WO1999026644A1 - Procede favorisant la proliferation et la differentiation des cellules hematopoitiques et mesenchymateuses - Google Patents

Procede favorisant la proliferation et la differentiation des cellules hematopoitiques et mesenchymateuses Download PDF

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WO1999026644A1
WO1999026644A1 PCT/US1998/025390 US9825390W WO9926644A1 WO 1999026644 A1 WO1999026644 A1 WO 1999026644A1 US 9825390 W US9825390 W US 9825390W WO 9926644 A1 WO9926644 A1 WO 9926644A1
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WO1999026644A9 (fr
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Kathleen Rodgers
Gere Dizerega
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University Of Southern California
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Priority to JP2000521846A priority Critical patent/JP2002507383A/ja
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Priority to CA002310852A priority patent/CA2310852A1/fr
Priority to AU17063/99A priority patent/AU1706399A/en
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Publication of WO1999026644A9 publication Critical patent/WO1999026644A9/fr

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    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
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    • 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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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells

Definitions

  • This present invention relates to methods for use in accelerating the proliferation and differentiation of hematopoietic and mesenchymal cells.
  • Bone marrow contains pluripotent stem cells that are capable of reconstituting either the hematopoietic system or a wide range of mesenchymal tissues.
  • the mechanisms by which hematopoietic and mesenchymal stem cells produce a range of progenitor cell types are quite dissimilar.
  • HPC hematopoietic progenitor cells
  • HLSPC hematopoietic lineage-specific progenitor cells
  • HPC transplantation therapy has been successful for a variety of malignant and inherited diseases and also provides myelopoietic support for patients undergoing high-dose chemotherapy or radiotherapy.
  • stem cell transplantation has been limited by several features. First, acquiring a sufficient quantity of stem cells to achieve benefit after transfusion requires either extensive, operative bone marrow harvests or extensive pheresis procedures. (Emerson, supra). Next, even under these circumstances, only a limited number of useful cells is obtained. Finally, mature blood cell regeneration after transfusion is slow, so that little direct therapeutic benefit is seen for periods of 1 to 3 weeks. (Emerson, supra).
  • ex vivo expansion may render inadequate HPC populations in peripheral blood and umbilical cord blood sufficient for autologous transplantation and adult allogeneic transplantation respectively.
  • ex vivo expansion of HPC will greatly increase their utility as gene therapy vehicles.
  • ex vivo expansion of HLSPC promises substantial clinical benefits, such as re-infusion of expanded populations of myeloid precursor cells to reduce the period of obligate neutropenia following autologous transplantation, the generation of natural killer cells for use in adoptive immunotherapy protocols, generation of megakaryocyte precursors to alleviate post-transplant-associated thrombocytopenia and more efficient generation of delivery systems for gene therapy. (Alcom and Holyoake, supra).
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte/macrophage colony stimulating factor
  • SCF stem cell factor
  • M-CSF macrophage colony-stimulating factor
  • interleukins 1, 3, 6, and 11 Reviewed in Takaku, J. Cancer Res. Clin. Oncol. 121:701-709, 1995; Holyoake, et
  • tumor necrosis factor ⁇ TNF- ⁇
  • TGF ⁇ transforming growth factor ⁇
  • MSC Mesenchymal stem cells
  • mesenchymal stem cells are pluripotent progenitor cells that possess the ability to differentiate into a variety of mesenchymal tissue, including bone, cartilage, tendon, muscle, marrow stroma, fat and dermis as demonstrated in a number of organisms, including humans (Bruder, et al., J. Cellul. Biochem. 56:283-294 (1994).
  • the formation of mesenchymal tissues is known as the mesengenic process, which continues throughout life, but proceeds much more slowly in the adult than in the embryo (Caplan, Clinics in Plastic Surgery 21 :429-435 (1994).
  • the mesengenic process in the adult is a repair process but involves the same cellular events that occur during embryonic development (Reviewed in Caplan, 1994, supra).
  • chemoattraction brings MSC to the site of repair where they proliferate into a mass of cells that spans the break. These cells then undergo commitment and enter into a specific lineage pathway (differentiation), where they remain capable of proliferating. Eventually, the cells in the different pathways terminally differentiate (and are no longer able to proliferate) and combine to form the appropriate skeletal tissue, in a process controlled by the local concentration of tissue-specific cytokines and growth factors (Caplan, 1994, supra).
  • MSC therapy can serve as a means to deliver high densities of repair- competent cells to a defect site when adequate numbers of MSC and MSC lineage- specific cells are not present in vivo, especially in older and/or diseased patients.
  • methods for rapidly producing large numbers of MSC are necessary. While MSC have been exposed to a number of growth factors in vitro, only platelet-derived growth factor (PDGF) showed mitotic activity (Caplan et al., 1994, supra), while none have been demonstrated to independently induce differentiation. Methods that increase the ex vivo proliferation and differentiation of MSC will greatly increase the utility of MSC therapy.
  • PDGF platelet-derived growth factor
  • MSCs from various species have been differentiated in vitro into colonies of osteoblasts, chondrocytes, and adipocytes in response to dexamethasone, 1,25 dihydroxyvitamin D 3 , or BMP -2. (Prockop, Science 276:71-74, 1997)
  • ex vivo culturing of MSC to produce chondrocytes can be used to resurface joint cartilage in patients with degenerative arthritis or in reconstractive plastic surgery in patients with osteoarthritis.
