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  Inhibitor and stimulator of stem cell proliferation and uses thereofUSPTO Application #: 20060166863Title: Inhibitor and stimulator of stem cell proliferation and uses thereof Abstract: Disclosed and claimed are methods for the isolation and use of stem cell modulating factors for regulating stem cell cycle and for accelerating the post-chemotherapy peripheral blood cell recovery. Also disclosed and claimed are the inhibitors and stimulators of stem cell proliferation. (end of abstract)
Agent: Nixon & Vanderhye, PC - Arlington, VA, US Inventors: Stephen D. Wolpe, Irena Tsyrlova USPTO Applicaton #: 20060166863 - Class: 514006000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai, Heavy Metal Containing (e.g., Hemoglobin, Etc.) The Patent Description & Claims data below is from USPTO Patent Application 20060166863. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The present invention relates to the use of modulators of stem cell proliferation for regulating stem cell cycle in the treatment of humans or animals with autoimmune diseases, aging, cancer, myelodysplasia, preleukemia, leukemia, psoriasis, acquired immune deficiency syndrome (AIDS), myelodysplastic syndromes, aplastic anemia or other diseases involving hyper- or hypo-proliferative conditions, as well as the use of such compounds for analgesia. The present invention also relates to a method of treatment for humans or animals anticipating or having undergone exposure to chemotherapeutic agents, other agents which damage cycling stem cells, or radiation exposure and for protection against such agents during ex vivo treatments. Finally, the present invention relates to the improvement of stem cell maintenance or expansion cultures for auto- and allo-transplantation procedures or for gene transfer, as well as for in vivo treatments to improve such procedures. BACKGROUND OF THE INVENTION [0002] Most end-stage cells in renewing systems are short-lived and must be replaced continuously throughout life. For example, blood cells originate from a self-renewing population of multipotent hematopoietic stem cells (HSC). Hematopoietic stem cells are a subpopulation of hematopoietic cells. Hematopoietic cells can be obtained, for example, from bone marrow, umbilical cord blood or peripheral blood (either unmobilized or mobilized with an agent such as G-CSF); hematopoietic cells include the stem cell population, progenitor cells, differentiated cells, accessory cells, stromal cells and other cells that contribute to the environment necessary for production of mature blood cells. Hematopoietic progenitor cells are a subset of stem cells which are more restricted in their developmental potency. Progenitor cells are able to differentiate into only one or two lineages (e.g., BFU-E and CFU-E which give rise only to erythrocytes or CFU-GM which give rise to granulocytes and macrophages) while stem cells (such as CFU-MIX or CFU-GEMM) can generate multiple lineages and/or other stem cells. Because the hematopoietic stem cells are necessary for the development of all of the mature cells of the hematopoietic and immune systems, their survival is essential in order to reestablish a fully functional host defense system in subjects treated with chemotherapy or other agents. [0003] Hematopoietic cell production is regulated by a series of factors that stimulate growth and differentiation of hematopoietic cells, some of which, for example erythropoietin, GM-CSF and G-CSF, are currently used in clinical practice. One part of the control network which has not been extensively characterized, however, is the physiological mechanism that controls the cycling status of stem cells (Eaves et al., Blood 78:110-117, 1991; Lord in Stem Cells (C. S. Potten, Ed.), Academic Press, NY, pp. 401-422, 1997). [0004] Early studies by Lord and coworkers showed the existence of soluble protein factors in normal and regenerating bone marrow extracts which could either inhibit or stimulate stem cell proliferation (reviewed in Lord & Wright, Blood Cells 6:581-593, 1980; Wright & Lorimore, Cell Tissue Kinet. 