Methods to enhance delivery and engraftment of stem cells including the identification of specific prostaglandin E2 receptors

Abstract

The receptor EP.sub.4 is identified as the PGE.sub.2 receptor that is most responsible enhancing the homing and engraftment of hematopoietic stem and progenitor cells. Treatment of graft sources and graft recipients with compounds that preferentially target the EP.sub.4 receptor provide effective methods of increasing engraftment success while minimizing adverse side effects that may be associated with therapies that include the use of less selective molecules such as PGE.sub.2 and dmPGE.sub.2. One effective molecule used in such therapies is 5-[(1E,3R)-4,4-difluoro-3-hydroxy-4-phenyl-1-buten-1-yl]-1-[6-(2H-tetrazol-5R-yl)hexyl]-2-pyrrolidinone or a pharmaceutically acceptable salt thereof (L-902, 688).

Claims

1. A method of increasing homing of a hematopoietic stem cell or hematopoietic progenitor cell, comprising the steps of: (a) contacting a hematopoietic stem cell or hematopoietic progenitor cell expressing at least one PGE2 EP4 receptor with at least one compound or a pharmaceutically acceptable salt thereof such that homing potential is increased in the contacted hematopoietic stem cell or hematopoietic progenitor cell, wherein the at least one compound or the pharmaceutically acceptable salt thereof: (i) preferentially interacts with the PGE2 EP4 receptor as compared to the PGE2 EP1, EP2, and EP3 receptors, and (ii) increases the homing of the contacted hematopoietic stem cell or hematopoietic progenitor cell when transplanted into a patient compared to a compound that does not preferentially interact with the PGE2 EP4 receptor, and (b) washing the contacted hematopoietic stem cell or hematopoietic progenitor cell, and wherein the amount of the compound contacting said hematopoietic stem cell or hematopoietic progenitor cell is about 0.001-10 microMolar.

2. The method according to claim 1, wherein said hematopoietic stem cell or hematopoietic progenitor cell is isolated from marrow, umbilical cord or peripheral blood.

3. The method according to claim 1, wherein contacting the compound with said hematopoietic stem cell or hematopoietic progenitor cell increases the homing of said cells.

4. The method according to claim 1, wherein contacting the compound with said hematopoietic stem cell or hematopoietic progenitor cell increases the engrafting potential of said cells.

5. The method according to claim 1, wherein the at least one compound is selected from the groups consisting of: 2-[3-[(1R,2S,3R)-3-hydroxy-2-[(E,3S)-3- hydroxy-5-[2-(methoxymethyl)phenyl-]pent-l-enyl]-5-oxocyclopentyl]sulfanylpropylsulfanyl]acetic acid; methyl 4-[2-[(1R,2R,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-4-[3-(methoxymethyl)phenyl-]but-1-enyl]-5-oxocyclopentyl]ethylsulfanyl]butanoate; 16-(3-Methoxymethyl)phenyl-w-tetranor-5-thiaPGE; 543-[(2S)-2-{(3R)-3-hydroxy-4-[3-(trifluoromethyl)phenyl]butyl}-5-oxopyrrolidin-1-yl]propyl]thiophene-2-carboxylate; [4′-[3-butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1,5-dihydro-[1,2,4]triazol-4-ylmethyl]-biphenyl-2-sulfonic acid (3-methyl-thiophene-2-carbonyl)-amide]; and ((Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-p-ent-l-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoic acid) or a pharmaceutically acceptable salt thereof.

6. The methods according to claim 1, wherein the at least one compound is 5-[(1E,3R)-4,4-difluoro-3-hydroxy -4-phenyl-1-buten-1-yl]-1-[6-(2H-tetrazol-5Ryl) hexyl]-2-pyrrolidinone or a pharmaceutically acceptable salt thereof.

7. The method according to claim 1, wherein said cell is contacted with said compound or pharmaceutically acceptable salt thereof for 15 minutes to 6 hours.

8. The method according to claim 1, further comprising administering the washed cell to the patient.

9. A method of increasing homing of a hematopoietic stem cell or hematopoietic progenitor cell, comprising the steps of: (a) contacting about 1-10 million hematopoietic stem cells or hematopoietic progenitor cells expressing at least one PGE2 EP4 receptor with at least one compound or a pharmaceutically acceptable salt thereof such that homing potential is increased in the contacted hematopoietic stem cell or hematopoietic progenitor cell, wherein the compound or the pharmaceutically acceptable salt thereof: (i) preferentially interacts with the PGE2 EP4 receptor as compared to PGE2 EP1, EP2, and EP3 receptors, and (ii) increases the homing potential of the contacted hematopoietic stem cell or hematopoietic progenitor cells compared to a compound that does not preferentially interact with the PGE2 EP4 receptor, and (b) washing the contacted hematopoietic stem cell or hematopoietic progenitor cell, and wherein the amount of the compound contacting said hematopoietic stem cells or hematopoietic progenitor cells is about 0.001-10 microMolar.

