting in a precipitate. The solid is collected by filtration and dried to afford 2-amino-6-(5- amino-2-methylphenyl)-8-methylpteridin-7(8H)-one as a green-yellow powder.
[00452] To a solution of 2-amino-6-(5-amino-2-methylphenyl)-8-methylpteridin-
7(8H)-one (30 mg, 0.079 mmol), 3-(4-methyl-1 H-imidazol-1 -yl)-5-(trifluoromethyl)benzoic acid (23 mg, 0.085 mmol), DIEA (50 ul, 0.29 mmol), and DMF (2 ml) is added HATU (30 mg, 0.079 mmol), and the mixture is stirred for 1 h at room temperature. The reaction mixture is diluted with EtOAc and washed with 5% aqueous Na2S203 solution, saturated aqueous NaHC03 solution, and brine. The organic layer is dried over MgS04 and concentrated under reduced pressure. The residue is purified by preparative LC MS to provide the title compound.
[00453] MS m/z 535.17 (M + 1 ).
Example 4: N-(3-(1 -Cyclopropyl-7-((1 ,3-dimethyl-1 H-pyrazol-5-yl)amino)-2-oxo-1 ,2- dihydropyrimido[4,5-d] rimidin-3(4H)-yl)-4-methylphenyl)-3-(trifluoromethyl)benzamide
Figure imgf000091_0001
[00454] N-(3-(7-Chloro-1 -cyclopropyl-2-oxo-1 ,2-dihydropyrimido[4,5-d]pyrimidin-
3(4H)-yl)-4-methylphenyl)-3-(trifluoromethyl)benzamide is prepared according to the precedure of Example 1 , substituting cyclopropylamine in place of methylamine in the first step.
[00455] A mixture of N-(3-(7-chloro-1 -cyclopropyl-2-oxo-1 ,2-dihydropyrimido[4,5- d]pyrimidin-3(4H)-yl)-4-methylphenyl)-3-(trifluoromethyl)benzamide (20 mg, 0.042 mmol), 5- amino-1 ,3-dimethylpyrazole (0.13 mmol), palladium acetate (2 mg, 0.009 mmol), xanthphos (7 mg, 0.012 mmol), cesium carbonate (30 mg, 0.098 mmol), and dioxane (2 ml) is heated at 150 oC under microwave irradiation with stirring for 30 min. The cooled reaction mixture was subjected to prep LCMS purification to provide the title compound.
[00456] MS m/z 577.1 (M + 1 )
Example 5: N-(3-(7-Amino-1 -methyl-2-oxo-1 ,2-dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-4- methylphenyl)-3-(trifluoro
Figure imgf000091_0002
[00457] 5-Bromo-2,4-dichloro-pyrimidine (2.41 g, 10.6 mmol) is slowly treated with methylamine (8 M in EtOH, 3.3 ml_) in THF (15 ml_) at about -20°C. After stirring for 30 minutes at about -20 °C, the reaction mixture is partitioned between CHCI3 and saturated NaHC03. The aqueous layer is extracted with additional CHCI3 twice and the combined organic layer is dried over MgS04, filtered and concentrated. The crude product is purified by column chromatography (Si02, EtOAc/Hexane = 3/7) to give 1 .76 g (75%) of (5-bromo-2- chloro-pyrimidin-4-yl)-methylamine as a white solid.
[00458] A mixture of (5-bromo-2-chloro-pyrimidin-4-yl)-methylamine (3.75 g, 16.9 mmol), tris(dibenzylidineacetone)dipalladium(0) (388 mg, 0.4 mmol), and tri-2- furylphosphine (777 mg, 3.3 mmol) in DMF is stirred for 20 minutes at room temperature and then tributylvinyltin (5.93 mL, 20.3 mmol) is added. After stirring for 16 hours at about 65°C, the reaction mixture is cooled to room temperature and stirred with a 10% aqueous solution of potassium fluoride (800 mL) and diethyl ether (600 mL) for 1 hour before filtering through a pad of Celite. The pad of Celite is rinsed with a further portion of diethyl ether (200 mL). The aqueous layer is separated and extracted with CHCI3. The combined organic extract is dried over MgS04 and concentrated under reduced pressure to give crude oil which is purified by flash column chromatography (Si02, EtOAc/Hx = 1/4) to afford (2- chloro-5-vinyl-pyrimidin-4-yl)-methylamine (2.63 g, 92%) as a white solid.
[00459] Ozone is bubbled through a solution of (2-chloro-5-vinyl-pyrimidin-4-yl)- methylamine (2.50 g, 14.7 mmol) in CHCI3/MeOH (15 mL/15 mL) for 30 minutes at -78°C and then a stream of argon is passed through the solution for 3 minutes at the same temperature. The reaction mixture is allowed to warm up to room temperature and treated with dimethyl sulfide (3.24 mL, 44.1 mmol). The reaction mixture is concentrated under reduced pressure to give colorless oil that is purified by flash column chromatography (Si02, EtOAc/Hx = 1/3) over silica gel to give 2-chloro-4-methylamino-pyrimidine-5-carbaldehyde (2.40 g, 95%) as a white solid.