  • treatment of MSC to differentiate into osteoclasts can be used for implantation into poorly healing bone.
  • MSC lineage-specific descendants of MSC
  • methods that enhance the proliferation of lineage-specific descendants of MSC including but not limited to bone marrow stromal cells, osteoclasts, chondrocytes, and adipocytes, will enhance the therapeutic utility of MSC therapy by increasing the concentration of lineage-specific cell types at appropriate repair sites.
  • the present invention fulfills a need in the art for methods that promote hematopoietic and mesenchymal stem and lineage-specific cell proliferation and differentiation by growth in the presence of angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (All), All analogues, All fragments and analogues thereof and All AT 2 type 2 receptor agonists.
  • AI angiotensin I
  • AI AI analogues
  • AI fragments and analogues thereof angiotensin II
  • AT 2 type 2 receptor agonists All AT 2 type 2 receptor agonists.
  • Figure 1 is a graph showing the effect of All on the phagocytic capability of murine macrophages.
  • Figure 2 is a graph showing the effect of All on the phagocytic capability of rat macrophages.
  • Figure 3 is a graph showing the effect of All on respiratory burst function in rat peritoneal macrophages.
  • Figure 4 is a graph showing the effect of All on respiratory burst function in human
  • Figure 5 is a graph showing the effect of AII(l-7) (SEQ. ID. NO:4) on respiratory burst function in rat peritoneal macrophages.
  • Figure 6 is a graph showing the effect of GSD 24B (SEQ ID NO:31) on respiratory burst function in rat peritoneal macrophages.
  • Figure 7 is a graph showing the effect of GSD 22 A (SEQ ID NO: 18) on respiratory burst function in rat peritoneal macrophages.
  • Figure 8 is a graph showing the effect of GSD 28 (SEQ ID NO:37) on respiratory burst function in rat peritoneal macrophages.
  • Figure 9 is a graph showing the effect of All on proliferation in response to pokeweed mitogen.
  • Figure 10 is a graph showing the effect of All on rat bone marrow cultures.
  • Figure 11 is a graph showing the effect of All on rat bone marrow cultures.
  • Figure 12 is a graph showing the effect of All on murine HSC cultures.
  • Figure 13 is a graph showing the effect of All on murine HSC cultures.
  • Figure 14 is a graph showing the effect of All on murine HSC cultures.
  • Figure 15 is a graph showing the effect of All on murine HSC cultures.
  • Figure 16 is a graph showing the effect of All on murine HSC cultures.
  • Figure 17 is a graph showing the effect of All on murine HSC cultures.
  • Figure 18 is a graph showing the effect of AII(l-7) (SEQ ID NO:4) on MSC proliferation.
  • Figure 19 is a graph showing the effect of GSD 22A (SEQ ID NO: 18) on MSC proliferation.
  • Figure 20 is a graph showing the effect of GSD 24B (SEQ ID NO:31) on MSC proliferation.
  • Figure 21 is a graph showing the effect of GSD 28 (SEQ ID NO:37) on MSC proliferation.
  • Figure 22 is a graph showing the effect of All on alkaline phosphatase expression by
  • Figure 23 is a graph showing the effect of All on GM-CSF secretion by mouse mesenchymal stem cells.
  • HPC refers to any hematopoietic pluripotent progenitor cells capable of giving rise to a wide variety of differentiated hematopoietic cell types.
  • cell types included within this definition are CD34 + bone marrow mononuclear cells (BMMC) (Berardi, et al, Blood 86:2123-2129, 1995), PBSC (Fritsch, et al, Bone Marrow Transplantation 17:169-178, 1996), cobblestone area forming cells (CAFC) (Lemieux, et al., Blood 86:1339-1347, 1995) and 5-FU BM cells (Alcom and Holyoake, Blood Reviews 10:167-176, 1996).
  • BMMC bone marrow mononuclear cells
  • CAFC cobblestone area forming cells
  • 5-FU BM cells Alcom and Holyoake, Blood Reviews 10:167-176, 1996.
  • HLSPC refers to hematopoietic lineage-specific progenitor cells, and includes the progeny of HPC that are committed to a cell-specific differentiation path.
  • MSC mesenchymal stem cells
  • proliferation encompasses both cell self renewal and cellular proliferation with accompanying differentiation.
  • “differentiation” refers to any cellular processes that distinguish a non-committed cell type from a more lineage committed cell type.
  • U.S. Patent No. 5,015,629 to DiZerega describes a method for increasing the rate of healing of wound tissue, comprising the application to such tissue of angiotensin II (All) in an amount which is sufficient for said increase.
  • the application of All to wound tissue significantly increases the rate of wound healing, leading to a more rapid re-epithelialization and tissue repair.
  • All refers to an octapeptide present in humans and other species having the sequence Asp-Arg-Nal-Tyr-Ile-His- Pro-Phe [SEQ ID ⁇ O:l].
  • the biological formation of angiotensin is initiated by the action of renin on the plasma substrate angiotensinogen.
  • AI angiotensin I
  • Angiotensinase which removes the C-terminal His-Leu residues from AI. All is a known pressor agent and is commercially available.
