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WO1999065299A1 - Compositions et procedes permettant l'expansion de cellules hematopoïetiques - Google Patents

Compositions et procedes permettant l'expansion de cellules hematopoïetiques Download PDF

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Publication number
WO1999065299A1
WO1999065299A1 PCT/US1999/014065 US9914065W WO9965299A1 WO 1999065299 A1 WO1999065299 A1 WO 1999065299A1 US 9914065 W US9914065 W US 9914065W WO 9965299 A1 WO9965299 A1 WO 9965299A1
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Prior art keywords
cells
mmol
cfu
vivo
leu
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PCT/US1999/014065
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English (en)
Inventor
Pradip Kumar Bhatnagar
James F. Callahan
Daniel Horowitz
Andrew G. King
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Smithkline Beecham Corporation
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Publication of WO1999065299A1 publication Critical patent/WO1999065299A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06043—Leu-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the present invention relates to the field of hematopoiesis in mammals, and more particularly to the use of certain novel compounds and known compounds for the in vitro, ex vivo, or in vivo expansion of hematopoietic cells
  • the invention provides a method for expanding the population of a culture of myelopoietic progenitor cells in vitro by ( 1 ) adding to the cell culture medium containing hematopoietic growth factors an effective amount of a compound of formula (I) to reversibly reduce the rate of differentiation of the cells, then (2) removing the compound of Formula (I) after a period of 1- 100 days, preferably 1-7 days, and (3) transplanting the cells into a patient in need thereof
  • the invention provides a method for expanding the population of myelopoietic progenitor cells in ⁇ o by administering to a patient in need thereof, an effective amount of a compound of Formula (I)
  • a further aspect of the invention provides a method for reversibly inhibiting myelopoiesis in mammalian tissue in v ivo and ex comprising administering to said tissue an amount of a compound of formula (I)
  • Additional aspects of this invention include assav methods for use in identifying or screening for compounds which reversibly inhibit hematopoietic progenitor cells, as described in detail below
  • the present invention provides a method for expanding the population of myelipoietic progenitor cells in vitro comprising adding to a culture medium with hematopoietic growth factors an amount of a compound which is glycinamide (H-Gly- NH2) or any tetra-, t ⁇ - or dipeptide analog which can be converted in serum to glycinamide (H-GIV-NH2)
  • Preferred compounds are those of Formula (I) H-A-B-C-Gly-NH 2
  • A is a bond, , or
  • B is a bond, . , " " x , ° “N , or
  • RI is H or C]_4alkyl
  • x- ⁇ -X R2 is H, , naphthyl, indoyl, or pyridyl
  • R3 is H, C ⁇ _4alkyl, cycloC3.8a.kyl, 0* , naphthyl, indoyl, or pyridyl; wherein the
  • C ⁇ _4 alkyl may be substituted by one or more X groups
  • X is H, OH, I, Br, NH 2 , N 3 , 0-C ⁇ _ 4 alkyl, C j ⁇ alkyl, or COPh;
  • H-Gly-NH 2 H-Tyr-Pro-Leu-Gly-NH 2 [SEQ ID NO: 1] H-Tic-Leu-Gly-NH 2
  • H-Tyr-Pro-Trp-Gly-NH 2 [SEQ ID NO: 2] H-Tyr-Pro-Phe-Gly-NH 2 [SEQ ID NO: 3] H-Phe-Pro-Leu-Gly-NH 2 [SEQ ID NO: 4] Hpp-Pro-Leu-Gly-NH 2 [SEQ ID NO: 5]
  • glycinamide H-Gly-NH 2 .
  • All alkyl groups may be straight or branched.
  • the compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active form. All of these compounds and diastereoisomers are contemplated to be within the scope of the present invention.
  • the peptides of the invention are prepared by the solid phase technique of Mer ⁇ field, J Am Chem Soc . 85 2149 ( 1964), or solution methods known to the art may be successfully employed The methods of peptide synthesis generally set forth in J. M Stewart and J D.