[00460] A solution of 2-chloro-4-methylamino-pyrimidine-5-carbaldehyde (1 .08 g,
6.3 mmol) and N-(3-amino-4-methyl-phenyl)-3-trifluoromethylbenzamide (2.04 g, 6.9 mmol) in MeOH (70 mL) is stirred for 2 hours at 45 °C and then treated with sodium
cyanoborohydride (1 .19 g, 18.9 mmol) and acetic acid (1 mL) sequentially. After stirring for 2 hours at room temperature, the reaction mixture is diluted with CHCI3 and washed with saturated NaHC03. The organic layer is dried over MgS04 and concentrated under reduced pressure. The residue is purified by flash column chromatography (Si02,
EtOAc/hexane = 1/2) to give N-{3-[(2-chloro-4-methylaminopyrimidin-5-ylmethyl)amino]-4- methylphenyl}-3-trifluoromethylbenzamide (1 .80 g, 64%) as a white solid.
[00461] To a stirred solution of N-{3-[(2-chloro-4-methylaminopyrimidin-5- ylmethyl)amino]-4-methylphenyl}-3-trifluoromethylbenzamide (559 mg, 1 .24 mmol) and triethylamine (693 μί, 4.97 mmol) in THF (15 mL) is added triphosgene (147 mg, 0.49 mmol) in THF (5 mL) at 0 °C, and the mixture is stirred for 30 minutes at room temperature. The precipitate is filtered off and the filtrate is stirred for 3 hours at 1 10 °C. The reaction mixture is then diluted with EtOAc and washed with saturated NaHC03. The organic layer is dried over MgS04 and concentrated under reduced pressure to give crude oil which is purified by flash column chromatography (Si02, EtOAc/hexane = 1/2) to give W-[3-(7-chloro- 2-OXO-1 ,4-dihydro-2H-pyrimido[4,5-d]pyrimidin-3-yl)-4-methylphenyl]-3- trifluoromethylbenzamide (420 mg, 71 %) as a white solid.
[00462] /V-[3-(7-chloro-2-oxo-1 ,4-dihydro-2H-pyrimido[4,5-d]pyrimidin-3-yl)-4- methylphenyl]-3-trifluoromethylbenzamide (1050 mg, mmol) is dissolved in 2 M ammonia solution in 2-propanol (30 mL) and the mixture is stirred for 16 hours at 90 °C in a sealed tube. The cooled reaction mixture is concentrated. A portion is purified by silica gel chromatography (2% methanol in ethyl acetate as eluant) to provide the title compound.
[00463] MS m/z 471 .3 (M + 1 )
Table I: Examples of Compounds of Formula I (Reagent A)
Figure imgf000093_0001
Examples of Reagent B (Compounds of Formula II)
[00464] The present invention is further exemplified, but not limited, by the following examples that illustrate the compounds of Formula II. These examples are also summarized in Table II.
Example B1 : 4-(2-(2-(benzo[b]thio hen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol
Figure imgf000094_0001
[00465] Synthesis of 2,6-dichloro-9-isopropyl-9H-purine (b): To 2,6-dichloro-9H- purine (a) (6.0 mmol) dissolved in anhydrous DMF (5.0 ml_) was slowly added sodium hydride (7.8 mmol) with stirring at rt over 2 hr. 2-iodopropane was added and the mixture was stirred for 16 hr. The mixture was concentrated. The residue was purified by column chromatography on silica gel, eluting with hexane/EtOAc (20:1 to 3:1 ) to afford the title compound as a white solid. 1 H NMR (500 MHz, CDCI3): δ 8.15 (s, 1 H), 4.91 (m, 1 H), 1 .63 (d, 6H).
[00466] Synthesis of 4-(2-(2-chloro-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol
(c): 2,6-dichloro-9-isopropyl-9H-purine (1 .1 mmol) was mixed with tyramine (1 .16 mmol) dissolved in i-PrOH (6 ml) and the mixture was stirred overnight. The reaction mixture was concentrated, and the residue purified by column chromatography on silica gel, eluting with hexane/EtOAc (5:1 to 1 :2) to afford the title compound as a white solid. 1 H NMR (500 MHz, CDCI3): δ 9.21 (br, 1 H), 8.49 (s, 1 H), 7.80 (s, 1 H), 7.10 (d, 2H), 6.73 (d, 2H), 4.87 (m, 1 H), 4.03 (t, 2H), 3.01 (t, 2H), 1 .68 (d, 6H); HRMS (El) calcd for C16H18CIN50 (M + H+) 332.1273, found 332.1278.
[00467] Synthesis of 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol (d): A flame-dried schlenk flask was charged with 4-(2-(2-chloro-9- isopropyl-9H-purin-6-ylamino)ethyl)phenol (0.62 mmol), thianaphthene-3-boronic acid (0.94 mmol), pd2(dba)3 (0.062 mmol), Cs2C03 (1 .25 mmol) and 1 ,3-bis(2,4,6-trimethylphenyl) imidazolium chloride (0.125 mmol). The flask was evacuated and backfilled with N2 and anhydrous 1 ,4-dioxane (2 ml_) was added. The flask was sealed and the reaction mixture was stirred at 80 °C for 24 hours. The reaction mixture was concentrated and purified directly by column chromatography on silica gel, eluting with hexane/EtOAc (20:1 to 1 :4) to afford the title compound as a yellowish solid.
[00468] The compound of Example B1 can be recrystallised using a
toluene/ethanol mixture and washed at room temperature with NaHC03 aqueous solution.