10. The method of claim 1, wherein the contacted hematopoietic stem cell or hematopoietic progenitor cell has increased engraftment when transplanted into a patient compared to a non-contacted hematopoietic stem cell or hematopoietic progenitor cell.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Graph illustrating that treatment of cells with EP.sub.4 agonist ex vivo specifically up regulates CXCR4 expression on CD34.sup.+ cells.

(2) FIG. 2. Graph illustrating that signaling via the EP.sub.4 receptor is responsible for up regulation of CXCR4 expression.

DESCRIPTION

(3) For the purposes of promoting an understanding of the principles of the novel technology, reference will now be made to the preferred embodiments thereof, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the novel technology is thereby intended, such alterations, modifications, and further applications of the principles of the novel technology being contemplated as would normally occur to one skilled in the art to which the novel technology relates.

(4) Unless stated otherwise the term, “therapeutically effective amount” refers to an amount of a pharmaceutically active compound that when administered to a human being or an animal patient or to a cell or collection of cells either alone or in combination with other pharmaceutically active ingredients or other components of medicaments that have a desirable effect on the physiological condition of a patient or the cell or collection of cells.

(5) Therapeutically effective, beneficial or efficacious doses of various compounds that preferentially bind to PGE.sub.2 EP.sub.4 receptors administered in vivo to either a human or an animal patient are in the range of between about 0.1 mg of the compound per Kg of body weight of the patient per day to about 100 mg of the compound per Kg of body weight of the patient per day.

(6) Compounds that preferentially bind to PGE.sub.2 EP.sub.4 receptors are compounds that have a higher affinity for the EP.sub.4 receptor than for any of the other three EP receptors namely EP.sub.1, EP.sub.1and EP.sub.3.

(7) Compounds that can be used to practice some embodiments of the invention include, but are not limited to, the following: 5-[(1E,3R)-4,4-difluoro-3-hydroxy-4-phenyl-1-buten-1-yl]-1-[6-(2H-tetrazol-5R-yl)hexyl]-2-pyrrolidinone also referred to as L-902,688 (Young, et al., 2004); 2-[3-[(1R,2S,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-5-[2-(methoxymethyl)phenyl]pent-1-enyl]-5-oxocyclopentyl]sulfanylpropylsulfanyl]acetic acid also referred to as ONO-AE1-329 (Suzawa et al., 2000); methyl 4-[2-[(1R,2R,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-4-[3-(methoxymethyl)phenyl]but-1-enyl]-5-oxocyclopentyl]ethylsulfanyl]butanoate also referred to as ONO-4819 (Maruyama et al., 2002; Ohta et al., 2009); 16-(3-Methoxymethyl)phenyl-ω-tetranor-5-thiaPGE.sub.1 (Maruyama et al., 2002); 5-{3-[(2S)-2-{(3R)-3-hydroxy-4[3-(trifluoromethyl)phenyl]butyl}-5-oxopyrrolidin-1-yl]propyl]thiophene-2-carboxylate also referred to as PF-04475270 (Luu et al., 2009); APS-999 Na (El-Nefiawy et al., 2005); [4′-[3-butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1,5-dihydro-[1,2,4]triazol-4-ylmethyl]-biphenyl-2-sulfonic acid (3-methyl-thiophene-2-carbonyl)-amide](Machwate et al., 2001); and ((Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoic acid) U. S. Patent application number US2005/0164992A1, Jul. 28, 2005, to Donde Y, Nguyen J H, Kedzie K M, Gil D M, Donello J E and Im W B.

(8) Unless stated otherwise the term “about” as used herein refers to range of value of plus or minus 10%, e.g., ‘about 1.0’ includes values between 0.9 and 1.1.

(9) Treatment of bone marrow cells, umbilical cord blood cells, mobilized peripheral blood cells or any hematopoietic cell graft to be used for hematopoietic transplantation with Prostaglandin E.sub.2 (PGE.sub.2) or any active analogue or metabolite of PGE.sub.2 or any E series prostaglandin with specificity for the PGE.sub.2 EP.sub.4 receptor, improves the homing, survival and proliferation of the transplanted hematopoietic stem cells. This treatment can be used to increase stem cell engraftment rates and thereby improve the efficiency of hematopoietic stem cell transplantation.