  • Each amino acid or peptide is suitably protected as known in the peptide art
  • the ⁇ -fluoroenylmethyloxycarbonyl group (Fmoc) or t-butoxycarbonyl (t-Boc) group are preferred for protection of the amino group, especially at the ⁇ -position
  • a suitably substituted carbobenzoxy group may be used for the ⁇ -amino group of lysine and benzyl group for the ⁇ and ⁇ carboxy groups of Asp and Glu respectively Suitable substitution of the carbobenzoxy protecting group is ortho and/or para substitution with chloro, bromo.
  • the protective groups are, most conveniently, those which are not removed by mild acid treatment These protective groups are removed by such methods as catalytic hydrogenation, sodium in liquid ammonia or HF treatment as known
  • the peptide is built up sequentially starting from the carboxy terminus and working toward the amino terminus of the peptide
  • Solid phase synthesis is begun by covalently attaching the C terminus of a protected amino acid to a suitable resin, such as benzhydrylamine resin (BHA), methylbenzhydrylamine resin (MBHA) or chloromethyl resin (CMR), as is generally set forth in U S Patent No 4,244,946 or phenyl acid amino methyl resin (PAM)
  • BHA or MBHA support resin is used if the carboxy terminus of the product peptide is to be a carboxamide
  • a CMR or PAM resin is generally used if the carboxy terminus of the product peptide is to be a carboxy group, although this may also be used to produce a carboxamide or ester
  • the protective group on the ⁇ -amino group is removed by mild acid treatment (l e t ⁇ fluoroacetic acid) Suitable deprotection, neutralization and coupling cycles known in the art are used to sequentially add amino acids without isolation of the intermediate, until the desired peptide has been formed.
  • the completed peptide may then be deblocked and/or split from the carrying resin in any order
  • Treatment of a resin supported peptide with HF or HBr/acetic acid splits the peptide from the resin and produces the carboxy terminal amino acid as a carboxylic acid or carboxamide
  • the CMR or PAM resin may be treated with an appropriate alcohol, such as methyl, ethyl, propyl, butyl or benzyl alcohol, in the presence of t ⁇ ethylamine to cleave the peptide from the resin and product the ester directly Esters of the peptides of this invention may also be prepared by conventional methods from the carboxylic acid precursor Typically, the carboxylic acid is treated with an alcohol in the presence of an acid catalyst Alternatively, the carboxylic acid may be converted to an activated acyl intermediate, such as an acid halide, and treated with an alcohol, preferably in the presence of a base
  • the preferred method for cleaving a peptide from the support resin is to treat the resin supported peptide with anhvdrous HF in the presence of a suitable cation scavenger, such as anisole or dimethoxybenzene
  • a suitable cation scavenger such as anisole or dimethoxybenzene
  • This method simultaneously removes all protecting groups, except a thioalkyl group protecting sulfur, and splits the peptide from the resin Peptides hydrolyzed in this way from the CMR and PAM resins are carboxylic acids, those split from the BHA resin are obtained as carboxamides
  • acylation is carried out upon the free amino group using the acyl halide, anhydride or activated ester, of the corresponding alkyl or aryl acid, in the presence of a tertiary amine
  • Mono-alkylation is carried out most conveniently by reductive alkylation of the amino group with an appropriate aliphatic aldehyde or ketone in the presence of a mild reducing agent, such a lithium or sodium cyanoborohyd ⁇ de
  • Dialkylation may be carried out by treating the amino group with an excess of an alkyl halide in the presence of a base
  • Solution synthesis of peptides is accomplished using conventional methods used to form amide bonds
  • a protected t-Boc amino acid which has a free carboxyl group is coupled to a protected amino acid which has a free amino group using a suitable coupling agent, such as N,N'-d ⁇ cyclohexyl carbodnmide (DCC), optionally in the presence of catalysts such as 1-
  • a compound of this invention is added to a culture medium containing one or more hematopoietic growth factor in an amount effective to reversibly reduce the rate of differentiation of the cells
  • the amount of peptide will greatly depend on the composition and cell count of the culture As one example, a suitable amount of peptide is between about 0 1 and about 500 macerograms/mL culture, preferably 10- 100 micrograms/mL ex vivo, or 0 1-100 milligrams/kg m vivo
  • hematopoietic growth factors are M-CSF, IL- 1 , IL-3, IL-6, IL- 1 1 , G-