  • a peptide agonist selective for the AT2 receptor (All has 100 times higher affinity for AT2 than ATI) has been identified. This peptide is p- aminophenylalanine6-AII ["(p-NH 2 -Phe)6- All)"] , Asp- Arg-Val-Tyr-Ile-Xaa-Pro-Phe
  • AII(l-7) All residues 1-7) or other fragments of All to evaluate their activity.
  • AII(l-7) elicits some, but not the full range of effects elicited by AIL Pfeilschifter, et al., Eur. J. Pharmacol. 225:57-62 (1992);
  • a preferred class of AT2 agonists for use in accordance with the present invention comprises All, All analogues or active fragments thereof having p-NH-Phe in a position corresponding to a position 6 of AIL
  • various nonpeptidic agents e.g., peptidomimetics
  • having the requisite AT2 agonist activity are further contemplated for use in accordance with the present invention.
  • the active All analogues, fragments of All and analogues thereof of particular interest in accordance with the present invention are characterized as comprising a
  • R ⁇ R ⁇ R ⁇ R ⁇ R ⁇ R ⁇ R ⁇ R 8 in which R 1 and R 2 together form a group of formula X-R A -R B -, wherein X is H or a one to three peptide group and a peptide bond between R A and R B is labile to aminopeptidase A cleavage;
  • R is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr;
  • R 4 is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer and azaTyr;
  • R 5 is selected from the group consisting of He, Ala, Leu, norLeu, Nal and Gly;
  • R 6 is His, Arg or 6- ⁇ H 2 -Phe
  • R > 7 is Pro or Ala
  • R is selected from the group consisting of Phe, Phe(Br), He and Tyr, excluding sequences including R 4 as a terminal Tyr group.
  • Compounds falling within the category of AT2 agonists useful in the practice of the invention include the AH analogues set forth above subject to the restriction that R 6 is p-NH 2 -Phe.
  • R A is suitably selected from Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala, Me 2 Gly, Pro, Bet, Glu(NH 2 ), Gly, Asp(NH ) and Sue.
  • R B is suitably selected from Arg, Lys, Ala, Om, Ser(Ac), Sar, D-Arg and D-Lys. Particularly preferred combinations for R ⁇ and R B are Asp- Arg, Asp-Lys, Glu- Arg and Glu-Lys.
  • Particularly preferred embodiments of this class include the following: AH, AIII or AH(2-8), Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]; AH(3-8), also known as desl-AIII or AIV, Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:3]; AII(l-7), Asp-Arg-Val-Tyr-Ile-His-Pro ⁇ SEQ ID NO:4]; AII(2-7).
  • Arg-norLeu-Tyr-Ile-His-Pro-Phe [SEQ ID NO: 12] and Arg- Val-Tyr-norLeu-His-Pro-Phe [SEQ ID NO: 13].
  • Still another preferred embodiment encompassed within the scope of the invention is a peptide having the sequence Asp- Arg-Pro-Tyr-Ile-His-Pro-Phe [SEQ ID NO:31].
  • AH(6-8), His-Pro-Phe [SEQ ID NO:14] and AH(4-8), Tyr-Ile-His-Pro-Phe [SEQ ID NO:15] were also tested and found not to be effective.
  • R 2 is selected from the group consisting of H, Arg, Lys, Ala, Om, Ser(Ac), Sar, D-Arg and D-Lys;
  • R 3 is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr;
  • R 4 is selected from the group consisting of Tyr, Tyr(PO 3 ) , Thr, Ser, homo Ser and azaTyr;
  • R 5 is selected from the group consisting of He, Ala, Leu, norLeu, Val
  • R 6 is His, Arg or 6-NH 2 -Phe;
  • R 7 is Pro or Ala; and R 8 is selected from the group consisting of Phe, Phe(Br), He and Tyr.
  • a particularly preferred subclass of the compounds of general formula II has the formula
  • R , R and R are as previously defined. Particularly preferred is angiotensin III of the formula Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]. Other preferred compounds include peptides having the structures Arg-Val-Tyr-Gly-His- Pro-Phe [SEQ ID NO: 17] and Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID NO: 18].
  • the fragment AH(4-8) was ineffective in repeated tests; this is believed to be due to the exposed tyrosine on the N-terminus.
  • the standard three-letter abbreviations for amino acid residues are employed. In the absence of an indication to the contrary, the L-form of the amino acid is intended. Other residues are abbreviated as follows:
  • AH and its analogues adopt either a gamma or a beta turn (Regoli, et al., Pharmacological Reviews 26:69 (1974).
  • neutral side chains in position R 3 , R 5 and R 7 may be involved in maintaining the appropriate distance between active groups in positions R 4 , R 6 and R 8 primarily responsible for binding to receptors and/or intrinsic activity.
  • Hydrophobic side chains in positions R 3 , R 5 and R 8 may also play an important role in the whole conformation of the peptide and/or contribute to the formation of a hypothetical hydrophobic pocket.
  • R 2 Appropriate side chains on the amino acid in position R 2 may contribute to affinity of the compounds for target receptors and/or play an important role in the conformation of the peptide. For this reason, Arg and Lys are particularly preferred as R 2 .
  • R 3 may be involved in the formation of linear or nonlinear hydrogen bonds with R 5 (in the gamma turn model) or R 6 (in the beta turn model). R 3 would also participate in the first turn in a beta antiparallel structure (which has also been proposed as a possible structure). In contrast to other positions in general formula I, it appears that beta and gamma branching are equally effective in this position. Moreover, a single hydrogen bond may be sufficient to maintain a relatively stable conformation. Accordingly, R may suitably be selected from Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr.