Example B15: 4-(2-(Pyridin-3-yl)- -isopropyl-9H-purin-6-ylamino)ethyl)phenol
Figure imgf000095_0001
[00469] Synthesis of 2-Amino-6-chloro-9-isopropyl-9H-purine (b): Sodium hydride
(1.5 g of 60% dispersion in mineral oil, 38 mmol) was added in portions over 10 min to a stirred suspension of 2-amino-6-chloro-9H-purine (5.34 g, 31 .5 mmol) in anhydrous DMF (50 mL) at rt. After 45 min, the mixture was cooled in an ice bath, then 2-iodopropane was added. The cooling bath was removed and the stirred mixture was allowed to warm to rt over 16 h. The mixture was cooled in ice, then water was added. The mixture was concentrated, and the residue was treated with hot ethyl acetate. The cooled mixture was filtered, and the filtrate was concentrated. The residue was purified by column
chromatography on silica gel, eluting with 0 to 50% EtOAc in hexane to afford the title compound as a solid. 1 H NMR (400 MHz, CDCI3) δ 7.83(s, 1 H), 5.17(s, 2H), 4.71 -4.66(m, 1 H), 1 .57(d, 6H). MS m/z 212.1 (M + 1 ).
[00470] Synthesis of 6-Chloro-2-iodo-9-isopropyl-9/-/-purine (c): 6-chloro-9- isopropyl-9H-purin-2-amine (2.68 g, 12.7 mmol) was dissolved in THF (64 mL) at rt. Iodine (1 .61 g, 6.25 mmol), CH2I2 (10.6 mL) and Cul (1 .27 g, 6.66 mmol) were added. The mixture was stirred for 5min at room temperature. Isopentyl nitrite (5.33 mL) was added. The reaction mixture was refluxed for 45 min, and was then cooled to room temperature.
Saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted with EtOAc three times. The combined organic phase was washed with brine, dried with MgS04 and concentrated. The residue was purified by column chromatography on silica gel, eluting with 0 to 30% ethyl acetate in hexane to afford the title compound as a solid. 1 H NMR (400 MHz, CDCI3) δ 8.09(s, 1 H), 4.95-4.88(m, 1 H), 1 .65(d, 6H). MS m/z 323.0 (M +
1 )- [00471] Synthesis of 6-Chloro-2-(pyridin-3-yl)-9-isopropyl-9H-purine (d): A round- bottom flask was charged with 6-chloro-2-iodo-9-isopropyl-9H-purine (1 .2 g, 3.7 mmol), pyridine-3-boronic acid 1 ,3-propanediol cyclic ester (0.91 g, 5.6 mmol), and
tetrakis(triphenylphosphine)palladium(0) (430 mg, 0.37 mmol). To this mixture was added toluene (60 ml), ethanol (6 ml) and aqueous sodium carbonate solution (2M, 15 ml). The flask was sealed and the reaction mixture was stirred at 80 °C for 4 h. Water was added to the cooled mixture, which was extracted with ethyl acetate (50 ml x 3). The organic fractions were combined, dried over sodium sulfate, and concentrated. The residue was purified by column chromatography on silica gel, eluting with 30 to 70% EtOAc in hexane to afford the title compound as a solid. 1 H NMR (400 MHz, CD30D) δ 9.60(d, 1 H), 8.90-8.87(m, 1 H), 8.68(s, 1 H), 8.67(d, 1 H), 7.63-7.60(m, 1 H), 5.12-5.05(m, 1 H), 1 .74(d, 6H). MS m/z 274.1 (M + 1 ).
[00472] Synthesis of 4-(2-(pyridin-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol (e): 6-chloro-9-isopropyl-2-(pyridin-3-yl)-9H-purine (300 mg, 1 .1 mmol) was suspended in anhydrous 2-propanol (40 ml_) in a pressure tube. Tyramine (300 mg, 2.2 mmol) was added. The tube was sealed and heated to 85 °C for 16 hr. The reaction was concentrated and the residue was purified by column chromatography on silica gel, eluting with 0 to 70% EtOAc in hexane to afford the title compound as a solid.
Example B123: 4-(2-(9-lsopropyl-2-(2-methyl-1 H-imidazol-1 -yl)-9H-purin-6- ylamino)ethyl)phenol
Figure imgf000096_0001
[0001] A microwave reaction tube was charged with 4-(2-(2-chloro-9-isopropyl-
9H-purin-6-ylamino)ethyl)phenol (30 mg, 0.091 mmol), 2-methyl-1 H-imidazole (59 mg, 0.73 mmol) and 0.5 ml of NMP. The sealed tube was heated under microwave irradiation at 240 °C for 2 hr. The reaction mixture was purified by reverse-phase HPLC (C18 column, eluting with ACN-H20 0.05% TFA) to afford the title compound as an off-white solid. Example B128: 4-(2-(2-(5- hloropyridin-3-yl)-9-isopropyl-9H^urin-6-ylamino)ethyl)phenol
Figure imgf000097_0001
[00473] Synthesis of 4-(2-(2-iodo-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol (b):
A mixture of 6-chloro-2-iodo-9-isopropyl-9H-purine (a) (1 .0 g, 3.1 mmol), tyramine (0.64 g, 4.65 mmol), triethylamine (0.63 g, 6.2 mmol) and 2-propanol (30 ml_) was heated at 85 °C for 2 hr. The reaction mixture was concentrated and saturated aqueous sodium
bicarbonate solution was added. The mixture was extracted with ethyl acetate (50 ml x 3). The combined organic layers were dried over sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography on silica gel (25 to 75% ethyl acetate in hexane eluant) to afford the title compound as a solid. MS m/z 424.1 (M + 1 ).