  • CSF, GM-CSF, LIF, TGF- ⁇ , MlPl- ⁇ , FLT3 hgand, SCF. TPO, MPIF-1, IL-3/GM, IL-3/G, FLT3/GM Preferred growth factors are combinations of SCF, IL- 1 1 , IL-3, IL-6, IL- 1 , G- CSF, GM-CSF, TPO, FLT3, M-CSF
  • the compounds of Formula (I) have a characteristic biological activity of reversibly inhibiting myelopoiesis in mammals, mammalian tissue, or other biological samples in vitro, ex vivo or in vivo These compounds may almost completely inhibit differentiation of myelopoietic cells, e g , colony forming units in culture (CFU-C), such as CFU-GM, when in contact with such cells
  • the compounds of this invention are useful in providing a protective function in cancer or other therapies which involve irradiation and/or cytotoxic drugs which function normally to kill proliferating cells, including proliferating stem cells
  • cytotoxic drugs which function normally to kill proliferating cells, including proliferating stem cells
  • the inventors have discovered that certain compounds are capable of inhibiting stem cell differentiation when the compounds are in contact with the stem cells ( thereby causing undifferenuated cells to pile up) These compounds also have a desirable attribute of reversing the inhibition by simply remov ing the compound from contact with the stem cells All of the compounds of this invention have a reversible action, I e .
  • the myeloprotectant compounds of this invention are also specific to normal hematopoietic stem cells and inhibit the proliferation of normal cells and not the proliferation of target cancer cells
  • This invention prov ides a method for rev ersiblv inhibiting myelopoiesis in mammals, mammalian tissue, including bone marrow and other samples, in vivo and e ⁇ vixo and in vitro This method entails administering to the mammal, tissue or sample an amount of a peptide of the formula (I) or glycinamide.
  • the myelopoietic colonies inhibited are CFU-C colony forming cells, and more specifically, CFU-GM colony forming cells
  • a mammalian subject preferably a human patient, undergoing chemotherapy or radiation may be administered the peptide so that the reversible inhibition of myelopoiesis occurs in vivo
  • the method may be accomplished ex vivo or in vitro
  • the mammalian tissue for example, bone marrow
  • an effective amount of myeloprotectant peptide of this invention substantially simultaneously with exposure of the tissue to radiation or chemotherapeutics. Inhibition of myelopoiesis is reversible when administration of the insult and the peptide, which occur ex vivo, cease
  • Treatment with a myeloprotectant of this invention may be necessary throughout the time period in which the insulting agent is present within the body Inhibition of normal hematopoietic stem cells during this period could protect normal cells while damaging only target cells such as cancer cells Because the peptides of this invention have demonstrated reversible inhibition, a more rapid rebound of hematopoietic stem cell proliferation is expected when administration of the myeloprotectant peptide is ceased (concurrently with, or shortly after administration of the chemotherapeutic or radiation is ceased)
  • the end result of treatment with the peptides of this invention as myeloprotective agents is to prevent neutropenia and or accelerate neutrophil/leukocyte recovery, and prevent stem cell loss due to myelosuppression
  • the advantage of such administration to the patient includes fewer infections due to leukopenia and the ability to use more aggressive chemotherapy regimens to kill cancer cells (dose intensification/increase dose frequency)
  • the term "effective amount” refers to that amount of the peptide of formula (I) which evokes a reversible inhibition of proliferating myelopoietic cells
  • the peptides of the invention may be administered to human or other mammalian patients by injection in the dose range of about 0 5 mg to about 1000 mg
  • a desirable dosage range is from about 5 to about 1000 mg
  • Oral administration dosages are desirably in the range of about 5 mg to about 2000 mg, desirably, for example, about 10 mg to 1000 mg per 70 kg body weight per day
  • the dose may be in the range of about 5 micrograms to about 200 mg per 70 kg body weight, for example about 100 micrograms to 100 mg over six days.
  • a concentration of the peptide of about 10 M to about 10 M in the extracellular fluid of the patient, preferably 10 M.
  • the effective amounts or dosages may be adjusted based on the amount of tissue being treated
  • an effective in vitro concentration is in the range of about 10 - " 8 M to 10 - " 5 M.