(10) By some estimates the success of peripheral blood stem cell transplantation requires administration of approximately 2 million CD34.sup.+ cells per kilogram of recipient patient body weight. Any agent, combination of agents or manipulations that increases the number of stem cells that can be collected, enhances their survival rates, enhances their ability to home to the appropriate marrow environment and/or enhances their self-renewal and proliferation rates will likely have a positive impact on the efficacy of hematopoietic transplantation procedures. The success of these procedures may be measured in terms of reduced patient morbidity and mortality. Numerous studies have been undertaken to try and expand the number of human hematopoietic stem cells within isolated grafts in ex vivo settings, with limited success (Broxmeyer, 2006; Haylock and Nilsson, 2007). Recently, the CXCR4 antagonist AMD3100 has been shown to enhance mobilization of stem cells (Broxmeyer, et al., 2005; Liles, et al., 2003) and in clinical trials, (Plerixafor; Mozibil) has been shown to enhance collection of mobilized stem cells when used in combination with G-CSF (DiPersio et al., 2007b; DiPersio et al., 2007a). Truncation of chemokines has been used as a method to enhance the body's ability to mobilize stem cells. Some of these methods have been patented, e.g., U.S. Pat. Nos. 6,080,398; 6,447,766B1; 639053B1; 6,713,052, each of which is incorporated by reference in its entirety. Their ability to more efficiently mobilize stem cells has also been reported (King, et al., 2001; Pelus, et al., 2004). A role for blocking the activity of a surface peptidase (CD26) has been reported as a method for enhancing the homing of hematopoietic stem cells (Christopherson, et al., 2004).

(11) A number of agents when used in combination with G-CSF have been reported to increase the number of hematopoietic progenitor cells that can be recovered (Pelus and Fukuda, 2007; Herbert, et al., 2007), however, the ability of these agents to mobilize the long-term repopulating stem cells, i.e., the stem cells with self-renewal activity, has not been uniformly demonstrated. A recent study has shown that pulse exposure of mouse bone marrow cells to 16,16 dimethyl pGE.sub.2 (dmpGE.sub.2) enhances engraftment of hematopoietic stem cells, however this study provides no evidence of mechanism of action and specifically states that the effect of PGE.sub.2 is not on cell homing (North, et al., 2007). It was unexpectedly demonstrated by Hoggatt, et al., 2009, that PGE.sub.2 increases the CXCR4 receptor on hematopoietic stem and progenitor cells, and that this increase is responsible for increasing the homing to the bone marrow niche, resulting in a subsequent increase in engraftment.

(12) It is generally believed that PGE.sub.2 interacts with 4 highly conserved G-protein coupled receptors (GPCR); EP.sub.1, EP.sub.2, EP.sub.3, and EP.sub.4 that account for the multiple, sometimes opposing effects attributed to PGE.sub.2 (Breyer, et al., 2001). EP receptor expression levels vary among different tissues, with EP.sub.3 and EP.sub.4 mRNA being most abundant (Sugimoto and Narumiya, 2007a) and EP.sub.2 mRNA expressed at lower levels than EP.sub.4 in most tissues (Katsuyama, et al., 1995). EP.sub.1 activates phospholipase C (PLC) via an unidentified G protein (Tsuboi, et al., 2002), which increases intracellular Ca.sup.2+ coupled to inositol phosphates resulting in activation of phosphokinase C (PKC) (Breyer, et al., 2001). EP.sub.3 receptor ligation results in inhibition of adenylate cyclase and decreased cAMP that is Gα.sub.i linked (Sugimoto, et al., 2007). Multiple EP.sub.3 splice variants have been identified and depending on C-terminal splicing, they can couple to multiple G proteins (Namba, et al., 1993). EP.sub.2 and EP.sub.4 both couple to G.sub.αs leading to adenylate cyclase activation and increased cAMP, activating protein kinase A (PKA), as well as Rap1, Rac 1, and PKCζ (PKC zeta), a unique isoform implicated in HSC function (Goichberg, et al., 2006). EP.sub.2 and EP.sub.4 are thought to have partially redundant roles in some systems, while in others they play distinct roles (Sugimoto and Narumiya, 2007). EP.sub.4 but not EP.sub.2 can activate the PI3K/Akt pathway in addition to adenylate cyclase (Fujino, et al., 2003). EP.sub.4 has a longer cytosolic domain allowing for more ligand dependent phosphorylation and more rapid desensitization (Nishigaki, et al., 1996) enabling a selective negative feedback loop (Sugimoto and Narumiya, 2007). Lastly, EP.sub.4 is internalized when activated, while EP.sub.2 is not (Desai, et al., 2000). As a consequence, EP.sub.2 and EP.sub.4 can have different roles based upon continuation or attenuation of signals generated by receptor activation (Breyer, et al., 2001). Treating with PGE.sub.2 often exhibits a “bell-shaped” dose-response curve suggesting a different repertoire of EP receptors is activated dependent upon PGE.sub.2 concentration (Hull, et al., 2004).