[00474] Synthesis of 4-(2-(2-(5-chloropyridin-3-yl)-9-isopropyl-9H-purin-6- ylamino)ethyl)phenol (c): Following the procedure of Example B15d, 4-(2-(2-iodo-9- isopropyl-9H-purin-6-ylamino)ethyl)phenol (b) was reacted with 5-chloropyridin-3-ylboronic acid. The crude product was purified by reverse-phase HPLC (C18 column, eluting with ACN-H20 0.05% TFA) to afford the title compound as an off-white solid.
Example B134: 4-(2-(6-(5-Fluoropyridin-3-yl)-1 -isopropyl-1 H-pyrazolo[3,4-d]pyrimidin-4- ylamino)ethyl)phenol
Figure imgf000097_0002
[00475] Synthesis of 4-(2-(6-chloro-1 -isopropyl-1 H-pyrazolo[3,4-d]pyrimidin-4- ylamino)ethyl)phenol (b): Following the procedure of Example B128b, 4,6-dichloro-1 - isopropyl-1 H-pyrazolo[3,4-d]pyrimidine (US3399196) (a) (0.184 g, 0.795 mmol) was reacted with tyramine. The crude residue was purified by silica gel chromatography (25 to 75% ethyl acetate in hexane eluant) to afford the title compound as a solid. MS m/z 332.1 (M +
1 )-
[00476] Synthesis of 4-(2-(6-(5-fluoropyridin-3-yl)-1 -isopropyl-1 H-pyrazolo[3,4- d]pyrimidin-4-ylamino)ethyl)phenol (c): Following the procedure of Example B15d, 4-(2-(6- chloro-1 -isopropyl-1 H-pyrazolo[3,4-d]pyrimidin-4-ylamino)ethyl)phenol (b) was reacted with 5-fluoropyridin-3-ylboronic acid. The crude residue was purified by reverse-phase HPLC (Ci8 column, eluting with ACN-H20 0.05% TFA) to afford the title compound as an off-white solid.
Example B141 : 4-(2-(2-(5-Fluoropyridin-3-yl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4- ylamino)ethyl)phenol
Figure imgf000098_0001
[00477] Synthesis of 2,4-dichloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidine (b):
Following the procedure of Example B15b, 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (0.5 g, 2.67 mmol) was reacted with 2-iodopropane. The crude residue was purified by silica gel chromatography (15 to 25% ethyl acetate in hexane eluant) to afford the title compound as a solid. MS m/z 230.2 (M + 1 ).
[00478] Synthesis of 4-(2-(2-chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4- ylamino)ethyl)phenol (c): Following the procedure of Example B128b, 2,4-dichloro-7- isopropyl-7H-pyrrolo[2,3-d]pyrimidine (b) (0.278 g, 1 .21 mmol) was reacted with tyramine. The crude residue was purified by silica gel chromatography (25 to 75% ethyl acetate in hexane eluant) to afford the title compound as a solid. MS m/z 331 .1 (M + 1 ).
[00479] Synthesis of 4-(2-(2-(5-fluoropyridin-3-yl)-7-isopropyl-7H-pyrrolo[2,3- d]pyrimidin-4-ylamino)ethyl)phenol (d): Following the procedure of Example B15d, 4-(2-(2- chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)ethyl)phenol (20 mg, 0.06 mmol) was reacted with 5-fluoropyridin-3-ylboronic acid. The crude residue was purified by reverse-phase HPLC (C18 column, eluting with ACN-H20 0.05% TFA) to afford the title compound as an off-white solid.
Example B153: (R)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-9H-purin-6- ylamino)ethyl)phenol
Figure imgf000099_0001
[00480] Synthesis of (R)-2,6-dichloro-9-(tetrahydrofuran-3-yl)-9H-purine (b): A solution of 5,7- 2,6-dichloro-9H-purine (400 mg, 2.12mmol), (S)-tetrahydrofuran-3-ol (88 mg, 2.5 mmol) and triphenylphosphine (1 .0 g, 3.8 mmol) in anhydrous THF (30 ml_) was treated at -78 oC with diisopropyl azodicarboxylate (856 mg, 4.23 mmol). The reaction was allowed to warm to rt and was stirred for 16 hr. Saturated aqueous sodium bicarbonate solution was added and the mixture was extracted with ethyl acetate. The organic layers were combined, dried over sodium sulfate, and concentrated. The residue was purified by silica gel chromatography (10 to 80% ethyl acetate in hexane eluant) to afford a white solid which consisted of the title compound contaminated with triphenylphosphoxide. MS m/z 258.0 (M + 1 ).
[00481] Synthesis of (R)-4-(2-(2-chloro-9-(tetrahydrofuran-3-yl)-9H-purin-6- ylamino)ethyl)phenol (c): Following the procedure of Example B128b, (R)-2,6-dichloro-9- (tetrahydrofuran-3-yl)-9H-purine (b) was reacted with tyramine. The crude reaction mixture was purified by reverse-phase preparative HPLC. MS m/z 360.1 (M + 1 ).