  • One of skill in the art may readily determine other appropriate dosages, depending on the mode of administration, and the level of aggressiveness of therapy required in the specific circumstance
  • compositions comprising as active ingredient one or more peptides of formula (I) as hereinbefore defined or physiologically compatible salts thereof, in association with a pharmaceutical camel or excipient
  • the compositions according to the invention may be presented, for example, in a form suitable for oral, nasal, parenteral or rectal administration
  • pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition
  • Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline and water
  • Solid carriers include starch, lactose calcium sulfate dihydrate, terra alba, magnesium stearate or stea ⁇ c acid, talc, pectin acacia, agar or gelatin
  • the carrier may also include a sustained release material such as
  • compositions of the peptides of this invention, or derivatives thereof may be formulated as solutions of lyophilized powders for parenteral administration
  • Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use
  • the liquid formulation is generally a buffered, isotonic, aqueous solution
  • suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution
  • Such formulation is especially suitable for parenteral administration, but mav also be used for oral administration and contained in a metered dose inhaler or nebulizer for insufflation It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate
  • a pulverized powder of the peptides of this invention may be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository for rectal administration
  • the pulverized powders may also be compounded with an oily preparation, gel, cream or emulsion, buffered or unbuffered, and administered through a transdermal patch Nasal sprays may be formulated similarly in aqueous solution and packed into spray containers either with an aerosol propellant or provided with means for manual compression
  • Dosage units of such pharmaceutical compositions containing the compounds of this invention preferably contain about 1 microgram to 2 g, for example 1 microgram to about 50 mg of the peptide ot formula ( I) or salt thereof
  • Progenitor cell as ⁇ >a ⁇ s Cells from expansion cultures were washed twice and mixed with 0 3%> agar in medium with growth factors This was added to Falcon 12 well plates at 0 5 mL per well at cell concentrations ranging from 5 x 10 ⁇ per well to 160 cells per well Plates were left at room temperature for about 10 min for agar to gel and were then placed in 37° C incubator at 5% 0 , and 6% C0
  • CFU-GM responsive to GM-CSF 10 ng/mL were counted after seven days as colonies greater than fifty cells per colonies
  • HPP(h ⁇ gh prohferative potential) responsiv e to interleukin 3 ( 10 ng/mL), interleukin 1 ( 10 ng /mL) and stem cell factor (50 ng/mL) were counted after fourteen days as colonies greater than 0 5 mm in diameter with one or more dense centers All other colonies grown with these factors with at least fiftv cells were counted as LPP(low prohferative potential)
  • A549 cells (ATCC CCL- 185) were added at 125 cells/well in McCO s medium with 20% fetal bovine serum in 0 3% agar After 10 ten days at 37°C 6% CO , colonies of greater than 50 cells were counted
  • Micro-Inhibition Assay One screening and identification method useful in the present invention may employ the step of screening test samples which detectably bind to a receptor for in vitro or in vivo inhibition of CFU-C colony formation in either a conventional 7 day CFU-GM assay (CFU-C Assay), well known to those skilled in the art, or in the novel, micro-screening assay as described herein
  • the micro-inhibition assay is a modification of the conventional assay and provides for an efficient and rapid screening and identification of inhibitors
  • the presence or amount of inhibition of CFU-C or CFU- GM colony formation can then be measured in order to identify those test samples which act as agonists