(13) Most current strategies to improve hematopoietic transplantation utilizing prostaglandin have used either native PGE.sub.2 or a long acting derivative of PGE.sub.2, 16,16 dimethylprostaglandin E.sub.2 (dmPGE.sub.2). These prostaglandin compounds are thought to activate all 4 EP receptors leading to the numerous downstream signaling events briefly described above. As demonstrated herein, the enhancement in homing and engraftment of hematopoietic stem and progenitor cells is due to up regulation of the CXCR4 receptor by treatment with prostaglandin. Specifically focusing on the EP receptor that is responsible for the increase in CXCR4, (the EP.sub.4 receptor) has the benefit of enhancing the grafting process without activating receptors that may be detrimental to the engraftment process and/or have other unknown possible deleterious consequences.

(14) Treating With An E4 Selective Agonist Affects Homing and Engraftment Efficiency

(15) Un-manipulated hematopoietic grafts or purified hematopoietic stem cell populations (e.g., SKL cells in mice or CD34.sup.+ cells in humans) are incubated with an EP.sub.4 specific agonist: 5-[(1E,3R)-4,4-difluoro-3-hydroxy-4-phenyl-1-buten-1-yl]-1-[6-(2H-tetrazol-5R-yl)hexyl]-2-pyrrolidinone (i.e. L-902,688), on ice or at room temperature at concentrations of 0.001-10 microMolar agonist per 1-10 million cells in 1 ml of culture medium, e.g. IMDM, for 15 minutes-6 hrs. After incubation, the cells are washed 3 times in sterile media or saline and administered to recipients, intravenously. L-902,688 was a generous gift from Merck Frosst (Kirkland, Canada) (Young, et al., 2004).

(16) Referring now to FIG. 1. This graph illustrates that treatment of cells with EP.sub.4 agonist ex vivo specifically up regulates CXCR4 expression on CD34.sup.+ cells. The insert shows cytometry histograms of CD34.sup.+ cells from cord blood samples showing significant up regulation of CXCR4 on the surface of CD34.sup.+ cells after pulse exposure to EP.sub.4 agonist (light line) and dmPGE.sub.2 (dark line) compared to isotype control (grey shaded area). The bar graph indicator data measured from such experiments graphed as a function of different compounds used dmPGE.sub.2, Butaprostone, sulprostone and EP.sub.4.

(17) Referring now to FIG. 2. This graph illustrates that signaling via the EP.sub.4 receptor is responsible for up regulation of CXCR4 expression. The insert shows data for 3 cord blood samples. Treatment with EP.sub.4 agonist ex vivo up regulates CXCR4 expression on CD34.sup.+ cells. The bar chart shows combined data for 8 samples. The bar chart data demonstrates that ex vivo treatment with EP.sub.4 agonist up regulates CXCR4 by about 3 fold. In the bar chart this effort is normalized to 100%. Pretreatment of cells with a specific antagonist of the EP 1, 2 and 3 receptor prior to exposure to EP.sub.4 antagonist had no effect on up regulation of CXCR4 whereas pre-incubation with a selective EP.sub.4 antagonist prior to exposure to EP.sub.4 agonist significantly blocked up regulation of CXCR4. The fact that up regulation of CXCR4 by an EP.sub.4 selective agonist is blocked by a selective EP.sub.4 antagonist, but not by EP1, 2, 3, antagonist, provides additional evidence that up regulation is mediated through the EP.sub.4 receptor.

(18) Additional embodiments include administering EP.sub.4 agonists (e.g., on the order of about 0.001-10 microMolar) to patients immediately prior to and daily afterwards after receiving a hematopoietic graft as a means of enhancing stem cell function. Therapeutic effective doses are amounts of the pharmaceutically active agent used either alone or in combination with another pharmaceutical agent or inert material that has a beneficial effect on a so treated human or animal patient. Such benefits may include, but are not limited to, increasing the effectiveness of other steps in a given treatment regimen.

(19) While the novel technology has been illustrated and described in detail in the figures and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the spirit of the novel technology are desired to be protected. As well, while the novel technology was illustrated using specific examples, theoretical arguments, accounts, and illustrations, these illustrations and the accompanying discussion should by no means be interpreted as limiting the technology. All patents, patent applications, and references to texts, scientific treatises, publications, and the like referenced in this application are incorporated herein by reference in their entirety.

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