[00482] Synthesis of (R)-4-(2-(2-(benzo[b]thiophen-3-yl)-9-(tetrahydrofuran-3-yl)-
9H-purin-6-ylamino)ethyl)phenol: Following the procedure of Example B15d, (R)-4-(2-(2- chloro-9-(tetrahydrofuran-3-yl)-9H-purin-6-ylamino)ethyl)phenol (c) was reacted with benzo[b]thiophen-3-ylboronic acid (22.3 mg, 0.125 mmol). The crude residue was purified by reverse-phase preparative HPLC to afford the title compound as an off-white solid.
Example B157: 2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9- yl)propan-1 -ol
Figure imgf000100_0001
[00483] Synthesis of methyl 2-(2,6-dichloro-9H-purin-9-yl)propanoate (b): A mixture of 2,6-dichloro-9H-purine (5.0 g, 26.5 mmol), methyl 2-bromopropanoate (5.3 g, 31 .7 mmol) and potassium carbonate (1 1 .0 g, 79.4 mmol) in anhydrous DMF (100 mL) was heated at 100 oC for 15h. Sat. aqueous sodium bicarbonate solution was added and reaction was extracted with ethyl acetate (150 ml X 3). The organic layers were combined, washed with brine, dried over sodium sulfate, and concentrated. The residue was purified by silica gel chromatography (10 to 80% ethyl acetate in hexane eluant) to afford the title compound as a white solid. MS m/z 275.0 (M + 1 ).
[00484] Synthesis of methyl 2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-chloro-9H-purin-
9-yl)propanoate (c): A mixture of methyl 2-(2,6-dichloro-9H-purin-9-yl)propanoate (b) (600 mg, 2.2 mmol), tryptamine (420 mg, 2.6 mmol) and 2-propanol (30 mL) was heated at 85oC in a sealed tube for 16 h. The reaction mixture was cooled to room temperature and concentrated. The residue was purified by silica gel chromatography (10 to 80% ethyl acetate in hexane eluant) to afford the title compound as a white solid. MS m/z 360.1 (M + 1 )-
[00485] Synthesis of methyl 2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3- yl)-9H-purin-9-yl)propanoate (d): A 150 ml pressure tube was charged with methyl 2-(6-(2- (1 H-indol-3-yl)ethylamino)-2-chloro-9H-purin-9-yl)propanoate (c) (300 mg, 0.75 mmol), 5- fluoropyridin-3-ylboronic acid (159 mg, 1 .1 mmol), tetrakis(triphenylphosphine)-palladium(0) (87 mg, 0.075 mmol), K3P04 (638 mg, 3.0 mmol), and anhydrous dioxane (15mL). The pressure tube was sparged with nitrogen and was sealed, then the reaction mixture was heated at 130 °C for 6 h with stirring. Water was added to the cooled mixture, and the mixture was extracted with ethyl acetate (50 ml x 3). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated. The residue was purified by silica gel chromatography (10 to 80% ethyl acetate in hexane eluant) to afford the title compound contaminated with a small amount of triphenylphosphine oxide. MS m/z 460.1 (M + 1 ). [00486] Synthesis of 2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H- purin-9-yl)propan-1 -ol: Lithium aluminum hydride (230 mg, 6.1 mmol) was added in portions to a 0 °C solution of methyl 2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3- yl)-9H-purin-9-yl)propanoate (282 mg, 0.61 mmol) in anhydrous THF (15 ml_). The stirred reaction mixture was allowed to warm to rt over 2h, then water was added carefully. The mixture was extracted with EtOAc (50 ml x 3). The organic fractions were combined, washed with brine, dried over sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (0 to 5% solvent B in dichloromethane; solvent B = 2M ammonia in methanol) to afford the partially purified title product. This was further purified by preparative TLC (5% solvent B in dichloromethane) to provide the title compound as a white solid.
Examples B157A & B157S: (R)-2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)- 9H-purin-9-yl)propan-1 -ol &(S)-2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)- 9H-purin-9-yl) ropan-1 -ol
Figure imgf000101_0001
[00487] (R/S)-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9- yl)propan-1 -ol was separated into the individual enantiomers using preparative chiral HPLC on a 21 x250 mm Lux-Cellulose-2 (Phenomenex) chiral column. A 3 mg/ml solution of the racemate in methanol was prepared and loaded onto the column with 0.5 ml solution per injection. The column was eluted with 85/7.5/7.5 hexane/ethanol/methanol at a flow rate of 20 mL/min for 25 min. Peaks 1 and 2 were eluted at 20 min and 22.5 min, respectively. Analytical chromatography was performed on a 4.6 x 100 mm Lux_Cellulose-2
(Phenomenex) chiral column, eluting with 90/5/5 hexane/ethanol/methanol at 1 mL/min for 20 min. Peaks 1 and 2 were eluted at 17.45 and 18.14 min, respectively. Example B157R: (R)-2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9- yl)propan-1 -ol
Figure imgf000102_0001
[00488] Synthesis of (R)-N-(2-(1 H - indol-3-y l)ethy I) -9- ( 1 -(benzyloxy)propan-2-yl)-2-
(5-fluoropyridin-3-yl)-9H-purin-6-amine (b): Following, in succession, the procedures of Example B153b (using 2,6-dichloro-9H-purine and (S)-1 -(benzyloxy)propan-2-ol as reactants), Example B153c (using tryptamine as reactant), and Example B153d (using 5- fluoropyridin-3-ylboronic acid as reactant), the title compound was obtained.