  • an assay for the screening or identification of other myeloprotectant compounds capable of reversible inhibition includes the following steps First, to measure the occurrence of, and degree of, inhibition, a test sample containing one or more test compounds and selected hematopoietic growth factors is contacted with bone marrow cells in soft agar (a semi-
  • the plate is then incubated under conditions which would permit the detection of inhibition of CFU-C CFU-C or CFU-GM colony growth
  • conditions include incubation at about 37°C humidified atmosphere of about 7% C02 for about 4 to 14 days
  • the degree of inhibition caused by the test sample is detected by measuring the conversion of the mitochond ⁇ al metabolism of (3-[4.5-d ⁇ methylth ⁇ azol-2-yl]-2,5-d ⁇ phenyl tetrazohum bromide), also known as MTT, to an insoluble formazan crystal which can be dissolved in sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Optical density is measured at 570 nm with a reference filter of 750 nm on an ELISA reader
  • the results of the assay with the compound are compared to those of a control, l e , bone marrow cells with growth factors and no compound, as well as marrow cells only Increased
  • PCLC reversibility of inhibition
  • the test sample is first contacted with bone ma ⁇ ow cells in a liquid medium with or without a test compound, in this instance a compound of formula (I), in a 96 well tissue culture plate for 1 to 4 days Marrow cells are washed with medium three times and recultured in either a conventional CFU-C assay or the above noted Micro-Inhibition assay
  • the degree of inhibition caused by the test sample is again detected by measuring MTT conversion, as an index of CFU-C proliferation, and measuring optical density at 570 nm with a reference filter of 750 nm on an ELISA reader, using the same controls, as described above
  • the assay methods may be further modified by preparing two or more additional test samples from the original test sample or samples that are determined to inhibit CFU-C colony formation activity These additional test samples contain a lesser number of test compounds than the original test sample from which they were
  • HPP 3-[(4-hydroxy)-phenyl]propton ⁇ c acid
  • MeTyr alpha-methyltyrosine
  • Tic(7-OH) 2-carboxy-7-hydroxytetrahydro ⁇ soqu ⁇ nohne
  • Example 1 A The compound of Example 1 A (220 mg) was treated with 4N HCl/dioxane at room temperature for 2 hours The reaction mixture was evaporated and purified by a bond elute column [0 1 % aqueous t ⁇ fluoroacetic acid (TFA)] to give 130 mg of the title compound MS (FAB) m/e 432 [M+H] + , HPLC k' 2 96 (PRP- 1 , gradient, A 0 1 % aqueous TFA B CH3CN-O 1 % TFA 5 507c B over 30 minutes, UV detection at 220 nm)
  • Example 2A The compound of Example 2A (350 mg) was treated with TFA at room temperature for 30 minutes The reaction mixture was evaporated and the residue treated with anhydrous hydrogen fluoride (HF) at 0°C for 1 hour The residue after evaporation of the HF was precipitated from ether Purification by preparative high pressure liquid chromatography (HPLC) (PRP-1 packing material [Hamilton], 127c CH3CN in H2O with 0 17e TFA) gave 78 mg of the title compound MS (ES) m/e 573 [M+H] + , HPLC k' 4 55 (PRP- 1 , gradient, A 0 17c aqueous TFA, B CH3CN-O 17c TFA, 5-50%> B over 30 minutes, UV detection at 220 nm)
  • the protected nonapeptide resin Boc-Tyr(BrZ)-Pro-Phe-Gly-BHA was prepared according to Example 13 on a 1 mmol scale After remov al of the N-terminal Boc group with 50% TFA in methylene chloride and neutralizing the resulting TFA salt with 57c DIEA in methylene chloride, the peptide was cleaved from the resin with removal of the side chain protecting groups by treatment with anhydrous liquid HF ( 10 mL) in the presence of anisole ( 1 mL) at 0°C for 1 hour After remo al of the HF under vacuum, the resin was washed with ethyl ether and air-dried The resin was then extracted with glacial acetic acid and the combined extracts were lyophilized to yield a crude peptide