  • R 4 conformational analyses have suggested that the side chain in this position (as well as in R and R ) contribute to a hydrophobic cluster believed to be essential for occupation and stimulation of receptors.
  • R 4 is preferably selected from Tyr, Thr, Tyr (PO 3 ) , homoSer, Ser and azaTyr.
  • Tyr is particularly preferred as it may form a hydrogen bond with the receptor site capable of accepting a hydrogen from the phenolic hydroxyl (Regoli, et al. (1974), supra).
  • Gly is suitable in position R 5 , it is preferred that the amino acid in this position be selected from He, Ala, Leu, norLeu, Gly and Val.
  • R 6 is His, Arg or 6-NH 2 -Phe.
  • the unique properties of the imidazole ring of histidine e.g., ionization at physiological pH, ability to act as proton donor or acceptor, aromatic character) are believed to contribute to its particular utility as R 6 .
  • conformational models suggest that His may participate in hydrogen bond formation (in the beta model) or in the second turn of the antiparallel stmcture by influencing the orientation of R 7 .
  • R 7 should be Pro in order to provide the most desirable orientation of R 8 .
  • both a hydrophobic ring and an anionic carboxyl terminal appear to be particularly useful in binding of the analogues of interest to receptors; therefore, Tyr and especially Phe are preferred for purposes of the present invention.
  • polypeptides of the instant invention may be synthesized by any conventional method, including, but not limited to, those set forth in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, 111. (1984) and J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, Academic Press, New York, (1973) for solid phase synthesis and E. Schroder and K. Lubke, The Peptides, Vol. 1, Academic Press, New York, (1965) for solution synthesis.
  • the disclosures of the foregoing treatises are inco ⁇ orated by reference herein.
  • these methods involve the sequential addition of protected amino acids to a growing peptide chain (U.S. Patent No. 5,693,616, herein inco ⁇ orated by reference in its entirety). Normally, either the amino or carboxyl group of the first amino acid and any reactive side chain group are protected. This protected amino acid is then either attached to an inert solid support, or utilized in solution, and the next amino acid in the sequence, also suitably protected, is added under conditions amenable to formation of the amide linkage. After all the desired amino acids have been linked in the proper sequence, protecting groups and any solid support are removed to afford the crude polypeptide. The polypeptide is desalted and purified, preferably chromatographically, to yield the final product.
  • peptides are synthesized according to standard solid-phase methodologies, such as may be performed on an Applied Biosystems Model 430A peptide synthesizer (Applied Biosystems, Foster City, Calif), according to manufacturer's instructions. Other methods of synthesizing peptides or peptidomimetics, either by solid phase methodologies or in liquid phase, are well known to those skilled in the art.
  • the peptides can be produced via recombinant DNA technologies. Techniques for recombinant production of the peptides of the invention are well known in the art and can be found in references such as Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991.
  • AH has been shown to increase the proliferation of a number of cell types in vitro, it does not necessarily increase the proliferation of all cell types. Studies have shown that AH accelerates cellular proliferation through the production
  • TGF ⁇ transforming growth factor ⁇
  • AI angiotensin I
  • AI analogues AI fragments and analogues thereof
  • AH angiotensin II
  • HPC are isolated from bone marrow, peripheral blood or umbilical cord blood. HPC is then selected for in these samples. HPC-enriched samples are cultured under appropriate growth conditions, in the presence of angiotensin II (AH), AH analogues, AH fragments and analogues thereof and or AH AT 2 type 2 receptor agonists. HPC proliferation is assessed at various time points during culture.
  • AH angiotensin II
  • HPC and HLSPC are isolated from bone marrow aspirates from the posterior iliac crest.
  • CD34 + HPC are isolated from the aspirate by attaching a biotinylated monoclonal antibody specific for CD34 (available from Becton Dickinson, Sunnyvale, CA, USA) to a streptavidin affinity column (Ceprate SC; CellPro, Bothell, WA, USA) and passing the aspirate through the column, followed by appropriate column washing and stripping, according to standard techniques in the art.
  • CD34 + HPC are suspended in culture medium and incubated in the presence of between 0.1 ng/ml and 1 mg/ml angiotensin II (AH), AH analogues,
  • AH fragments and analogues thereof and/or AH AT 2 type 2 receptor agonists are expanded for a period of between 8 and 21 days and cellular proliferation with accompanying differentiation is assessed via phase microscopy following standard methylcellulose colony formation assays (Berardi, et al., supra) at various points during this time period. Similarly, "self-renewal" of HPC is assessed periodically by reactivity to an antibody directed against CD34 + .
  • HPC that have been cultured in the presence of angiotensin II (AH), AH analogues, All fragments and analogues thereof and/or AH AT type 2 receptor agonists are used for autologous transplantation, to reconstitute a depleted hematopoietic system.
  • AH angiotensin II
  • the cells Prior to transplantation, the cells are rinsed to remove all traces of culture fluid, resuspended in an appropriate medium and then pelleted and rinsed several times. After the final rinse, the cells are resuspended at between 0.7 x 10 6 and 50 x 10 6 cells per ml in an appropriate medium and reinfused into a subject through intravenous infusions.