[00489] Synthesis of (R)-2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-
9H-purin-9-yl)propan-1 -ol (c): A solution of (R)- N-(2-(1 H-indol-3-yl)ethyl)-9-(1 - (benzyloxy)propan-2-yl)-2-(5-fluoropyridin-3-yl)-9H-purin-6-amine (b) (0.15 g, 0.29 mmol) in DCM (10 ml) was treated with BCI3 (1 M, 2.9 ml, 2.9 mmol) in DCM (10 ml) at -78 °C for 2 hr. 1 N aqueous sodium hydroxide solution was added, and the mixture was extracted with DCM. The combined organic extracts were dried over sodium sulfate, filtered, and concentrated and the residue was purified by silica gel column chromatography (5% MeOH in DCM eluant) to provide the title compound. MS m/z 432.2 (M + 1 ).
Example B157S: (S)-2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9- yl)propan-1 -ol
Figure imgf000102_0002
[00490] Following the procedure of Example B157R, but employing (R)-1 -
(benzyloxy)propan-2-ol in place of (S)-1 -(benzyloxy)propan-2-ol, the title compound was prepared. MS m/z 432.2 (M + 1 ). Example B161 : 4-(2-(6-(5-Fluoropyridin-3-yl)-1 -isopropyl-1 H-imidazo[4,5-c]pyridin-4- ylamino)ethyl)phenol
Figure imgf000103_0001
[00491] Synthesis of 4,6-dichloro-1 -isopropyl-1 H-imidazo[4,5-c]pyridine (b):
Following the procedure of Example B15b, 4,6-dichloro-1 H-imidazo[4,5-c]pyridine (J. Het. Chem. 1965, 196-201 ) (0.19 g, 1 .0 mmol) was reacted with 2-iodopropane. The residue was purified by silica gel chromatography (25 to 35% ethyl acetate in hexane eluant) to afford the title compound as a solid. MS m/z 230.2 (M + 1 ).
[00492] Synthesis of 4-(2-(6-chloro-1 -isopropyl-1 H-imidazo[4,5-c]pyridin-4- ylamino)ethyl)phenol (c): A mixture of 4,6-dichloro-1 -isopropyl-1 H-imidazo[4,5-c]pyridine (b) (40 mg, 0.17 mmol), tyramine (120 mg, 0.86 mmol), and 2-butanol (2 mL) was heated under microwave irradiation at 140 °C for 8 hr. The mixture was concentrated and the residue was purified by reverse-phase HPLC (C18 column, eluting with ACN-H20 0.05% TFA) to afford the title compound as an off-white solid. MS m/z 331 .1 (M + 1 ).
[00493] Synthesis of 4-(2-(6-(5-fluoropyridin-3-yl)-1 -isopropyl-1 H-imidazo[4,5- c]pyridin-4-ylamino)ethyl)phenol (d): A 5 ml microwave reaction vial was charged with 4-(2- (6-chloro-1 -isopropyl-1 H-imidazo[4,5-c]pyridin-4-ylamino)ethyl)phenol (c): (17 mg, 0.051 mmol), 5-fluoropyridin-3-ylboronic acid (72 mg, 0.51 mmol), and
tetrakis(triphenylphosphine)palladium(0) (36 mg, 0.031 mmol). To this mixture was added toluene (1 ml), ethanol (0.5 ml) and aqueous sodium carbonate solution (2M, 0.5 ml). The vial was sealed and the reaction mixture was stirred at 140 °C under microwave irradiation for 2 hours. Water was added to the cooled mixture, which was extracted with ethyl acetate (5 ml x 3). The organic fractions were combined, dried over sodium sulfate, and
concentrated. The residue was purified by reverse-phase HPLC (C18 column, eluting with ACN-H20 0.05% TFA) to afford the title compound as an off-white solid. Example B177: 4-(2-(5-(5-Fluoropyridin-3-yl)-3-isopropyl-3H-imidazo[4,5-b]pyridin-7- ylamino)ethyl)phenol
Figure imgf000104_0001
[00494] Synthesis of 5,7-dichloro-3-isopropyl-3H-imidazo[4,5-b]pyridine (b):
Following the procedure of Example B15b, 5,7-dichloro-3H-imidazo[4,5-b]pyridine (J. Med. Chem., 2007, 50, 828-834) (0.1 18 g, 0.624 mmol) was reacted with 2-iodopropane. The crude product mixture was purified by silica gel chromatography (25 to 35% ethyl acetate in hexane eluant) to afford a mixture of the title compound (major) and an isomeric product as a solid. MS m/z 230.2 (M + 1 ).
[00495] Synthesis of 4-(2-(5-chloro-3-isopropyl-3H-imidazo[4,5-b]pyridin-7- ylamino)ethyl)phenol (c): The product mixture containing 5,7-dichloro-3-isopropyl-3H- imidazo[4,5-b]pyridine (b) (40 mg, 0.17 mmol), tyramine (120 mg, 0.87 mmol), and 2- propanol (2 ml_) was heated in a sealed vial at 140 °C for 72 hr. The mixture was concentrated, and the residue was purified by preparative TLC (1 :2 hexanes/ethyl acetate eluant) to afford the title compound as an off-white solid. MS m/z 331 .1 (M + 1 ).
[00496] Synthesis of 4-(2-(5-(5-fluoropyridin-3-yl)-3-isopropyl-3H-imidazo[4,5- b]pyridin-7-ylamino)ethyl)phenol (d): Following the procedure of Example B161 d, 4-(2-(5- chloro-3-isopropyl-3H-imidazo[4,5-b]pyridin-7-ylamino)ethyl)phenol (c) (15 mg, 0.047 mmol) was reacted with 5-fluoropyridin-3-ylboronic acid. The crude residue was purified preparative TLC (1 :1 hexanes/ethyl acetate eluant) to afford the title compound as an off- white solid.