  • the protected nonapeptide resin Boc-Tyr(BrZ)-Pro-Trp-Gly-BHA was prepared according to Example 13 on a 1 mmol scale After removal of the N-terminal Boc group with 50% TFA in methylene chloride and neutralizing the resulting TFA salt with 57c DIEA in methylene chloride, the peptide was cleaved from the resin with removal of the side chain protecting groups by treatment with anhydrous liquid HF ( 10 mL) in the presence of anisole ( 1 mL) at 0°C for 1 hour After removal of the HF under vacuum, the resin was washed with ethyl ether and air-dried The resin was then extracted with glacial acetic acid and the combined extracts were lyophilized to yield a crude peptide The resulting crude peptide was purified by prep HPLC (PRP-1, gradient, A 0 17c aqueous TFA B CH3CN-O 1 % TFA, 5-507c B over 20 minutes) to
  • Example 5B The compound of Example 5B ( 140 mg) was treated with 4N HCl/dioxane at room temperature for 1 hour The reaction mixture was evaporated and evaporated from toluene Purification by prep HPLC (PRP- 1 , 5-30% gradient over 20 minutes of CH3Cn in H2 ⁇ with 0 17c TFA) gave 100 mg of the title compound MS (ES) m e 436 [M+H] + , HPLC k' 3 43 (PRP-1, gradient, A 0 17c aqueous TFA B CH3CN-O 17c TFA, 5-507c B over 30 minutes, UV detection at 220 nm)
  • Example 6A The compound of Example 6A (500 mg, 1 61 mmol) in DMF was treated with Boc-Tyr-OH (500 mg, 1 77 mmol), HOBt (240 mg, 1 77 mmol), DIEA (620 microhters, 3 50 mmol) and EDC «HC1 (339 mg, 1 77 mmol) at room temperature for 24 hours
  • the reaction mixture was evaporated and the residue was taken up into EtOAc and extracted with 100 mL of 57c citric acid followed by 5%> NaHC ⁇ 3
  • the material was then purified by flash chromatography (silica gel, 1007c EtOAc) to give 430 mg (0 802 mmol) of the titled compound MS(ES) m/e 537 [M+H] + , 535 [M-H] "
  • Example 6B The compound of Example 6B (430 mg) was treated with 10 mL of 2M NH4/MeOH at room temperature for 48 hours The reaction was evaporated and treated with 4N HCl/dioxane at room temperature for 1 hour The reaction mixture was evaporated and evaporated from toluene Purification of prep HPLC (PRP- 1 , 5-507c gradient over 20 minutes of CH3CN in H 2 0 with 0 1 % TFA) gave 126 mg of the title compound MS (ES) m/e 422 [M+H] + , 420 [M-H] " , HPLC k' 2 98 (PRP- 1 , gradient, A 0 17c aqueous TFA B CH ⁇ CN-0 1 % TFA, 5-507c B over 30 minutes, UV detection at 220 nm)
  • Example 7A The compound of Example 7A (266 mg) was treated with 4N HCl/ dioxane at room temperature for 40 minutes The reaction mixture was evaporated and evaporated from toluene The mixture was treated with H2 and 57c Pd/C under Parr conditions for 3 hours The reaction was filtered through cehte and purified by a bond elute column (0 1 % aqueous TFA) to give 80 mg of the title compound MS(ES) m/e 447 [M+H] + , 445 [M-H] " HPLC k 1 87 (PRP- 1 , gradient, A 0 17c aqueous TFA B
  • Example 7B The compound from Example 7B ( 16 mg) was treated with 3 mL 0.1N HCl and 54 microhters (0 54 mmol) NaN ⁇ 2 at 0°C for 10 minutes after which time 360 mL (0 360 mmol) NaN3 was added The reaction mixture reacted at 0°C for 1 hour and evaporated. The reaction was purified by prep HPLC (PRP- 1 , 10-307c gradient over 20 minutes of CH3CN in H20 with 0 1 % TFA) to give 1 .9 mg of the title compound. MS(ES) m e 473 [M+H] + , 471 [M-H] " , HPLC k' 5 02 (PRP- 1 , gradient. A 0 1 7c aqueous TFA B. CH3CN- 0 17c TFA, 5-507c B over 30 minutes, UV detection at 220 nm).
  • Example 9B The compound of Example 9B (0.15 g, 0.36 mmol) was treated with 4N HCl/dioxane for 0.5 hours. After the reaction was evaporated to dryness, the residue in DMF was treated with Boc-Tyr-OH ( 1 10 mg, 0.36 mmol), HOBt (50 mg, 0 36 mmol), EDC'HCl (70 mg, 0.36 mmol) and DIEA (0.25 mL, 1 44 mmol) at room temperature for 24 hours The reaction mixture was evaporated, and the residue was washed with 20 mL of H2 ⁇ and recrystalhzed from chloroform diethyl ether ( 1 mL/10 mL) to give 0.19 g of the titled compound.