  • subject peripheral blood samples are evaluated for increases in the number of HPC, HLSPC, and more mature blood cells at various time points by standard flow cytometry and cell sorting techniques. (Talmadge, et al., supra).
  • a method of increasing ex vivo MSC and lineage-specific mesenchymal cell proliferation by exposure to angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT type 2 receptor agonists is disclosed.
  • Experimental conditions for the isolation, purification and in vitro growth of lineage- specific mesenchymal cells, such as bone-marrow derived stromal cells, have been reported (Johnson and Dorshkind, Blood 68(6):1348-1354 (1986); hereby inco ⁇ orated by reference in its entirety).
  • MSC are isolated from bone marrow aspirates from the posterior iliac crest and/or femoral head cancellous bone, purified, resuspended in appropriate growth medium, counted and diluted to an appropriate concentration to seed in tissue culture plates. Purified MSC are cultured in an appropriate growth medium and growth conditions in a humidified atmosphere. The cells are allowed sufficient time to attach to the tissue culture dish, whereupon
  • Adherent cells are placed in growth medium at 37°
  • angiotensinogen AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and/or AH AT 2 type 2 receptor agonists.
  • the cells are expanded for a period of between 2 and 21 days and cellular proliferation is assessed at various time points during this time period. Subsequent medium changes are performed as needed. When the primary cultures are nearly confluent, the cells are harvested for reinfusion into a subject. Cells are examined microscopically to verify the absence of contamination.
  • the cells are rinsed to remove all traces of culture fluid, resuspended in an appropriate medium and then pelleted and rinsed several times. After the final rinse, the cells are resuspended at between 0.7 x 10 6 and 50 x 10 6 cells per ml in an appropriate medium and reinfused into a subject through intravenous infusions. Subjects are evaluated for MSC proliferation in vivo by means of a repeat diagnostic bone marrow aspirate and biopsy to be compared with the original aspirate and biopsy.
  • in vivo proliferation is assessed by reactivity to an antibody directed against a protein known to be present in higher concentrations in proliferating cells than in non- proliferating cells, such as proliferating cell nuclear antigen (PCNA, or cyclin).
  • PCNA proliferating cell nuclear antigen
  • Such antibodies are commercially available from a number of sources, including Zymed Laboratories (South San Francisco, California).
  • isolated MSC are placed into Dulbecco's medium MEM (DMEM-LG) (Gibco, Grand Island, NY, USA).
  • DMEM-LG Dulbecco's medium MEM
  • the cells are purified by a series of steps. Initially, the cells are pelleted and resuspended in complete medium. The cells are centrifuged through a 70% Percoll (Sigma, St. Louis, MO, USA) gradient at 460X g for 15 minutes, the top 25% of the gradients are transferred to a tube containing 30 ml of complete medium and centrifuged to pellet the cells, which will then be resuspended in complete medium, counted and diluted to seed in 100-mm plates at 50 x 10 6 nucleated cells per plate.
  • DMEM-LG Dulbecco's medium MEM
  • purified MSC are cultured in complete
  • cells are allowed to attach for 3 days, whereupon non-adherent cells are removed by changing the culture medium. Cellular proliferation of adherent cells and the presence of normal MSC mo ⁇ hology are assessed by phase microscopy at various time points during the subsequent growth period. Subsequent medium changes are performed every four days.
  • the primary cultures are nearly confluent, the cells are detached with 0.25% trypsin containing 0.1 mM EDTA (Gibco) and either diluted and replated as second passage cells, or used for reinfusion into a subject.
  • cells are rinsed free of culture fluid using Tyrode's solution (Gibco). After the final
  • the Tyrode's solution is removed and the cells are preferably placed into TCI 99 medium (Gibco) supplemented with 1% serum albumin.
  • the cells are rinsed a number of times with this medium and after the final rinse MSC are resuspended in TCI 99 plus 1% serum albumin. Subsequently, MSC are injected slowly intravenously over 15 minutes. Evaluation of subsequent bone marrow aspirates are conducted up to 8 weeks after injection.
  • assessment of the in vivo proliferative effect of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT type 2 receptor agonists on MSC and mesenchymal lineage-specific cells is done by histochemical evaluations of various tissues.
  • in vivo proliferation of MSC and mesenchymal lineage-specific cells is assessed by reactivity to an antibody directed against a protein known to be present in higher concentrations in proliferating cells than in non-proliferating cells, such as proliferating cell nuclear antigen (PCNA, or cyclin).
  • PCNA proliferating cell nuclear antigen
  • the effect of the angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT type 2 receptor agonists on HPC, HLSPC, MSC and mesenchymal lineage-specific cell differentiation are assessed by examination of changes in gene expression, phenotype, mo ⁇ hology, or any other method that distinguishes a cell undergoing differentiation from a progenitor cell.
  • MSC are incubated with angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof or AH AT 2 type 2 receptor agonists as described above. MSC are then tested for the production of colony stimulating factors into the culture supernatant as a demonstration of MSC differentiation.
  • the colony stimulating factor tested for is granulocyte-macrophage colony stimulating factor.
  • macrophage differentiation to an elicited or activated state is assessed after exposure to angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof or AH AT 2 type 2 receptor agonists, as described above.
  • the macrophages are assessed for phagocytotic ability by any of the well known art methods, including but not limited to determination of the number of macrophages that have ingested opsonized yeast particles, and the number of yeast per macrophage ingested.