[00497] By repeating the procedures described in the above examples, using appropriate starting materials, the following compounds of Formula II, as identified in Table 11 , are obtained. Table II. Exmepfied Examples of Compounds of Formula II (Reagent B)
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
109
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
[00498] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.
SEQUENCE LISTING
SEQ ID NO: 1
gcggcataga gaccgactta atttcaagag aattaagtcg gtctctatgc cgcttttttg 60 g 61
SEQ ID NO: 2
cgcgccaaaa aagcggcata gagaccgact taattctctt gaaattaagt cggtctctat 60 gccgc 65
SEQ ID NO: 3
ggcttctttg atgttgcatt aattcaagag attaatgcaa catcaaagaa gccttttttg 60 g 61
SEQ ID NO: 4
cgcgccaaaa aaggcttctt tgatgttgca ttaatctctt gaattaatgc aacatcaaag 60 aagcc 65

Claims

CLAIMS WE CLAIM :
1 . An ex vivo process for producing a population of ex vivo produced platelets (exPLTs) comprising a Step C, which comprises:
contacting a population of ex vivo produced megakaryocytes (exMKs) with Reagent B and thrombopoietin (TPO), wherein Reagent B is a compound of Formula
Ila:
Figure imgf000135_0001
or a pharmaceutically acceptable salt, or stereoisomer thereof; wherein
L is selected from -NR5A(CH2)2_3- -NR5A(CH2)2NR5B-, -NR5A(CH2)2S- - NR5ACH2CH(OH)- and -NR5ACH(CH3)CH2-; wherein R5A and R5B are independently selected from hydrogen and C1 -4alkyl;
RT is selected thiophenyl, furanyl, benzoimidazolyl, isoquinolinyl,
imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyrazolyl, pyridinyl, imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, pyrrolyl and thiazolyl; wherein said thiophenyl, furanyl, benzoimidazolyl, isoquinolinyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyrazolyl, pyridinyl, imidazolyl, pyrrolidinyl, pyrazinyl, pyridazinyl, pyrrolyl or thiazolyl of is optionally substituted by 1 to 3 radicals independently selected from halo, cyano, d- 4alkyl, halo-substituted-d^alkyl, C1-4alkoxy,-S(O)0-2R8a, and -C(0)OR8A, wherein R8A is selected from hydrogen and Ci-4alkyl;
R2 is selected from -S(0)2NR6AR6B, -NR6AC(0)N R6BR6C, phenyl, pyrrolopyridinyl, indolyl, thiophenyl, pyridinyl, triazolyl, 2-oxoimidazolidinyl, pyrazolyl, and indazolyl; wherein
R6A, R6B and R6C are independently selected from hydrogen and Ci-4alkyl; and
said phenyl, pyrrolopyridinyl, indolyl, thiophenyl, pyridinyl, triazolyl, oxoimidazolidinyl, pyrazolyl, or indazolyl of R2 is optionally substituted with 1 to 3 radicals independently selected from hydroxy, halo, methyl, methoxy, amino, - 0(CH2)NNR7AR7B, -OS(0)2NR7AR7B and -NR7AS(0)2R7B; wherein R7A and R7B are independently selected from hydrogen and C1 -4alkyl; R3 is selected from hydrogen, d^alkyl and biphenyl; and
R4 is selected from C1-10alkyl, C1-4alkenyl, oxetanyl, tetrahydrofuranyl, cyclohexyl, (oxopyrrolidinyl)ethyl, tetrahydropyranyl, phenyl, and benzyl, wherein said Ci-i0alkyl, d- 4alkenyl, oxetanyl, tetrahydrofuranyl, cyclohexyl, (oxopyrrolidinyl)ethyl,
tetrahydropyranyl, phenyl, and benzyl of R4 can be optionally substituted with 1 to 3 radicals independently selected from hydroxy, C _4alkyl and halo-substituted-C^alkyl.
2. The process of claim 1 , wherein the population of exPLTs represents at least a 2-fold increase of exPLTs per exMKs when compared to no contacting with Reagent B.
3. The process according to claim 1 or 2, further comprising a Step A for producing the exMKs from hematopoietic stem cells (HSC), wherein Step A comprising:
contacting a population of HSC with Reagent A and TPO, wherein Reagent A is of Formula I:
Figure imgf000136_0001
or a pharmaceutically acceptable salt, or stereoisomer thereof; wherein
L is -NHC(0)- or -C(0)NH-;
Ri is selected from hydrogen, d^alkyl, phenyl, and d-6heteroaryl, wherein the Ci-4alkyl, phenyl or C5-6heteroaryl is unsubstituted or substituted by 1 to 2 substituents independently selected from halo, cyano, d^alkyl, halo-substituted d-4alkyl, d- 4alkoxyC1-4alkyl, d_4alkoxy, C3.6cycloalkyl and C4.6heterocycloalkyl, wherein the C3. ecycloalkyl or C4.6heterocycloalkyl is further unsubstituted or substituted by 1 to substituents independently selected from halo, hydroxy, oxo, d_4alkylcarbonyl, or d- 4alkyoxy;
R2 is d-4alkyl or C3-6cycloalkyl;
R3 is d-4alkyl or d_4alkenyl;
R4 is hydrogen or C5-6heteroary, unsubstituted or substituted by d_4alkyl; and R5 is halo, d_4alkyl, halo-substituted d-4alkyl, d_4alkoxy-substituted Ci-4alkyl, d-4alkoxy, and halo-substituted d_4alkoxy.