  • Example 9C The compound of Example 9C (0 18 g, 0 32 mmol) was treated with NH3/MeOH solution (2.0 mmol, 20 mL) for 18 hours. The reaction was evaporated, and the residue was treated with HCl/dioxane (4N, 4 mL) for 1 hour. After the reaction was evaporated to dryness, the residue was further purified with HPLC (PRP column, 207c of CH3CN in H2 ⁇ with 0.1 % TFA) to give 86 mg of the titled compound.
  • HPLC PRP column, 207c of CH3CN in H2 ⁇ with 0.1 % TFA
  • the compound of 10A (0.25 g, 0.70 mmol) was treated with 4N HCl/dioxane for 0.5 hours. After the reaction was evaporated to dryness, the residue in DMF was treated with Boc-Tyr-OH (210 mg, 0.70 mmol), HOBt ( 100 mg, 0 70 mmol), EDC»HC1 (140 mg, 0.70 mmol) and DIEA (0.96 mL, 2.80 mmol) at room temperature for 24 hours. The reaction mixture was evaporated, and the residue was washed with 20 mL of H2 ⁇ and recrystallized from chloroform/diethyl ether ( 1 mL/10 mL) to give 0.31 g of the title compound.
  • Boc-Ala(2-Naphthyl)-Pro-Leu-Glv-NH2 Boc-ala(2-Naphthyl)-OH ( 169 mg, 0 5 mmol) in DMF was treated with H-
  • Example 11 A The compound of Example 11 A (160 mg, 0.27 mmol) was treated with HCl/dioxane (4N, 4 mL) for 1 hour. After the reaction was evaporated to dryness, the residue was recrystallized twice from methanol/diethyl ether (1 mL/10 L) to give 101 mg of the title compound MS (ES) m e [M+H] + 482; HPLC k' 4.77 (PRP- 1 , gradient, A: 0.1 % aqueous TFA B: CH3CN-O 17c TFA, 10-507c B over 30 minutes, UV detection at 220 nm).
  • Example 12 Preparation of H-Trp-Pro-Leu-Glv-NH2 TSEQ ID NO:141
  • Boc-Trp-OH (152 mg, 0 5 mmol) in DMF was treated with H-Pro-Leu- Gly-NH2 (Bachem, 147 mg, 0 5 mmol), HOBt (77 mg, 0 5 mmol), EDC'HCl (96 mg, 0 5 mmol) and DIEA (0 43 mL, 2 0 mmol) at room temperature for 24 hours The reaction mixture was evaporated The residue was washed with H2O and recrystallized from chloroform diethyl ether (1 mL/10 mL) to give 0 18 g of the title compound
  • peptides of formula (I) may be prepared by the following method Peptide amides are synthesized by solid phase peptide synthesis using benzhydrylamine resin as the support Protected amino acids are added sequentially starting from the carboxyl terminus until the desired sequence is obtained
  • the Boc group is used for protection of the alpha-amino group
  • Side chain functional groups are protected as follows arginine and histidine, tosyl (Tos), cysteine, p-methylbenzyl (MeBn), se ⁇ ne and threonine, benzyl ether (Bn), lysme, p-chlorocarbobenzyloxy (C1Z), glutamic acid and aspartic acid, benzyl ester (OBn), tyrosine, p-bromocarbobenzyloxy (BrZ) Removal of the Boc group was accomplished by treatment with 507o TFA in methylene chloride Neutralization of the amme-TFA salt was accomplished by treatment with
  • Example 14 Effect of glycinamide on a human lung carcinoma cell line.
  • A549 cells (ATCC CCL- 185) were added at 125 cells/well in McCOY's medium with 207c fetal bovine serum in 0 37c agar After 10 ten days at 37°C , 67c C0 2 , colonies of greater than 50 cells were counted
  • Example 15 Effect of glycinamide on normal mouse bone marrow.
  • glycinamide Dilutions of glycinamide are made in phosphate buffered saline (PBS) with 0 01 % bovine serum albumin
  • PBS phosphate buffered saline
  • Different concentrations of glycinamide were added to wells in a maximum volume of 25 microhters
  • Non adherent bone marrow cells (4 x 10 ⁇ cells/ 200 ul) were added in agar with 250 Units of M-CSF
  • Example 17 Effect of glycinamide on the expansion of LPP from Sca-1+ cells.