20:191-203, 1987; Marshall & Lord, Intl. Rev. Cyt. 167:185-261, 1996). These activities were designated stem cell inhibitor (SCI) and stem cell stimulator (SCS), respectively. [0005] To date, no candidate SCS molecules have been purified from bone marrow extracts prepared as described by Lord et al. (reviews referenced above). Purification of either SCI or SCS from primary sources was not accomplished due to the difficulties inherent in an in vivo assay requiring large numbers of irradiated mice. In an attempt to overcome these problems Pragnell and co-workers developed an in vitro assay for primitive hematopoietic cells (CFU-A) and screened cell lines as a source of the inhibitory activity (see Graham et al., Nature 344:442-444, 1990). As earlier studies had identified macrophages as possible sources for SCI (Lord et al., Blood Cells 6:581-593, 1980), a mouse macrophage cell line J774.2 was selected (Graham et al., Nature 344:442-444, 1990). The conditioned medium from this cell line was used by Graham et al. for purification; an inhibitory peptide was isolated which proved to be identical to the previously described cytokine macrophage inflammatory protein 1-alpha (MIP-1.alpha.). Receptors for MIP-1.alpha. have been cloned; like other chemokine receptors, these MIP-1.alpha. receptors are seven-transmembrane domain (or "G-linked") receptors which are coupled to guanine nucleotide (GTP) binding proteins of the G.sub.inhibitory subclass ("G.sub.i") (reviewed in Murphy, Cytokine & Growth Factor Rev. 7:47-64, 1996). The "inhibitory" designation for the G.sub.i subclass refers to its inhibitory activity on adenylate cyclase. [0006] MIP-1.alpha. was isolated from a cell line, not from primary material. While Graham et al. observed that antibody to MIP-1.alpha. abrogated the activity of a crude bone marrow extract, other workers have shown that other inhibitory activities are important. For example, Graham et al. (J. Exp. Med. 178:925-932, 1993) have suggested that TGF.beta., not MIP-1.alpha., is a primary inhibitor of hematopoietic stem cells. Further, Eaves et al. (PNAS 90:12015-12019, 1993) have suggested that both MIP-1.alpha. and TGF.beta. are present at sub optimal levels in normal bone marrow and that inhibition requires a synergy between the two factors. [0007] Recently, mice have been generated in which the MIP-1.alpha.. gene has been deleted by homologous recombination (Cook et al., Science 269:1583-1585, 1995). Such mice have no obvious derangement of their hematopoietic system, calling into question the role of MIP-1.alpha. as a physiological regulator of stem cell cycling under normal homeostatic conditions. Similarly, although transforming growth factor beta (TGF.beta.) also has stem cell inhibitory activities, the long period of time it takes for stem cells to respond to this cytokine suggests that it is not the endogenous factor present in bone marrow extracts; further, neutralizing antibodies to TGF.beta. do not abolish SCI activity in bone marrow supernatants (Hampson et al., Exp. Hemat. 19:245-249, 1991). [0008] Other workers have described additional stem cell inhibitory factors. Frindel and coworkers have isolated a tetrapeptide from fetal calf marrow and from liver extracts which has stem cell inhibitory activities (Lenfant et al., PNAS 86:779-782, 1989). Paukovits et al. (Cancer Res. 50:328-332, 1990) have characterized a pentapeptide which, in its monomeric form, is an inhibitor and, in its dimeric form, is a stimulator of stem cell cycling. Other factors have also been claimed to be inhibitory in various in vitro systems (see Wright & Pragnell in Bailliere's Clinical Haematology 5:723-739, 1992 (Bailliere Tinadall, Paris); Marshall & Lord, Intl. Rev. Cyt. 167:185-261, 1996). [0009] Tsyrlova et al., SU 1561261 A1, disclosed a purification process for a stem cell proliferation inhibitor. [0010] Commonly owned applications WO 94/22915 and WO 96/10634 disclose an inhibitor of stem cell proliferation, and are hereby incorporated by reference in their entirety. [0011] To date, none of these factors have been approved for clinical use. However, the need exists for effective stem cell inhibitors. The major toxicity associated with chemotherapy or radiation treatment is the destruction of normal proliferating cells which can result in bone marrow suppression or gastrointestinal toxicity. An effective stem cell inhibitor will protect these cells and allow for the optimization of these therapeutic regimens. Just as there is a proven need for a variety of stimulatory cytokines (i.e., cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-I1, IL-13, IL-14, IL-15, G-CSF, GM-CSF, erythropoietin, thrombopoietin, stem cell factor, flk2/flt3 ligand, etc., which stimulate the cycling of hematopoietic cells) depending upon the clinical situation, so too it is likely that a variety of