  • the respiratory burst activity of leukocytes is assessed after exposure to angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof or AH AT 2 type 2 receptor agonists, as described above.
  • the leukocytes are assessed for respiratory burst activity by any method known in the art, including but not limited to the ability to generate hydrogen peroxide via the respiratory burst system.
  • angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof or AH AT 2 type 2 receptor agonists are used to increase in vivo HPC, HLSPC, MSC and mesenchymal lineage-specific cell proliferation.
  • AH, AH analogues, AH fragments and analogues thereof and AH AT type 2 receptor agonists may be administered orally, parentally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.
  • parenteral as used herein includes, subcutaneous, intravenous, intramuscular, intrastemal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneally.
  • a method of increasing in vivo HPC, HLSPC, MSC and lineage-specific mesenchymal cell proliferation by exposure to angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT type 2 receptor agonists is disclosed, either in the presence or absence of other growth factors and
  • the dosage regimen for increasing in vivo proliferation or differentiation of HPC, HLSCP, MSC and lineage-specific mesenchymal cell with angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT type 2 receptor agonists is based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods.
  • Dosage levels of the order of between 0.1 ng/ml and 100 mg/ml angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, angiotensin II (AH), AH analogues, AH fragments and analogues thereof and/or AH AT 2 type 2 receptor agonists are useful for all methods of use disclosed herein.
  • the angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT type 2 receptor agonists may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions).
  • the angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT 2 type 2 receptor agonists may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc.
  • angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT 2 type 2 receptor agonists can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other agents.
  • the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.
  • the angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT 2 type 2 receptor agonists are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration.
  • the compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration.
  • the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, com oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.
  • Other adjuvants and modes of administration are well known in the pharmaceutical art.
  • the carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
  • angiotensinogen AI
  • AI analogues, AI fragments and analogues thereof, AH, AH analogues, AH fragments and analogues thereof and AH AT type 2 receptor agonist is administered topically.
  • a suitable topical dose of active ingredient of angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, AH, All analogues, AH fragments and analogues thereof and or AH AT type 2 receptor agonists is 1 mg/ml administered twice daily.
  • the active ingredient may comprise from 0.001% to 10% w/w, e.g., from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from
  • Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or
  • the present invention by providing a method for enhanced proliferation of
  • HPC and HLSPC will greatly increase the clinical benefits of HPC transplantation.
  • MSC and mesenchymal lineage-specific cells will greatly increase the utility of MSC therapy in the repair of skeletal tissues such as bone, cartilage, tendon and ligament. More rapid production of large numbers of MSC and mesenchymal lineage-specific cells will permit more efficient delivery of high densities of these cells to a defect site and more rapid in vivo amplification in the local concentration of stem and lineage-specific cells at an appropriate repair site.
  • the method of the present invention also increases the potential utility of HPC and HLSPC as vehicles for gene therapy in hematopoietic disorders, as well as MSC and mesenchymal lineage-specific cells as vehicles for gene therapy in skeletal disorders by more efficiently providing a large number of such cells for transfection, and also by providing a more efficient means to rapidly expand transfected HPC, HLSPC, MSC and mesenchymal lineage-specific cells.
  • Resident peritoneal macrophages have very little phagocytic activity.
  • peritoneal macrophages were harvested from C57B1/6 mice or Sprague Dawley rats and resuspended at a concentration of 1 x 10 cells/ml in phosphate buffered saline (PBS). Five separate 0.5 ml cell aliquots were placed on a glass coverslip in a 35 mm petri dish. Prior to incubation, either 0.5 ml of PBS, AH, or AH analogues or fragments at between 1-1000 ug/ml final concentration was added to the individual cover slips.
  • PBS phosphate buffered saline
  • the dishes containing the cover slips were then incubated at 37°C for 4 hours, after which the cover slips were washed 3 to 6 times with PBS.
  • Opsonized yeast particles (Sigma Chemical Co.) (yeast opsonized with adult serum from the same species as that under study) were added to the cover slips and incubated for 2 hours, after which the cover slips were again washed with PBS and inverted onto a glass slide.
  • the number of macrophages that ingested yeast and the number of yeast per macrophage ingested was then determined microscopically. At least 100 macrophages per coverslip were counted.
  • Table 5 describes the AH analogues and fragments used in these studies.
  • AII(l-7)-0 90 3 0 0 3.2 0.06 100 100 21 5 0 20.6 0.53 1000 90 30 10 0 30.8 0.85
  • GSD22A-0 90 1 0 0 1.1 0.02 100 100 26 5 0 23.7 0.59 1000 100 22 1 0 18.7 0.40
  • GSD 24-B-O 100 2 0 0 2.0 0.04 100 100 25 5 0 23.1 0.58 1000 100 10 0 0 9.1 0.18
  • the respiratory burst of leukocytes is one component of the mediator system used to kill bacteria. As with phagocytosis, the level of this respiratory burst activity in resident macrophages is low. With differentiation, either to an elicited (inflammatory) or activated state, this functional activity is significantly elevated. Studies were conducted to assess the effect of in vitro exposure of murine or rat peritoneal macrophages and human peripheral blood mononuclear cells (PBMC) to various concentrations of AH on the
  • the murine or rat peritoneal cells were harvested by lavage with 5-15 ml of cold PBS with 0.5% bovine serum albumin.