  • Sca- 1+ cells purified from bone marrow from BDFl female mice were cultured with IL-1 , IL-3, IL-6, SCF. GM-CSF. G-CSF and varying concentrations of glycinamide. After incubation at 37°C in 57c O2 and 67c CO2 cultures were assayed for LPP at 4,7,10 and 13 days after start of incubation.
  • Example 18 Effect of glycinamide on the expansion of HPP from Sca-1+ cells.
  • Sca-1+ cells ,purified from bone marrow from BDFl female mice were cultured with IL-1, IL-3, IL-6, SCF, GM-CSF, G-CSF and varying concentrations of glycinamide. After incubation at 37°C in 5% 0 2 and 6% C0 2 cultures were assayed for HPP at 4,7,10 and 13 days after start of incubation.

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Abstract

La présente invention concerne l'hématopoïèse chez des mammifères. L'invention concerne plus particulièrement à l'utilisation de certains composés récemment découverts ainsi que de composés déjà connus, laquelle utilisation vise à l'expansion in vitro, ex vivo, ou in vivo de cellules hématopoïétiques.
PCT/US1999/014065 1998-06-19 1999-06-18 Compositions et procedes permettant l'expansion de cellules hematopoïetiques WO1999065299A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7192972B2 (en) * 2002-03-22 2007-03-20 Eisai Co., Ltd. Hemiasterlin derivatives and uses thereof
US7585976B2 (en) 2002-03-22 2009-09-08 Eisai Co., Ltd. Hemiasterlin derivatives and uses thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0637448A1 (fr) * 1993-07-19 1995-02-08 Iscofar Sas Di Paolo E. Ghirardi Combinaisons de la glycinamide ou de la N-acétylglycinamide avec des composés analgésique

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0637448A1 (fr) * 1993-07-19 1995-02-08 Iscofar Sas Di Paolo E. Ghirardi Combinaisons de la glycinamide ou de la N-acétylglycinamide avec des composés analgésique

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Title
BHATNAGAR P. K., ET AL.: "STRUCTURE-ACTIVITY RELATIONSHIPS OF NOVEL HEMATOREGULATORY PEPTIDES.", JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 39., no. 19., 13 September 1996 (1996-09-13), US, pages 3814 - 3819., XP000611484, ISSN: 0022-2623, DOI: 10.1021/jm960099d *
CALLAHAN et al., "Identification of Glycinamide as the Active Principal Responsible for the Effects of Tyr-MIF on Hematopoietic Progenitor Cells", BLOOD, 15 November 1998, Vol. 92, No. 10, Suppl. 1, Part 1-2, page 581A, XP002930009. *
HOROWITZ et al., "Ex Vivo Expansion of Hematopoietic Cells with Glycinamide", BLOOD, 15 November 1998, Vol. 92, No. 10, Suppl. 1, Part 1-2, page 311B. XP002930008. *
SMITH A. G., ET AL.: "INHIBITION OF PLURIPOTENTIAL EMBRYONIC STEM CELL DIFFERENTIATION BYPURIFIED POLYPEPTIDES.", NATURE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 336., no. 6200., 1 December 1988 (1988-12-01), United Kingdom, pages 688 - 690., XP000955109, ISSN: 0028-0836, DOI: 10.1038/336688a0 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7192972B2 (en) * 2002-03-22 2007-03-20 Eisai Co., Ltd. Hemiasterlin derivatives and uses thereof
US7528152B2 (en) 2002-03-22 2009-05-05 Eisai Co., Ltd. Hemiasterlin derivatives and uses thereof in the treatment of cancer
US7585976B2 (en) 2002-03-22 2009-09-08 Eisai Co., Ltd. Hemiasterlin derivatives and uses thereof
US8129407B2 (en) 2002-03-22 2012-03-06 Eisai Inc. Hemiasterlin derivatives and uses thereof in the treatment of cancer
US8633224B2 (en) 2002-03-22 2014-01-21 Eisai Co., Ltd. Hemiasterlin derivatives and uses thereof in the treatment of cancer

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