inhibitory factors will be needed to address divergent clinical needs. [0012] Further, there is a need to rapidly reverse the activity of such an inhibitor. The original studies of Lord et al. (reviews referenced above) demonstrated that the inhibitory activity could be reversed by addition of the stimulatory activity. While a variety of stem cell stimulatory cytokines has been identified (see above), none has been demonstrated to represent the activity described by Lord and coworkers as being present in bone marrow extracts and of being able to reverse the activity of the inhibitor. [0013] Hematopoietic progenitors and stem cells primarily reside in the bone marrow in normal adults. Under certain conditions, for example chemotherapy or treatment with cytokines such as G-CSF, large numbers of progenitors and stem cells egress into the peripheral blood, a process referred to as "mobilization" (reviewed in Simmons et al., Stem Cells 12 (suppl 1): 187-202, 1994; Scheding et al., Stem Cells 12 (suppl 1):203-211, 1994; Mangan, Sem. Oncology 22:202-209, 1995; Moolten, Sem. Oncology 22:271-290, 1995). Recent published data suggest that the vast majority of mobilized progenitors are not actively in cell cycle (Roberts & Metcalf, Blood 86:1600-1605, 1995; Donahue et al., Blood 87:1644-1653, 1996; Siegert & Serke, Bone Marrow Trans. 17:467-470, 1996; Uchida et al., Blood 89:465-472, 1997). [0014] Hemoglobin is a highly conserved tetrameric protein with molecular weight of approximately 64,000 Daltons. It consists of two alpha and two beta chains. Each chain binds a single molecule of heme (ferroprotoporphyrin IX), an iron-containing prosthetic group. Vertebrate alpha and beta chains were probably derived from a single ancestral gene which duplicated and then diverged; the two chains retain a large degree of sequence identity both between themselves and between various vertebrates (see FIG. 16A). In humans, the alpha chain cluster on chromosome 16 contains two alpha genes (alpha, and alpha.sub.2) which code for identical polypeptides, as well as genes coding for other alpha-like chains: zeta, theta and several non-transcribed pseudogenes (see FIG. 16B for cDNA and amino acid sequences of human alpha chain). The beta chain cluster on chromosome 11 consists of one beta chain gene and several beta-like genes: delta, epsilon, G gamma and A gamma, as well as at least two unexpressed pseudogenes (see FIG. 16C for cDNA and amino acid sequences of human beta chain). [0015] The expression of these genes varies during development. In human hematopoiesis, which has been extensively characterized, embryonic erythroblasts successively synthesize tetramers of two zeta chains and two epsilon chains (Gower I), two alpha chains and two epsilon chains (Gower II) or two zeta chains and two gamma chains (Hb Portland). As embryogenesis proceeds, the predominant form consists of fetal hemoglobin (Hb F) which is composed of two alpha chains and two gamma chains. Adult hemoglobin (two alpha and two beta chains) begins to be synthesized during the fetal period; at birth approximately 50% of hemoglobin is of the adult form and the transition is complete by about 6 months of age. The vast majority of hemoglobin (approximately 97%) in the adult is of the two alpha and two beta chain variety (Hb A) with small amounts of Hb F or of delta chain (Hb A.sub.2) being detectable. [0016] Several methods have been used to express recombinant hemoglobin chains in E. coli and in yeast (e.g., Sessen et al., Meth. Enzymol. 231:347-364, 1994; Looker et al., Meth. Enzymol. 231:364-374, 1994; Ogden et al., Meth. Enzymol. 231:374-390, 1994; Martin de Llano et al., Meth. Enzymol. 231:364-374, 1994). It has thus far not been possible to express isolated human alpha chain in high yields by recombinant methods (e.g., Hoffman et al., PNAS 87:8521-8525, 1990; Heman et al., Biochem. 31:8619-8628, 1992). Apparently, the isolated alpha chain does not assume a stable conformation and is rapidly degraded in E. coli. Co-expression of beta chain with alpha chain results in increased expression of both (Hoffman et al. and Hernan et al., op. cit.). While the alpha chain has been expressed as a fusion protein with a portion of the beta chain and a factor Xa recognition site (Nagai & Thorgersen, Meth. Enzymol. 