  • the human PBMC were harvested by venipuncture from normal human volunteers and isolated from peripheral blood by Ficoll Hypaque density centrifugation. After isolation, the cells were resuspended at
  • Human PBMC were collected from normal volunteers and isolated via Ficoll Hypaque (Sigma Chemical, St. Louis) density centrifugation. After isolation of the buffy coat, the cells were washed 3X to remove the Ficoll Hypaque, counted in trypan blue (0.01%) and resuspended at a concentration of 1 x 10 6 cells/ml in RPMI 1640
  • Bone marrow cells were harvested from the femur and tibia of female Sprague Dawley rats by flushing the bones with Dulbecco's Minimal Essential Medium-High Glucose ("DMEM-HG") with a syringe and an 18 gauge needle. These cells were cultured in 24 well plates at 5 x 10 3 cells/mm 2 in DMEM- HG containing selected lots of fetal calf serum (FCS) and antibiotics (complete medium) at 37°C incubator containing 5% CO in air. Twenty-four hours after the initiation of the cultures the medium and nonadherent cells were aspirated and fresh medium was added. To each of these several wells, complete medium with (1 to 100
  • HPC HPC were harvested from C57B1/6 mice by immunomagnetic affinity chromatography and placed in semi-solid cultures with optimal growth medium. At various times after initiation of culture, the formation of colonies and the size of the colonies (number of cells/colony) were assessed microscopically.
  • mice Female C57B1/6 mice were purchased from Simonson and used as a source of bone marrow cells in this study.
  • the bone marrow was harvested, either from untreated mice or from mice injected with 5-fluorouracil (5-FU) (3 mg/mouse; approximately 150 mg/kg) in the tail vein 48 hours before harvest, from the femur and tibia of mice by flushing with phosphate buffered saline (PBS), pH 7.4, containing 2% fetal bovine serum (FBS) with a 21 -gauge needle.
  • PBS phosphate buffered saline
  • FBS fetal bovine serum
  • the reagents for immunomagnetic labeling were purchased from Stem Cell Technologies, Inc. (Vancouver, BC). Biotin-labeled monoclonal antibodies to the following murine lineage-specific cell surface antigens were included in a cocktail for HPC enrichment and used according to the manufacturer's instructions: CD5 (Ly-1), CD45-R (B220), CDl lb (Mac-1), Myeloid Differentiation Antigen (Gr-1) and
  • a column containing a stainless steel matrix was prepared by washing the matrix with PBS followed by washing with PBS containing 2% protein.
  • the immunomagnetically-labeled bone marrow cells were loaded onto the column and unlabeled cell-containing medium (enriched HPC) was collected in the flow through
  • the enriched HPC cell fractions were diluted into a semi-solid medium containing 0.9% methylcellulose in alpha minimal essential medium (alpha MEM), 30%) fetal calf serum, 1% bovine serum albumin, 10 "4 M 2-mercaptoethanol, 2 mM L- glutamine, and 2% conditioned medium containing colony stimulating factors.
  • alpha MEM alpha minimal essential medium
  • bovine serum albumin fetal calf serum
  • bovine serum albumin fetal calf serum
  • 10 "4 M 2-mercaptoethanol 2 mM L- glutamine
  • conditioned medium containing colony stimulating factors.
  • the conditioned medium was supernatant from splenocyte cultures (1 x 10 6 cells/ml)
  • ⁇ g/ml were added in a small volume to the wells of microtiter dishes, to which
  • the cells were incubated at 37°C and 5% CO 2 for 14 days. At day 14 only,
  • mice untreated (normal) mice treated with 10 ⁇ g/ml (18 macroscopic colonies) and 100 ⁇ g/ml AH (100 macroscopic colonies). Microscopic evaluation of the cells was
  • Figures 12-14 and 16 represent the number of colonies containing more than a certain number of cells/colony as a function of the duration and concentration of AH exposure ( Figures 12-14 for normal cells; Figure 16 for 5-FU treated cells.)
  • Figures 15 and 17 represent the number of cells per colony seen after incubation of from normal ( Figure 15) or 5-FU treated ( Figure 17) mice with various concentrations of AH as a function of time. The results clearly demonstrate that HPC colony size increases proportionately with exposure to increased concentrations of AH, and thus that AH increases HPC proliferation.
  • Example 6 Effect of All Analogues and Fragments on Rat Mesenchymal Stem Cell Proliferation These studies were conducted to determine the effect that inclusion of AH analogues and fragments in the cell culture of MSC would have on the proliferation of these cells.
  • Bone marrow cells were harvested from the femur and tibia of female Sprague Dawley rats by flushing the bones with Dulbecco's Minimal Essential Medium-High Glucose ("DMEM-HG”) with a syringe and an 18 gauge needle.
  • DMEM-HG Dulbecco's Minimal Essential Medium-High Glucose
  • Example 7 Differentiation of MSC that have undergone proliferation in the presence ofAII.
  • Mesenchymal stem cells isolated from bone marrow and grown under appropriate conditions can express characteristics of multiple cell types, including cells involved in the generation of bone, cartilage, muscle and tendons.
  • Osteogenic cells (cells that can form bone tissue) express the enzyme alkaline phosphatase when cultured in medium that drives them toward their osteogenic differentiation.
  • Bone marrow from female Sprague Dawley rats were harvested by flushing the femur with medium.