4. The process according to claim 3, in Step A, wherein the population of exMKs represents about a 2.5-fold increase of exMKs per HSC when compared to no contacting with Reagent A.
5. The process according to any one of claims 1 to 4, further comprising:
Step B1 : gathering the quantity of exMKs; and
Step B2: re-suspending the quantity of exMKs.
6. The process according to any one of claims 1 to 5, in Step C, wherein the cell culturing medium optionally further comprising a matrix metalloproteinases (MMP) inhibitor.
7. An ex vivo process for producing a population of ex vivo produced platelets (exPLTs), wherein the process comprising:
A1 ) providing a population of hematopoietic stem cells (HSC);
A) contacting HSC with Reagent A to yield a population of ex vivo produced megakaryocytes (exMKs), and
C) contacting the population of exMKs with ex vivo produced platelets
(exPLTs);
wherein
Regent A is 3-(7-((2,6-dimethylpyridin-3-yl)amino)-1 -methyl-2-oxo-1 ,2- dihydropyrimido[4,5-d]pyrimidin-3(4H)-yl)-N-(3-(trifluoromethyl)phenyl)-4- vinylbenzamide; and
Reagent B is of Formula lib:
Figure imgf000137_0001
or a pharmaceutically acceptable salt, or stereoisomer thereof; wherein
L is selected from -NH(CH2)2_3- -NH(CH2)2S- and -NHCH2CH(OH)- RT is selected from thiophenyl, benzoimidazolyl, imidazopyridinyl, benzothiophenyl, pyrimidinyl, pyridinyl, imidazolyl, and pyridazinyl; each of which is unsubstituted or substituted by 1 to 2 radicals independently selected from halo, cyano, d-4alkyl, halo-substituted-d-4alkyl, d-4alkoxy, and -S(O)0-2R8a, wherein R8A is d-4alkyl;
R2 is selected from phenyl, indolyl and pyrrolopyridinyl, each of which is unsubstituted or substituted with 1 to 2 radicals independently selected from halo, hydroxy, methyl, methoxy, -0(CH2)nNR7aR7b, -OS(0)2NR7aR7b, and - NR7aS(0)2R7b; wherein R7a and R7b are independently selected from hydrogen and d-4alkyl; and
R4 is selected from Ci-i0alkyl, d^alkenyl, oxetanyl, tetrahydrofuranyl, wherein the d-ioalkyl or d^alkenyl is unsubstituted or substituted with hydroxyl.
8. The process according to claim 7, wherein Reagent B is selected from
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)phenol; N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- amine;
N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6- amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin-6-amine;
2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1 - ol;
N-(2-(1 H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; N-(2-(1 H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine; and
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(2-methyl-1 H-indol-3-yl)ethyl)-9H-purin- 6-amine.
9. The process according to claim 7 or 8, further comprising the steps of:
B-1 ) pelleting the population of exMKs; and
B-2) re-suspending the population of exMKs in a medium suitable for Step d
10. The process according to any one of claims 7 to 9, wherein the population of exPLTs represents at least about 2000 exPLT per HSC.
1 1 . The process according to any one of claims 7 to 10, wherein the population of exPLTs represents at least about 7.5-fold increase in yield of ex-platelets per HSC when compared to no contacting with Reagent A and Reagent B. .
12. A cell population comprising exPLTs obtained by the method of any one of claims 1 to 1 1 .
13. The composition according to claim 12, wherein exPLTs expressing CD41 a+ CD42b+ composed at least 20% of the sum of the number of platelets and viable nucleated cells.
14. A composition comprising exPLTs and Reagent B in a pharmaceutically acceptable medium, wherein Reagent B is selected from
4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; 4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)phenol; N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- amine;
N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6- amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin-6-amine;
2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1 - ol;
N-(2-(1 H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; N-(2-(1 H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine; and
2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(2-methyl-1 H-indol-3-yl)ethyl)-9H-purin- 6-amine.
15. A cell composition comprising isolated exPLTs, wherein the composition is
characterized by the presence of Reagent B, wherein Reagent B is selected from
4-(2-(2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6-ylamino)ethyl)phenol; N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6- amine;
N-(2-(1 H-indol-3-yl)ethyl)-2-(benzo[b]thiophen-3-yl)-9-sec-butyl-9H-purin-6- amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine;
N-(2-(1 H-indol-3-yl)ethyl)-9-sec-butyl-2-(5-fluoropyridin-3-yl)-9H-purin-6-amine;
2-(6-(2-(1 H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1 - ol;
N-(2-(1 H-indol-3-yl)ethyl)-2-(5-fluoropyridin-3-yl)-9-isopropyl-9H-purin-6-amine; N-(2-(1 H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine; and 2-(5-fluoropyridin-3-yl)-9-isopropyl-N-(2-(2-methyl-1 H-indol-3-yl)ethyl)-9H-purin- 6-amine.
16. A method of treating thrombocytopenia in a subject in need thereof, comprising
administering a sufficient amount of exPLTs produced by the process of any one of claims 1 to 1 1 .
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