231:347-364, 1994) it is expressed as an insoluble inclusion body under these conditions. [0017] Both the beta chain and the alpha chain contain binding sites for haptoglobin. Haptoglobin is a serum protein with extremely high affinity for hemoglobin (e.g., Putnam in The Plasma Proteins--Structure, Function and Genetic Control (F. W. Putnam, Ed.) 2:1-49 (Academic Press, NY); Hwang & Greer, JBC 255:3038-3041, 1980). Haptoglobin transport to the liver is the major catabolic pathway for circulating hemoglobin. There is a single binding site for haptoglobin on the alpha chain (amino acids 121-127) and two on the beta chain (amino acid regions 11-25 and 131-146) (Kazim & Atassi, Biochem J.197:507-510, 1981; McCormick & Atassi, J. Prot Chem. 9:735-742, 1990). [0018] Biologically active peptides with opiate activity have been obtained by proteolytic degradation of hemoglobin (reviewed in Karelin et al., Peptides 16:693-697, 1995). Hemoglobin alpha chain has an acid-labile cleavage site between amino acids 94-95 (Shaeffer, J. Biol. Chem. 269:29530-29536, 1994). [0019] Kregler et al. (Exp. Hemat. 9:11-21, 1981) have disclosed that hemoglobin has an enhancing activity on mouse bone marrow CFU-C progenitor colonies. Such assays demonstrate effects on CFU-GM and CFU-M progenitor populations as opposed to stem cells such as CFU-MIX. The authors observed activity in both isolated alpha and beta chains of hemoglobin. This activity was abolished by treatment with N-ethylmaleimide, which suggested to Kregler et al. that sulfhydryl groups were necessary. This observation, coupled with the fact that the stimulatory activity was resistant to trypsin digestion, suggested to Kregler et al. that the C-terminal hydrophobic domain or "core" region was responsible for the activity. Moqattash et al. (Acta. Haematol. 92:182-186, 1994) have disclosed that recombinant hemoglobin has a stimulatory effect on CFU-E, BFU-E and CFU-GM progenitor cell number which is similar to that observed with hemin. Mueller et al. (Blood 86:1974, 1995) have disclosed that purified adult hemoglobin stimulates erythroid progenitors in a manner similar to that of hemin. [0020] Petrov et al. (Bioscience Reports 15:1-14, 1995) disclosed the use of a "nonidentified myelopeptide mixture" in the treatment of congenital anemia in the W.sup.v/W.sup.v mouse. The mixture increased the number of spleen colonies, especially those of the erythroid type. [0021] Heme and hemin have been extensively examined with regard to their influences on hematopoiesis (see Sassa, Semin. Hematol. 25:312-320, 1988 and Abraham et al., Intl. J. Cell Cloning 9:185-210, 1991 for reviews). Heme is required for the maturation of erythroblasts; in vitro, hemnin (chloroferroprotoporphyrin IX--i.e., heme with an additional chloride ion) increases the proliferation of CFU-GEMM, BFU-E and CFU-E. Similarly, hemin increases cellularity in long-term bone marrow cultures. [0022] "Opiates" are substances with analgesic properties similar to morphine, the major active substance in opium. Opiates can be small organic molecules, such as morphine and other alkaloids or synthetic compounds, or endogenous peptides such as enkephalins, endorphins, dynorphins and their synthetic derivatives. Endogenous opiate peptides are produced in vivo from larger precursors--pre-proenkephalin A for Met- and Leu-enkephalins, pre-proopiomelanocortin for .alpha., .beta., and .gamma. endorphins, and pre-prodynorphin for dynorphins A and B, .alpha.-neoendorphin and .beta.-neoendorphin. In addition, peptides with opiate activity can be obtained from non-classical sources such as proteolysis or hydrolysis of proteins such as .alpha. or .beta. casein, wheat gluten, lactalbumin, cytochromes or hemoglobin, or from other species such as frog skin (dermorphins) or bovine adrenal medulla. Such peptides have been termed "exorphins" in contrast to the classical endorphins; they are also referred to as atypical opiate peptides (Zioudrou et al., JBC 254:2446-2449, 1979; Quirion & Weiss, Peptides 4:445-449, 1983; Loukas et al., Biochem. 22:4567-4573, 1983; Brantl, Eur. J. Pharmacol. 106:213-214, 1984; Brantl et al., Eur. J. Pharmacol. 111:293-294, 1985; Brand et al., Eur. J. Pharmacol. 125:309-310, 1986; Brantl & Neubert, TIPS 7:6-7,1986; Glamsta et al., BBRC 184:1060-1066, 1992; Teschemacher, Handbook Exp. Pharmacol. 104:499-428, 1993; Petrov et al., Bioscience Reports 15:1-14, 1995; Karelin et al., Peptides 16:693-697, 1995). Other endogenous peptides, such as the Tyr-MIF-1 family, have also been shown to have opiate activity (Reed et al., Neurosci. Biobehav. Rev. 18:519-525, 1994). Continue reading... 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