  • the cells were placed in culture dishes 9 cm 2 in diameter, allowed to adhere overnight, and then placed in DMEM-LG medium containing antibiotics and 10% fetal calf serum together with varying concentrations of AIL At various times after culture initiation, the cells were washed with Tyrode's buffer and placed in osteogenic medium (DMEM-LG containing 10% fetal calf serum, 100 nM dexamethasone and 0.05 mM ascorbic acid) for 4 days prior to assessment of the level of alkaline phosphatase activity per well.
  • DMEM-LG 10% fetal calf serum, 100 nM dexamethasone and 0.05 mM ascorbic acid
  • alkaline phosphatase substrate solution 50 mM glycine, pH 10.5, containing 1 mM magnesium chloride and 2.5 mM p-nitrophenyl phosphate
  • the buffer was removed from the culture and mixed with 1 ml of IN sodium hydroxide to stop the reaction.
  • the absorbance of the resultant mixture at 405 nm was then determined via spectroscopy.
  • the level of alkaline phosphatase activity is expressed as the level of absorbance per culture dish.
  • GM-CSF granulocyte-macrophage-colony stimulating factor
  • Bone marrow cells were harvested from the femur and tibia of C57B1/6 mice (Battin and Kingdom, Gilroy, CA) with saline containing 2% fetal calf serum. Thereafter, the red blood cells were lysed by brief exposure to a hypotonic ammonium chloride solution. The nucleated cells were then resuspended in 3 ml of 5X phosphate buffered saline (PBS; pH 7.2) and rapidly layered over 7 ml of PercoU (70% PercoU gradient separation). This combination of nucleated bone marrow cells and PercoU were centrifuged at 1800 ⁇ m for 20 minutes.
  • PBS 5X phosphate buffered saline
  • the cells in the upper 25% of the PercoU gradient were harvested and washed 3 times with PBS to remove excess PercoU.
  • the cells were then resuspended at 4 x 10 5 cells/ml in alpha minimal essential medium containing 10% horse serum, 10% fetal calf serum and 5 x 10 "5 M 2-mercaptoethanol. These cells were cultured overnight in 24-well plates to allow adherence to the tissue culture plastic. Thereafter, the non-adherent cells were removed by washing with PBS and the medium was replaced with fresh medium containing various concentrations of angiotensin II (AH) (0.01- 100 ⁇ g/ml). Twenty four, 48, or 72 hours after addition of AH, supernatants were harvested and frozen until the time of assay.
  • AH angiotensin II

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Abstract

La présente invention répond à un besoin, dans l'état actuel de la technique, de procédés favorisant la prolifération et la différentiation de cellules souches hématopoïétiques et mésenchymateuses, ou de cellules spécifiques à une lignée, par une croissance en présence d'angiotensinogène, d'angiotensine I (AI), d'analogues d'angiotensine I, de fragments d'angiotensine et de leurs analogues, d'angiotensine II (AII), d'analogues d'angiotensine II, de fragments d'angiotensine II et de leurs analogues, et d'agonistes des récepteurs AT2 de type 2 de l'angiotensine II.
PCT/US1998/025390 1997-11-26 1998-11-24 Procede favorisant la proliferation et la differentiation des cellules hematopoitiques et mesenchymateuses WO1999026644A1 (fr)

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US8557958B1 (en) 2012-06-18 2013-10-15 Tarix Pharmaceuticals Ltd. Compositions and methods for treatment of diabetes
US8633158B1 (en) 2012-10-02 2014-01-21 Tarix Pharmaceuticals Ltd. Angiotensin in treating brain conditions
US9333233B2 (en) 2014-02-25 2016-05-10 Tarix Pharmaceuticals Ltd. Methods and compositions for the delayed treatment of stroke
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WO2021023698A1 (fr) 2019-08-02 2021-02-11 Lanthiopep B.V Agonistes du récepteur de l'angiotensine 2 (at2) destinés à être utilisés dans le traitement du cancer

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US7288522B1 (en) 1998-02-09 2007-10-30 The University Of Southern California Method of promoting erythropoiesis
WO1999040106A3 (fr) * 1998-02-09 1999-09-23 Univ Southern California Procede pour promouvoir l'erythropoiese
US7745411B2 (en) 1998-02-09 2010-06-29 University Of Southern California Methods for promoting erythropoiesis
US6239109B1 (en) 1998-02-09 2001-05-29 University Of Southern California Method of promoting erythropoiesis
WO1999040106A2 (fr) * 1998-02-09 1999-08-12 University Of Southern California Procede pour promouvoir l'erythropoiese
WO1999058140A1 (fr) * 1998-05-11 1999-11-18 University Of Southern California Procede d'augmentation du taux de survie des globules blancs apres une chimiotherapie
US6258778B1 (en) 1998-07-13 2001-07-10 University Of Southern California Methods for accelerating bone and cartilage growth and repair
US6916783B2 (en) 1998-07-13 2005-07-12 University Of Southern California Methods for accelerating bone and cartilage growth and repair
US6177407B1 (en) 1998-08-13 2001-01-23 University Of Southern California Methods to increase blood flow to ischemic tissue
US6730775B1 (en) 1999-03-23 2004-05-04 University Of Southern California Methods for limiting scar and adhesion formation
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AU1706399A (en) 1999-06-15
WO1999026644A9 (fr) 2000-05-25

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